EFFECT OF SOLVENT, IONIC STRENGTH AND

EFFECT OF SOLVENT IONIC STRENGTH AND

METAL IONS ON THE PHOTOLYSIS OF RIBOFLAVIN

AND ITS NANOPARTICLES

Thesis

Presented by

Zubair Anwar Pharm D M Phil (BMU) R Ph

for the degree of

Doctor of Philosophy

Baqai Medical University

Department of Pharmaceutical Chemistry

Faculty of Pharmaceutical Sciences

Baqai Medical University Karachi

Pakistan June 2017

AUTHORS DECLARATION

I Zubair Anwar hereby state that my PhD thesis titled ldquoEffect of Solvent Ionic

Strength and Metal Ions on the Photolysis of Riboflavin and its Nanoparticlesrdquo is my own

work and has not been submitted previously by me for taking any degree from Baqai

Medical University or anywhere else in the countryworld

At any time if my statement is found to be incorrect even after my Graduation the

university has the right to withdraw my PhD degree

Name of Student Zubair Anwar

PLAGIARISM UNDERTAKING

I solemnly declare that the research work presented in the thesis titled ldquoEffect of

Solvent Ionic Strength and Metal Ions on the Photolysis of Riboflavin and its Nanoparticlesrdquo

is solely my research work with no significant contribution from any other person Small

contributionhelp wherever taken has been duly acknowledged and that complete thesis

has been written by me

I understand the zero tolerance policy of the HEC and Baqai Medical University

towards plagiarism Therefore I as an Author of the above titled thesis declare that no

portion of my thesis has been plagiarized and any material used as reference is properly

referred cited

I undertake that if I am found guilty of any formal plagiarism in the above titled

thesis even after the award of PhD degree the University reserves the rights to withdraw

revoke my PhD degree and that HEC and the University has the right to publish my

name on the HEC University website on which names of students are placed who

submitted plagiarized thesis

Student Author Signature

Name Zubair Anwar

CERTIFICATE OF APPROVAL

This is to certify that the research work presented in this thesis entitled ldquoEffect of

Solvent Ionic Strength and Metal Ions on the Photolysis of Riboflavin and its Nanoparticlesrdquo was conducted by Mr Zubair Anwar under the supervision of Prof Dr Iqbal Ahmad

No part of this thesis has been submitted anywhere else for any other degree This

thesis is submitted to the Department of Pharmaceutical Chemistry Baqai Institute of

Pharmaceutical Sciences Baqai Medical University in partial fulfillment of the

requirements for the degree of Doctor of Philosophy in the field of Pharmaceutical

Chemistry Department of Pharmaceutical Chemistry Baqai Institute of

Pharmaceutical Sciences Baqai Medical University Karachi

Student Name Zubair Anwar Signature ___________

Examination Committee

a) External Examiner 1 Name Signature ___________

(Designation amp Office Address)

_____________________________

b) External Examiner 2 Name Signature ___________

_____________________________

c) Internal Examiner Name Signature ___________

_____________________________

Supervisor Name _______________________ Signature ___________

Name of DeanHOD _____________________ Signature ___________

ABSTRACT

The present investigation is based on the study of the evaluation of the following

factors on the photolysis of riboflavin (RF) in aqueousorganic solvents

1 Solvent Effect on the Photolysis of RF

The kinetics of photolysis of RF in water (pH 70) and in organic solvents

(acetonitrile methanol ethanol 1-propanol 1-butanol ethyl acetate) has been studied

using a multicomponent spectrometric method for the assay of RF and its major

photoproducts formylmethylflavin and lumichrome The apparent first-order rate

constants (kobs) for the reactions range from 319 (ethyl acetate) to 461times10minus3

minminus1

(water) The values of kobs have been found to be a linear function of solvent dielectric

constant implying the participation of a dipolar intermediate along the reaction pathway

The degradation of this intermediate is enhanced by the polarity of the medium This

indicates a greater stabilization of the excited-triplet state of RF with an increase in

solvent polarity to facilitate its photoreduction The rate constants for the reaction show a

linear relation with the solvent acceptor number showing the magnitude of solutendashsolvent

interaction in different solvents It would depend on the electronndashdonating capacity of the

RF molecule in organic solvents The values of kobs are inversely proportional to the

viscosity of the medium as a result of diffusion-controlled processes

2 Ionic Strength Effects on the Photodegradation Reactions of RF

It involves the study of the effect of ionic strength on the photodegradation

reactions (photoreduction and photoaddition) of RF in phosphate buffer (pH 70) using

the specific multicomponent spectrometric method mentioned above The rates of

photodegradation reactions of RF have been found to be dependent upon the ionic

strength of the solutions at different buffer concentrations The values of kobs for the

photodegradation of RF at ionic strengths of 01ndash05 M (05 M phosphate) lie in the range

of 735ndash3032 times 10minus3

minminus1

Under these conditions the rate constants for the formation

of the major products of RF lumichrome (LC) by photoreduction pathway and

cyclodehydroriboflavin (CDRF) by photoaddition pathway are in the range of 380ndash

1603 and 170ndash607 times 10minus3

minminus1

respectively A linear relationship has been observed

between log kobs and radicμ1+radicμ A similar plot of log kko against radicμ yields a straight line

with a value of ~+1 for ZAZB indicating the involvement of a charged species in the rate

determining step NaCl promotes the photodegradation reactions of RF probably by an

excited state interaction The implications of ionic strength on RF photodegradation by

different pathways and flavinndashprotein interactions have been discussed

3 Metal Ion Mediated Photolysis of RF

The effect of metal ion complexation on the photolysis of RF using various metal

ions (Ag+ Ni

3+) has been

studied Ultraviolet and visible spectral and fluorimetric evidence has been obtained to

confirm the formation of metal-RF complexes The kinetics of photolysis of RF in metal-

RF complexes at pH 70 has been evaluated and the values of kobs for the photolysis of RF

and the formation of LC and LF (0001 M phosphate buffer) and LC LF and CDRF

(02ndash04 M phosphate buffer) have been determined These values indicate that the rate of

photolysis of RF is promoted by divalent and trivalent metal ions The second-order rate

constants (kprime) for the interaction of metal ions with RF are in the order Zn

2+ gt Mg

gt Mn2+

gt Cu2+

gt Cd2+

gt Fe2+

gt Ca2+

gt Fe3+

gt Co2+

gt Ni2+

gt Ag+ In phosphate buffer

(02-04 M) an increase in metal ion concentration leads to a decrease in the formation of

LC compared to that of CDRF by different pathways The values of kobs for the photolysis

of RF have been found to increase with a decrease in fluorescence intensity of RF The

photoproducts of RF formed by pathways have been identified and the mode of

photolysis of RF in metal-RF complexes has been discussed

4 Preparation Characterization and Formation Kinetics of RF-Ag NPs

Riboflavin conjugated silver nanoparticles (RFndashAg NPs) have been prepared by

photoreduction of Ag+ ions and characterized by UVndashvisible spectrometry

spectrofluorimetry dynamic light scattering (DLS) atomic force microscopy (AFM) and

FTIR spectrometry These NPs exhibit a surface plasmon resonance (SPR) band at 422

nm due to the interaction of RF and Ag+ ions The fluorescence of RF is quenched by Ag

NPs and the total loss of fluorescence is due to complete conversion of RF to RFndashAg NPs

conjugates FTIR studies indicate the appearance of an intense absorption peak at

2920 cmndash1

due to the interaction of RF and Ag DLS has shown the hydrodynamic radii

(Hd) of RFndashAg NPs in the range of 579ndash722 nm with polydispersity index of 275ndash290

AFM indicates that the NPs are spherical in nature and polydispersed with a diameter

ranging from 57 to 73 nm The effect of pH ionic strength and reducing agents on the

particle size of NPs has been studied At acidic pH (20ndash62) aggregation of RFndashAg NPs

occurs due to an increase in the ionic strength of the medium The rates of formation of

RFndashAg NPs on UV and visible light irradiation have been determined in the pH range of

80ndash105 and at different concentration of Ag+ ions The photochemical formation of RFndash

Ag NPs follows a biphasic firstndashorder reaction probably due to the formation of Ag NPs

in the first phase (fast) and the adsorption of RF on Ag NPs in the second phase (slow)

ACKNOWLEDGEMENTS

ldquoO My Lord Increase Me in My Knowledgerdquo

ldquoO Allah I Ask You for Knowledge that is of Benefitrdquo

(Quran 20114)

I am highly thankful to ALLAH ALL MIGHTY who gave me courage in all

difficulties and provided me strength to overcome the problems during this work

All and every kind of respect to the prophet Hazrat Muhammad (صلى الله عليه وسلم) for

complete and endless guidance and knowledge

Words are limited and are inoperative to express my gratitude to my dignified

supervisor Prof Dr Iqbal Ahmad TI Department of Pharmaceutical Chemistry for his

supervision keen interest and above all giving his valuable time throughout the course of

this work His personality and individuality has been a source of permanent motivation

throughout my study period and research work He not only groomed me with his

valuable suggestions and moral support but also guided me at every step during my

research work My deepest regards are due for his time and efforts

I am highly thankful to Professor Dr Syed Fazal Hussain CEO and Professor

Dr Shaukat Khalid Dean Faculty of Pharmaceutical Sciences for providing me an

opportunity to be a part of their organization and to complete my degree in this

institution

I am very thankful to Professor Dr Moinudin (Late) for providing me the

materials for this study

I am very thankful to Associate Professor Dr Sofia Ahmad Chairperson

Department of Pharmaceutics Associate Professor Dr Muhammad Ali Sheraz

Chairman Department of Pharmacy Practice for their encouragement innovative ideas

and support during this work

I am highly thankful to Professor Dr Syed Abid Ali and Professor Dr Raza

Shah International Center for Chemical and Biological Sciences HEJ Research Institute

of Chemistry for their guidance and help in my research work

I acknowledge with sincere thanks to Associate Professor Dr Kiran Qadeer

Chairperson Department of Pharmaceutical Chemistry Associate Professor Dr Raheela

Bano and Associate Professor Dr Adeel Arsalan Department of Pharmaceutics for their

kind support in my Ph D studies

I am thankful to Ms Tania Mirza Ms Saima Zahid Ms Sadia Kazi Ms Sadia

Ahmed Zuberi Ms Nafeesa Mustan Ms Marium Fatima Khan Ms Qurat-e-Noor

Baig and Mr Muhammad Ahsan Ejaz for their moral support

I am very grateful to Mrs Professor Dr Iqbal Ahmad for her affection during my

visits which gives me motivation to do hard work and to be consistent

I feel prodigious contentment to pay my sincere and exclusive benediction to

Ms Adeela Khurshid and Aqeela Khurshid for their moral and ethical support

I am highly thankful to Mr Syed Haider Abbas Naqvi Mr Shahzaib

Ms Samina Sheikh Ms Perveen Nawaz Ms Syeda Mahwish Kazmi Ms Laiba

Saleem Sultan Ms Laraib Saleem Sultan Ms Kinza Khan Ms Zuni and Ms Nazia

Ishaque for their love care and support

I am thankful to Mr Sajjad Ali Mr Anees Hassan Mr Wajahat Mr Mohsin

Ali and Mr Azharuddin for providing their technical services during my research work

In the last but not the least I would like to thank and express my gratitude to My

Father (Muhammad Anwar) Late Mother (Gul) Beloved Brother (Zeeshan

Anwar) Sisters (Shahbana Anwar and Rizwana Anwar) Sister-in-Law (Bushra

Ejaz) my Nephews (Musa Alam Essa Alam and Hassan Alam) and my Nieces

(Inshrah Hamna Anushay Aymen) for their moral support kindness and

encouragement throughout my life

To my beloved parents

and my niece

Anushay Zeeshan

CONTENTS

Chapter Page

ABSTRACT vi

ACKNOWLEDGEMENTS ix

I INTRODUCTION

11 INTRODUCTION 2

12 BIOCHEMICAL IMPORTANCE 2

13 CHEMICAL STRUCTURE OF RIBOFLAVIN 5

14 PHYSICAL PROPERTIES OF RIBOFLAVIN 7

15 CLINICAL USES 8

16 ABSORPTION FATE AND EXCRETION 9

17 THERAPEUTIC USES 10

18 PHARMACOKINETICS 10

19 LITERATURE ON RIBOFLAVIN 11

II ANALYTICAL METHODS USED FOR THE

DETERMINATION OF RIBOFLAVIN

21 SPECTROPHOTOMETRIC METHOD 13

211 UV-visible Spectrometry 13

212 Spectrofluorimetric Method 17

213 Infrared Spectrometry 23

214 Mass Spectrometry 23

22 CHROMATOGRAPHIC METHODS 25

221 High-Performance Liquid Chromatography (HPLC) 25

222 Liquid Chromatography 30

223 Ion Chromatography 31

23 ELECTROCHEMICAL METHODS 32

24 PHOTOCHEMICAL METHODS 34

25 ENZYMATIC ASSAY 35

26 FLOW INJECTION ANALYSIS (FIA) METHOD 36

III PHOTOCHEMISTRY OF RIBOFLAVIN

31 INTRODUCTION 38

32 ANAEROBIC PHOTOREACTIONS 39

33 AEROBIC PHOTOREACTIONS 42

34 TYPES OF PHOTOCHEMICAL REACTIONS 43

341 Photoreduction 43

3411 Intramolecular photoreduction 43

3412 Intermolecular photoreduction 46

342 Photodealkylation 50

343 Photoaddition Reactions 51

344 Photooxidation 52

345 Photosenstization Reactions 52

346 Photostabilisation Reactions 57

347 Factors Affecting Photochemical Reactions of Riboflavin 59

3471 Radiation source 59

3472 pH effect 60

3473 Buffer effect 61

3474 Effect of complexing agents 63

3475 Effect of quenchers 66

3476 Effect of solvent 67

3477 Effect of ionic strength 68

3488 Effect of formulation 68

IV INTRODUCTION TO NANOPARTICLES AND

APPLICATIONS TO RIBOFLAVIN

41 INTODUCTION 71

42 RIBOFLAVIN AND NANOTECHNOLOGY 73

421 Photosenstizer 73

422 Stabilizer 74

423 Photoluminescent 74

424 Biosensor 76

425 Target Drug Delivery 79

426 Photochemical Interaction 80

427 Colorimetric Sensor 82

OBJECT OF PRESENT INVESTIGATION 83

PROPOSED PLAN OF WORK 84

V MATERIALS AND METHODS

51 MATERIALS 86

52 REAGENTS 88

53 METHODS 89

531 Thin-Layer Chromatography (TLC) 89

532 pH Measurements 90

533 Fourier Transform Infrared (FTIR) Spectrometry 90

534 Ultraviolet and Visible Spectrometry 92

535 Fluorescence Spectrometry 92

536 Dynamic Light Scattering (DLS) 93

537 Atomic Force Microscopy (AFM) 93

538 Photolysis of Riboflavin Solutions 94

5381 Choice of reaction vessel 94

5382 Choice of radiation source 94

539 Methods of Photolysis of Riboflavin 96

5391 Photolysis in aqueous and organic solvents 96

5392 Photolysis at various ionic strengths 96

5393 Photolysis in the presence of metal ions 96

5310 Assay of Riboflavin and Photoproducts 97

5311 Calculation of Molar Concentrations in the Spectrometric Assay of RF

and Photoproducts

53111 Two-component spectrometric assay (additive absorbances) 100

53112 Three-component spectrometric assay (additive absorbances) 101

VI SOLVENT EFFECT ON THE PHOTOLYSIS OF RIBOFLAVIN

61 INTRODUCTION 106

62 RESULT AND DISCUSSION 108

621 Photoproducts of RF 108

622 Spectral Characteristics 108

623 Assay of RF and Photoproducts 111

624 Kinetics of Photolysis 116

625 Effect of Solvent 128

626 Effect of Dielectric Constant 131

627 Effect of Viscosity 132

628 Mode of Photolysis 132

VII IONIC STRENGTH EFFECTS ON THE

PHOTODEGRADATION REACTIONS OF RIBOFLAVIN IN

AQUEOUS SOLUTION

71 INTRODUCTION 135

72 RESULTS AND DISCUSSION 138

722 Spectral Characteristics of Photolysed Solutions 152

723 Kinetics of RF Photolysis 152

724 Fluorescence Studies 156

725 Ionic strength Effects 160

VIII EFFECT OF METAL IONS ON THE PHOTODEGRADATION

REACTIONS OF RIBOFLAVIN IN AQUEOUS SOLUTION

81 INTRODUCTION 165

821 Photoproducts of Metal-RF Complexes 170

822 Spectral Characteristics of Metal-RF-Complexes 171

823 Spectrometric Assay of RF and Photoproducts in Photolyzed

Solutions

824 Fluorescence Characteristics of Metal-Flavin Complexes 181

825 Kinetic of Photolysis of Metal-Flavin Complexes 181

826 Mode of Interaction of Metal Ions with RF 213

IX PHOTOCHEMICAL PREPARATION CHARACTERIZATION

AND FORMATION KINETICS OF RIBOFLAVIN

CONJUGATED SILVER NANOPARTICLES

91 INTRODUCTION 217

921 Characterization of RF-Conjugated Ag NPs 220

9211 Optical studies 220

9212 Spectral characteristics of RF-Ag NPs 220

9213 Fluorescence characteristics of RF 222

9214 FTIR studies 224

9215 Dynamic light scattering (DLS) 228

9216 Atomic force microscopy (AFM) 230

922 Factors Affecting the Particle Size of RF-Ag NPs 230

9221 pH 232

9222 Ionic strength 232

923 Kinetics of Formation of RF-Ag NPs Conjugates 235

924 Mode of Photochemical Interaction of RF and Ag+ Ions 241

CONCLUSIONS 248

REFERENCES 252

AUTHORrsquoS BIODATA 321

No LIST OF FIGURES Page

11 Chemical structures of riboflavin (1) and its analogues (flavin

mononucleotide (2) and flavin adenine dinucleotide (3))

12 Conversion of RF to FMN and FAD 6

31 Scheme for the photodegradation pathways of RF 40

32 Formation of αndashketone from flavin 45

33 Photoreduction of flavins to form 15ndashdihydrogen flavin and alkyl

adducts in the presence of unsaturated hydrocarbons

51 FTIR spectrum of riboflavin 91

52 Spectral emission of HPLN lamp 95

61 Absorption spectra of RF photolyzed in methanol at 0 30 60 90

and 120 min

62 Kinetic plots for the photolysis of RF in water

RF () FMF () LC () LF(diams)

63 Kinetic plots for the photolysis of RF in acetonitrile

RF () FMF () LC ()

64 Kinetic plots for the photolysis of RF in methanol

65 Kinetic plots for the photolysis of RF in ethanol

RF () FMF () LC ()

66 Kinetic plots for the photolysis of RF in 1ndashpropanol

RF () FMF () LC ()

67 Kinetic plots for the photolysis of RF in 1ndashbutanol

RF () FMF () LC ()

68 Kinetic plots for the photolysis of RF in ethyl acetate

RF () FMF () LC ()

69 Apparent firstndashorder plot for the photolysis of RF

(5 times 10ndash5

M) in water (pH 70)

610 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5

acetonitrile

methanol

ethanol

1ndashpropanol

1ndashbutanol

ethyl acetate

616 Plot of kobs for the photolysis of RF versus dielectric constant (x)

ethyl acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol ()

methanol (+) acetonitrile () water

617 Plot of lnkobs for the photolysis of RF versus acceptor number (x)

618 Plot of kobs for the photolysis of RF versus inverse of viscosity(x)

619 Plot of dielectric constant versus inverse of viscosity 130

71 Absorption spectra of the photolysed solutions of RF (5 times 10ndash5

M) at pH 70 (a) at zero and (b) at 05 M ionic strength

72 Plots of fluorescence of RF solutions (pH 70) versus ionic

strength at different buffer concentrations (diams) 0001 M () 0025

M () 005 M (times) 01 M () 02 M (∆) 03 M () 04 M ()

73 A plot of log kobs versus radicμ1 + radicμ at 05 M phosphate buffer 161

74 A plot log k1k0 () and k3k0 () versus radicμ at 05 M phosphate

buffer

81 The photoreduction and photoaddition pathways of riboflavin

82 Formation of the metalndashRF complex 168

83 Absorption spectra of RF (5 times 10ndash5

M) (pH 70) (____

) in the

presence of metal ions (1times 10ndash3

M) (ndashndashndash) (a) Fe2+

ions (b) Zn2+

ions and (c) Cu2+

84 The percent decrease in fluorescence intensity of RF solutions

(pH 70 0001 M phosphate buffer) in the presence of metal ions

() Ni2+

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

ions () Mg2+

() Zn2+

ions and () Fe3+

85 Excitation spectrum of RF (5 times 10ndash5

M) (pH 70) (a)

Fluorescence spectra of RF pure RF (b1) RF + Fe2+

ions (1 times 10ndash

3 M) (b2) RF + Fe

2+ ions (2 times 10

ndash3 M) (b3)

86 Firstndashorder plots for the photolysis of RF (0001 M phosphate

buffer pH 70) in the presence Ag+ ions at different

concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()

buffer pH 70) in the presence Fe2+

ions at different

buffer pH 70) in the presence Cu2+

ions at different

buffer pH 70) in the presence Zn2+

ions at different

buffer pH 70) in the presence Mg2+

ions at different

concentrations (M times 104) (diams) 10 () 20 () 30 () 40 () 50

buffer pH 70) in the presence Pb2+

ions at different

buffer pH 70) in the presence Ni2+

ions at different

buffer pH 70) in the presence Ca2+

ions at different

buffer pH 70) in the presence Mn2+

ions at different

buffer pH 70) in the presence Cd2+

ions at different

buffer pH 70) in the presence Co2+

ions at different

843 A plot of kobs for the photolysis of RF versus fluorosecne loss

in the presence of different metal ions () Ni2+

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams)

ions () Pb2+

ions () Mg2+

ions () Zn2+

ions () Fe3+

844 Scheme for the photolysis of RF in metalndashRF complex 215

91 Photodegradation pathway of RF 219

92 Colour change for the formation of RFndashAg NPs from yellow

green to brown

93 Absorption spectra of RF and RFndashAg NPs 223

94 Excitation spectrum of RF (green colour) and Fluorescence

spectra of RFndashAg NPs at different time 0 min (blue) 60 min

(black) 120 min (pink) 180 min (orange) 240 min (dark blue)

300 min (purple)

95 A plot of fluorescence loss versus time (h) for the formation of

RFndashAg NPs

96 FTIR spectrum of RF (a) and RFndashAg NPs (b) 227

97 Dynamic light scattering measurements of RFndashAg NPs 229

98 AFM micrograph (25 times 25 microm) of RFndashAg NPs 231

99 Absorption spectra of RFndashAg NPs at different pH values 20

(black) 40 (red) 60 (blue) 80 (green) 100 (pink) 120 (light

green)

910 Absorption spectra of RFndashAg NPs at different ionic strengths

(mM) 01 (black) 10 (red) 50 (blue) 100 (light green) 500

(purple) 100 (green) 250 (dark blue) 500 (maroon) 1000

(pink)

911 A plot of log absorbance versus time for the formation of RF-Ag

912 A scheme for the formation of Ag NPs (first phase) and the

adsorption of RF on the surface of Ag NPs (second phase)

913 Plots of k1 () (left hand side) and k2 () (right hand side) versus

pH for the formation of RF-Ag NPs in UV light

pH for the formation of RF-Ag NPs in visible light

915 Plots k1 () (left hand side) and k2 ()(right hand side) versus

Ag+ ion concentrations (mM) for the formation of RF-Ag NPs in

UV light

visible light

No LIST OF TABLES Page

41 Definition of Nanoparticles (NPs) and Nanomaterials

(NMs) according to different Organizations

52 Molar Absorptivities (Mminus1

cmminus1

) of RF and

Photoproducts

61 Rf values and Fluorescence of RF and Photoproducts 109

62 Concentrations of RF and Photoproducts in Water

(pH 70)

63 Concentrations of RF and Photoproducts in Acetonitrile 112

64 Concentrations of RF and Photoproducts in Methanol 113

65 Concentrations of RF and Photoproducts in Ethanol 113

66 Concentrations of RF and Photoproducts in 1ndashPropanol 114

67 Concentrations of RF and Photoproducts in 1ndashButanol 114

68 Concentrations of RF and Photoproducts in Ethyl acetate 115

69 Apparent FirstndashOrder Rate Constants for the Photolysis

of Riboflavin (kobs) in Organic Solvents and Water

71 Concentrations of RF and Photoproducts in 01 M

Phosphate Buffer (pH 70) at 01 M Ionic Strength

Phosphate buffer

(pH 70) at 02 M ionic strength

715 Concentrations of RF and Photoproducts in 03 M 146

726 Apparent FirstndashOrder Rate Constants (kobs) for the

Photodegradation of Riboflavin in the presence of

Phosphate Buffer (pH 70) at different Ionic Strength

(01ndash05M) for the formation of Lumichrome (k1)

Lumiflavin (k2) and Cyclodehdroriboflavin (k3)

81 Concentration of RF (M times 105) and LC (M times 10

5) (0001

M Phosphate Buffer) in the presence of 10ndash50 times 10ndash4

M Metal Ions Concentration

82 Concentration of RF (M times 105) and CDRF (M times 10

5) (02

5) (04

84 Apparent Firstndashorder Rate Constants (kobs) for the

Photolysis of RF in the Presence of Various Metal Ions at

pH 70 (0001 M Phosphate Buffer) for the formation of

LC (k1) LF (k2) and the SecondndashOrder Rate Constants

for the Interaction of RF and Metal Ions (kʹ)

pH 70 (02 M Phosphate Buffer) for the Formation of

LC (k1) LF (k2) CDRF (k3) and the SecondndashOrder Rate

Constants for the Interaction of RF and Metal Ions (kʹ )

86 Apparent Firstndashorder Rate Constants (kobs) for the 209

Constants for the Interaction of RF and Metal Ions (kʹ)

91 First-order Constants for the Photoinduced Electron

Transfer Reaction of RF and Ag+ ions (k1) and

Adsorption of RF at Ag NPs Surface (k2) in UV and

visible light at 001 mM of Ag+ Ion Concentration

Adsorption of RF at Ag NPs (k2) in UV and visible light

at Different Ag+ ion Concentrations

CHAPTER I

INTRODUCTION TO RIBOFLVAIN

11 INTRODUCTION

Riboflavin (RF) (1) (Fig 11) belongs to the family of vitamin B complex and is

also called as vitamin B2 It belongs to the chemical class of yellow coloured flavins

(isoalloxazines) RF was named due to its color which is derived from the Latin word

ldquoFlavinsrdquo meaning ldquoyellowrdquo It was discovered by the isolation of a heatndashstable fraction

from yeast that contained a yellow growth factor This factor after purification was

named riboflavin (Emmett and Luros 1920) Warburg and Christian (1931) isolated RF

from yeast as a coenzyme complex and named it as an antioxidant ferment The

physiological role of the yellow growth factor was later shown by Warburg and Christian

(1932) who described It as ldquoold yellow enzymerdquo composed of an apoenzyme and a

yellow factor as coenzyme The coenzyme was found to have an isoalloxazine ring (Stern

and Holiday 1934) and a phosphate containing sidendashchain ie riboflavinndash5rsquondashphosphate

(Theorell 1934) that was found to be essential for the human metabolism growth and

health RF was first synthesized by Kuhn et al (1935) and Karrer et al (1935) It is

synthesized by most of the green plants bacteria fungi and the richest sources of the

vitamin are meat legumes dairy products and eggs (Ortega et al 2004)

12 BIOCHEMICAL ROLE

RF plays a critical role in the body energy production in the form of flavin

mononucleotide (FMN) (2) or flavin adenine dinucleotide (FAD) (3) (Fig 11) When RF

is converted into FAD and FMN forms as coenzymes it is attached to protein enzymes

and allows oxygenndashbased energy production to occur Proteins with FAD or FMN

attached to them are often referred to as flavoproteins (Rivlin 2007 Moffat 2013)

CH2O P

Fig 11 Chemical structures of riboflavin (1) and its analogues (flavin mononucleotide (2)

and flavin adenine dinucleotide (3))

These flavoproteins are found throughout the body and particularly in that

location where oxygenndashbased energy production is constantly needed (Merrill et al

RF plays an important role in maintaining the supplies of other B vitamins One

of the pathways used in the body to produce vitamin B3 (niacin) is by conversion of the

amino acid tryptophan This conversion is accomplished with the help of an enzyme

kynureninendashmonondashoxygenase and RF in its FAD form RF is the precursor of the two

flavocoenzymes (FMN and FAD) required by the two flavoproteins of the mitochondrial

electron transport chain (McCormick 1989)

Glutathione reductase is a FAD ndashdependent enzyme that precipitates in the redox

cycle of glutathione The glutathione redox cycle plays a major role in protecting

organisms from reactive oxygen species Glutathione reductase requires FAD to

regenerate two molecules of reduced glutathione (an antioxidant) from oxidized

glutathione (Beutler 1969)

Xanthine oxidase is another FAD dependent enzyme that catalyzes the oxidation

of hypoxanthine and xanthine to uric acid Uric acid is one the most effective waterndash

soluble antioxidant in the blood RF deficiency can result in decreased xanthine oxidase

activity reducing blood uric acid levels (Bohles 1997) Recent studies on migraine

patients show some evidence that indicates impaired mitochondrial oxygen metabolism in

the brain that may play a role in the pathology of migraine headaches

13 CHEMICAL STRUCTURE OF RIBOFLAVIN

Chemically RF is 78-Dimethyl-10-[(2S3S4R)-2345-

tetrahydroxypentyl]benzo[g]pteridine-24-dione (British Pharmacopoeia 2016) The

planar isoalloxazine ring not only provides the basic structure for RF but also for the

naturally occurring phosphorylated coenzymes that are derived from RF These

coenzymes include FMN FAD and flavin coenzymes linked covalently to specific tissue

proteins generally at the 8ndashα methyl position of the isoalloxazine ring RF exists in the

cationic and anionic forms with the pKas of 19 and 102 (Moffat et al 2013)

respectively and due to strong conjugated system it has a high molar absorptivity as well

as high fluorescence characteristics due to the presence of a strong conjugated system

(Rivlin 2007) RF in the presence of flavokinase and FMN phosphatase is converted into

FMN which is further converted into FAD by the action of FAD pyrophosphorylase and

pyrophosphatase (Powers 2003) (Fig 12) Initially flavokinase which is biosynthetic

enzyme initiates the phosphorylation of RF from ATP for the formation of FMN This

FMN in small portion is used as a coenzyme and the major portion of FMN is further

combined with a second ATP molecule for the formation of FAD The formation of FAD

is catalysed by FAD synthetase and these flavins are further covalently attached to the

different tissues after the formation of FAD (Powers 2003)

CH2O P

FlavokinaseFMN Phosphatase

FAD Pyrophosphorylase Pyrophosphatase

Thyroid Harmone

Fig 12 Conversion of RF to FMN and FAD

14 PHYSICOCHEMICAL PROPERTIES OF RIBOFLAVIN

The physicochemical properties of RF that affect its stability or the physiological

functions are as follows (Moffat et al 2013 Sweetman 2009 British Pharmacopoeia

Empirical formula

C17H20N4O6

Molar mass 3764

Crystalline form fine needles

Melting point 278 to 282 oC

ndash112 to ndash122o

pH of saturated solution ~6

pKa 19 102 (20o)

Redox potential

(riboflavindihydroriboflavin) pH 70 ndash0208 V

Solubility mg 100 ml

Water 33ndash606

Absolute ethanol 045

Acetone chloroform ether benzene insoluble

Absorption maxima (pH 70) 223 267 373 and 444 nm

Fluorescence emission (pH 70) 520 nm

Principle infrared peaks (KBr disk) 1544 1575 1641 1715 1235

1070 cmndash1

15 CLINICAL USES

RF is used in both clinical and in therapeutic conditions It is also used in the

phototherapy of a condition termed as neonatal jaundice RF in high doses with betandash

blockers is used in the treatment of migraine (Sandor et al 2000 Schoenen et al 1998)

It has been used in the management of the muscle pain RF along with the UV light is

effective against the pathogens that cause disease while present in the blood (Goodrich et

al 2006 Kumar et al 2004) RF is also used in the treatment of the corneal disorder

named keratoconus (Spoerl et al 2004a 2004b)

RF as a precursor of FMN and FAD shows a powerful antioxidant activity It

provides protection against peroxidase of lipids in glutathione redox cycle (Dutta 1993)

The breakdown of lipid peroxidase is mediated by glutathione peroxidase and it requires

reduced form of glutathione (GSH) which results in the regeneration of the oxidized form

of glutathione (GSSG) by glutathione reductase a FAD containing enzyme If

glutathione reductase activity is compromised then the GSH concentration is decreased

which serves as a substrate for glutathione reductase and glutathione Sndashtransferase This

results in decrease in the degradation of lipid peroxides and xenobiotic substances

(Rivilin and Dutta 1995) It has also been found that in RF deficiency glucosendash6ndash

phosphate dehydrogenase activity is also stopped (Taniguvhi and Harm 1983 Dutta et

al 1995) Miyazawa et al (1983 1984) stated that in RF deficiency the oxidant defense

system is compromised and if the RF supplement is taken then the oxidant response

system is progressively improved Deficiency of RF is also related to the lipid

peroxidation and on the use of its supplement the process is restricted (Taniguchi and

Harm 1983 Dutta et al 1995)

Deficiency of RF in animals and humans is found to be protective against malaria

(Kaikai and Thurnham 1983 Das et al 1988) Glactoflavin and 10ndash(4ʹndashchlorophenyl)ndash

3ndashmethlflavin are isoalloxazine derivatives that are inhibitors of glutathione reductase

and possess antimalarial activity (Becker et al 1990 SchonlebenndashJanas et al 1996)

RF is also involved in the regulation and metabolism of homocysteine (HC) HC

is mainly involved in cardiovascular peripheral vascular and cerebrovascular diseases

(Graham et al 1997) The conversion of Nndash5ndashmethyltetrahydrofolate to methionine

which is a condashsubstrate for HC and FAD are required by methyltetrahydrofolate

reductase for the conversion of Nndash5 10ndashmethylenetetrahydrofolate to Nndash5ndash

methylatetrahydrofolate For this conversion RF is required for the effective utilization of

dietary folic acid In the patients who are homozygous for genetic mutation RF controls

the HC metabolism (Rozen 2002 Yamada et al 2001) In USA it was reported that as

the dietary intake of RF increases the concentration of serum HC decreases (Ganji and

Kafai 2004)

16 ABSORPTION FATE AND EXCRETION

RF is readily absorbed from upper gastrointestinal tract by a specific transport

mechanism in which phosphorylation of the vitamin to FMN takes place (Jusko and

Levy 1975) RF is distributed to all tissues but its concentration is uniformly low and

little amount is stored in the body If RF is taken according to its daily requirement then it

is only excreted up to 9 in urine but if it is taken more than the daily requirement then

it is excreted in urine in the unchanged form If RF is present in the feces it is due to the

synthesis of the vitamin by intestinal microorganism (Tillotson and Karcz 1977) In the

case of boric acid poisoning RF forms a complex with boric acid and this promotes

urinary excretion that may induce riboflavin deficiency (Roe et al 1972)

17 THERAPEUTIC USES

RF at its nutritional doses is helpful in the treatment of cataracts in combination

with other B vitamins (Niacin B3) (Sperduto et al 1993) It is also used in the treatment

of sicklendashcell anemia (Ajayi et al 1993) and also in the treatment of HIV infection (Tang

et al 1996)

RF is used in the treatment of its deficiency a condition called as ariboflavinosis

It is also used in other nutritional disorders Recent randomized controlled trial of highndash

dose RF (400 mgday) in patients suffering migraine headaches showed significant

reductions in attack frequency and illness days (Schoenen et al 1998)

18 PHARMACOKINETICS

RF is mainly found in nature in the form of FMN and FAD It is used for the food

fortification RF and FMN are the principal nutritional supplement forms of riboflavin

with riboflavin being the major form Coenzyme forms of RF (FMN FAD) that are not

covalently bound to proteins are released from proteins in the acid environment of the

stomach (Zempleni et al 1996)

FMN and FAD are converted to RF in the small intestine via the action of

pyrophosphatase and phosphatase It is mainly absorbed in the proximal small intestine

by the saturable system The presence of the bile salts appears to facilitate absorption of

RF (Nath 2000)

19 LITERATURE ON RIBOFLAVIN

Books (Chemistry Biochemical Function and Clinical Uses)

Chapters in Books

Dyke (1965) Penzer and Radda (1971) Dollery (1999) Chapman et al (2002) Rivlin

and Pinto (2001) Baxter (2003) Delgado and Remers (2004) Rivlin (2007)

Reviews

Penzer and Radda (1967) Hemmerich (1976) Walsh (1980) Heelis (1982 1991)

Powers (2003) Ahmad and Vaid (2006)

Chemical and Photostability

Macek (1960) Garrett (1967) Hashmi (1973) DeRitter (1982) Allwood and Kearney

(1998)

Chromatography and Assay

Bolliger and Konig (1969) HoffmanndashLa Roche (1970) Hashmi (1973) Shah (1985)

Song et al (2000) Eitenmiller et al (2008)

Physiochemical Data

British Pharmacopeia (2016) United States Pharmacopeia (2009) Moffat et al (2013)

Sweetman (2009) OrsquoNeil (2013)

CHAPTER II

ANALYTICAL TECHNQIUES USED FOR THE

DETERMINATION OF RIBOFLAVIN AND

RELATED COMPOUNDS

Several analytical methods have been used for the determination of riboflavin

(RF) and related compounds in pure solutions pharmaceutical preparations and

biological samples These methods are described in the following sections

21 SPECTROMETRIC METHODS

211 UVndashVisible Spectrometry

The method reported for the determination of RF in British Pharmacopoeia (BP)

(2016) involves the measurement of the absorbance of aqueous solutions at 444 nm and

calculating the concentration using the value of A (1 1cm) as 328 However since RF

is sensitive to light the major problem associated with the determination of RF in

photodegraded solutions is the presence of its photoproducts that interfere at the

absorption wavelength Ghasemi and Abbasi (2005) have determined RF in vitamin B

preparations containing folic acid thiamin and pyridoxine using a multicompartment

spectrometric method This method is based on the measurement of absorbance in the pH

range of 20 to 120 at 25 oC using parallel factor analysis (PARAFA) The calibration

curves were found to be linear in the concentration range of 4ndash22 1ndash20 6ndash26 and 4ndash20

mg Lndash1

for pyridoxine riboflavin thiamin and folic acid respectively This method

shows recovery of 906ndash107 for each vitamin The kinetics of photodegradation of

RF as a function of pH has been studied using a multicomponent spectrometric method

for the determination of RF and its photoproducts formylmethylfalvin (FMF)

lumichrome (LC) and lumiflavin (LF) formed by intramolecular photoreduction reaction

(Ahmad and Rapson 1990 Ahmed et al 2004a) The photolysis of FMF a major

intermediate in the photodegradation of RF has also been studied by the application of

this method (Ahmed et al 1980 2006ab 2008 2013) These methods have also been

used for the study of thermal degradation (Ahmad et al 1973) and photodegradation of

RF by photoaddition reactions (Ahmad et al 2004b 2005 2006 2010) Some other

applications of these methods include the study of the buffer effect (Ahmad et al 2014

Sheraz et al 2008) solvent effect (Ahmad et al 2015) ionic strength (Ahmad et al

2016) and metal ion effect (Ahmad et al 2017) on the photodegradation of RF

A multindashcomputed flow method for the determination of RF and B vitamins in

pharmaceutical products has been reported by Rocha et al (2003) At 997 confidence

interval the calibration curve was found to be linear for RF The average recovery

obtained for the commercial and pharmaceutical products lies between 956 and 100

Mohamed et al (2011) developed a derivative and multivariate spectrometric

method for the determination of pharmaceutical preparations containing a mixture of RF

and other B vitamins in the wavelength range of 200ndash500 nm using a

01 M HCl solution The results showed a linear response in the range of

25 to 90 microg mLndash1

with a recovery range of 961 to 1012 and 970 to 1019 for the

derivative and multivariate methods respectively A method involving spectrometric

determination based on total absorbance measurement of a complex mixture containing

folic acid (FA) RF pyridoxine (PY) and thiamine (TH) has been developed by partial

least regression The calibration matrix constructed for FA RF PY and TH determined

their concentration in the ranges between 102ndash143 microg mLndash1

102ndash102 microg mLndash1

and 600ndash200 microg mLndash1

respectively The estimated detection limits

of 008 microg mLndash1

009 microg mLndash1

045 microg mLndash1

and 017 microg mLndash1

have been found for FA

RF PY and TH respectively (Aberasturi et al 2002)

A comparison between FTndashNIRS and UVndashvis spectrometry for the evaluation of

mixing kinetics for the assay of a low quantity of RF in tablets has been made NIRS is a

nonndashdestructive technique which is used for the analysis of pharmaceutical dosage forms

In this study binary mixtures of microcrystalline cellulose and RF were used to prepare

tablets by direct compression The partial least square regression fit method was used to

build the prediction model The assay of RF was carried out by NIR transmission and the

results were compared with those of the UVndashvis spectrometry method and found that

NIR spectroscopy is faster nonndashdestructive and shows less variability in results (Bodson

et al 2006)

A study has been carried out for the simultaneous spectrometric determination of

FA TH RF and PY in artificial mixtures using multivariate calibration method The

calibration curves were found to be linear in the concentration range of 04ndash150 07ndash30

02ndash11 and 08ndash30 microg mlndash1

for FA TH RF and PY respectively The optimization of

calibration matrices by PLSndashI method was carried out by absorption spectra of quaternary

mixtures The recovery for these vitamins was found to be 95ndash105 (Ghasemi and

Vosough 2002)

The simultaneous multicomponent spectrometric determination of FA TH RF

and PY using doublendashdivisorndashratio spectra derivative zero crossing method has been

carried out for the assay of these vitamins in synthetic mixtures This method was based

on the derivative signals of the ratio spectra employing double divisor The spectral

measurements were carried out in the range of 225ndash475 nm The calibration curves were

found to be linear in the concentration range of 1ndash26 microg mlndash1

4ndash50 microg mlndash1

and 6ndash42 microg mlndash1

for FA TH RF and PY respectively in phosphate buffer (pH 580)

(Ghasemi et al 2004)

The simultaneous determination of waterndashsoluble vitamins (TH PY RF and CA)

in binary ternary and quaternary mixtures has been carried out by two spectrometric

methods (derivative and multivariate methods) The derivative method was divided into

first derivative and first derivative of ratio spectra method and multivariate method into

classical least squares and principal component regression method These methods were

based on the spectrometric measurements of the vitamins in 01 M HCl in the wavelength

range of 200 to 500 nm The methods showed good linearity in the concentration range of

25ndash90 microg Lndash1

with a regression in the range of 09991ndash09999 The mean recovery

( recovery) for derivative and multivariate methods ranged from 9611 (plusmn12)ndash

1012 (plusmn10) and 970 (plusmn05)ndash1019 (plusmn13) respectively (Mohamed et al

The principle of surface Plasmon resonance with onndashchip measurements has been

developed for the quantification of RF in milkndashbased products It has been carried out by

the determination of excess RF binding protein (RBP) that was free after complexation

with RF molecules In this method the modification was done at N(3) position to

introduce an ester group for the binding of amino groups at the surface of the chip RF

content in the milk based products was measured in comparison with the calibration

curve obtained from the standard RF with optimized RBP LOD and LOQ were found to

be 234 microg Lndash1

and 70 microg Lndash1

respectively for the 160 microLndash1

injections (Caelen et al

A catalytic photokinetic method has been developed for the microdetermination

of RF and riboflavin 5primendashphosphate This method is based on the rate of photoreduction of

these compounds by EDTA The rate of photoreduction was monitored by spectrometry

by the formation of ferroin The ferroin was produced by the reduction of Fe (III) via a

1ndash5 dihydro form of RF in the presence of 110ndashphenanthroline This method shows

linearity in the concentration range of 3 times 10ndash8

to 96 times 10ndash7

M (PerezndashRuiz et al 1987)

212 Spectrofluorimetry

Spectrofluorimetry is the method used for the assay of RF and its preparations

United States Pharmacopeia (USP) (2016) The method involves the measurement of

fluorescence of RF solution at 530 nm The concentration of RF solution is calculated by

comparing it with the USP reference standard taking 440 nm as the excitation

wavelength

A spectrofluorimetric method has been developed for the determination of RF in

tablets The emission and excitation wavelength used were 535 and 435 respectively

This method was found to be linear for RF in the concentration range of

with regression of 09978 The mean recovery was found to in the range

of 93ndash102 with a coefficient of variation of 232 (Junqing 1997)

One of the methods for the assay of RF in total parenteral nutrition (TPN) for

neonates involves the measurement of its fluorescence in the range of 400ndash700 nm using

360 nm as the excitation wavelength (Ribeiro et al 2011) RF flavin mononucleiotide

(FMN) and flavinadenine dinucleotide (FAD) have been quantified in human plasma at

530 nm using capillary electrophoresis and laser induced fluorimetry The 4 and 9

withinndashday and betweenndashday coefficient of variance values have been reported for RF

with a linear calibration falling in the concentration range of 03 and 1000 mol Lndash1

(Hustad et al 1999)

Synchronous fluorescence spectrometry has been used for the determination of

TH RF and PY in commercial preparations (Garcia et al 2001) RF and PY have been

determined using acetate buffer (pH 6) by a sensitive fluorimetric method The

concentration found lies in the range of 10ndash500 microg mLndash1

with a standard deviation

between 046 to 1002 and the recovered amount in the range 976 to 1012

(Mohamed et al 2011) RF determination in commercial preparations such as skimmed

milk 2 partially skimmed homogenized milk 2 partially skimmed chocolate and

nonndashfat dry milk has been made using fluorimetry with the help of extracted samples

Depending on the product assayed the RSD lies between 171 to 316 with a recovery

range between 90 to 110 (Rashid and Potts 2006) The analysis of RF in anchories

has also been carried out by synchronous spectrofluorimetry by the measurement of

fluorescence spectra in 300ndash600 nm region The excitation and emission slit widths were

set to 5 mm and the difference in wavelengths was 65 nm Fluorescence measurements

were carried out by peak area base of 430 to 509 nm and recovery was found to be higher

than 908 (LoperndashLayton et al 1998) A synchronous spectrofluorimetric method has

been developed for the simultaneous determination of vitamin B2 and B6 in beverages

The limits of detection have been found to be 002ndash006 mg Lndash1

and 012ndash036 mg Lndash1

B2 and B6 respectively (TorresndashSequeiros et al 2001)

A spectrofluorimetric study has been conducted for the evaluation of interaction

between RF and isolated protein from egg white at different pH values It has been found

that in phosphate buffer (01 M pH 70) the complex formation between RF and protein

(11) occurs with an association constant (Ka) of 77 times 107 M

ndash1 The complex was

dissociated in the presence of sodiumndashdodecyl sulphate (0033 ) with a rate constant of

4 times 10ndash2

secndash1

at 29 oC The binding affinity of RF to protein has been found to decrease

in the pH range of 70ndash40 and below pH 40 the binding affinity does not exist The

fluorimetric studies showed that carboxyl group 1ndash2 tryptophan residues and 2ndash3

disulphide bridges are necessary for binding The quantum yield (Φ) and energy transfer

from tyrosine to tryptophan have been calculated by excitation of the complex at 280 and

295 nm (Murthy et al 1976)

An investigation has been carried out on the molecular interaction between

quinine sulfate (QS) and RF by fluorimetry and UVndashvis spectrometry It has been found

that in the presence of QS the RF fluorescence is quenched At different temperatures

(294 301 307 314 oK) the thermodynamic parameters enthalpy change (∆H) and Gibbs

energy change (∆G) were determined via a Vanrsquot Hoff equation By calculating all these

thermodynamic parameters it was found that hydrogen bond helps in the stabilization of

the complex The critical energy transfer distance (Ro) was calculated as 4047 oA and

this showed that efficient resonance energy transfer takes place between QS (donor) and

RF (acceptor) Cyclic voltammetry (CV) of QS and RF complex showed that electron

transfer occurs in the excited singlet state (Patil et al 2011)

A fluorimetric method has been developed for the simultaneous determination of

TH PY and RF in pharmaceutical multivitamin formulations In this method TH

determination is based on the measurement of thiochrome formed by oxidation using Nndash

bromosuccinimide (NndashBS) in isopropanol whereas pyridoxine and RF measurements

were made in phosphate buffer (pH 70) For TH PY and RF sensitivity ranges were

found to be 15ndash35 05ndash25 and 04ndash20 microg mlndash1

respectively (Barary et al 1986)

A fluorimetric method for the determination of RF in hemoglobinndashcatalyzed

enzymatic reaction has been developed In this method two reactions occur

photochemical reaction of RF and hemoglobin catalyzed enzymatic reaction This

method has been found to be linear in the concentration range of 50 times 10ndash9

mol Lndash1

and the detection limit is 305 times 10ndash9

mol Lndash1

For 11 determination of 70 times 10ndash2

mol Lndash1

the RSD of measurements is 23 (XiaondashYan et al 2002)

A multivariate method for the rapid determination of caffeine caramel (class III

and IV) and RF in energy drinks using synchronous fluorimetry has been developed The

synchronous spectra are measured in the wavelength range of 200ndash500 nm Partial least

squares (PLS) models are created by the determination of the analyte with HPLC with a

fluorescence detector This method has been found to be linear in the concentration range

of 02ndash42 025ndash525 04ndash100 and 0007ndash0054 mg Lndash1

for caffeine caramel and RF

respectively (Ziak et al 2014) In nutritional beverages the simultaneous determination

of FA and RF have been carried out by synchronous fluorescence measurments In this

method FA has been detected by treating it with H2O2 plus Cu (II) (oxidation system) to

form pterinendash6ndashcarboxylic acid that is fluorescent The method shows good linearity in

the concentration range of 100ndash250 microg Lndash1

and 1ndash250 microg Lndash1

and the detection limits of

20 and 0014 microg Lndash1

for FA and RF respectively (Wang et al 2011)

A synchronous spectrofluorimetric method has been developed for the

simultaneous determination of RF and PY Synchronous scanning is carried out at ∆λ of

58 nm The measurements were carried out in phosphate buffer (pH 70) Two peaks have

been found at 526 and 389 nm in the synchronous fluorescence spectra for RF and PY

respectively The method shows linearity in the concentration range of 0ndash10 microg mlndash1

and recovery of 935ndash1057 for RF and PY respectively

(Li et al 1992)

The determination of RF in blood in newborn babies and their mothers has been

carried out by a spectrofluorimetric microndashmethod It is based on the hydrolysis of blood

in tridichloroacetic acid medium separation of RF and FMN on florisil column and

measurements by spectrofluorimetry by standard additional method after elution with

collidine buffer This method shows a sensitivity of 001 microg mlndash1

in the blood sample of

05ndash10 ml with an average concentration of 171 plusmn 24 microg100 ml and 142 microg100 ml of

RF in new born baby and women respectively (Knobloch et al 1978)

A synchronous fluorimetric method has been used for the simultaneous

determination of B1 B2 and B6 It is difficult to analyse them individually as their spectra

overlap and to overcome this problem parallel factor analysis (PARAFA) is used to

enhance the resolution of the overlapped spectra of the mixture The excitation

wavelength was in the range of 200ndash500 nm and ∆λ was in the range of 20ndash120 nm In

this study PARAFA has been established and applied to the synthetic and commercial

samples of the vitamins (Ni and Cai 2005) Synchronous fluorescence spectrometry in

organized media has been used for the determination of TH RF and PY in

pharmaceuticals in the presence of bisndash2ndashethoxyndashsulfosuccinate sodium salt (AOT)

micelles It has been found that RSD for repeatability is less than 14 and the LOD

has been found to be 12 microg Lndash1

10 microg Lndash1

for TH PY and RF respectively

(Garcia et al 2001)

Artificial neural network and LavenvergndashMarquardt backndashpropagation tanning

have also been used for the simultaneous determination of B1 B2 and B6 In this method

fluorescence were measured out at 15 wavelengths which were considered as

characteristic of artificial neural network The mean recoveries were found to be 9986

9980 and 9949 for B1 B2 and B6 respectively with RSDs of 17 16 and 17

respectively for these vitamins (Wu and He 2003)

213 InfrandashRed Spectrometry

A study has been carried out for the determination of femtosecond time resolved

infrared spectroscopy in vibrational response of RF in dimethyl sulfoxide (DMSO) for

photoexcitation at 387 nm In this study the vibrational cooling of the excited electronic

state was evaluated and its characterization was carried out by a time constant of 40 plusmn

01 ps The characteristic pattern of excited state vibrational frequencies of RF is useful

for its determination and identification in the spectral region of 1000 to 1740 cmndash1

calculation for vibrational spectra of ground and excited singlet state was carried out by

HartreendashFock (HF) and configuration interaction signals (CIS) methods It has been

found that upon photooxidation of RF the double bond position C(4a) and N(5)

disappeared (Wolf et al 2008)

214 Mass Spectrometry

Depending on the molecular fragmentation laser desorption mass spectrometry

(LDMS) has been developed for the analysis of RF TH HCl retinoic acid (RA) ascorbic

acid (AA) and PY HCl vitamins in commercial preparations (McMahon 1985) A

triplendashquad mass spectrometric method (LCUVMSndashMRM) has also been designed for

the determination of RF and other B vitamins in multivitamin and multimineral

supplements using a photodiode array detector (PAD) The method is simple as it does

not involve sample cleaning (Chen and Wolf 2007) Another method employed for the

determination of RF and other B vitamins is by comparing peaks of labeled vitamins with

those of unlabelled vitamins using LCndashisotopes dilution mass spectrometry (LCIDMS)

(Chen et al 2007) Electrondashspray ionization mass spectrometry (ESIMS) has been

employed for the determination of RF PY CF nicotinamide (NA) and taurine (TU) in

energy drinks Linear calibration curves have been observed in the range 08 to 15

with a recovery of 81 to 106 (Aranda and Morlock 2006) The analysis of waterndash

soluble vitamins in an infant formula has been performed using ultrandashperformance liquid

chromatographyndashtanden mass spectrometry (UPLCndashMSMS) The vitamins are extracted

using BEH Shield RP 18 column and the recovery range for RF has been found to be

818 to 106 using methanol and ammonium acetate (aqueous) as mobile phase

(Zhang et al 2009)

Planar chromatographicndashmultiple detection with confirmation by electrospray

ionization mass spectrometric method has been carried out for the simultaneous

determination of vitamin B2 B6 B3 caffeine and taurine in energy drinks For the

analysis of caffeine 10 samples of energy drinks and six samples of beverages were

prepared after degassing on ultrasonic bath for 20 min Chromatographic separation and

multindashwavelength scanning is carried out at 261 and 275 nm for B3 and caffeine

fluorescence measurements at 366400 and 313340 nm for RF and pyridoxine

respectively and 325 nm for taurine after post column chromatographic derivatization by

ninhydrin The overall recoveries for these vitamins and other substances have been

found to be in the range of 81ndash105 The intermediate precision for B2 B6 B3 caffeine

and taurine is in the range of 36ndash74 28ndash63 25ndash44 21ndash29 and 05ndash40

respectively Mass confirmation for each substance is carried out by MS in positive

electrospray ionization (ESI) positive scan mode except for taurine in negative mode

(Aranda and Morlock 2006)

A simple and precise method has been designed using HPLCndashMS for the assay of

RF in crude products The analysis has been carried out using methanol and water as

mobile phase and all the components have been separated and identified efficiently using

a C18 column (Guo et al 2006)

22 CHROMATOGRAPHIC METHODS

221 High Performance Liquid Chromatography (HPLC)

A simultaneous method for the determination of various B vitamins including RF

involves reverse phase liquid chromatography using the ionndashpair technique The

separation of the vitamin (RF at 280 nm) has been carried out at pH 36 using methanol

and water (1585 vv) with triethylamine (005) as a mobile phase The average

recovery for RF has been found to be 982 to 10202 with RSD of 102ndash55 (Li

2002) HPLC has been employed to study the chemical stability of total parenteral

nutrition (TPN) containing several vitamins using diode array detector RF PY AA and

other B vitamins are separated using Bondapak (C18 column) and methanolwater (2773

vv) as mobile phase with 14 sodium 1ndashhexanesulfonate for ionndashpair formation

(Ribeiro et al 2011) The RPndashHPLCndashdiode arrayfluorescence detector using ODS

column has been employed for the assay of multivitamins preparations containing RF and

other B vitamins The gradient elution system is used for the determination of RF (Chen

et al 2009)

Another reverse phase HPLC method reported for the determination of water

soluble vitamins in nutraceuticals has been reported This method quantitatively

determines the amount of RF PY cyanocobalamin (CA) and FA using gradient elution

The quantities of RF PY CA and FA determined by UV detection have been found to be

013 mgg 0235 mgg 00794 mgg and 00966 mgg respectively Recoveries for the

method have been found to be in the range of 986 to 1005 with RSD values of less

than 1 (Perveen et al 2009)

Stability studies of certain pharmaceutical preparations containing vitamins have

been carried out using a reverse phase HPLC method The detection has been made at

280 nm using gradient elution with a mobile phase of 0015 M sodium salt of 1ndashhexane

sulphonic acid and methanol Vitamins B2 B6 B3 and B1 show 151 199 63 and 427

min retention time respectively with coefficient correlation values of 0999 (Thomas et

al 2008)

Yantih et al (2011) reported a validated HPLC method for the quantitative

determination of vitamins in syrups containing multivitamins RF TH HCl NA and PY

HCl are separated using a C18 column with 10 microm particle size The separation of the

effluent is achieved within 20 min monitored at 280 nm using a mixture of methanolndash

acetic acid (1) and sodium salt of 1ndashhexane sulphonic acid in the ratio of 2080 vv as

mobile phase

The stability of total parenteral nutrition containing multivitamins has been

studied using a HPLC method NA is determined using UV detector where as PY and RF

5primendashphosphate via fluorescence detection without pretreatment of the sample FA and TH

are quantified using UV detector after prendashcolumn enrichment Detection of vitamin C

(AA) is done by determining the concentration of AA as well as dehydroascorbic acid

(DHA) DHA is determined by fluorescence detection after it was converted to a

quinoxaline (Van der Horst et al 1989)

The determination of total RF phosphates by immobilized sweet potato and

phosphatase (prendashcolumn reactor) has been carried out by a chromatographic method

Hydrolysed RF is eluted using methanol as a mobile phase and the measurements are

carried at 280 nm This method shows good linearity in the concentration range of 05ndash

500 nmol mlndash1

for total RF phosphates The LOD has been found to be 25 pmol mlndash1

an average transformation of RF phosphates to RF to be 97 The intrandash and interndashday

precisions ( RSD) have been found to be 12 and 26 respectively (Yamato et al

The simultaneous determination of waterndashsoluble vitamins (TH RF NA PY

CA FA) in multivitamin pharmaceutical formulations and biological fluids (urine blood

serum) has been carried out by HPLC A Phenomenex Luno C18 column with gradient

elution (CH3COONH4CH3OH (991 vv) H2OCH3OH (5050 vv)) and flow rate of

05 ml minndash1

has been used The detection is carried out by PDA detector at a wavelength

of 280 nm LOD for these vitamins has been found to be 16ndash34 ng with a linearity range

of 25 ng microLndash1

In this method theobromine (2 ng dlndash1

) is used as internal standard (IS)

The mean recoveries () have been found to be in the range of 846ndash103

(Chatzimichalakis et al 2004)

A study has been carried out for the determination of RF by HPLC in RF depleted

urine samples as calibration and control matrix In this method 1 mg mlndash1

of RF in RF

depleted urine is used to validate the HPLC method with fluorescence detection This

method shows good linearity in the concentration range of 10ndash5000 ng mlndash1

coefficients of variations for intrandash and interndashday precision have been found to be 39 and

9 respectively (Chen et al 2005)

An HPLC method has been developed for the simultaneous determination of

vitamin B1 B2 B6 and sorbic acid in Alvityl syrup The samples are diluted with water

and separated by C18 column with a mobile phase of 1ndashsodiumhexane sulfonate (8 mmol)

solution containing triethylamine (025 ml) acetic acid (92 ml) and methanol The

detection for these compounds is carried out at 280 nm This method shows good

linearity in the concentration range of 002ndash04 ng mlndash1

002ndash04 ng mlndash1

0007ndash01

and 003ndash06 002ndash04 ng mlndash1

for vitmain B1 B2 B6 and sorbic acid

respectively (Yang et al 2010)

The determination of B1 and B2 has been carried out in four vitamin glucose

calcium particles for children by HPLC In this method a ORBAXndashEclipse XDBndashC18

column with a mobile phase of 1ndashheptane solution (0005 mol Lndash1

) containing acetic acid

(05 ) and triethylamin (005) has been used The detection is carried out at 260 nm

and the flow rate was 1 ml minndash1

This method shows good linearity in the concentration

range of 713ndash2296 microg mlndash1

for B1 and B2 respectively The

recoveries for B1 and B2 have been found to be 1011 and 1014 respectively with a

RSD of 06 (Yuan et al 2008)

A reversed phase ionndashpair HPLC method has been developed out for the

determination of TH RF PY and NA in the chewable tablets of vitamins The water

microndashBondapak C18 column is used with a mobile phase of sodium hexane sulfonate buffer

(0005 M) and methanol The detection is carried out at 280 nm and the method shows

good linearity in the concentration range of 06ndash288 microg mlndash1

15ndash45

microg mlndash1

for TH RF PY and NA respectively Mean recoveries

have been found to be 1008 1003 998 and 992 for TH RF PY and NA

respectively with RSDs of 14 12 05 and 09 respectively (Xinhe et al

The determination of vitamin Bndashcomplex (TH RF NA nicotinc acid (NC) PY

cyanocobalamin (CA) and FA) has been carried out by HPLC in pharmaceutical

preparations (multivitamin formulations) and biological fluids (blood serum and urine)

after sold phase extraction (SPE) In this method a Phenomenex luna C18 column is used

and gradient elution is carried out at a ratio of 991 of CH3COONH4CH3OH (005 M)

and H2OCH3OH (5050 vv) with a flow rate of 08 ml minndash1

with detection using a

photodiode array (PDA) detector at 280 nm The method showed good linearity upto

25 ng microL with a detection limits in the range of 16ndash34 ng for each vitamin

A HPLC method has been developed and used for the determination of RF and

aromatic amino acids in the form of shrimp hydrolysates This method is based on the

acid hydrolysis (01 M HCl) of RF followed by an enzymatic digestion and protein

precipitation by trichloroacetic acid A Chrom SEPSS C18 column (5 microm) column with a

mobile phase of ammonium acetate (5 mM) and methanol (7228 vv) at a flow rate of

10 ml minndash1

has been used The method shows good linearity reproducibility accuracy

and LOD in the studied range (BuenondashSolano et al 2009)

RF has been determined in milk and nonndashdiary imitation milk during refrigeration

by HPLC with UV detection The content of RF has been found to be in the range of

for cows milk and nonndashdiary imitation milks

respectively These open containers when stored in a refrigerator (8 oC) in the dark the

loss of RF content ranged from 160ndash234 and 125ndash165 in cows milk and nonndash

diary imitation milk respectively (Munoz et al 1994)

222 Liquid Chromatography (LC)

A ionndashpair RP liquid chromatographic (IPndashRPndashLC) method has been developed

for the determination of RF in cooked sausages In this method the sausage samples have

been subjected to acid and enzymatic hydrolysis The samples are directly injected

without any purification and concentration treatment into the column In this method

heptansulfonic acid (5 mM pH 27) and acetronitrile (7525 vv) are used as a mobile

phase The intrandash and interndashday precisions have been found to be 13 and 26

respectively with LOD of 0015 mg100 g This method shows a mean recovery of gt 95

(Valls et al 1999)

The selective detection of RF has been made by liquid chromatography with a

series of dualndashelectrode electrochemical detectors In this method two electrodes

(upstream downstream) are held at ndash04 V and +01 V versus SCE This method shows

good linearity in the concentration range of 4 ngndash26 microg with a LOD of 4 ng There is no

interference in absorbance and electrochemical detection of RF in the presence of 13

different vitamins (Hou and Wang 1990)

223 Ion Chromatography (IC)

Ion chromatography (IC) with photochemical fluorimetry (PCF) has been used for

the determination of RF in health protection products The chromatographic separation is

carried out at a Low Pac AsHndashHC column using NaOH (40 mmol Lndash1

) as the mobile

phase The column effluents are subjected to UVndashirradiation (245 nm) to transform RF

into a strongly fluorescent component and detection is carried out by spectrofluorimetry

This method shows good linearity in the concentration range of 10ndash100 mg Lndash1

with LOD

of 05 ng Lndash1

The means recovery for RF was found to be 10146 plusmn 25 (Cao et al

23 ELECTROCHEMICAL METHODS

Cyclic voltammetry and differential pulse voltammetric (DPV) methods with

glass electrode have been employed to investigate the electrochemical behavior of RF

The sensitivity of RF peaks and the detection accuracy is enhanced using glass electrode

made up of poly (3ndashmethylthiophene) Diffusivity (Do) and the electron transfer number

lsquonrsquo using cyclic measurements have been found to be 0000026 cm2s and 2 respectively

DPV has been used for the quantitative determination of RF with a detection limit of

50 times 10ndash8

mol Lndash1

A linear peak current in the range of 1 times 10ndash7

to 2 times 10ndash4

mol Lndash1

along with a RSD of 15 has been determined (Zhang et al 2010)

A simultaneous electrochemical method has been developed for the determination

of waterndashsoluble vitamins by the use of a pretreated glassy carbon electrode (PGCE)

PGCE has been prepared by potential cycling (ndash08 to +10 V) and voltammetry is carried

out following anodic oxidation (18 V) Increase in electrochemical responses and wellndash

defined peaks (Epa = ndash0073 V Epc = 0044 V) of certain waterndashsoluble vitamins have

been achieved using PGCE (Gu et al 2001) In pharmaceutical dosage forms a

voltammetric method has been described for the determination of RF and LndashAA Using

GCE both the compounds have been investigated for their electrochemical behavior at

pH 68 (KH2PO4Na2HPO4) The concentration range for the determination of RF is

15 times 10ndash6

ndash3 times 10ndash5

M giving an anodic peak at ndash047 where as for LndashAA acid it is

15 times 10ndash4

M with a peak at +035 V (Mielech 2003)

Square wave adsorptive stripping voltammetry (SWASV) is another method that

has been used for the assay of RF A mercury film electrode (MFE) is used in this

method Subsequent reductive stripping step is carried out at pH 12 after RF has been

adsorbed at 00 V (AgAgCl) A 8 precision has been found with a recovery over 90

and the limit of detection to be 05 nmolL (Economou and Fielden 2005)

The electrochemical determination of RF on glass carbon cyclic voltammetry

electrode has been studied by using cyclic voltammetry This electrode is activated by 80

mol Lndash1

HNO3 solution with an electrode potential in the range of +06 ~ +20 V The

adsorption scanning has been studied in the range of 08 ~ 70 V by changing the RF

concentration from 60 times 10ndash8

mol Lndash1

at 90 mVsec RF shows

characteristics reversible adsorption at the carbon electrode and the calibration curve is

linear in the concentration range of 60 times 10ndash8

mol Lndash1

with a LOD of

10 times 10ndash8

mol Lndash1

(Yang et al 2001)

The voltammetric determination of RF and Lndashascorbic acid (LndashAA) has

simultaneously been carried out in multivitamin pharmaceutical preparations The

electrochemical behavior of RF and LndashAA has been studied in the presence of phosphate

buffer (pH 60) using a glassy carbon electrode RF and LndashAA gave anodic peaks at

ndash 047 and + 035 V versus SCE respectively The oxidation peaks are directly related to

the concentrations of RF and LndashAA This method has been found to be useful for the

determination of RF and LndashAA in the concentration ranges of 15 times 10ndash4

and 15 times 10ndash4

M respectively (Mielech 2003)

24 PHOTOCHEMICAL METHODS

RF and RF 5rsquondashphosphate have been assayed by photochemical method using

injection flow technique Photondashreduction of both the compounds has been carried out

using ethylenediaminetetraacetic acid A linear curve has been obtained at low

concentration using chemiluminescent hydrogen peroxidendashluminol reaction RF a result

of photochemical process has been observed to form 1 5ndashdihydro derivative obtained by

the peroxidation of hydrogen peroxide A linear calibration curve has been obtained in

the concentration range of 1 times 10ndash7

to 3 times 10ndash6

mol Lndash1

(PerezndashRuiz et al 1994)

RF in photodegraded samples and aged vitamin preparations has been determined

by a stabilityndashindicating photochemical method This method is based on the conversion

of RF into lumichrome (LC) in alkaline solution under a control set of conditions (ie

light intensity pH temperature distance and time of exposure) In these conditions the

twondashthird of the RF is converted in to LC and the concentrations of RF in degraded

solutionssamples is determined by the RFLC ratio In this method the photolysed

solution of RF are adjusted to pH 20 and extracted with chloroform The determination

of LC and lumiflavin (LF) is carried out by a twondashcomponent spectrometric method at

356 and 445 nm respectively This method shows a percent recovery of 99 to 101 with

a precision of around 2 (Ahmad et al 2015)

25 ENZYMATIC ASSAYS

The homogenousndashtype enzymendashRF complex based determination of RF and its

binder protein has been performed using synthetic enzymendashbiotin and avidinndashRF

conjugates Amount dependant addition of RF binding protein (RBP) in the determination

of RF results in reversal of observed inhibition and enzymendashbiotin conjugate activity In

the mixture free RF addition results in rendashinhibition of the activity which has been found

concentration dependant Glucose 6ndashphosphate dehydrogenase adenosine deaminase and

alkaline phosphate are the three enzymes determined in this process Significant

inhibition of the catalytic activity of the enzyme has been observed (gt 90 ) when

enzymendashbiotin conjugates were determined using avidinndashRF conjugate binding and the

process has been reversed when RBP was added (Kim et al 1995)

A RF assay based on homogenous type enzyme linked determination has been

developed This method is based on the ability of binding of either analyte vitamin

molecule or glucose 6ndashphosphate dehydrogenasendash3ndashcarboxymethylflavin conjugate on

limited RBP sites which have previously been immobilized using sepharose particles

The catalytic activity of the conjugate is increased significantly Detactability has been

observed using optimal conditions An effect of pH and different organic solvents with

different proportions on the reaction has been studied The ratio of protein binding sites

to the conjugates has been found as the main factor on which the calibration curve

sensitivity and the detection limit for the assay depends The proposed method based on

the RBP sites agrees well with the selectivity and results of the method

(Cha and Meyerhoff 1987)

26 FLOW INJECTION ANALYSIS (FIA) METHOD

The flow injection analysis with chemiluminescence (CL) detection has been

carried out for the determination of RF In this method reduction of RF is carried out with

chromium VI which results in the formation of chromium III The chromium III reacts

with luminal and H2O2 in alkaline solution to produce CL The CL intensity is related to

the concentration of RF which has been found to be linear in the concentration range of

10 times 10ndash10

mol Lndash1

with a detection limit of 30 times 10ndash11

mol Lndash1

method shows a mean recovery of 1013 with a RSD of 18 (Xie et al 2005)

The various analytical methods used for the assay of RF in pharmaceutical

preparations food materials and biological fluids have been described in the above

sections The specificity and sensitivity of these methods would depend on the nature of

the samples vitamin content interference accuracy requirement and other factors The

fluorimetric methods are inherently more sensitive than the spectrometric and

chromatographic methods for the assay of RF in different systems However

spectrometric and chromatographic methods are widely used for the assay of RF in

pharmaceutical preparations

CHAPTER III

PHOTOCHEMISTRY OF RIBOFLAVIN

31 INTRODUCTION

Riboflavin (RF) (1) is a photosensitive compound and therefore its stability in

the pharmaceutical preparations may alter when exposed to light (ie UV light visible

light sunlight) Various studies have been carried out on the photostability of RF in

pharmaceutical preparations (Macek 1960 Deritter 1982 Ahmad and Vaid 2006) and

parenteral nutrition (Allwood 1984 Allwood and Kearny 1998 Buxton et al 1983

Chen et al 1983 Ribeiro et al 2011 Smith and Metzler 1963 Martens 1989

Yamaoka et al 1995 Min and Boff 2002 Casini et al 1981 Asker and Habib 1990

Loukas et al 1995 1996)

RF undergoes a number of photochemical reactions in aqueous solution which

include intramolecular and intermolecular photoreduction photodealkylation (Ahmad

and Vaid 2006 Ahmad et al 2004ab 2013 2014 2015 Heelis 1982 1991 Sheraz et

al 2014b Song 1971) intramolecular and intermolecular photoaddition (Ahmad et al

2004b 2005 2006a Sheraz et al 2014ab) photooxidation (Jung et al 1995)

photosensitization (Huang et al 2004 2006) and photostabilization reactions (Ahmad et

al 2008 2011 2016a Habib and Asker 1991 Sheraz et al 2014b) When RF is

exposed to light it degraded into a number of photoproducts which include

formylmethylflavin (FMF) (4) lumichrome (LC) (5) lumiflavin (LF) (6)

carboxymethylflavin (CMF) (7) cyclodehydroriboflavin (CDRF) (8) 23ndashbutanedione

(9) and isoalloxazine ring cleavage products (Ahmad and Vaid 2006 Ahmad et al

1980 2004ab 2005 2006ab 2008 2009 2010ab 2011 2013 2014 2015ab 2016ab

Cairns and Metzler 1971 Smith and Metzler 1963 McBride and Metzler 1967 Heelis

et al 1980 1991 Schuman Jorns et al 1975 Sheraz et al 2014ab Song et al 1965

Treadwell et al 1968) In the presence of divalent anions (HPO42ndash

SO42ndash

) RF undergoes

photoaddition reactions to form CDRF and in the absence of divalent anions it follow

normal photolysis pathway to form FMF LC and LF A scheme for the photodegradation

pathways is given in Fig 31

Two main types of photoreactions including anaerobic and aerobic photoreactions

are discussed below

32 ANAEROBIC PHOTOREACTIONS

RF at neutral pH when exposed to light results in the fading of yellow colour by

the formation of leucodeuteroflavin The leucodeuteroflavin leads to the formation of

deutroflavin by dehydrogenation caused by oxygen The deuteroflavin in alkaline

solution is converted into LF (Kuhn and WagnerndashJauregg 1934) In the first step of

photodegradation reaction the 2ndashhydroxy group of RF sidendashchain is oxidized to a keto

group to form 78ndashdimethylndash10ndashformylmethyl isoalloxazine (FMF) (4) (Smith and

Metzler 1963) which leads to the formation of LC (5) in acidic and LC (5) and LF (6) in

alkaline solutions (Song et al 1965)

RF photolysis depends on the presence of an electron donor (photoreduction) or in

the absence of an electron donor (photobleaching) The irradiation of an aqueous solution

of RF in the presence of disodium ethylenediamine (EDTA) leads to the loss of colour

but when this solution is exposed to oxygen the colour is regained (Oster et al 1962)

excited singlet state excited triplet state

(8) (5)

(7) (6)

r phot

oadditi

intramolecular photodealkylation

intramolecular photoreduction

[O] neutral and alkaline pH

acid neutral and alkaline pH

Fig 31 Scheme for the photodegradation pathways of RF

This photoreduction of RF in the presence of an external donor results in the

intermolecular reduction of the isoalloxazine ring (Enns and Burgess 1965) whereas

photobleaching is due to the intramolecular reduction of isoalloxazine nucleus by the

ribose sidendashchain (Holmstrom and Oster 1961) This leads to the formation of a 2ndashketo

compound (deutroflavin) that was predicted by Karrer et al (1935)

Under anaerobic and aerobic conditions a variety of alcoholic type sidendashchains on

N(10) position of the isoalloxazine nucleus is photobleached At neutral pH the anaerobic

photolysis of these flavins leads to the formation of alloxazine and a cyclic intermediate

which is oxygen sensitive The ratio of these two degradation products depends on the

length of the sidendashchain Under anaerobic photolysis conditions the primary secondary

and tertiary alcoholic groups attached on the side chain lead to the formation of

aldehydes ketones and regenerated alcohols respectively (Moore and Bayler 1969)

RF and other flavins containing N(10)ndashsubstituted isoalloxazine rings when

irradiated in alcohol and alcoholndashwater mixtures result in the formation of FMF and LC

(Moore and Ireton 1977) Another photoproduct (78ndashdimethylndash10(1ndashdeoxyndashDndasherythrondash

2primendashpentolosyl) isoalloxazine) of RF is formed by its photolysis in the pH range of 4ndash10

and its formation is similar to that of FMF (Cairns and Metzler 1971) At neutral pH

another photoproduct (4primendashketoflavin) of RF is formed like LC and this product is not

easily quenched by the addition of potassium iodide This product is formed by the

abstraction of 2prime and 4primendashα hydrogens in the excited ring (Cairns and Metzler 1971)

Heelis et al (1980) proposed that the triplet state [3RF] of RF is involved in the formation

of FMF below neutral pH whereas an increase in the rate of photolysis of RF at higher

pH is due to the anion radical This anion radical increased the rate of photodegradation

as compared to that at neutral pH (neutral radical)

33 AEROBIC PHOTOREACTIONS

RF on exposure to light in the presence of oxygen forms LC and LF (Kuhn and

WagnerndashJauregg 1934 Holmstrom and Oster 1961 Strauss and Nickerson 1961) and

also results in the breakdown of ribityl side chain (Oster 1951 Shimizu 1955

Fukumachi and Sakurai 1955) This aerobic photolysis of RF and other flavins at acid

pH is said to be a case of general acidndashbase catalysis The degradation rate of aerobic

photolysis is dependent on the buffer components (Halwer 1951)

In aerobic photolysis of RF FMF (deuteroflavin) is an intermediate which on

further photolysis leads to the formation of LF (Svobodova et al 1953) During the

aerobic photolysis of RF at alkaline pH another photoproduct carboxymethylflavin

(CMF) is also formed This photoproduct is formed by the photooxidation of 2ndashcarbonyl

of the sidendashchain of FMF by peroxides (H2O2) (Fukumachi and Sakurai 1955) During

the aerobic photolysis of RF the acidity of the aqueous solution increases due to the

formation of formic acid by the oxidation of the sidendashchain Anaerobic photolysis at pH

72 gives the same product distribution on 28 of photobleaching as that at 50 of

bleaching in aerobic photolysis This shows greater photobleaching of RF on aerobic

photolysis as compared to that of the anaerobic photolysis (Treadwell et al 1968)

In the presence of macormolecules (ie polyvinyl pyrrolidine (PVP) polysorbate

80 sodium dodecyl sulfate (SDS)) the rate of aerobic photobleaching is increased This

increase in the rate of photobleaching is due to the reversible binding of excited RF [RF]

to macromolecules which leads to the formation of the triplet state [3RF] This catalytic

effect of polymer is due to the protection of [3RF] by polymer from quenching by oxygen

(Kostenbauder et al 1965) Under aerobic photolysis RF at pH greater than 60 in the

presence of divalent phosphate (HPO42ndash

) anion or sulfate (SO42ndash

) anion leads to the

intramolecular photoaddition reaction which results in the formation of

cyclodehydroriboflavin (CDRF) (Schuman Jorns et al 1975)

34 TYPES OF PHOTOCHEMICAL REACTIONS

Flavins undergo a variety of photochemical reactions which occurs separately as

well as simultaneously These reactions depend on the nature of flavin and the reaction

conditions Flavins undergo both intermolecular and intramolecular reactions

(Hemmerich 1976 Heelis 1982) Different types of photochemical reactions are

discussed in the following sections

341 Photoreduction

RF undergoes intramolecular as well as intermolecular photoreduction as

discussed below

3411 Intramolecular photoreduction

RF undergoes anaerobic photoreduction in the absence of external electron donor

by the process of intramolecular disproportination This disproportination results in the

oxidation of ribityl sidendashchain and leads to the reduction of isoalloxazine ring

(Holmstrom and Oster 1961 Moore et al 1963 Radda and Calvin 1964) This

reduction in the isoalloxazine ring results in the degradation of the RF which leads to the

formation of FMF LC and LC (Smith and Metzler 1963) This photoreduction or

photodehydration leads to the dehydrogenation of ribityl sidendashchain with the formation of

ketonic or aldehydic functional group in the ribityl sidendashchain (Cairns and Metzler

1971) The intramolecular photoreduction of flavinRF is dependent on the pH and on the

cationic triplet [3RFH

+] and neutral triplet [

3RF] species which react differently (Cairns

and Metzler 1971)

A study has been carried out on the kinetic isotope effect on flavin (10) which

results in the replacement of αndashhydrogen in the ribityl sidendashchain (11) However no

hydroxyl hydrogen replacement has been observed (Moore and Bayler 1969 Moore and

Ireton 1977) In this reaction the αndashhydrogen removal from αndashCH results in the

formation of an intermediate biradical (12) which then disproportionate to form an

αndashketone (13) (Fig 32)

Intramolecular photoreduction of flavinRF involves singlet excited state [1RF]

and the triplet excited state [3RF] (Cairns and Metzler 1971) In an intramolecular

hydrogenndashtransfer reaction the ribityl side chain should be condashplanar with isoalloxazine

ring system (Song and Kurtin 1969) The intramolecular photoreduction rate is

dependent on the solvent polarity and this could be due to the conformational changes in

the ribityl side chain in different solvents (Moore and Ireton 1977 Ahmad et al 2015)

Fig 32 Formation of αndashketone from flavin

3412 Intermolecular photoreduction

Flavins (10) in the presence of amino acids αndashhydroxyndashcarboxylic acids thiols

aldehydes unsaturated hydrocarbon (Knappe and Hemmerich 1972 1976) and αndash

substituted acetic acids (Ahmad and Tollin 1981a) results in the photoredcution that

leads to the formation of 15ndashdihydrogen flavin (H2Flred) (14) or its alkyl adducts

(RndashFlredH) ((15)ndash(17)) (Fig 33)

This H2Flred is reoxidized in the presence of oxygen (O2) to form hydrogen

peroxide (H2O2) and oxidized flavin (Eq 31) (Massey et al 1973)

H2Flred + O2 H2O2 + Flox

Intermolecular photoreduction of flavins has two different mechanisms In the

first step the photoreduction occurs by initial one electron involvement by transferring

from the substrate to the flavin and leads to the formation of flavosemiquinone radical

Fl hv 1Fl

FlH + R1Fl + RH

Fl- + RH+1Flo + RH

Fig 33 Photoreduction of flavins to form 15ndashdihydrogen flavin and alkyl adducts in the

presence of unsaturated hydrocarbons

Photoreduction of flavins in presence of carboxylate anions or substrates is

expressed by the following equation

Fl hv 1Fl

Fl- + RCOO1Fl + RCOO-

R + CO2RCOO-

In this mechanism when flavin is exposed to light it is converted into the excited

singlet state (Eq 35) The excited singlet state when reacts with the carboxylate substrate

(Eq 36) leads to the formation of radicals (ie Flndash and RCOO

) The carboxylic radical

forms an alkyl radical and carbon dioxide (CO2) (Eq (37))

Photodegradation products are formed when two semiquinone radicals

disproportionate to form one reduced and the other oxidized flavin (Eq (38)) or by

radical addition

HFl + HFl

H2Flred + Flox

ProdcutsR

RFlredHHFl + R

Fritz et al (1987) presented a mechanism for the photoreduction of flavins in the

presence of external donor (EDTA) at pH 70 When the flavin is exposed to light it is

excited from the ground state to the excited singlet state (Eq (311))

1FloFl hv

This excited singlet state [1Fl] then through internal conversion is deactivated to

the ground state with release of heat energy (Eq (312))

1Fl oFlic

The flavin singlet excited state is converted into flavin excited triplet state through

intersystem crossing (Eq (313))

1Fl 3Flisc

Triplet excited state [3Fl] may be deactivated with release of heat energy by

coming back to ground state (Eq (314))

3Fl oFl+ heat

In the presence of a quencher the excited triplet state is quenched which leads to

the conversion of triplet state to the ground state with release of energy (Eq (315))

3Fl + oxygen quencher oFl + heat

When [3Fl] reacts with EDTA the flavin is reduced and EDTA is oxidized

(Eq (316))

3Fl + EDTAoFlred + EDTAox

The reoxidation of [oFlred] form occurs in the presence of oxygen which leads to

the formation of ground state flavin [oFl] and peroxide (Eq (317))

oFlred + O2

oFl + H2O2

342 Photodealkylation

Photodealkylation of flavins occurs via an intramolecular mechanism which is

due to the involvement of excited singlet and triplet states (Gladys and Knappe 1974)

Flavin photodealkylation occurs due to the simultaneously breakage of N(10)ndashC(1ʹ) and

C(2ʹ) bond via a direct proton transfer in cisndashperiplanar confirmation that leads to the

formation of LC (Hemmerich 1976) When flavins are photolysed in acetonitrile it

results in the formation of LC (5) and the corresponding alkene or cycloalkene (Gladys

and Knappe 1974)

pH 70hv

(1) (5)

Photodealkylation occurs by two mechanisms The first step involves homolytic

fission of the N(10)ndashC(1ʹ) bond in the biradical intermediate (Moore and Ireton 1977)

However the second step results by a synchronous process that does not involve radical

intermediates (Song 1971) The photodealkylation of RF takes place by the excited

singlet state which leads to the formation of LC (5) and its formation is not retarded by

the addition of triplet state quenchers (Cairns and Metzler 1971) It has been found that

intramolecular photodecarboxylation and dealkylation of flavins is mediated by excited

singlet and triplet state reactions (Gladys and Knappe 1974 Knappe 1975)

Carboxymethyl flavin (CMF) (flavinndash10ndashacetic acid) (7) is formed by the excited triplet

state which results in the formation of a biflavin intermediate This biflavin intermediate

when exposed to light forms LC (5) and other products (Knappe 1975)

343 Photoaddition Reactions

The solvent (R=H or alkyl) when introduced at position Cndash6 or Cndash9 positions of

the benzenoid subnucleus leads to the formation of hydroxy or alkoxyndashdindashhydroflavins

(Eq 318) as an intermediate (Schollnhammer and Hemmerich 1974) When ammonia or

cyanide is introduced in the system containing the flavin the reaction occurs by the attack

of a nucleophile (CNndash NH3

ndash) on the excited triplet state (Traber et al 1981a) These

reactions involve intermolecular photoaddition to RF

1Fl + CH3OH CH3O-Fl redH

The intramolecular photoaddition reactions are similar to that of the

photodehydration of flavin (Schollnhmmer and Hemmerich 1974) These reactions lead

to the formation of CDRF via autoxidation of an intermediate (dihydroriboflavin)

(Schuman Jorns et al 1975) This reaction occurs due to the presence of a nucleophilic

group in the ribityl sidendashchain It has been proposed that in this reaction the addition of a

proton takes place at N(1) and simultaneous deprotonation at C(9) position This leads to

the formation of a stable compound 15ndashdihydrondash9ndashalkoxylndashflavin which is then

converted into the CDRF by the process of autoxidation (Fig 31)

Quenching studies have been carried out to evaluate the involvement if [1Fl] and

[3Fl] states in the reactions of flavins It has been found that excited singlet state of flavin

is involved in photoaddition reaction while excited triplet state is involved in the normal

photolysis (photoreduction) reaction The excited singlet state reaction is dominant when

the triplet state is quenched ie oxygen quenching The photoaddition reaction occurs in

the presence of divalent anions (HPO42ndash

SO42ndash

) above pH 60 This photoaddition

reaction occurs by the formation of a flavinndashdivalent complex that results in the

C(4)O(2ʹα) interaction to form the cyclic product CDRF (8)

344 Photooxidation

Flavins in the presence of oxygen initiate the oxidation of a number of

compounds such as amino acids (Penzer 1970) indoleacetic acid (AmatndashGuerri et al

1990) cyanocobalamin (Hussain 1987) retinol (Futterman and Rollins 1973) bilirubin

(Sanvordeker and Kostenbauder 1974) lipids (Chan 1977) DNA and nucleotides

(Speck et al 1975) and phenothiazines (Uekama et al 1979)

Photooxidation of flavins occurs by electron abstraction from the substrate by

radical mechanism These substrate radicals and flavosemiquinone radicals react and

inhibit the radical back reaction (Vaish and Tollin 1971) Flash photolysis studies have

been carried out to determine the rate of photooxidation of flavin semiquinone radicals It

has been found that the neutral semiquinone radical is unreactive to oxygen as compared

to that of the anionic form of the flavin radical

345 Photosenstization Reactions

RF when exposed to light forms singlet oxygen species from triplet oxygen by

excited triplet state of RF [3RF] and triplet oxygen annihilation mechanism This plays an

important role in the photosensitized reactions (Choe et al 2005 Jung et al 2007)

RFhv 1RF

3RF1RF isc

3RF RF + 3O2

Aerobic RFndashsensitized photodegradation of the endocrine disruptor

44rsquondashisopropylidenebisphenol (BPA) and of similar compounds like 26ndashdibromophenol

and 26ndashdimethyl phenol has been studied in water and waterndashmethanol mixtures by

continuous photolysis using visible light the uptake of oxygen being detected by

polarography stationary and time resolved fluorescence spectroscopy time resolved near

IR phosphorescence detection and laser flash photolysis techniques Bisphenols (BPs)

quench the excited singlet and triplet states of RF and have rate constants near to the

diffusion limit BPs and dissolved molecular oxygen are added in similar concentration

and they competitively quench the excited triplet state of RF As a result of this reaction

singlet molecular oxygen (O2 (1∆g)) and superoxide radical anions (O2

ndash) are produced by

electron and energy transfer The photooxidation products of BPA resulting from

oxidation dimerization and fragmentation have been identified These reactions indicate

that BPs in natural water are photodegraded under environmental conditions in the

presence of an adequate photosenstizer (Barbieri et al 2008)

RF is sensitive to light but it is relatively stable during thermal and nonndashthermal

food processing RF can accept and donate a pair of hydrogen atoms Under the influence

of light RF acts as a photosensitzer or prooxidant for food components During the

photosensitization of RF there is production of reactive oxygen such as singlet oxygen

hydroxyl radical superoxide anion and hydrogen peroxide Reactive oxygen and radicals

produced in this process potentiate the decomposition of proteins lipids carbohydrates

and vitamins RF acts as an excellent photosenstizer for singlet oxygen formation (Choe

and Min 2006)

RF is present in the eye as a normal component and which when exposed to light

triggers photosensitizing activity When this photosensitized RF is influenced by short

wavelength light below 400 nm it damages vitamin C that is present in the lens for the

inhibition of the photosensitization process (Rochette et al 2000)

It has been observed that RF photosensitized singlet oxygen oxidation of vitamin

D is not observed in samples without RF stored in a dark room and also in those samples

containing RF that are stored in dark Vitamin D containing RF is oxidized under the

influence of light Singlet oxygen quenched rate of αndashtocopherol is 250 times 108 M

ndash1s

ndash1 and

for ascorbic acid it is 223times107 M

ndash1s

ndash1 (King and Min 1998)

RF when exposed to light forms LC and LF and this formation is also influenced

by the pH RF when exposed to neutral or acidic pH form LC and when it is exposed to

basic pH it forms LF This conversion of RF to LF and LC is due to the type 1

mechanism of RF photosensitized reaction and singlet oxygen is also involved in the

conversion of RF to LF and LC The rates of reaction of RF LF and LC with singlet

oxygen are 966 times 108 850 times 10

8 and 821 times 10

ndash1s

ndash1 respectively (Huang et al

A study has been carried out on the RF sensitized decomposition of ascorbic acid

(AA) under the influence of light and it has been found that light and RF increases

photodecomposition of AA The photosensitizing activity of RF methylene blue and

protoporphyrin IX is 21511 at Indash2 ppm at different pH values (75 60 and 45) and the

rate constants for the reactions of AA are 663times108 577times10

8 and 527times10

ndash1s

ndash1 It has

been found that RF and methylene blue sensitize photooxidation of AA cyestine shows

strong antioxidant activity that is concentration dependent Alanine and phenylalanine

(01 ) show antioxidant effect on the RF sensitized photooxidation of AA and

prooxidant effect on the methylene blue sensitized photooxidation Tyrosine at 01

concentration shows prooxidant effect on both RF and methylene blue sensitized

photooxidation of AA but tryptophan (01 ) shows antioxidant or prooxidant effect on

the photooxidation of AA depending on the storage time (Jung et al 1995)

The photodegradation of tryptophan in oxygen saturated aqueous solution

resulting in the generation of reactive oxygen species 1O2 OH H2O2 and O2

ndash is

sensitized by RF Photodegradation experiments have been runs with 14

CndashRF and 14

Cndash

tryptophan The photoproducts have been separated by Sephadex Gndash15 and C18ndashHPLC

and detected as aggregate forms of RF indolic products associated to flavins indolic

products of molecular weight higher than tryptophan formyl kynurenine and other

tryptophan photoproducts (Silva et al 1994)

RF and amino acids such as phenylalanine tryptophan leucine isoleucine and

valine are present in milk RF as a photosensitzer results in the destruction of essential

amino acid by the process of oxidation It has been found that in aqueous samples that

contain increased concentration of trolox (TX) and AA show an increased head space

oxygen depletion and this is due to the oxidation of trolox AA and amino acid in the

presence of RF HPLC has shown that trolox and ascorbic acid decrease the

photodegradation of phenylalanine tryptophan and tyrosine and this is due to the

presence of trolox and AA acting as singlet oxygen quenchers of tryptophan and tyrosine

(Reddy 2008)

The effect of pH and ionic micelles on the rate of formation of products on the

irradiation of RF in the presence of tryptophan has been studied by absorption and

fluorescence spectroscopy In anaerobic conditions the formation of RFndashtryptophan

complex is inhibited in acid solution by the addition of anionic (sodium dodecylsulphate)

and cationic (cetyltrimethylammonium bromide) micelles In the presence of RF the

oxidation of tryptophan is faster in alkaline solutions than in acid solutions (Silva et al

A study has been carried out in the presence of flavins as sensitizers on the

photooxidation of substituted phenols under aerobic condition to determine the fate of

synthetic chemicals in environment RF is easily decomposed to form LC by the

influence of several minutes illumination with simulated sunlight It has been found that

LC is extremely stable toward sunlight and it is the major flavin component in natural

water The order of photolysis rate is pndashmethoxyphenol gt pndashchlorophenol gt phenol gt

nitrophenol in the LC sensitized photodecomposition of substituted phenols It has been

found that the total organic carbon (TOC) is decreased from the reaction solutions of all

the phenols except pndashnitrophenol (Tatsumi et al 1992)

In the presence of RF 4ndashhydroxyquinolone (4ndashOHQ) and 8ndashhydroxyquinolone

(8ndashOHQ) are photooxygenated under the influence of visible light in watermethanol

(91 vv) mixture RF in this reaction acts as a dye sensitizer Both of the quinolones are

transparent under the influence of visible light but 8ndashOHQ has five time faster

degradation than that of 4ndashOHQ The kinetic data shows that 4ndashOHQ degrades by the

mechanism of superoxide radical anion where as 8ndashOHQ degrades by the mechanism of

singlet molecular oxygen along with superoxide radical anion RF as a sensitizer is

photodegraded under the influence of visible light and is regenerated in the presence of

either of these two quinolones by an electron transfer process that produces superoxide

radical anion (O2-) (Criado et al 2003)

The aerobic irradiation of methanolic solutions either of phenol type compounds

pndashphenylphenol (PP) pndashnitrophenol (NP) and phenol (Ph) or other phenolic derivatives

pndashchlorophenol (CIP) and pndashmethoxyphenol (MeOP) in the presence of RF as sensitizer

results in the photodegradation of ArOH and the sensitizer A complex mechanism is

involved in the photodegradation of ArOH in which superoxide radical anion (O2ndash

singlet molecular oxygen (O2 (1∆g)) is involved This mechanism is highly dependent on

the concentration of ArOH (Haggi et al 2004)

346 Photostabilisation Reactions

The effect of certain stabilizers on the aerobic photobleaching of RF has been

examined under the influence of fluorescent light The greatest photostabilizing effect is

seen by disodium ethylenediamine (EDTA) which is followed by thiourea

methylparaben DLndashmethionine and sodium thiosulfate The photostabilizing effect of

these compounds increases with an increase in their concentration The photodegradation

of RF solutions is influenced by pH and buffer species and EDTA (Asker and Habib

The quantum efficiency (Φ) of RF under aerobic conditions has been determined

by a microirriadiation method It has been found that the initial quantum yield of RF is

independent of light intensity wavelength of light and concentration The quantum

efficiency of RF is decreased in the presence of phenols and there is linear relation

between Hammettrsquos Sigma values and rates of photodegradation As compared to

phenols benzyl alcohol and benzoic acid are ineffective as photochemical stabilizers

The photodegradation of RF is enhanced by cinnamyl alcohol which acts as an electron

donor (Shin et al 1970)

A study has been carried out on the photostablization of RF in liposomes in

aqueous solution under various irradiation conditions liposomal composition

concentration pH and ionic strength It has been found that the photostability of RF is

increased in the presence of neutral and positively charged liposomes and by increasing

the concentration of dimyristoylndashphosphatidylcholine (DMPC) in the composition of

liposome The photostability of RF in the presence of 5ndash8 mM DMPC increases up to 23

fold as compared to a control buffer solution It has been found that the pH of the

medium effects the photostability of RF and the ionic strength of solution does not affect

The photodegradation of RF follows firstndashorder kinetics in the presence and absence of

liposomes (Habib and Asker 1991)

A study has been carried out on the formulation of liposomal preparations of RF

with a change in the concentration of phosphatidylcholine (PC) showing an increase in

their entrapment efficiency from 26 to 42 Physical characterization of these liposomes

has been carried out by dynamic light scattering (DLS) and atomic force microscopy

(AFM) RF encapsulated in liposomes when subjected to visible light follows firstndashorder

kinetics for its degradation RF and its photoproduct (LC) in liposomes were assayed by a

twondashcomponent spectrometric method at 356 and 445 nm and to compensate for the

interference of liposomal components an irrelevant absorption correction method was

used It has been found that with an increase in PC concentration from 1215ndash1485 mM

the rate of RF photodegradation is decreased This decrease in the rate is due to the

interaction of RF with PC and its reductive stabilization (Ahmad et al 2015b)

347 Factors Affecting Photochemical Reactions of RF

There are a number of factors which affect the photochemical reactions of RF

These factors are discussed below

3471 Radiation source

In the photolysis reactions of drugs the radiation source plays an important role

RF in the milk when exposed to sunlight degraded around 30 in 30 mins (Wishner

1964) In the powder forms RF is much stable as compared to that of the solution form in

which when exposed to light it is degraded into different photoproducts (FMF LC LF

CMF etc) (Ball 2006 Cairns and Metzler 1971 Smith and Metzler 1963 Ahmad and

Vaid 2006 Treadwell et al 1968 Ahmad et al 2004ab 2005 2006ab 2008 2009

2010 2011 2013ab Sheraz et al 2014a McDowell 2000) Different studies have been

carried out on the photolysis of RF using low and high intensity radiation sources

(Ahmad et al 2004a 2006 Ahmad and Rapson 1990 Becker et al 2005 Dias et al

2012 Mattivi et al 2000 Sato et al 1982) A comparison has been made on the effect

of UV and visible radiation on the rate of photolysis of RF (Ahmad et al 2004 2006)

The photoproducts formed in both cases are similar however the rate of reaction is

higher in the case of UV radiation as compared to the visible light This increase in rate is

due to the intensity of UV radiation (219plusmn012 times 1018

qsndash1

) as compared to that of visible

light (114 plusmn01 times 1017

qsndash1

(125 W) (Ahmad et al 2004a)

A study has been carried out on RF tablets exposed to a xenon lamp emitting in

the range of 300ndash800 nm It has been found that the greater colour change in samples

(yellow to green) was at 250 Wm2 after initial exposure to xenon lamp This change in

colour (yellow to green) is due to the visible light gt 400 nm and only LC was found as

the degradation product (SuendashChu et al 2009)

3472 pH effect

The pH of an aqueous solution influences the photodegradation reactions of RF

and its photoproducts The major photoproducts FMF and LC are formed in both the

acidic and alkaline pH while LF is formed in the pH range of 70 to 120 The formation

of all these products is due to the oxidation of the ribityl sidendashchain CMF βndashketoacid

and a diketo compound are minor photoproducts CMF is formed at pH 10ndash120 while

βndashketoacid and the diketo compound are formed at pH 100ndash120 The βndashketoacid and the

diketo compound are formed by the cleavage of the isoalloxazine ring by the alkaline

hydrolysis of RF (Song et al 1965 Treadwell et al 1968 Ahmad et al 2004a 2013

Ahmad and Rapson 1990) LC and LF are formed by the excited triplet state via an

intermediate photoproduct FMF (Ahmad and Rapson 1980 Ahmad et al 2004ab 2005

2006ab 2008 2009 2010 2011 2013ab) LC is stable at lower pH as compared to that

of higher pH which is due to its protonation at lower pH However LF is further

degraded at pH 140ndash146 to form 78ndashdimethylisoalloxazine anionic

methylisoalloxazine and quinoxaline derivatives (12ndashdihydrondash2ndashketondash167ndashtrimethylndash

1Hndashquinoxalinendash2ndashone) by cleavage of the isoalloxaine ring (Penzkofer et al 2011)

Another photoproduct (23ndashbutanedione) of RF which has buttery smell is formed in 01

M phosphate buffer at different pH (450 650 850) after light exposure This product is

formed via a ribityl sidendashchain cleavage through the effect of anion singlet oxygen (Jung

et al 2007)

A detailed study has been carried out on the photolysis of RF in the pH range of

10ndash120 It has been found that under UV and visible light the maximum stability is

achieved at pH 50ndash60 which is due to the lower redox potential of RF at this pH The

rate of photolysis at pH 100 is 80 fold higher as compared to that of 50 which is due to

the higher redox potential and higher reactivity of the flavin triplet state at this pH Above

pH 100 the rate of photolysis decreases due to the anion formation of RF (Ahmad et al

2004a)

3473 Buffer effect

The photolysis of RF has been found to be influenced by the kind and

concentration of the buffer used Several studied have been carried out on the catalytic

effect of buffers ie phosphate acetate and carbonate (Schuman Jorns et al 1975

Ahmad et al 2004ab 2005 2006 2010 2013) However borate (Ahmad et al 2008)

and citrate (Ahmad et al 2011) have a photostabilizing effect on RF In borate buffer RF

forms a complex with borate ion to inhibit its photolysis The divalent citrate ions

decrease the fluorescence of RF due to quenching of the excited singlet state and thus

decrease the rate of photolysis The trivalent citrate ions show a greater stabilizing effect

due to the quenching of the excited triplet state (Ahmad et al 2008 2011) Acetate

(pH 38ndash56) and carbonate (pH 92ndash108) buffers exert a catalytic effect on the

photolysis of RF The acetatendash and carbonatendashcatalyzed reactions represent bell shaped

and steep curve type kndashpH profiles respectively The rate of photolysis of RF has been

found to be catalyzed by HCO3ndash and CO3

2ndash ions in the alkaline solution and there is a

major role of CO32ndash

ions in the catalysis of RF (Ahmad et al 2014a)

The intramolecular photoreduction and photoaddition reactions of RF in the

presence of phosphate buffer have been studied in detail The analysis of RF and its

photoproducts of both reactions (CDRF FMF LC LF) is carried out by a

multicomponent spectrometric method It has been found that H2PO4ndash and HPO4

2ndash species

of phosphate buffer play a major role in the degradation of RF The H2PO4ndash species are

involved in the photoreduction reaction to form LC and LF while HPO42ndash

(02 M ge)

catalyze the photoaddition reaction to from CDRF (Ahmad et al 2005) The effect of

pH buffer and solvent viscosity on the aerobic and anaerobic photolysis of FMF has been

studied It has been found that the rate of photolysis under aerobic conditions is higher at

pH 40 and above pH 100 The rate of photolysis at alkaline pH is higher due to

sensitivity of flavin triplet state to alkaline environment The rate of photolysis of FMF is

linearly increased with the inverse of solvent viscosity (Ahmad et al 2013)

3474 Effect of complexing agents

In the presence of divalent species (ie HPO42ndash

tartarte succinate

malonate) RF is rapidly degraded via an intramolecular photoaddition pathway through

the formation of a RFndashdivalent ion complex (Schuman Jorns et al 1975 Ahmad et al

2004b 2005 2006 2010) The rate of photodegradation is lower in the case of organic

species (Ahmad et al 2010) In the presence of sulfate anions the rate of photolysis is

much higher as compared to that of phosphate anions This is probably due to the

formation of a strong divalent anion complex higher electronegative character and higher

amount of anionic species in the case of sulfate (Schuman Jorns et al 1975 Ahmad et

al 2010) These reactions can be expressed (Ahmad et al 2005 Ahmad and Vaid 2006)

as follows

RF [1RF] LC

hv H2PO4-

[3RF][1RF] RFH2

phosphateleucodeutroflavin

O2 FMF + side-chain products

FMFhv LC + side-chain products

FMFHOH LC + LF + side-chain products

In the presence of HPO42ndash

RF undergoes photoaddition reaction involving the

formation of a RFndashHPO42ndash

complex which on the absorption of light forms an excited

singlet state [1RF] [

1RF] is then converted into a dihydroflavin intermediate which upon

autoxidation gives CDRF

RFHPO4

RF-HPO42- hv [1RF]

complex

dihydroflavin autoxidation[1RF]intermediate

A study has been carried out on the effect of caffeine complexation on the

photolysis of RF in the pH range of 20ndash105 The rate of photolysis decreases with an

increase in the caffeine concentration which shows that caffeine exerts inhibitory effect

on the photolysis of RF It has been found from the kndashpH profile that initially the rate of

photolysis increase upto 100 and at pH 20 and 105 the lower photolysis rates are due to

the ionization of RF The interaction of RF with caffeine gives a bell shape curve in the

pH range of 30ndash60 and then a sigmoid curve in the pH range of 70ndash100 This shows

that a decrease in the rate of photolysis of RF in the presence of caffeine is due to

monomeric interaction and complex formation between RF and caffeine (Ahmad et al

A photodegradation study of RF (50 times 10ndash5

M) in phosphate buffer (02ndash10 M)

in the presence and absence of caffeine (250 times 10ndash4

M) has been carried out at pH 60ndash

80 In the presence of phosphate buffer RF undergoes photoreduction and photoaddition

reactions simultaneously that result in the formation of LC and CDRF respectively as

the major photoproducts It has been found that an increase in phosphate concentration

leads to greater formation of CDRF The formation of CDRF in the presence of caffeine

is enhanced by the photoaddition reaction due to suppression of the photoreduction

pathway of RF (Sheraz et al 2014a)

Fluorimetric studies have been carried out on RFndashcyclodextrin (CD) complex

formation using a nonndashlinear least square model Differential scanning calorimetry (DSC)

and 1H NMR spectrometry have been used for the confirmation of a RFndashβndashCD complex

in the solid state and in aqueous solution respectively (Loukas and Vraka 1997)

Spectroscopic and solubility methods have been used to study inclusion complex

formation of hydroxypropylated αndash βndash and γndashCD with RF and alloxazine Alloxazine

which is an analog of RF has been used to evaluate the role of ribityl and methyl

substituent in complexation It has been found that the cavity of hydroxypropylndashβndashCD is

appropriate for the formation of stable RF complexes Because of van der Waals forces

and hydrogen bonding these complexes were stabilized 1H NMR and computer modeling

was used to confirm the insertion of RF in the CDndashcomplex (Terekhova et al 2011a)

A thermodynamic study has been carried out on the inclusion complex formation

of αndash βndash and γndashCD with RF and alloxazine The influence of reagents structure on the

complex formation has been related to thermodynamic parameter (K ∆cG0 ∆cH

0 ∆cS

It has been found that αndashCD shows less bonding affinity to RF and alloxazine as

compared to βndashCD This binding is associated with negative enthalpy and entropy

changes that involve van der Waals forces and hydrogen bonding Ribityl sidendashchain

prevents the penetration of RF in the macrocyclic cavity (Terekhova et al 2011b) Nonndash

inclusion complexes between RF and CD have been prepared to investigate the molecular

interaction between βndashCD (HPβndashCD) and their anticancer activity UVndashvis and NMR

spectrometry fluorimetry and DSC have been used for the physiochemical

characterization of these formulations The interaction between RF and CD has been

evaluated by molecular dynamics simulation cytotoxicity of RFndashCD against prostate

cancer by inndashvitro cell culture tests It has been found that there are no physicochemical

changes in RF on complexation with βndashCD and HPβndashCD At low concentration βndashCD

and HPβndashCD interaction is due to hydrogen bonding between flavinoid and external ring

of CDs RFndashCDs complexes have increased RF solubility and antitumor activity (de

Jesus et al 2012)

3475 Effect of quenchers

In pharmaceutical preparations of RF the external quenchers are added for the

improvement of quantum yield of photochemical reactions without the fluorescence

quenching of RF (Holmstrom et al 1961) A variety of external quenchers have been

used to deactivate the RF excited states These includes βndashcarotene and lycopene

(Cardoso et al 2007) glutathione and Dndashmannitol (Baldursdottir et al 2003) phenol

(Song and Metzler 1967) polyphenols (ie catechin epigallocatechin rutin) (Bucker et

al 2005) potassium iodide (Baldursdottir et al 2003) purine derivatives (ie uric acid

xanthine hypoxanthine) (Cardoso et al 2005) vitamin B6 (Natera et al 2012)

tocopherols (Cardoso et al 2007) xanthone derivatives (Hiraku et al 2007) 14ndash

diazabicylol [222] octane 25ndashdimethylfuran (Bradley et al 2006) ascorbic acid and

sodium azide In RF solution ascorbic acid quenches both the singlet oxygen and the

excited triplet states of RF whereas sodium azide only quenches singlet oxygen (Huang

et al 2004)

3476 Effect of solvent

Solvent polarity affects the rate of photolysis of RF due to conformational

changes in ribityl sidendashchain of RF in organic solvents (Moore and Ireton 1977) RF is

more stable in less polar solvents (Koziol 1966a) while in alcohol and alcoholndashwater

mixtures exposed to light it is degraded to FMF and LC (Moore and Ireton 1977) LC

has been found to be the major photoproduct of RF in organic solvents (ie acetic acid

acetone dioxane ethanol pyridine) (Koziol 1966ab Koziol and Knobloch 1965) The

rate of photodegradation of RF in greater in organic solvent as compared to aqueous

solution (Koziol 1966a Koziol and Knobloch 1965) This may be due to the effect of

physical properties of the solvents (ie viscosity polarity etc) (Ahmad et al 2006

2013a Ahmad and Fasiullah 1990 1991 Moore and Ireton 1977)

The photodegradation of RF is also influenced by the quality of water (ie D2O

distilled water) The rate of photodegradation is higher in D2O (66) as compared to that

of the distilled water (40) (Huang et al 2004) UVndashvisible spectrometric methods have

been used to study the effect of aqueous and organic solvent on the photolysis of FMF

(Ahmad et al 1990 1991 2006 2013a) It has been found that the photolysis of FMF

does not follow firstndashorder kinetics in organic solvents and water The rate of photolysis

of FMF is dependent on the dielectric constant and increases with an increase in the

dielectric constant of the solvent (Ahmad et al 2013a)

A study has recently been made on the photolysis of RF in water (pH 70) and in

organic solvents (ie acetonitrile methanol ethanol 1ndashpropanol 1ndashbutanol ethyl

acetate) using a multicomponent spectrometric method The rate of photolysis of RF is a

linear function of solvent dielectric constant due to the participation of a dipolar

intermediate in the reaction pathway (Ahmad and Tollin 1981a) The rate of photolysis

also shows that with an increase in electron acceptor (EA) number the rate of photolysis

is increased This shows the degree of solutendashsolvent interaction in the reaction (Ahmad

et al 2015a)

3477 Effect of ionic strength

The effect of ionic strength (01ndash05 M) on the photodegradation reactions

(photoreduction and photoaddition) of RF in phosphate buffer (pH 70) has been studied

The results show that with an increase in the ionic strength the rate of photolysis of RF is

also increased The effect of phosphate buffer concentrations (01ndash05 M) on the

phororeduction and photoaddition pathways of RF has also been evaluated An increase

in buffer concentration leads to an increase in the photodegradation of RF by both

pathways In the presence of NaCl the excited singlet state of RF forms an exciplex with

NaCl which leads to the formation of photoproducts at a faster rate (Ahmad et al 2016a)

3488 Effect of formulation

There are various formulation characteristics such as source (ie synthetic

biosynthetic natural) irradiation (ie occasional continuous) tablet processing (ie

direct compression wet granulation) that affect the photochemical reactions The change

in colour in synthetic powder samples on irradiation was found gradual while in

biosynthetic samples the change was instant at a radiation of greater than 450 kJm2

(SuendashChu et al 2009) In solid dosage forms RF colour change is due to the phenomena

of photochromism This change in colour is only on the surface and does not affect RF

quantitatively (SuendashChu et al 2008 2009)

The photostability of RF could be improved by encapsulating it in liposomes The

stability of RF in liposomal preparations depends on the composition of liposomes pH of

the preparation and concentration of ingredients (Habib and Asker 1991 Chauhan and

Awasthi 1995 SenndashVarma et al 1995 Arien and Dopuy 1997 Loukas 1997 Ionita

and Ion 2003 Bhowmik and Sil 2004 Ahmad et al 2015b) Dimyristoylndash

phosphatidylcholine (DPC) concentration affects the photostability of RF An increase in

DPC concentration leads to an increase in the photostability of RF (Habib and Asker

1991 Loukas 2001)

CHAPTER IV

INTRODUCTION TO NANOPARTICLES AND

41 INTODUCTION

The word nano is derived from a Greek word dwarf and nanometer is

onendashbillionth of a meter (10ndash9

m) The word nanotechnology (NT) was first used by Norio

Taniguchi in Japan in 1974 (Royal Society 2004) Eric Drexler (1986 1992) who is

known to be a God Father of NT defined NT as a molecular nanotechnologyprocess

which deals with the transfer of molecules and atoms to the nanoscale products NT is a

vast term and it deals with more than one disciplines based on the scientific and

technological principles for the design preparation and characterization of nanomaterials

(NMs) (Farokhzad and Langer 2009 Ferrari 2005 Fox 2000 Jiang et al 2007

BrannonndashPeppas and Blanchette 2004 Sinha et al 2006 Uchegbu 2006) It is also

defined as the activity which is aimed to understand the natural laws on the level of

nanoscale (Balzani 2005) NT is referred as science technology and engineering for the

preparation of NMs on the scale of 1ndash100 nm (Alexis et al 2010) In NT NMs are

defined as any small material or object which itself behaves as a simple single unit for

transportation and exhibiting its properties These NMs cover the range of 100ndash2500 nm

Ultrafine particles are in the size range of 1ndash100 nm and their physical and chemical

properties depend on the nature of material through which they are prepared NPs are the

engineered structures with a diameter of less than 100 nm and are prepared by the

physical and chemical process with many definite properties (Gwinn and Vallyathan

2006) Different organizations have defined NPs which is given in Table 41

Table 41 Definition of Nanoparticles (NPs) and Nanomaterials (NMs) according to

different Organizations (Horikoshi and Serpone 2013)

Organization NPs NMs

International Organization

for Standardization (ISO)

1ndash100 nm ndash

American Society of

Testing and Materials

(ASTM)

Ultrafine particle whose

length in 2 or 3 places is

1ndash100 nm

National Institute of

Occupational Safety and

Health (NIOSH)

Particle diameter in the range

of 1ndash100 nm or fiber

spanning range in 1ndash100 nm

Scientific Committee on

Consumer Products (SCCP)

At least one dimension in

nanoscale

Internal structure or one

side in nanoscale range

British Standards Institution

All the dimensions are in the

nanoscale range

Bundesanstalt fuumlr

Arbeitsschutz und

Arbeitsmedizin (BAuA)

nanoscale range

Material consisting of a

nanostructure or a

nanosubstance

There are certain limitations which have been applied to NT as the utilization of

materials with structural orientation between the atom and at the molecular scale but at

least the dimensions must be in the nanoscale range (Rao and Cheetham 2001 Rao et al

2002 Jortner and Rao 2002) NPs are gaining importance in modern science and

technology due to the ability of a scientist to manipulate their properties according to

onersquos requirements

42 RIBOFLAVIN AND NANOTECHNOLOGY

Riboflavin (RF) has been used as a photosensitizer stabilizer of nanoparticles

(NPs) biosensor and for other purposes in nanotechnology These aspects are described

in the following sections

421 Photosenstizer

A study has been made for the photosensitization of colloidal ZnO NPs with RF

and the determination was carried out by absorption fluorescence and time resolved

fluorescence spectrometry RF is strongly adsorbed on the ZnO NPs surface and the

association constants have been obtained by fluorescence quenching The Rehem Weller

equation has been used for the calculation of free energy change (∆Get) for the electron

transfer reaction (Vaishnavi and Renganathan 2012)

RF acts as a photosensitizer in the photooxidation of impurities present in water

courses lakes and seas It is known that RF interacts with aromatics sorbed on silica

sediments or on suspended silica particles In a study the characterization and

modification of silica NPs has been carried out by the condensation of silanol groups of

the particles with Endashcinnamic alcohol This reaction has been confirmed by FTIR solid

state 13

C and 29

Si cross polarization magic angle spinning (CPMAS) NMR and also by

the reduction of specific surface area measured by BET thermal analysis and

fluorescence spectrometry It has been found that RF fluorescence is quenched in the

presence of Endashcinnamic alcohol in aqueous media or in suspensions The quenching may

be due to the formation of 11 complexes between ground state of RF and free or

adsorbed cinnamic alcohol This complex formation has been confirmed by density

functional theory (DFT) calculations in aqueous medium and also by RF fluorescence

quenching on the addition of cinnamic alcohol (Arce et al 2014)

422 Stabilizer

Gold (Au) NPs are stabilized by using RF against trisndashbufferndashinduced

aggregation In the presence of Hg2+

ions RF could be released from AundashNPs surface

resulting in the formation of a RFndashHg2+

complex and leading to the aggregation of Aundash

NPs in trisndashbuffer This aggregation depends upon the concentration of Hg2+

ions This

method helps in the detection of Hg2+

ions in the concentration range of 002ndash08 microM

with the detection limit of 14 nM It indicates that Hg2+

ions shows good selectivity over

other metal ions (Cu2+

K+) (Xu et al

423 Photoluminescence

A study has been carried out on the interaction of luminescent water soluble ZnS

NPs with flavin RF quenched ~60 of the photoluminescence of ZnS NPs but FMN

and FAD showed different quenching pattern of photoluminescence under these

conditions It has been found that there is no effect on luminescence intensity of ZnS NPs

when flavin are bonded with proteins such as glucose oxidase (scavenging of

photogenertaed electron of ZnS NPs by the flavin molecules may be attributed) to the

decrease in luminescence intensity The quenching of ZnS NPs with flavin shows a linear

SternndashVolmer plot and SternndashVolmer constants are decreased in the order of Ksndashv(RF) gt

Ksndashv(FAD) gt Ksndashv(FMN) This study gives a beneficial protocol for the fluorimetric

determination of RF content in biological systems (Chatterjee et al 2012)

The grapheme oxide (GO)ndashRF hybrids have been decorated by AgndashNPs with

different compositions Scanning electron microscopy of GOndashRndashAg shows a helical

fibrillar morphology that is different from the bar and wrinkled sheet of R and GO

respectively The FTndashIR spectra show that GO gives a supra molecular complex with R

and AgndashNPs that are stabilized by R and GO The UVndashvis spectra of these complexes

show a larger shift of surface Plasmon band from 390 to 570 nm The spectra of cellular

dichorism show a sudden change in the GOndashRndashAg system as compared to the GOndashR

system for a weight ratio of GO to R of 13 This suggests that AgndashNPs are enveloped in

GOndashR hybrid and R moieties The photoluminescence intensity of R is increased in the

GOndashR hybrids as compared to that of GOndashRndashAg ones The dcndashconductivity is increased

for GOndashR hybrids by the magnitude of addition of AgndashNPs Characteristics curves for

GOndashRndashAg (GOR) show negative differential resistance due to charge trapping on the

silver of NPs followed by stabilization by R (Routh et al 2012)

424 Biosensor

A study has been carried out for the fabrication and testing of RF as a biosensor

It is based on the use of Cr doped SnO2 NPs The CrndashSnO2 NPs are prepared by the

microwave irradiation method using different chromium concentrations (0ndash5 ww) In

this study the magnetic studies have also been carried out which show that only 3 wv

Crndashdoped nanondashSnO2 particles have ferromagnetic properties at room temperature It has

also been found that CrndashSnO2 NPs modified electrode response to RF is linear in the

concentration range of 02 times 10ndash6

to 10times 10ndash4

M with a limit of detection of 107 nM This

fabricated sensor shows good antindashinterference ability against electroactive species and

metal ions Hence it has proved to be beneficial for the determination of RF in

pharmaceutical samples (Lavanya et al 2013) The in vitro detection of RF has been

carried out by a RF binding aptamer (RBA) in combination with gold NPs (AuNPs)

These RBAndashAuNPs conjugates respond colorimetrically in the presence of RF This

method has been used as a model study to check the modification of aptamer sequence

effect on the RBAndashAuNPs stability and their response to the specific target The length of

the aptamer affects RBAndashAuNPs stability as observed by dynamic light scattering and

UVndashaggregation kinetic studies (Chavez et al 2008)

A simple and sensitive electrode has been prepared which is based on nickel

oxide NPsRFndashmodified glass carbon (NiONPsRFG) for the determination of hydrogen

peroxide This electrode is immersed in the RF solution for 5 to 300 seconds and the

projected molecules are immobilized on the surface of the electrode as a thin film This

electrode shows well defined redox couples in the pH range of 2 to 10 having surface

confined properties The results obtained from this electrode show that RF is adsorbed on

the surface of NiO NPs The surface coverage and hetergenous electron transfer rate

constants (ks) of RF immobilized on NiOndashGC electrode are 483 times 10ndash11

molcm2 and

54s respectively This sensor has a powerful electrocatalytic activity for H2O2 reduction

The sensitivity catalytic rate constant (kcat) and limit of detection of this electrode for the

reduction of H2O2 are 24 nA microM 73 (plusmn02) times 10ndash3

Mndash1

sndash1

and 87 nM respectively and

found to be linear in the concentration range up to 30 mM (Roushani et al 2013)

The composite film of Au fine particles and RF are used for the circular dichorism

(CD) studies in the visible region It has been found that the chiral molecules bound on

the surface of Au particles are not essential for Plasmonndashinduced CD and composite

films that contain a dye and glucose in place of Au particles and RF induced signal of CD

at wavelengths of their absorption maixma The polarity of CD is altered by using

different enantiomer of glucose (Kosaka et al 2012)

A simple novel sensitive and selective aptasensor has been developed for the

detection of cocaine an addictive drug by using an electrochemical transduction method

This sensor has been constructed by the covalent immobilization of Ag NPs (aptasensor

functionalized) on a nanocomposite (MWCNTsILChit) for the sensing interface that

improves the performance characteristics and conductivity of the aptasensor and

increases the loaded amount of the aptamer DNA sequence RF for the first time has been

used as a redox probe for the development of an aptasensor to detect cocaine In this

study it has been found that Ag NP leads to speed up the electron transfer kinetics that is

related to the reduction of RF The differential pulse voltammteric (DPV) signal of RF is

decreased with the increased concentration of cocaine in the range of 2 nMndash2 5 microM with

a limit of detection of 150 pM (Roushani and Shahdostndashfard 2015)

Membranes of nafionndashRF have been constructed and characterized by scanning

electron microscopy transmission electron microscopy UVndashvisible spectroscopy and

cyclic voltametry The average diameters of prepared NPs are 60 nm and these

membranes exhibit quasindashreversible electrochemical behavior with a potential of ndash562 plusmn1

mV by using a gold electrode By studying electrochemical parameters of this system it

has been found that the system has good and stable electron transfer properties In this

study horsereddish peroxide (HRP) has been immobilized on the RFndashnafion membrane

and electrochemical behavior of HRP has been found to be quasindashreversible with a

potential of 80 plusmn5 mV This film shows good catalytic activity via the reduction of H2O2

(RezaeindashZarchi et al 2008)

The NPs of ferric oxide (Fe3O4) and binary mixture of Fe3O4 via an ionic liquid

1ndashhexylndash3ndashmethylimidazolium bromide (ILndashFe3O4) have been prepared and used for the

adsorption of ascorbic acid (AA) folic acid (FA) and RF The morphology and size of

NPs have been studied by transmission electron microscopy Xndashray diffraction

thermogravimetric analysis and FTIR spectroscopy The immersion technique is used for

the determination of pH of the point of zero charge (pHpze) for both NPs This

determination is based on experimental curves and results obtained are under the

operational condition (40 mg of NPs contact time 10 mins initial concentration of

vitamins 20 mgL) The thermogravimetric analysis shows that Freundlich model lies on

the equilibrium data as compared to that of DubininndashRadushkevich model The

adsorption capacities of RF FA and AA are 48 225 and 69 mgg respectively of

adsorbent These capacities are dependent upon the pH of the solution chemical structure

of the adsorbent and temperature The pseudondashfirst order and pseudondashsecond order

kinetic models have been predicted by the comparative analysis of rate parameters

correlation coefficient and equilibrium adsorption capacity It has also been found that

the adsorption of FA and AA is endothermic and could be desorbed from ILndashFe3O4 NPs

at pH 30 by using NaCl for the recyclization of NPs (Kamran et al 2014)

The free radical polymerization of Nndashisopropylacrylamide is used for the

preparation of hybrid hydrogels of RF and poly(Nndashisopropylndashacrylamide) (PNIPAAM)

N Nˊndashmethylene bisacrylamide is used as a cross linker for RF in the concentration

range of 1ndash3 mM It has been found that the invariance of storage (Gˊ) and loss (Gˊˊ)

moduli at a wide range of angular frequency and Gˊ gt Gˊˊ for RFndashPNIPAAM systems

behave like a gel in a hybrid state The Gˊ and Gˊˊ are decreased with an increase in RF

concentration but this decrease is four times higher in case of Gˊ than that of Gˊˊ As

compared to PNIPAAM gels RFndashPNIPAAM gels have higher critical strain value that

increase with an increases in RF concentration This indicated that RF acts as a

supramolecular crossndashlinker and the intensity of RndashPNIPAAM gels increases with an

increase in RF concentration This variation with temperature and different pH shows a

higher intensity with temperature The maximum intensity is at ~ 30 oC which is due to

coilndash tondashglobule transition of PNIPAAM gels and could be used for temperature

detection as a probe (Chakraborty et al 2014)

425 Target Drug Delivery

In the malignant cells of human breast and prostate cancers the RF receptors are

overexpressed and these cells contain potential surface markers that are important for

targeted delivery of drugs and for the imaging of molecules In a study the fabrication

and characterization of core shell NCs having gold NPs (Au NPs) and coating of RF

receptor poly (amido amine) dendrimer has been carried The aim of this study was to

design NCs as a cancer targeted imaging material which is based on its surface Plasmon

resonance of Au NPs Atomic force microscopy (AFM) is utilized as a technique for

probing the binding interaction between NCs and RF binding protein (RFBP) in solution

The AFM technique also enables the precise measurement of the height of Au NPs before

and after chemisorptions of RF conjugated dendrimer as 135 and 205 nm respectively

This binding of RFndashBP to the Au NPs dendrimer results in the increase of height (267

nm) which then decreases 228 nm after coincubted with RF as a competitive ligand for

supporting interaction of Au NPs dendrimer and its target protein (Witte et al 2014)

The RF behavior adsorbed on Ag NPs and its interaction with serum albumins

(BSA HSA) has been studied The plasmonic features of the formed complexes by

RFBSAHAS and Ag NPs with an average diameter of 100 (plusmn 20 nm) have been

studied by UVndashvis absorption spectrometry The stability structure and dynamics of

serum albumins have been studied by using steadyndashstate and time resolved fluorescence

spectrometry The effectiveness of energy transfer reaction mechanisms between Ag NPs

and RF has been predicted and the mechanism of the reaction has also been proposed It

is illustrated by the participation of Ag NPs by the redox process of RF and RFndashserum

albumin interaction in Ag NPs complexes (Voicescu et al 2013)

426 Photochemical Interaction

The interaction and formation of a complex between RF and Ag NPs has been

studied by fluorescence spectrometry UVndashvis spectrometry and TEM AgNO3 and

trisodium citrate (TSC) have been used for the preparation of Ag NPs by the process of

chemical reduction By this method NPs of the size of 20 nm have been obtained with a

surface Plasmon resonance band at 426 nm The absorption maxima of RF (264 374 444

nm) shift significantly in the presence of Ag NPs due to the chemical interaction of Ag

NPs and RF The fluorescence of RF solutions is quenched by the addition of Ag NPs

and that may be due to the rapid adsorption of RF on AgNPs (Mokashi et al 2014)

The evaluation of the optical behavior of RF in aqueous solution in the presence

of Ag NPs has been made This Ag NPs were prepared by the oxidation and reduction

method and found that absorption intensity of RF was found to be enhanced It has been

found that when Ag NPs are added to an aqueous solution of RF the 372 and 444 nm

peaks are red and blue shifted respectively The fluorescence studies show that as the Ag

NPs concentration is increased the fluorescence intensity of RF solution is quenched

(Zhang et al 2011)

The NPs of copper have been prepared by the photoirradiation of doped solndashgel

silica by mixing Cu2+

ions ethylenediamine tetraacetic acid (EDTA) and RF into the solndash

gel solution of tetramethoxysilane (TMS) The absorption maxima of RF and Cu2+

EDTA is found to be at 442 nm and Cu2+

ndashEDTA at 740 nm respectively When the

photoirradaition is carried out the solndashgel silica develop reddish brown colour with an

absorption band around 580 nm because of Plasmon band CundashNPs Copper NPs are also

formed by solndashgel silica doped with lumichrome (LC) and lumiflavin (LF) The

photostability of the flavin dyes have been found to be in the order of LC gt LF gt RF in

solndashgel silica with Cu2+

ions The fluorescence intensities of LC LF and RF are reduced

by the photoirradiation of the solndashgel silica doped with Cu2+

ions without flavin dyes

(Noguchi et al 2011)

A study has been carried out on RFndashconjugation with ZnO NPs and their potential

application in jaundice The conjugation between RF and ZnO NPs has been confirmed

by UVndashvis spectrometry and photolumisence (PL) intensity In the RFndashconjugated NPs

the crystallinity and functional groups have been confirmed by Xndashray diffraction (XRD)

analysis and FTIR spectroscopy respectively Fieldndashemission scanning electron

microscopy (FESEM) and highndashresolution transmission electron microscopy (HRTEM)

have been used for the determination of the diameter of conjugated RFndashZnO NPs The

NPs shows significant ameliorative efficiency against the stress of jaundice at cellular

and molecular level in mice (Bala et al 2016)

427 Colorimetric Sensor

A study has been carried out to prepare Ag NPs using βndashcyclodextrin (βndashCD)ndash

grafted citrate as a stabilizer and reducer These NPs have been characterized by UVndashvis

spectrometry Xndashray diffraction and transmission electron microscopy (TEM) It has been

found that in the presence of RF the aggregation of Ag NPs occurs to a greater extent as

evident by the colour change (yellow to red) The formation of inclusion complexes

between RF and βndashCDndashgrafted citrate have been confirmed by 1H NMR spectroscopy

The interaction between βndashCD and RF is due to hydrogen bonding Ag NPs have been

used to develop a colorimetric sensor for the detection of RF This colorimetric

sensorprobe shows good response (selectivity and sensitivity) with 167 nM detection

limit for RF (Ma et al 2016)

OBJECT OF PRESENT INVESTIGATION

Vitamins are essential micronutrients required for the normal human growth

development and maintenance They are part of the enzyme systems and are involved in

the transformation of energy and for the regulation of metabolism A lack of the vitamins

results in clinical manifestations known as deficiency diseases In view of their

pharmaceutical importance it is necessary to ensure their stability in vitamin

formulations Riboflavin (RF) a component of vitamin B-complex is a photosensitive

compound and may degrade in vitamin formulations to give inactive products Several

studies have been carried out to investigate the photodegradation of RF and the effect of

factors enhancing or inhibiting these reactions These factors include pH solvent light

intensity buffers ionic strength metal ions etc Extensive work has been carried out on

the effects of pH light intensity and buffers on the photodegradation of RF However

some aspects still need to be investigated to understand the photochemical behavior of

RF under different conditions The object of present investigation is to conduct studies on

aspects such as the effect of solvent characteristics (ie dielectric constant and viscosity)

ionic strength and metal ions on the photodegradation of RF So far no quantitative and

kinetic studies have been carried out on these aspects and this work would facilitate the

formulation chemist in the development of better and more stable vitamin formulations

for the benefit of the users Moreover this work would provide a better insight into the

mechanism of RF photodegradation in aqueous and organic media In addition to this an

attempt would also be made to prepare RF nanoparticles and to study their spectrometric

fluorimetric and kinetic behavior under different experimental conditions

PROPOSED PLAN OF WORK

A brief outline of the proposed plan of work on various aspects of the photolysis

of riboflavin (RF) is presented as follows

1 Selection of appropriate radiation vessel and the radiation source for the

photolysis of RF in aqueous and organic solvents

2 Photolysis of RF in aqueous and organic solvents and identification of the

photoproducts in different media

3 Assay of RF and photoproducts by a suitable stability-indicating assay method

such as multicomponent spectrometric method or a HPLC method

4 Photolysis of RF in aqueous solution at different ionic strength of buffer species

at specific pH values

5 Photolysis of RF in aqueous solution at specified pH values in the presence of

different metal ions (eg Fe3+

Ag+ etc)

6 Evaluation of the kinetics of photolysis reactions as mentioned under No 24 5

7 Development of correlations between rate constants and dielectric

constantviscosityionic strengthmetal ion concentration

8 Determination of rate constants for the interaction of RF and metal ions at specific

pH values and proposed mechanism of interaction

9 Study of the photochemical formation and characterization of RF conjugated

silver (Ag) nanoparticles (NPs)

10 Evaluation of the effect of pH irradiation wavelengths (UV and visible light) and

concentration of Ag+ ions on the formation kinetics of RFndashAg NPs

CHAPTER V

MATERIALS AND METHODS

51 MATERIALS

Riboflavin 78-Dimethyl-10-[(2S3S4R)-2345-tetrahydroxypentyl]benzo[g]pteridine-

24-dione Merck

C17H20N4O6 Mr 3764

It was found to be chromatographically pure Rf 037 (1ndashbutanolndashacetic acidndash

water 415 vv organic phase silica gel G) [lit (Treadwell et al 1968) Rf 036] and

was stored in the dark in a refrigerator

Lumiflavin (7810ndashTrimethylisoalloxazine) Sigma

C13H12N4O2 Mr 2563

Lumiflavin was stored in a light resistant container in the dessicator below 0 degC

Lumichrome (78ndashDimethylalloxazine) Sigma

C12H10N4O2 Mr 2423

It was stored in the dark in a refrigerator

Formylmethylflavin (7 8ndashDimethylndash10ndashformylmethylisoalloxazine)

C14H12N4O3 Mr 2843

Formylmethylflavin was synthesized according to the method of Fall and Petering

(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute

methanol dried in vacuum and stored in the dark in a refrigerator

Carboxymethylflavin (78ndashdimethylflavinndash10ndashacetic acid)

C14H12N4O4 Mr 3003

It was prepared by the method of Fukumachi and Sakurai (1954) by aerobic

photolysis of riboflavin in alkaline solution in the presence of 30 H2O2 The material

was purified by column chromatography with Whatman CC31 cellulose powder using 1ndash

butanolndash1ndashpropanolndashacetic acidndashwater (5030218 vv) as the solvent system (Ahmad et

al 1980)

Cyclodehydroriboflavin

C17H18O6N4 Mr 3744

Cyclodehydroriboflavin was prepared by the method of Schuman Jorns et al

(1975) via aerobic photolysis of riboflavin in phosphate buffer (20 M) and recrystallized

by acetic acid (20 M)

Method of Preparation of Nanoparticles

RFndashconjugated Ag NPs were prepared by the photoreduction method A 001mM

AgNO3 solution was prepared in 50 ml in a screw capped transparent glass bottle to

which 50 ml of 0002 mM of RF solution was added To this solution 3 to 5 drops of

NaOH (18 mM) were added (pH 80ndash105) and it was placed in a thermostat bath

maintained at 25 plusmn 1oC the solution was irradiated with a Philips HPLN 125 W high

pressure mercury vapor fluorescent lamp (emission at 405 and 435 nm the later band

overlapping the visible absorption maximum of RF at 444 nm (British Pharmacopoeia

2016)) horizontally fixed at a distance of 25 cm from the center of the bottle The

solution was also irradiated with a Philips TUV 30 W UV tube vertically fixed at a

distance of 25 cm from the center of the bottle Samples were withdrawn at various

intervals for absorbance measurements The solutions were irradiated till there was no

change in absorbance at the maximum (422 nm)

Metal Salts

The various metal salts used in this study were obtained from Merck and are as

follows

AgNO3 (999) FeSO47H2O (999) MgSO4H2O (995) CaSO42H2O

(999) Fe2(SO4)3H2O (970) CuSO45H2O (999) NiCl26H2O (980)

ZnSO47H2O (990) PbSO4 (980) CdSO4H2O (999) MnSO4H2O (999)

CoSO47H2O (999)

52 REAGENTS

All reagents and solvents (1ndashbutanol 997 acetonitrile 998 ethanol 998

ethyl acetate 995 methanol 999) were of analytical grade obtained from

BDHMerck The following buffer systems were used KCl + HCl pH 20 CH3COONandash

CH3COOH pH 45 and KH2PO4ndashNa2HPO4 pH 70 The ionic strength was kept constant

in each case unless otherwise stated

Freshly boiled glassndashdistilled water was used throughout the work

53 METHODS

In photochemical studies care was taken to protect the solutions from light during

the experimental work The photolysis chromatography and assay procedures of

riboflavin were carried out in a dark chamber provided with a safe light All the solutions

of riboflavin were freshly prepared for each experiment to avoid any photochemical

change

531 ThinndashLayer Chromatography (TLC)

The details of TLC systems including the adsorbents and solvents used for the

separation and identification of riboflavin and its photoproducts are as follows

Adsorbent a) Silica gel GF 254 precoated plates (Merck)

b) Whatman Mirogranular CC41 cellulose

(Merck)

Layer thickness 250ndashmicrom

Solvent systems Z1 1ndashbutanolndashacetic acidndashwater (415 vv

organic phase) silica gel G (Treadwell et al

Z2 1ndashbutanolndashacetic acidndashwater (415 vv

organicphase) cellulose powder (Ahmad et

al 1980)

Z3 1ndashbutanolndash1ndashpropanolndashacetic acidndashwater

(5030218 vv) cellulose powder

(Ahmad et al1980)

Z4 Chloroform-Methanol (92 vv) cellulose

powder (Schuman Jorns et al 1975)

Temperature 25ndash27 degC

Location of spots UV light 254 and 365 nm (UVtech lamp UK)

532 pH Measurements

The pH measurements of the solutions were carried out with an Elmetron LCD

display pH meter (modelndashCP501 sensitivity plusmn 001 pH units Poland) using a

combination electrode The calibration of the electrodes was automatic in the pH range

10ndash140 (25 degC) using the following buffer solutions

Phthalate pH 4008 phosphate pH 6865 disodium tetraborate pH 9180

533 Fourier Transform Infrared (FTIR) Spectrometry

The purity and identity of riboflavin used in this study was confirmed by FTIR

spectrometry using a Nicolet iS5 FTIR spectrometer (Thermo Fisher Scientific USA)

The IR spectrum was collected in the range of 4000ndash600 cmndash1

at a resolution of 4 cmndash1

using OMNIC software (version 90) and is shown in Fig 51

FTIR analysis of RF-conjugated silver nanoparticles was carried out by using a

Nicolet iS5 FTIR spectrometer (Thermofischer Scientific USA) in the range of 4000

to 400 cm-1

The sample was centrifuged at 15000 rpm (60 min) and the supernatant

Fig 51 FTIR spectrum of riboflavin

Wavelength (cmndash1

was discarded while the residue was dried for analysis The dried sample was used for the

measurement of the spectrum in transmission mode as a function of wavenumber (cm-1

OMNIC 90 software was used to process data

534 Ultraviolet and Visible Spectrometry

The absorbance measurements and spectral determinations on pure and

photolysed solution of riboflavin were carried out on a Thermoscientific UVndashVis

spectrophotometer (Evolution 201 USA) using matched silica cell of 10 mm path length

The cells containing the solutions were always employed in the same orientation using

appropriate control solutions in the reference beam The baseline was automatically

corrected by the builtndashin baseline memory at the initializing period Autondashzero

adjustment was made by onendashtouch operation The wavelength calibration was carried

out automatically by the instrument The absorbance scale was periodically checked

using the following calibration standards

Absorbance scale 0050 g l of K2Cr2O7 in 005 M H2SO4

Absorbance at 257 nm = 0725 350 nm = 0539 plusmn 0005 (Rand

Riboflavin solution pH 40 (acetate buffer)

A (1 1 cm) at 444 nm = 328

(British Pharmacopoeia 2016)

535 Fluorescence Spectroscopy

Fluorescence measurements were carried out by using Spectromax 5 flourimeter

(Molecular Devices USA) and Jasco Spectrofluorimeter (FPndash8500 Japan) with a Xenon

arc lamp

The measurements were carried out by using a 10 mm quartz cell and the

excitation and emission wavelengths were adjusted to 374 and 520 nm respectively

(United State Pharmacopoeia 2016) The fluorescence intensity was recorded in relative

fluorescence units using a pure 005 mM RF solution (pH 70) as a standard

536 Dynamic Light Scattering (DLS)

DLS measurements were carried out by Laser Spectroscatter-201 system (RiNA

GmbH Berlin Germany) having a He-Ne laser source providing 690 nm light source with

an output power range of 10-50 mW The measurements were performed by an

autopiloted run of 50 measurements in 20s at room temperature (25 oC) The RF

conjugated Ag NPs as such or filtered through a 022 microm filter (Millipore USA) were

placed in a SUPRASIL reg cell (15 mm light path) for measurements (Hameed et al

2014) at a fix scattering angle of 90o and the scattered light was collected

Autocorrelation functions were performed using a program CONTIN to measure the

hydrodynamic radius (RH) distribution The Einstein-Stokes equation was used to relate

RH to the diffusion coefficient The PMgr v301p17 software was used for the analysis of

537 Atomic Force Microscopy (AFM)

The sample was prepared by pouring 10 microl of the desired solution on freshly

cleaned mica for 2-3 min which was then rinsed with Milli-Q water and dried with

nitrogen (Shah et al 2014) Agilent 5500 AFMSFM microscope was used to obtain

images immediately operating the instrument in tapping mode using soft silicon probes

(NCL nominal length = 225 microm mean width-38 microm and nominal resonance frequency =

190 KHz nominal force constant = 48 Nm) The images of the RF-conjugated silver

nanoparticle solutions were measured at random spot surface sampling

538 Photolysis of Riboflavin solutions

5381 Choice of reaction vessel

In the photochemical work a reaction vessel is to be chosen on the basis of the

absorption characteristics of the reactants and the transmission characteristics of the

reaction vessel The aqueous solutions of riboflavin absorbs at 223 267 373 and 444 nm

in the UV and visible region (British Pharmacopeia 2016) therefore a pyrex vessel can

be used for absorption above 300 nm region Pyrex vessels have previously been used for

the photolysis of riboflavin (Ahmad et al 2004a 2004b 2005 2006 2008 2009 2010)

5382 Choice of radiation source

Riboflavin exhibits a strong peak at 444 nm in the visible region This necessities

a radiation source with strong emission in this region Philips HPLN highndashpressure

mercury vapour fluorescent lamp strongly emits at 405 and 436 nm The 436 nm

wavelength is close to the major absorption maximum of riboflavin (444 nm) This

radiation source has previously been used by Ahmad et al (2004a 2004b 2005 2006

2008 2009 2010) for the photolysis of riboflavin The spectral power distribution of the

fluorescent lamp is shown in Fig 52

Fig 52 Spectral emission of HPLN lamp

539 Methods of Photolysis of Riboflavin

5391 Photolysis in aqueous and organic solvents

A 3ndash5 times 10minus5

M solution of RF (100 ml) was prepared in water (pH 70 0001 M

phosphate buffer) or in organic solvents in volumetric flasks (Pyrex) and immersed in a

water bath maintained at 25plusmn1degC The solution was exposed to a Philips HPLN 125 W

highndashpressure mercury lamp (emission bands at 405 and 435 nm the later band overlaps

the 444 nm band of RF (British Pharmacopoeia 2016)) fixed at a distance of 25 cm from

the center of the flask for a period of 2ndash3 h depending upon the nature of the solvent

used Samples of photolyzed solution were withdrawn at various time intervals for

thinndashlayer chromatographic separation and spectrometric assay of RF and photoproducts

5392 Photolysis at various ionic strength

A 10minus4

M aqueous solution of RF (100 ml) at pH 70 (01ndash05 M phosphate

buffer) with varying ionic strengths (01ndash05 M at each buffer concentration) was

prepared in a Pyrex flask and placed in a water bath maintained at 25 plusmn 1 degC and

proceeded further as stated above

5393 Photolysis in the presence of metal ions

A 5 times 10ndash5

M aqueous solutions of RF at pH 70 (0001ndash04 M phosphate buffer)

containing different metal ions at various concentrations (10ndash50 times 10ndash4

M) were

prepared in 100 mL Pyrex flasks and proceeded further as stated in section 5391

5310 Assay of RF and Photoproducts

RF and its major photoproducts in degraded solutions (aqueous and organic

solvents and in the presence of metal ions) detected by TLC were assayed using a

specific multicomponent spectrophotometeric method previously developed by Ahmad

and Rapson (1990) and Ahmad et al (2004b) The methods are based on the prendash

adjustment of photolysed solutions to pH 20 (02M HClndashKCl buffer) chloroform

extraction (3 times 10 ml) to remove the photoproducts lumichrome (LC) and lumiflavin (LF)

and their determination after chloroform evaporation and dissolution of the residue at pH

45 (02 M acetate buffer) by a twondashcomponent assay at 445 nm and 356 nm The

aqueous phase was assayed for RF and formylmethylflavin (FMF) by a twondashcomponent

assay at 445 nm and 385 nm and for RF FMF and cyclodehydroflavin (CDRF) at 445

410 and 385 nm Using this method it is possible to determine the concentrations of RF

and its major photoproducts (FMF CDRF LC LF) in photolysed solutions

The analytical scheme for the assay of RF and its photoproducts (Ahmad and

Rapson 1990 Ahmad et al 2004a) is given in Scheme 51 The molar absorptivites of

RF and photoproducts used in this study are reported in Table 52

5311 Calculation of Molar Concentrations in the Spectrometric Assay of RF and

Photoproducts

The assay of RF FMF CDRF LC and LF was carried out by onendashcomponent

twondashcomponent or threendashcomponent spectrometeric methods using specific wavelengths

and molar absorptivities given in Table 52 The methods of calculation of molar

concentrations are described as follows

Scheme 51 Assay of riboflavin and photoproducts

The assay of RF and photoproducts in photodegraded solutions (pH 2ndash11)

containing nonndashdegraded RF and several products has been carried out by prendashadjusted

of the solution to pH 20 and extracted with chloroform The variations in the

composition of the photoproducts in different reactions are monitored by TLC

RF and Photoproducts

Aqueous phase Chloroform extract

RF FMF minor components LC (acid photolysis)

Twondashcomponent assay (RF FMF) at 445 and

385 nm

Single component assay at 356 nm

LF LC ( neutral and alkaline

photolysis)

Twondashcomponent assay at 445 and 356

Threendashcomponent assay (RF FMF CDRF)

at 445 385 and 410 nm

photolysis)

Twondashcomponent assay at 445 nm and

356 nm

Assumed not to interfere in the assay

Table 52 Molar Absorptivities (Mminus1

cmminus1

) of RF and Photoproducts

(Ahmad and Rapson 1990 Ahmad et al 2004b)a

Compound pH 356 nm 385 nm 410 nm 445 nm

Riboflavin 20 97 804 125

Formymethylflavin 20 164 114 47

Cyclodehydroriboflavin 20 86 118 391

Lumichrome 45 108 013

Lumiflavin 45 74 104

a The values of molar absorptivities of RF and photoproducts were confirmed by using

pure reference compounds

Onendashcomponent assay

When a compound follows Beer Law its absorbences at a particular wavelength

are additive and therefore on the choice of a suitable wavelength (eg absorption

maximum) it is possible to calculate the concentration of the compound by applying the

following equation

A1 = 1a1 1C (51)

A1 is the absorbance at wavelength λ

1a1 is the absorptivity at waelenght λ

1C is the concentration of component 1

Using the same absorption cell in the measurement

A1 = 1ε1 1C (52)

1ε1 is the molar absorptivityndashcell path product used in the calculations

53111 Twondashcomponent spectrometric assay (additive absorbances)

In a twondashcomponent assay absorbance measurements on the solutions are made

at two selected wavelengths and the concentrations are determined by solving two

simultaneous equations

A1 = 1ε1 1C + 2ε1 2C (53a)

A2 = 1ε2 1C + 2ε2 2C (53b)

A1 is the absorbance at wavelength λ1

1ε1 is absorptivityndashcell path product for component 1 at wavelength λ1

1C is concentration of component 1

Equations (53a) and (53b) are solved for 1C and 2C as follows

1C = (2ε2 middot A1 ndash 2ε1 middot A2)(1ε1 middot 2ε2 ndash 2 ε1 middot 1ε2) (54a)

2C = (1ε1 middot A2 ndash 1ε2 middot A1)(1ε1 middot 2ε2 ndash 2 ε1 middot 1ε2) (54b)

53112 Threendashcomponent spectrometric assay (additive absorbances)

A threendashcomponent assay involves the measurement of absorbances of solutions

at three selected wavelengths and the concentrations of individual components are

determined by solving three simultaneous equations using matrix methods The

measurements of A1 A2 A3 at λ1 λ2 λ3 are carried out for the determination of 1C 2C

and 3C

A1 = 1ε1 1C + 2ε1 2C + 3ε1 3C (55a)

A2 = 1ε2 1C + 2ε2 2C + 3ε2 3C (55b)

A3 = 1ε3 1C + 2ε3 2C + 3ε3 3C (55c)

Wavelength Absorbance Absorbance Sum

λ1 A1 1 ε 1 1C + 2 ε 1 2C + 3 ε 1 3C

λ2 A2 1 ε 2 1C + 2 ε 2 2C + 3 ε 2 3C

λ3 A3 1 ε 3 1C + 2 ε 3 2C + 3 ε 3 3C

The matrix equation is as follows

A1 1ε1 2ε1 3ε1 1C

A2 = 1ε2 2ε2 3ε2 = 2C

A3 1ε3 2ε3 3ε3 3C

(AM) (ASM) (CM)

AM = Absorbance matrix

ASM = Absorbance sum matrix

CM = Concentration matrix

The solution of eq 55d for each concentration involves the replacement of the

particular column in the absorbance sum matrix in its determinant form and by dividing

the resultant by absorbance sum matrix (ASM) again in its determinant form

A1 2ε1 3ε1 1ε 1 2ε 1 3ε1

1C = A2 2ε2 3ε2 1ε2 2ε2 3ε2

A3 2ε3 3ε3 1ε3 2ε3 3ε3

1 ε 1 A1 3 ε 1

1ε 1 2ε 1 3ε1

2C = 1 ε 2 A2 3 ε 2 1ε2 2ε2 3ε2

1 ε 3 A3 3 ε 3 1ε3 2ε3 3ε3

1ε1 2ε1 A1

1ε 1 2 ε 1 3 ε 1

3C = 1ε2 2ε2 A2 1 ε 2 2 ε 2 3 ε 2

1ε3 2ε3 A3 1 ε 3 2 ε 3 3 ε 3

The above matrices are expanded to determine the concentration of the three components

using Laplacersquos method

A1 2ε2 3ε2

2ε3 3ε3

ndash 2 ε 1

A2 3ε 2

A3 3ε3

+ 3 ε 1

A2 2ε2

A3 2ε3

ASM expanded

A1(2ε 23ε3ndash3ε22ε3)ndash2ε1(A23ε3ndash3ε2A3)+3ε1(A22ε3ndash2ε2A3)

ASM expanded

1ε1(A23ε3ndash3ε2A3)ndashA1(1ε23ε3ndash3ε21ε3)+3ε1(1ε2A3ndashA21ε3)

ASM expanded

1ε1(2ε2A3ndashA22ε3)ndash2ε1(1ε2A3ndashA21ε3)+A1(1ε22ε3ndash2 ε 21ε3)

ASM expanded

CHAPTER VI

SOLVENT EFFECT ON THE PHOTOLYSIS OF

RIBOFLAVIN

61 INTRODUCTION

The influence of solvents on the rates of degradation of drugs is an important

consideration for the formulation chemist The effects of dielectric constant and viscosity

of the medium may be significant on the stability of pharmaceutical formulations

Theoretical basis of the effects of solvent on the rates and mechanism of chemical

reactions has been extensively dealt by many workers (Amis and Hinton 1973 Buncel et

al 2003 Connors et al 1986 Heitele 1993 Laidler 1987 Reichardt et al 1988

Sinko 2006 Yoshioka and Stella 2000) The effect of dielectric constant on the

degradation kinetics and stabilization of chloramphenicol (Marcus and Taraszka 1959)

barbiturates (Ikeda 1960) methanamine (Tada 1960) ampicillin (Hou and Poole 1969)

prostaglandin E2 (Roseman et al 1973) chlorambucil (Owen and Stewart 1979) 2ndash

tetrahydropyranyl benzoate (Hussain and Truelove 1979) indomethacin (Ghanem et al

1979) aspirin (Baker and Niazi 1983) phenoxybenzamine (Adams and Kostenbauder

1985) azathioprine (Singh and Gupta 1988) polypeptides (Brennan and Clarke 1993)

neostigmine (Yoshioka and Stella 2000) triprolidine (Mao et al 2000)

10ndashmethylisoalloxazine (Ahmad and Tollin 1981) formylmethylflavin (Ahmad et al

2006) levofloxacin (Ahmad et al 2013a) moxifloxacin (Ahmad et al 2014) and

norfloxacin (Ahmad et al 2015) has been reported The viscosity of the medium may

also affect the stability of a drug A linear relation has also been found between the rate

constant and the inverse of solvent viscosity for the photodegradation of 10ndash

methylisoalloxazine (Ahmad and Tollin 1981) formylmethylflavin (Ahmad et al

2013b) levofloxacin (Ahmad et al 2013a) moxifloxacin (Ahmad et al 2014) and

norfloxacin (Ahmad et al 2015) in organic solvents

Some kinetic studies of the photolysis of riboflavin (RF) in carboxylic acids

(Koziol 1966 Szezesma and Koziol 1977) alcoholic solvents (InsinskandashRak et al

2012 Moore and Ireton 1977 Schmidt 1982 Song and Metzler 1967) and pyridine

(Kurtin et al 1967) have been conducted However the method used for the

determination of RF is based on the measurement of absorbance at 445 nm without any

consideration of the interference caused by photoproducts formed during degradation

Thus the kinetic data obtained may not be accurate and specific methods may be required

for assay of RF in degraded solutions (Ahmad and Rapson 1990 Ahmad and Vaid

2006) Studies on the photolysis of formylmethylflavin (FMF) a major intermediate in

the photolysis sequence of the RF in organic solvents have been conducted (Ahmad et

al 2006a Ahmad et al 2013b) Solvent effects on flavin electron transfer reactions have

been found to be significant (Ahmad and Tollin 1981 Sheraz et al 2014a) The present

work involves a detailed study of the kinetics of photolysis of RF in a wide range of

organic solvents using a specific multicomponent spectrometric method for the assay of

RF and photoproducts (Ahmad and Rapson 1981 Ahmad and Vaid 2006 Sheraz et al

2014b) and to develop correlations between the kinetic data and solvent parameters such

as dielectric constant and viscosity These considerations are important in the formulation

of drugs with different polar character using condashsolvents and those whose oxidation is

viscosity dependent to achieve stabilization

The details of the experimental work involved in the study are given in section

53 (Chapter 5)

62 RESULT AND DISCUSSION

621 Photoproducts of RF

TLC of the photolysed solutions of RF in organic solvents on cellulose plates

using the solvent systems (Z1) and (Z3) showed the presence of FMF and LC as the main

photoproducts of this reaction CMF was also detected as a minor oxidation product of

FMF in these solvents (Ahmad et al 2006a 2013b) These products have been identified

by comparison of their fluorescence emission and Rf values with those of the authentic

compounds The formation of FMF and LC as the main photoproducts of RF in organic

solvents have previously been reported (Ahmad et al 2006a 2013b Koziol 1966) The

formation of LC in organic solvents may take place through FMF as an intermediate in

the photolysis of RF as observed in the case of aqueous solutions (Ahmad et al 2004

2006a 2013b Ahmad and Rapson 1990) The fluorescence intensity of the

photoproducts on TLC plates is an indication of the extent of their formation in a

particular solvent during the irradiation period In aqueous solutions (pH 70) LF is also

formed in addition to FMF and LC as previously reported (Ahmad et al 2004 Song and

Metzler 1967) The Rf values of RF and photoproducts are reported in Table 61

622 Spectral Characteristics

RF exhibits absorption maxima in organic solvents in the region of 440ndash450 nm

344ndash358 nm and 270ndash271 nm (Koziol 1966) A typical set of absorption spectra for the

photolysis of RF in methanol is shown in Fig 61

Table 61 Rf values and Fluorescence of RF and Photoproducts

Solvent System Fluorescence

Riboflavin 034 048 027 yellow green

Formylmethylflavin 057 070 069 yellow green

Lumichrome 063 067 064 Sky blue

Lumiflavin 035 052 040 yellow green

Carboxymethylflavin 019 037 020 yellow green

Cyclodehydroriboflavine

045 Non-

fluorescent a1ndashButanolndashethanolndashwater (702010 vvv Silica gel G) (Ahmad et al 1980)

b1ndashButanolndashacetic acidndashwater (401050 vvv organic phasecellulose powder CC41)

(Ahmad et al 1980)

c1ndashButanolndash1ndashpropanolndashacetic acidndashwater (5030218 vvv cellulose powder CC41)

(Ahmad et al 1980)

d Chloroform-Methanol (92 vv cellulose powder CC41) (Schuman Jorns et al 1975)

e See section 721 for TLC identification of CDRF

Fig 61 Absorption spectra of RF photolyzed in methanol at 0 30 60 90

and 120 min

250 300 400 500 600

Wavelength (nm)

There is a gradual loss of absorbance around 445 nm with a shift of the 358 nm

peak to 350 nm with time due to the formation of LC (λmax in methanol 339 nm)

(Sikorski et al 2003) the major of RF in organic solvents LC is formed through the

mediation of FMF an intermediate in the photolysis of RF (Song and Metzler 1967)

FMF has an absorption spectrum similar to that of RF due to the presence of a similar

chromophoric system and therefore it could not be distinguished from the absorption

spectrum of RF in organic solvents

The photolyzed solutions of RF have been assayed at pH 20 (02 M KClndashHCl

buffer) by extraction of LC with chloroform and its determination at pH 45 (02 M

acetate buffer) at 356 nm The aqueous phase was used to determine RF and FMF by a

twondashcomponent assay at 385 and 445 nm corresponding to the absorption maxima of

these compounds The molar concentrations of RF and its photoproducts FMF LC and

LF determined in the photolysis reactions in aqueous solution (pH 70) by the method of

Ahmad and Rapson (1990) are reported in Table 62 In the case of organic solvents the

photolysed solutions were evaporated under nitrogen at 40 oC the residue dissolved in

pH 20 buffer and the solution extracted with chloroform as stated above The RF and

FMF were determined at 384 and 445 nm and LC separately at 356 nm The results of the

assay of these compounds in organic solvents are reported in Table 63-68 The assay

method shows uniformly increasing values of FMF and LC in the photolysis reactions

with an almost constant molar balance with time indicating a good reproducibility of the

method

Table 62 Concentrations of RF and Photoproducts in Water (pH 70)

(Mtimes 105)

0 300 000 000 000 300

30 263 028 008 004 305

60 229 060 012 007 308

90 197 078 023 009 309

120 173 086 030 012 311

Table 63 Concentrations of RF and Photoproducts in Acetonitrile

(Mtimes 105)

0 300 000 000 300

30 269 023 012 304

60 239 040 021 308

90 213 058 031 304

120 194 066 045 311

Table 64 Concentrations of RF and Photoproducts in Methanol

(Mtimes 105)

0 300 00 00 300

30 255 036 015 306

60 215 058 029 308

90 201 071 032 306

120 191 079 037 312

Table 65 Concentrations of RF and Photoproducts in Ethanol

(Mtimes 105)

0 300 000 000 300

30 273 017 014 306

60 245 032 024 310

90 223 042 036 308

120 199 049 052 306

Table 66 Concentrations of RF and Photoproducts in 1ndashPropanol

(Mtimes 105)

0 300 000 000 300

30 268 020 015 305

60 245 031 028 307

90 223 040 039 304

120 202 049 050 302

Table 67 Concentrations of RF and Photoproducts in 1ndashButanol

(Mtimes 105)

0 300 000 000 300

30 269 022 012 303

60 245 037 022 304

90 222 052 031 307

120 204 060 039 309

Table 68 Concentrations of RF and Photoproducts in Ethyl acetate

(Mtimes 105)

0 300 000 000 300

30 275 017 011 308

60 251 031 023 309

90 227 037 039 306

120 208 046 050 304

Since the concentration of FMF (an intermediate product in the photolysis reactions) and

determined in aqueous and organic solvents is less than 1 times 10ndash5

M due to its loss to LC

and LF CMF a minor oxidation product of FMF in organic solvents (Ahmad et al

2006) accounting to less than 1 (Ahmad et al 2013) does not interfere with the assay

method

624 Kinetics of Photolysis

The photolysis of RF in aqueous solution (Ahmad et al 2004 2014a Song and

Metzler 1967) and in organic solvents (Kurtin et al 1967 Song and Metzler 1967)

follows firstndashorder kinetics The kinetic plots for the photolysis of RF in water and

organic solvents (Fig 62ndash68) show that LC is the final product in these reactions as

observed by previous workers (Ahmad et al 2004a InsinskandashRak et al 2012 Moore

and Ireton 1977) The firstndashorder plots for the photolysis of RF in water and organic

solvents are shown in Fig 69ndash615 and the rate constants (kobs) determined from the

slopes of these plots range from 319 (ethyl acetate) to 461times10minus3

minminus1

(water)

(correlation coefficients 0997ndash0999) (Table 69) The values of kobs increase with an

increase in the dielectric constant indicating the influence of solvent on the rate of

reaction The value for the photolysis of RF in aqueous solution (pH 70 0005 M

phosphate buffer) is also included for comparison A plot of kobs for the photolysis of RF

as a function of solvent dielectric constant is presented in Fig 616 It shows that the rate

constants are linearly dependent upon the solvent dielectric constant Similarly a linear

relation has been found between the values of kobs and the solvent acceptor number

indicating the degree of solutendashsolvent interaction (Fig 617)

Fig 62 Kinetic plots for the photolysis of RF in water

Fig 63 Kinetic plots for the photolysis of RF in acetonitrile

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

Fig 64 Kinetic plots for the photolysis of RF in methanol

Fig 65 Kinetic plots for the photolysis of RF in ethanol

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

Fig 66 Kinetic plots for the photolysis of RF in 1ndashpropanol

RF () FMF () LC ()

Fig 67 Kinetic plots for the photolysis of RF in 1ndashbutanol

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

Fig 68 Kinetic plots for the photolysis of RF in ethyl acetate

RF () FMF () LC ()

0 30 60 90 120

times10

Time (min)

Fig 69 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5

M) in water

(pH 70)

acetonitrile

0 20 40 60 80 100 120 140

Time (min)

times10

0 20 40 60 80 100 120 140

Time (min)

M times

methanol

ethanol

0 20 40 60 80 100 120 140

Time (min)

times10

0 20 40 60 80 100 120 140

M times

Time (min)

1ndashpropanol

1ndashbutanol

0 20 40 60 80 100 120 140

times10

Time (min)

0 20 40 60 80 100 120 140

Time (min)

M times

ethyl acetate

0 20 40 60 80 100 120 140

times10

Time (min)

Table 69 Apparent FirstndashOrder Rate Constants for the Photolysis of Riboflavin

(kobs) in Organic Solvents and Water

Solvents Dielectric

constant (isin)

(25 oC)

Acceptor

Number

Inverse

viscosity

(mPasndash1

(25 oC)

kobs times 103 min

ndash1

plusmnSDa

Ethyl acetate 602 171 2268 319plusmn014

1ndashButanol 178 368 0387 328plusmn013

1ndashPropanol 201 373 0514 334plusmn016

Ethanol 243 371 0931 345plusmn015

Methanol 326 413 1828 364plusmn017

Acetonitrile 385 189 2898 381plusmn016

Water 785 548 1123 461plusmn025

aSD standard deviation

Fig 616 Plot of kobs for the photolysis of RF versus dielectric constant (x) ethyl

acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol () methanol (+) acetonitrile

() water

00 100 200 300 400 500 600 700 800

Dielectric constant

s times

Fig 617 Plot of lnkobs for the photolysis of RF versus acceptor number (x) ethyl

() water

00 100 200 300 400 500 600 -70

Solvent acceptor number

s times

In order to observe the effect of viscosity on the rate of photolysis a plot of kobs versus

inverse of solvent viscosity was constructed (Fig 618) It showed a linear relation

between the two values indicating the influence of solvent viscosity on the rate of

reaction These results are supported by the fact that a plot of dielectric constant versus

inverse of viscosity of organic solvents is linear (Fig 619) However the values of kobs

for RF in ethyl acetate and water do not fit in the plot probably due to different behaviors

of RF in acetate (compared to alcohols) and water (eg degree of hydrogen bonding)

625 Effect of Solvent

It is known that the solvents could influence the degradation of drugs depending

on the solvent characteristics and solutendashsolvent interactions Solvents may alter the rate

and mechanism of chemical reactions (Abraham 1985 Amis and Hinton 1973 Laidler

1987 Parker 1969 Reichardt 1982 Sheraz et al 2014) and thus play a significant role

in the stabilization of pharmaceutical products (Connors et al 1986) Pharmaceutical

formulations of ionizable compounds such as RF may be stabilized by an alteration in the

solvent characteristics A suppression of the ionization of a drug susceptible to

degradation in water may be achieved by the addition of a cosolvent (eg alcohol

propylene glycol glycerin) This would result in the destabilization of the polar excited

state and therefore a decrease in the rate of reaction as observed in the case of many

drugs (Wypych 2001) The use of organic solvents as cosolvent can have a

photostabilizing effect on the product as a result of a change in the polarity and viscosity

of the medium (Tonnesen 2001)

Fig 618 Plot of kobs for the photolysis of RF versus inverse of viscosity

(x) ethyl acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol () methanol

(+) acetonitrile () water

10 15 20 25 05 30 00

Viscosity (mPa s)-1

times 1

Fig 619 Plot of dielectric constant versus inverse of viscosity

00 05 10 15 20 25 30 35

Viscosity (mPas)-1

These considerations are important in the formulation of drugs with different polar

characters and those whose oxidation is viscosity dependent These aspects with respect

to the photolysis of RF as a model compound used in the clinical treatment of neonatal

jaundice (Tan 1996) keratoconus (Caporossi et al 2010) and HIV infection (Montessori

et al 2004) would now be considered and correlations would be developed between the

solvent characteristics and the rate of reaction

626 Effect of Dielectric Constant

The rate of degradation reactions between ions and dipoles in solution depends on

the bulk properties of the solvent such as the dielectric constant Any change in the

dielectric constant of a solvent can lead to variation in the energy of activation (ΔG) and

hence in the rate constants (Yoshioka and Stella 2000) This can be applied to the

degradation of RF since its rate of photolysis is a linear function of dielectric constant

This can be explained on the basis of the participation of a polar intermediate in the

reaction pathway to facilitate the reaction (Ahmad et al 2006a Ahmad and Tollin

1981) The rate of RF photolysis is affected by solvent polarity probably due to changes

in the conformation of the ribityl side chain in different solvents (Moore and Ireton

1977) Quenching of flavin excited triplet state [3FL] by oxygen during the reaction has

been suggested (Ahmad et al 2006a InsinskandashRak et al 2012) and this may affect the

rate of RF photolysis However under the present reaction conditions (ie solvents in

equilibrium with the air) the firstndashorder plots are linear for RF solutions photolyzed up to

30 and the values of kobs are relative to these conditions The electronndashdonating

capacity of a molecule (eg fluoroquinolone RF) is affected by the nature of the solvent

(Ahmad et al 2015 Peng et al 2014) and hence its rate of degradation The acceptor

number is a measure of the ability of solvents to share electron pairs from suitable donors

(Schmidt and Sapunov 1982 Wypych 2001) and this could affect the rate of photolysis

The results obtained and the degradation behavior of RF in organic solvents suggest that

the stability of such polar drugs can be improved by alteration of dielectric constant of

the medium

627 Effect of Viscosity

The viscosity of the medium can also influence the rate of degradation

particularly of an oxidizable drug The photolysis of RF involves oxidation of the ribityl

side chain (Moore and Ireton 1977) and thus may be affected by the solvent viscosity

The values of kobs for RF in ethyl acetate and water do not follow the relation (Fig 5)

probably due to its different structural orientation (Moore and Ireton 1977) and degree of

hydrogen bonding (Sikorski et al 2003) compared to those of the organic solvents The

behavior of RF in organic solvents indicates that the viscosity of the medium suppresses

the rate of photolysis probably as a result of solute diffusionndashcontrolled processes

(Ahmad and Tollin 1981 Turro et al 2010) It has been observed that the flavin triplet

state [3RF] quenching depends on solvent viscosity (Ahmad and Tollin 1981) and that

would affect the rate of reaction Similar effects of viscosity have been observed on the

photooxidative degradation of formylmethylflavin (Ahmad et al 2013b) and

fluoroquinolones (Ahmad et al 2013a 2014b 2015)

628 Mode of Photolysis

The photochemistry of RF has widely been studied by several workers and the

various modes of its photodegradation reactions (ie intramolecular and intermolecular

photoreduction photodealkylation and photoaddition) have been discussed (Ahmad et

al 2006a 2013b Ahmad and Vaid 2006 Choe et al 2005 Heelis 1982 1991 Sheraz

et al 2014a) The pathway of RF degradation in organic solvents appears to be similar to

that of the aqueous solution involving intramolecular photoreduction followed by sidendash

chain cleavage (Ahmad and Vaid 2006) However the rate of the reaction is solvent

dependent due to the participation of a dipolar intermediate (Ahmad and Tollin 1981)

whose degradation is promoted by polar environment and suppressed by nonpolar media

It has been observed by laser flash photolysis that the reduction of [3FL] in organic

solvents proceeds through the mediation of the dipolar intermediate according to the

following reaction (Ahmad and Tollin 1981)

3FL + AH (F

σndash hellip H hellip A

σndash+) FLH (61)

The flavin semiquinone radical [FLH] undergoes further reactions to give an

oxidized and a reduced flavin (Eq (62)) The reduced flavin is then oxidized by air to

form degraded products (Eq (63))

2FLHbull FL + FLH2 (62)

FLH2 degraded FL + side chain products (63)

The extent of the photolysis reaction to form radicals is controlled by the degree

of solutendashsolvent interaction The polar character of the reaction intermediate would

determine the rate of reaction and the rate would be higher in solvents of greater polarity

Thus the solvent characteristics play an important role in determining the rate of RF

degradation An appropriate combination of waterndashalcohol mixture would be a suitable

medium for the stabilization of RF and drugs of similar character

CHAPTER VII

IONIC STRENGTH EFFECTS ON THE

PHOTODEGRADATION REACTIONS OF

RIBOFLAVIN IN AQUEOUS SOLUTION

71 INTRODUCTION

The ionic strength of a solution can have a significant effect on the rate of a

chemical reaction and is known as the primary kinetic salt effect The relationship

between the rate constant and the ionic strength for an aqueous solution at 25 oC may be

expressed by the BronstedndashBjerrum equation (Bronsted 1922 Bjerrum 1924)

log k = log ko + 102 Z

B radicmicro (71)

where ZA and Z

B are the charges carried by the reacting species in solution micro the

ionic strength k the rate constant of degradation and ko the rate constant at infinite

dilution A plot of log kko against radicmicro should give a straight line of slope 102 Z

Eq (71) is valid for ionic solutions up to micro = 001 At higher concentrations (micro le 01) the

BronstedndashBjerrum equation can be expressed as

B radicmicro (1 + β radicmicro) (72)

In Eq (72) the value of β depends on the ionic diameter of the reacting species

and is often approximated to unity

If the rate constants for a chemical reaction are determined in the presence of a

series of different concentrations of the same electrolyte then a plot of log k against

under root of ionic strength is linear even in the case of solutions of high ionic strength

(Florence and Attwood 2006) The influence of ionic strength on the kinetics of drug

degradation and chemical reactions has been discussed by several workers (Florence and

Attwood 2006 Lachman et al 1986 Carstensen 2000 Guillory and Post 2002 Sinko

2006 Yoshioka and Stella 2000 Laidler 1987 Koppenol 1980) Ionic strength has

been found to effect the aggregation kinetics of TiO2 (French et al 2009) and the

stability of Ag nanoparticles (Badawy et al 2010) The primary salt effects on the rates

and mechanism of chemical reactions have been discussed (Frost and Pearson 1964

Corsaro 1977)

In drug degradation and stability studies the reactions are normally carried out at

a constant ionic strength to minimize its effect on the rate of reaction (Sankara et al

1999 Stankovicova et al 1999 Yeh 2000 Chadha et al 2003 Jumaa et al 2004

Ahmad et al 2004a) However a large number of studies have been conducted to

evaluate the influence of ionic strength on the kinetics of chemical (Pramar and Gupta

1991 Hoitink et al 2000 Zang and Pawelchak 2000 Matos et al 2001 Miranda et al

2002 Alibrandi et al 2003 Sato et al 2003 Aloisi et al 2004 Lallemand et al 2005

Rexroad et al 2006) and photodegradation of drug substances (Khattak et al 2012) The

ionic strength effects have important implications in photoinduced electron transfer

reactions and the binding ability of proteins to flavin species (Fukuzumi and Tanaka

1988) Laser flash photolysis studies of the kinetics of electron transfer between flavin

semiquinone and fully reduced flavins and horse rate cytochrome c have shown that the

presence of a charged phosphate group in the Nndash10 ribityl side chain leads to small ionic

strength effects on the rate constant whereas a charged group attached to the

dimethylbenzene ring produces a large ionic strength effect (Ahmad and Cusanovich

1981) Attempts have been made to describe the dependence of bimolecular rate

constants on ionic strength for small molecules and protein interactions (Ahmad and

Cusanovich 1981 Ahmad et al 1982 Hazzard et al 1987 1988 Watkins et al 1994

Zhong and Zewail 2001) A temperature dependent study of the effect of ionic strength

on the photolysis of riboflavin (RF) has been conducted RF undergoes biphasic

photolysis with a lowndashintensity light source In higher ionic strength phosphate buffer

(031 M) an initial faster phase is followed by a slower second phase and vice versa in

lower ionic strength buffer (005 M) (Sato et al 1984) In the presence of higher

concentration (gt 01 M) of divalent phosphate anions (HPO42ndash

) and pH values above 60

the normal course of RF photolysis (photoreduction) involving 10ndashdealkylation to form

formylmethyflavin (FMF) lumiflavin (LF) and lumichrome (LC) (Ahmad et al 2004b)

is shifted to photoaddition to yield cyclodehydroriboflavin (CDRF) (Schuman Jorms et

al 1975 Ahmad et al 2005) The present study involves the evaluation of ionic strength

effects on the photodegradation of RF with a change in the mode of reaction at higher

buffer concentrations These effects may significantly influence the rates and mechanism

of RF degradation reactions flavinndashprotein interactions and the kinetics of electron

transfer reactions The study of ionic strength effects is also necessary since the single

and multivitamin parenteral and total parenteral nutrition (TPN) preparations containing

RF are isotonic and the amount of NaCl present (09 wv) may influence the stability

of RF on exposure to light The effects of ionic strength on a change in the mode of

photodegradation of RF need to be investigated Some related work on the effect of

factors such as pH (Ahmad et al 2004b) buffer (Ahmad et al 2013 2015ab) and light

intensitywavelengths (Ahmad et al 2006) on the photodegradation of RF has been

reported

53 (Chapter 5)

72 RESULTS AND DISCUSSION

An important consideration in kinetic studies is the use of a specific assay

procedure to determine the desired compounds in the presence of degradation products

The multicomponent spectrometric method used in this study is capable of simultaneous

determination of RF and its photoproducts with reasonable accuracy (Ahmad et al

2004a) It has the advantage of determining these compounds without mutual

interference Under the present reaction conditions (ie simultaneous photolysis and

photoaddition reactions) the photodegraded solutions of RF contain a mixture of RF

FMF LF LC and CDRF as photoproducts as detected by TLC (Section 531) on

comparison with the Rf values and fluorescence of difference compound and reported

previously (Ahmad et al 1990 2004ab) Therefore a specific rapid and accurate

method is required for the assay of such a complex mixture The method used for this

purpose (Ahmad et al 2004b) fulfils these requirements and has previously been applied

to the assay of these compounds during the kinetic studies of photodegradation of RF

(Ahmad et al 2004a 2009 2010 2013 2015) Such an analysis cannot be carried out

rapidly by HPLC methods The assay of RF and photoproducts in various reactions

carried out at pH 70 with an ionic strength of 01ndash05 (01ndash05 M phosphate buffer) is

reported in Table 71ndash725

Table 71 Concentrations of RF and Photoproducts in 01 M Phosphate Buffer

(pH 70) at 01 M Ionic Strength

(M times105)

0 500 00 00 00 500

30 451 019 021 010 501

60 398 039 045 019 506

90 373 053 059 022 507

120 340 064 071 027 508

(M times105)

0 500 00 00 00 500

30 446 020 022 016 504

60 386 044 049 021 508

90 332 069 073 029 509

120 309 076 081 035 501

(M times105)

0 500 00 00 00 500

30 435 020 031 016 502

60 381 039 052 029 505

90 331 065 071 035 508

120 288 078 089 046 501

(M times105)

0 500 00 00 00 500

30 417 031 035 020 503

60 361 054 058 031 504

90 308 069 082 043 507

120 269 081 099 052 508

(M times105)

0 500 00 00 00 500

30 404 032 044 022 502

60 336 056 075 036 505

90 290 068 097 047 507

120 245 079 118 059 501

(M times 105)

0 500 00 00 00 00 500

30 435 015 016 039 008 513

60 378 026 028 048 020 508

90 329 035 046 071 030 511

120 280 048 060 092 042 522

Table 77 Concentrations of RF and Photoproducts in 02 M Phosphate buffer

(M times 105)

0 500 00 00 00 00 500

30 416 024 036 057 006 539

60 353 040 059 075 016 543

90 293 079 081 134 028 615

120 251 089 091 175 034 640

(M times 105)

0 500 00 00 00 00 500

30 386 023 032 059 006 500

60 307 040 056 083 014 511

90 239 059 069 119 021 516

120 194 064 081 131 033 503

(M times 105)

0 500 00 00 00 00 500

30 369 030 036 062 009 506

60 280 045 060 093 023 501

90 217 060 073 122 033 509

120 153 071 089 145 048 506

(M times 105)

0 500 00 00 00 00 500

30 338 036 046 074 009 503

60 238 055 081 112 014 510

90 164 064 116 131 027 502

120 119 073 126 149 037 504

(M times 105)

0 500 00 00 00 00 500

30 398 016 031 045 010 510

60 327 031 055 066 022 508

90 267 042 065 085 041 503

120 224 050 076 101 049 506

(M times 105)

0 500 00 00 00 00 500

30 367 027 037 056 013 504

60 286 047 051 096 020 511

90 221 059 069 120 031 513

120 178 057 082 139 044 509

(M times 105)

0 500 00 00 00 00 500

30 354 024 049 059 014 504

60 236 049 069 108 038 508

90 168 068 076 139 049 503

120 108 078 096 158 060 509

(M times 105)

0 500 00 00 00 00 500

30 295 040 051 100 015 506

60 160 056 108 143 033 505

90 097 069 121 168 045 502

120 076 075 132 177 051 506

(M times 105)

0 500 00 00 00 00 500

30 282 046 060 106 006 511

60 145 076 088 154 037 505

90 079 091 104 175 051 509

120 052 100 110 200 057 507

(M times 105)

0 500 00 00 00 00 500

30 397 029 026 035 017 504

60 309 036 049 076 037 507

90 239 048 061 105 051 504

120 180 067 075 126 062 508

(M times 105)

0 500 00 00 00 00 500

30 361 029 042 047 023 508

60 256 048 056 095 047 512

90 183 061 077 118 063 502

120 127 073 095 145 071 514

(M times 105)

0 500 00 00 00 00 500

30 314 032 050 075 035 506

60 195 055 090 113 050 513

90 130 070 108 133 062 508

120 075 085 130 145 071 506

(M times 105)

0 500 00 00 00 00 500

30 292 042 052 079 039 504

60 148 069 083 135 066 511

90 078 093 103 155 076 509

120 042 103 114 163 084 506

(M times 105)

0 500 00 00 00 00 500

30 217 049 070 113 055 504

60 113 060 096 157 074 509

90 057 073 106 178 086 511

120 024 082 117 187 093 506

(M times 105)

0 500 00 00 00 00 500

30 425 013 028 027 009 502

60 338 032 041 065 024 509

90 251 045 074 091 043 514

120 157 066 085 135 059 512

(M times 105)

0 500 00 00 00 00 500

30 313 041 046 085 019 506

60 214 056 068 115 047 509

90 140 072 085 150 057 506

120 099 081 096 164 067 507

(M times 105)

0 500 00 00 00 00 500

30 298 037 062 075 030 506

60 179 061 079 125 056 511

90 099 076 097 155 075 502

120 049 088 108 169 087 508

(M times 105)

0 500 00 00 00 00 500

30 249 049 068 099 036 501

60 099 071 118 145 067 509

90 049 082 128 167 077 506

120 023 088 137 178 086 512

(M times 105)

0 500 00 00 00 00 500

30 210 062 086 126 026 508

60 078 088 112 179 049 506

90 034 094 120 190 069 509

120 013 099 132 201 080 511

The assay results show that a good molar balance is achieved during the reactions

indicating the accuracy and precision of the method in the determination of RF and

photoproducts

722 Spectral Characteristics of Photolysed Solutions

The absorption spectra of RF determined during a photolysis reactions at pH 70

with an ionic strength of 01 and 05 show a decrease in absorbance at the maximum at

445 (Ahmad and Rapson 1990 Ahmad et al 2004a) indicating the gradual loss of RF

and an increase in absorbance around 356 nm (Ahmad et al 2004a) indicating the

formation of LC in the reaction (Fig 71) There is no change in the shape of absorption

spectra with a change in the ionic strength of the solutions However the variations in

ionic strength affect the magnitude of spectral changes for instance an increase in ionic

strength shows a greater decrease in absorbance at 445 nm and a greater increase in

absorbance at 356 nm This supports the view that an increase in ionic strength leads to

an increase in the rate of photolysis reactions

723 Kinetics of RF Photolysis

A large number of studies have been conducted on the photolysis of RF under

different conditions (Ahmad et al 2004ab 2005 Schuman Jorms et al 1975 Sato et

al 1984) It has been established that the photolysis of RF in aqueous solution follows

firstndashorder kinetics (Ahmad et al 2004b 2005 2015ab Song et al 1965) In this study

the effect of ionic strength on the phorodegradation of RF under different conditions has

been studied

Fig 71 Absorption spectra of the photolysed solutions of RF (5 times 10ndash5

M) at pH 70

(a) at zero and (b) at 05 M ionic strength

Considering the photolysis of RF as parallel firstndashorder reactions leading to the

formation of LC (k1) and LF (k2) as final products by phororeduction and CDRF (k3) as

final product by photoaddition pathways the values of the rate constants k1 and k2 can be

calculated as previously reported (Ahmad et al 2004a 2010) These reactions can be

expressed as follows

The mathematical treatment of the analytical data to determine k1 k2 k3 for these

reactions is given by Frost and Pearson (1964) Using the concentration values of RF

LC LF and CDRF and RF0 for the initial concentration

ndashdRFdt = k1 RF + k2 RF + k3 RF = (k1+ k2+ k3) RF = kobs RF (73)

kobs= k1+ k2+ k3 (74)

ln (RF0RF) = kobst (75)

RF = RF0 endashkt

Similarly

dLCdt = k1 RF0 endashkt

LC = + constant (78)

LC = LC0 + (1 ndash endashkt

) (79)

LF = LF0 + (1 ndash endashkt

) (710)

CDRF = CDRF0 + (1 ndash endashkt

) (711)

If LC0 = LF0= CDRF0 = 0 the equation simplifies and is readily seen that

LFLC = k2 k1 CDRFLC = k3 k1 (712)

LC LF CDRF = k1 k2 k3 (713)

The products are in constant ratio to each other independent of time and initial

concentration of the reactant The method has been applied to the determination of rate

constants for all the three primary processes in the pure liquidndashphase pyrolysis of

αndashpinene (Fuguitt and Hawkins 1947)

The values of k1 k2 k3 determined as a function of the ionic strength at different

phosphate buffer concentrations along with k1k3 ratios are reported in Table 726 The

values of k1 show a greater increase compared to those of k3 with an increase in ionic

strength at a constant buffer concentration It has been observed that a change in k1k3

ratios in favor of k1 occurs with a change in ionic strength This indicates that the ionic

strength has a greater effect on k1 (photoreduction pathway) leading to the formation of

k2 RF0 kobs

k3 RF0 kobs

ndash RF0 endashkt

k1 RF0

LC The mechanism of promotion of the rate of photoaddition reactions (k3) of RF by Clndash

is not clear

The values of apparent firstndashorder rate constants (kobs) (Table 726) for the overall

photodegradation of RF in reactions carried out at a phosphate buffer concentration of 01

M (photoreduction pathway) (Ahmad et al 2004b) indicate the effect of ionic strength

on this particular reaction However the photodegradation reactions carried out at

phosphate buffer concentrations above 01 M involve both photoreduction and

photoaddition pathways the latter due to the buffer effect (Ahmad et al 2005 Schuman

Jorns et al 1975) Under these conditions the values of kobs for RF would not distinguish

the ionic strength effects on the rates of the two distinct reactions where as the individual

rate constants (k1 k2 for photoreduction pathway and k3 for photoaddition pathway)

would indicate the effect of ionic strength on these reactions The values of rate constants

are relative and have been observed under controlled conditions of light intensity and

other factors

724 Fluorescence Studies

RF exhibits an intense yellow green fluorescence at 520 nm in aqueous solution

(United States Pharmacopoeia 2016) that vanishes in strongly acidic and alkaline

solutions due to ionization of the molecule (Weber 1950) In order to observe the effect

of NaCl on the fluorescence intensity of RF fluorescence measurements were made on

5times10minus5

M RF solutions (pH 70) at different ionic strengths at constant buffer

concentrations (Fig 72) These results indicate that at a 0001 M buffer concentration

there is a 334 to 422 loss of florescence at 01 to 05 M ionic strength

Table 726 Apparent FirstndashOrder Rate Constants (kobs) for the Photodegradation

of Riboflavin in the presence of Phosphate Buffer (pH 70) at different Ionic

Strength (01ndash05M) for the formation of Lumichrome (k1) Lumiflavin (k2) and

Cyclodehdroriboflavin (k3)

Buffer

Concentration

Strength

kobs times 103

(minndash1

k0 times 103

(minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

01 01 201 079 145 055 ndashndash ndashndash

02 301 210 090 ndashndash ndashndash

02 01 276 085 139 063 072 193

02 485 284 070 144 197

03 715 407 102 198 205

04 978 535 177 255 209

05 1190 684 201 321 213

03 01 445 120 224 109 111 201

02 825 425 151 185 229

03 1185 632 240 265 238

04 1505 835 253 345 242

05 1860 1042 296 521 248

04 01 525 135 259 127 121 214

02 1150 501 282 226 221

03 1571 756 370 325 232

04 2030 1115 487 466 239

05 2491 1279 561 522 245

05 01 735 141 380 166 170 222

02 1250 660 285 277 238

03 1891 991 478 402 246

04 2421 1220 615 482 253

05 3032 1603 638 607 264

Fig 72 Plots of fluorescence of RF solutions (pH 70) versus ionic

strength at different buffer concentrations (diams) 0001 M () 0025 M

() 005 M (times) 01 M () 02 M (∆) 03 M () 04 M () 05 M

0 01 02 03 04 05 06

Ionic Strength (M)

With an increase in buffer concentration (01ndash05 M) there is a gradual increase in the

loss of florescence reaching a value of 271 to 332 at 01 to 05 M ionic strength

respectively in 05 M buffer concentration Since phosphate buffer also quenches the

florescence of RF (Ahmad et al 2005) a combined effect of buffer and NaCl is being

observed at each buffer concentration with an increase in ionic strength This is in

agreement with a previous observation that NaCl (01 M) quenches the fluorescence of

RF solutions (Ellinger and Holden 1944) Since the kinetic results show an increase in

rate with an increase in ionic strength at each buffer concentration the loss of florescence

cannot be attributed exclusively to the excited singlet state quenching and some

interaction between RF and NaCl may be stipulated This could be analogous to the

excited singlet state quenching of RF by complexation with HPO42minus

ions leading to the

formation of CDRF by the photoaddition pathway (Schuman Jorms et al 1975) On the

basis of the kinetic results it can be suggested that a similar mechanism may operate

between RF and NaCl as explained below In the present case RF on the absorption of

light is promoted to the excited singlet state [1RF] (Eq (714)) [

1RF] could react with Cl

ions to form an excited state complex (exciplex) as suggested for the exited state

reactions of organic compounds (Turro et al 2010) (Eq (715)) and observed in the case

of [1RFndashHPO4

2minus] complex leading to the formation of CDRF (Ahmad et al 2004b) In

both cases RF complexation with Clminus ions observed in the present study or with HPO4

2minus

ions (Ahmad et al 2005) results in the quenching of fluorescence involving the [1RF]

state as well as an acceleration of the photodegradation process The role of Clminus

appears to be analogous to that of the HPO42 minus

ions in promoting the rate of degradation

of RF This would lead to the formation of the photoproducts of RF (eg LC) (Eq (716))

RF [1RF] (714)

[1RF] + NaCl [

1RFhelliphellipCl

ndash] + Na

+ (715)

[1RF helliphellipCl

ndash] Photoproducts (716)

Clminus appears to form a nonndashfluorescent complex with the ground state RF molecule

by static quenching as suggested in the case of quinine (Gutow 2005) Thus the role of

Clminus ions in the photodegradation of RF is to promote the degradation of RF by different

pathways

725 Ionic Strength Effects

In order to correlate the rate constants for the photodegradation of RF by

photoreduction (LC LF) and photoaddition (CDRF) pathways with ionic strength the log

values of rate constants (kobs) were plotted against radicμ1 + radicμ (Eq (72)) which yielded

straight lines indicating a linear relationship Extrapolation to zero ionic strength yielded

the value for k0 the rate constant for the photodegradation of RF at zero ionic strength

(Fig 73) Further plots of log k1k0 and k3k0 against radicμ (Eq (71)) gave straight lines

with a positive slope of 102 ZAZB (Fig 74) shown for a typical photodegradation

reaction of RF at 05 M buffer concentration (ionic strength 01ndash05 M) The rate

constant k2 for the formation of LF by photoreduction pathway is a minor reaction and

has been neglected The number of unit charges ZAZB can be calculated from the slope

of the plots

B = 105 102 = 103 = ~ + 1 (for k1)

B = 161 102 = 157 = ~ + 160 (for k3)

exciplex

Fig 73 A plot of log kobs versus radicμ1 + radicμ at 05 M phosphate buffer

Fig 74 A plot log k1k0 () and k3k0 () versus radicμ at 05 M phosphate buffer

000 010 020 030 040

radicμ1 + radicμ

000 010 020 030 040 050 060 070 080

radicμ

The values of ZAZB (+1) for photoreduction suggest that a charged species is

involved in the rate determining step of the reaction (k1) It has been earlier suggested by

flash photolysis experiments that the flavin triplet reduction takes place via a dipolar

intermediate (Ahmad and Tollin 1981) as follows

[3F + F F ỏndashndashndashndashndashndashndash F ỏ+

] (717)

The degree to which this intermediate proceeds to form the products would be

affected by the interaction with NaCl at a particular ionic strength The higher the ionic

strength the greater is the interaction leading to the degradation and hence an increase in

the rate of the reaction A positive slope of the reaction indicates an increase in the rate of

reaction between similarly charged species as a result of an increase in the ionic strength

of the solution The degradation of RF by the photoaddition pathway also involves the

participation of a charged species in the form of a [1RFndashHPO4

2minus] complex Although Eq

(71) is essentially true for dilute solutions an effect due to ionic strength is in fact

observed at higher concentrations (Florence and Attwood 2006) as found in the present

case Since the value of ZAZB for the photoaddition reaction (k3) is 080 This value is not

an integer suggesting a complex mode of reaction between RF buffer species and Clminus

ions It has been suggested (Schuman Jorms et al 1975) that the photoaddition pathway

is not affected by the ionic strength These authors studied the analytical photochemistry

of RF by absorbance changes at the λmax at 445 nm Their analytical data may not be

reliable due to the fact that all the photoproducts of RF absorb at this wavelength and an

accurate assay of RF is not possible Thus any kinetic data obtained may not represent the

true rate constants for the reactions involved

+H ndashH+

The present study involves a specific analytical method to determine RF

accurately in the presence of various photoproducts and therefore the rate constants

derived from such analytical data would be reliable as reported in several previous

studies (Ahmad et al 2004a 2009 2010 2013 2015)

The effect of ionic strength has also been observed in studies carried out on the

photolysis of RF and related reactions under conditions different from those of the

present work These include the biphasic photolysis of RF in the ionic strength range of

003ndash046 M using phosphate buffer (pH 74) (Sato et al 1984) the photolysis of RF in

the presence of magnesium perchlorate at pH 70 (Schuman Jorns et al 1975) and the

alkaline hydrolysis of 67ndashdimethylndash9ndashformylmethylisoalloxazine (an intermediate in the

photolysis of RF) under various conditions of ionic strength and pH (Song et al 1965)

Ionic strength effects play a significant role in studies involving flavinndashprotein

interactions A charged phosphate group attached to the dimethylbenzene ring of flavins

has been found to produce a large ionic strength effect on the rate of interaction (Ahmad

et al 1981) The kinetics of electron transfer reactions and the binding ability of flavins

to proteins are dependent upon the ionic strength due to electrostatic interactions (Ahmad

et al 1981 1982 Hazzard et al 1987 Meyer et al 1984 Hurley et al 1999) and may

be significantly influenced at large values of ionic strength

CHAPTER VIII

METAL ION MEDIATED PHOTOLYSIS

REACTIONS OF RIBOFLAVIN

81 INTRODUCTION

Riboflavin (RF) (1) (Fig 81) is a photosensitive compound

(British Pharmacopoeia 2016) which undergoes degradation in aqueous solution on

exposure to light (Ahmad et al 2004a Astanov et al 2014 Sheraz et al 2014) The

degradation takes place by different mechanisms depending upon the reaction conditions

(pH buffer kind and concentration light intensity and wavelengths aerobic or anaerobic

condition) (Heelis 1982 1991 Ahmad and Vaid 2006) The photolysis of RF in aqueous

solution leads to the formation of a number of compounds including formylmethylflavin

(FMF) (2) lumichrome (LC) (3) lumiflavin (LF) (4) carboxymethylflavin (CMF) (5)

and cyclohdehydroriboflavin (CDRF) (6) by photoreduction and photoaddition pathways

given in Chapter 3 (Smith and Metzler 1963 Treadwell et al 1968 Cairns and Metzler

1971 Ahmad and Rapson 1990 Ahmad et al 2004ab 2008 2010) (Fig 31) The

kinetics of photolysis reactions of RF has been evaluated (Ahmad et al 2004a Cairns

and Metzler 1963 Ahmad et al 2004b 2008 2010 2014 2016) using specific

spectrometric methods (Ahmad and Rapson 1990 Ahmad et al 1980 2004ab 2014)

Flavins are known to interact with metal ions to form complexes For example

10ndashmethylisoalloxazine forms a complex with Cu+ ions (Hemmerich et al 1965 Yu and

Fritchie Jr 1975) RF with monovalent ions (Ag+) (Weber 1950 Wade and Fritchie Jr

1973) divalent ions (Fe Cu Cd Mg Mn Co Ni Zn Ru) (Isaka 1955 Isaka and Ishida

1953 Sakai 1956 Garland Jr and Fritchie Jr 1974 Mortland and Lawless 1983

Kaim et al 1999 Hussain et al 2006 Jabbar et al 2014) and trivalent ions (Cr3+

(Rutter 1958 Varnes et al 1971) flavin mononucleotide (FMN) with divalent ions (Mg

Ca Sr Ba Mn Co Cu Zn Cd) (Sigel et al 1995) and trivalent ions (Fe3+

) (Mortland

additi

photoreduction(1)

(4) (5)

H+ OH-

Fig 81 The photoreduction and photoaddition pathways of riboflavin (RF)

and Lawless 1984) flavin dinucleotide with Hg2+

and Cd2+

ions (Picaud and Desbois

2006) and flavin analogues (3ndashmethylndash10ndashphenylisoalloxazine and 3ndashmethylndash10ndash

phenylndash5ndashdeazaisoalloxazine) with Mg2+

and Zn2+

ions (Fukuzumi et al 1985

Fukuzumi and Kojima 2008) Structural characteristics (Wade and Fritchie Jr 1973

Isaka and Ishida 1953 Kaim et al 1999 Clarke et al 1979 1980) and redox reactivity

(Kaim et al 1999 Fukuzumi and Kojima 2008 Fukuzumi and Okhubo 2010) of the

metalndashflavin complexes have been studied in detail

It has been shown (Kaim et al 1999 Fukuzumi and Kojima 2008 Clarke et al

1978) that metal centres can bind to flavin in the N(5)ndash C(4a)ndashC(4)ndashO(4) site to form a

planar fivendashmembered chelate ring (Fig 82) Electrochemical and spectroscopic data on

the structural features of these complexes have been reported (Kaim et al 1999

Fukuzumi and Kojima 2008 Clarke et al 1978) The metalndashflavin interactions have

important implications in the electron transfer reactivity of flavins in biological systems

(Kaim et al 1999)

The aerobic photolysis of RF is promoted by Fe2+

in the decreasing order of reactivity The anaerobic photolysis of RF is

promoted by Fe3+

ions and inhibited by Fe2+

and Cu2+

ions (Sakai 1956) RF catalyzes

the photooxidation of Fe2+

(oxygen dependent) and photoreduction of Fe3+

(inhibited by

oxygen) Both ions have been found to quench the fluorescence of RF (Rutter 1958)

Metalndashflavin complexes presumably involve extensive charge transfer from metal d

orbitals to flavin π orbitals (Varnes et al 1971)

(1) (81)

Rearrangment

4a5 44a

Fig 82 Formation of the metalndashRF complex

The fluorescence of RF is quenched by Ag+ ions various divalent ions and Fe

ions due to the formation of nonndashfluorescent metalndashRF complexes (Weber 1950 Isaka

1955 Isaka and Ishida 1953 Varnes et al 1971) The quenching of excited singlet states

of organic molecules by metal ions has been observed (Kemlo and Shepherd 1971) [41]

ions promote photolysis of RF strongly followed by the effect of Fe3+

and Zn2+

ions Ag+ ion inhibits the photolysis of RF (Sakai 1956)

Trace quantities of metallic impurities in pharmaceuticals may catalyze the

degradation of drug substances (British Pharmacopoeia 2016) particularly in the

presence of light These processes occur by onendashelectron oxidative reactions and result in

an increase in the rate of formation of radicals that lead to the degradation products

Oxidative reactions are often initiated by metal ions such as Fe3+

These metal ions act as initiators since they are capable of acting as radicals in their

oxidation states for example Cu 2+

ion has 27 electrons and it requires one electron to

complete the electron pair The metal ion can react with a drug to form radicals

+ RH M(nndash1)+

+ H+ + R

˙ (81)

The radical can then participate in the propagation cycle or can react with a

hydroperoxide to catalyze the degradation

+ RʹOOH M(nndash1)+

+ H+ + RʹO2

˙ (82)

RʹOOH could be a hydroperoxide of the drug (eg RF) itself or of some other

component present in the system (Connors et al 1982) Thus the metal ion can directly

react with oxygen to form an oxygen radical which can then initiate an autoxidation

reaction The metal ion can also form a complex with oxygen to produce a peroxy radical

or it can react with a drug (eg RF) to form a radical to initiate a photochemical chain

reaction

The object of this work is to conduct a study of the photolysis of RF in metalndashRF

complexes using various metal ions to identify the photoproducts to determine the

absorption and fluorescence characteristics and to evaluate the influence of metal ions on

the kinetics of photolysis reaction at different buffer concentrations It may have

important implications in the understanding of the reactivity of flavoenzymes since these

complexes are known to modify the redox reactivity of enzymes in the biological system

The experimental details involved in these studies are presented in 53

(Chapter 5)

821 Photoproducts of MetalndashRF Complexes

The TLC studies of the photolyzed solutions of various metalndashRF complexes

indicated the formation of FMF an intermediate product LC LF and CMF (solvent

systems (Z1) and (Z2)) (Section 531) at low buffer concentration and FMF LC LF

CMF and CDRF (solvent system (Z3)) as the sidendashchain products of RF at pH 70 on

comparison of the Rf values and fluorescence emission (RF FMF LF CMF yellow

green LC skyblue) and CDRF (red colour) with those of the authentic compounds The

fluorescence intensity of the spots of these photoproducts varied with the concentration of

metal ions An increase in metal ion concentration leads to an increase in the

concentrations of the photoproducts as a result of enhancement in the rate of photolysis

All these photoproducts have previously been observed in the photolysis of RF

(Ahmad et al 2004a 2008 Smith and Metzler 1963 Treadwell et al 1968 Cairns and

Metzler 1971 Ahmad and Rapson 1990 Isaka 1955) Divalent ion impregnated silica

gel G TLC plates have been used for the separation of RF and other B vitamins on the

basis of complexation (Bushan and Parshad 1994)

822 Spectral Characteristics of MetalndashRFndashComplexes

The spectral characteristics of free RF and metalndashRF complexes have been

studied The UV and visible absorption spectra of some typical complexes (Fe2+

and Cu2+

) are shown in Fig 83 Aqueous solutions of RF (pH 70) exhibit absorption

maxima at 223 267 374 and 444 nm (British Pharmacopoeia 2016) On the addition of

ions to RF solution a big spectral change is observed in the UV and visible region

with disappearance of the 445 maximum and increase in absorption in the 200ndash400 nm

region The greater effect of Fe2+

ions (1 times 10ndash3

M) at a high concentration (20 fold)

compared to that of RF (5 times 10ndash5

M) on the spectral changes of RF is probably due to the

11 RFndashFe2+

complex formation as well as the chemical reduction of RF resulting in the

loss of the 445 nm band RF is easily chemically reduced by electron donors such as

sodium dithionite (Na2S2O4) (Burn and OrsquoBrien 1959) with a loss in absorption at 445

nm due to the disappearance of the N(5)ndashC(4a)ndashC(10a)ndashN(1) conjugated system (Fig

82) as a results of the formation of RFH2 molecule

RF + 2Fe2+ +2HRFH2 + 2Fe3+

Fig 83 Absorption spectra of RF (5 times 10ndash5

M) (pH 70) (____

) in the presence of

metal ions (1times 10ndash3

ions (b) Zn2+

ions and (c) Cu2+

On the contrary the changes in the absorption spectra of RF are not very

prominent in the presence of Zn2+

and Cu2+

ions (Fig 83) These spectral changes could

result from disturbance in the conjugated system of the pteridine ring in RF as mentioned

above A slight increase in the absorption of RF in the presence of Cu2+

ions appears to

be due to an increase in the intensity of colour as a result of RFndashCu2+

complex formation

Similar minor changes in the absorption spectra of RF have been observed in the

presence of other divalent ions studied Such spectral changes have previously been

observed in the spectra of metalndashRF complexes (Isaka and Ishida 1953 Fukuzumi et al

1985) These changes in the absorption spectra of RF are not very prominent in the

presence of Zn2+

and Cu2+

ions These spectral changes could result from disturbance in

the conjugated system of the pteridine ring in RF Such changes have previously been

observed in the absorption spectra of metalndashRF complexes (Isaka and Ishida 1953

Fukuzumi et al 1985)

It is well known that various metal ions bind to flavins in the N(5)ndashC(4a)ndashC(4)ndash

O(4) chelate site to form planar 5ndashmembered redoxndashactive αndashiminoketo chelate rings

(81) (Fig 82) (Kaim et al 1999 Fukuzumi and Kojima 2008 Kemlo 1977) [28 37

40] Electrochemical and spectroscopic data on the structural features of these metalndash

flavin complexes have been reported (Kaim et al 1999 Fukuzumi and Kojima 2008

Kemlo 1977) Since O(4) and N(5) atoms of the αndashiminoketo function in the chelate ring

of RF are connected in a asymmetric πndashconjugated system the redoxndashactive metal

chelate undergoes rearrangement of the C(4)ndashC(4a) bond to a symmetrical (C(4a)ndashC(4))

form (82) (Fig 82) as suggested for αndashdiimines (Juris et al 1988 Constable 1989

Greulich et al 1996) and αndashdiketones (Burns and McAuliffe 1979) This would result in

the disappearance of the πndashconjugated system affecting the UVndashabsorption maxima (444

nm) of the complex The gradual loss of these maxima with an increase in metal ion

concentration (Fig 83) is indicated by a shift in the equilibria to form the symmetrical

metalndashRF complex (82) through the intermediate form (81) (Fig 82)

823 Spectrometric Assay of RF and Photoproducts in Photolyzed Solutions

The assay of RF and photoproducts (FMF LC LF CDRF) in the photolyzed

solutions of metalndashRF complexes (pH 70) has been carried out by a multicomponent

spectrometric method extensively used for the assay of RF and photoproducts in the

photolysis reactions of RF (Ahmad et al 1980 2004a 2008 2014 2016 Ahmad and

Rapson 1990) The pH of the photolyzed solutions is adjusted to pH 20 to form the

protonated species of RF and FMF (Suelter and Metzler 1960) and the solutions are

extracted with chloroform to remove LC and LF followed by their twondashcomponent assay

at 356 and 445 nm The aqueous phase is used to assay RF and FMF (at low buffer

concentration 0001 M) (Table 81) or RF FMF and CDRF (at high buffer

concentrations 02ndash04 M) (Table 82ndash83) by a twondashcomponent assay at 385 and 445

nm or a threendashcomponent assay at 385 410 and 445 nm respectively CMF is a minor

oxidation product of FMF (Ahmad et al 2004a) (Fig 81) and is not accounted in the

assay The metal ions at the concentrations used do not interfere in the assay The assay

method gives good molar balance of RF and photoproducts with a RSD of plusmn5 as

observed in earlier studies (Ahmad and Rapson 1980 Ahmad et al 2014 2016)

Table 81 Concentration of RF (M times 105) and LC (M times 10

5) (0001 M Phosphate

Buffer) in the presence of 10ndash50 times 10ndash4

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 485 008 489 006 486 007 489 006 490 005

120 470 014 477 012 472 015 478 014 479 012

180 447 026 454 023 458 020 466 019 468 018

0 500 000 500 000 500 000 500 000 500 000

60 472 015 463 017 457 019 450 019 446 022

120 442 032 431 037 421 035 413 038 398 046

180 416 045 398 049 384 052 371 059 355 066

0 500 000 500 000 500 000 500 000 500 000

60 474 014 461 018 453 022 442 026 432 030

120 450 027 424 032 413 036 393 047 373 055

180 418 039 389 044 365 055 346 065 324 076

0 500 000 500 000 500 000 500 000 500 000

60 472 014 462 017 442 025 429 030 421 033

120 444 024 423 033 395 044 375 052 354 061

180 414 039 385 052 352 067 322 075 295 086

0 500 000 500 000 500 000 500 000 500 000

60 474 015 465 018 464 019 459 021 441 028

120 450 024 436 029 430 031 415 036 389 048

180 427 036 407 045 393 051 358 062 339 068

0 500 000 500 000 500 000 500 000 500 000

60 474 015 459 019 450 022 440 026 427 032

120 450 026 422 038 403 044 386 051 363 065

180 417 041 381 056 355 066 338 071 309 081

0 500 000 500 000 500 000 500 000 500 000

60 489 006 475 013 472 014 472 014 467 016

120 465 016 449 024 446 026 443 027 437 029

180 437 032 427 036 419 039 414 041 408 045

0 500 000 500 000 500 000 500 000 500 000

60 475 014 467 017 463 018 457 021 451 023

120 449 024 434 030 429 033 411 040 406 042

180 426 035 407 040 390 047 374 054 363 060

0 500 000 500 000 500 000 500 000 500 000

60 472 015 463 019 453 022 442 025 433 030

120 443 029 428 035 412 042 390 051 373 057

180 416 039 394 047 371 057 342 068 322 076

0 500 000 500 000 500 000 500 000 500 000

60 474 014 473 016 463 019 457 021 441 028

120 447 027 444 028 428 034 416 039 390 051

180 429 036 411 042 391 051 375 057 346 068

0 500 000 500 000 500 000 500 000 500 000

60 478 011 476 013 472 015 466 017 464 019

120 454 022 450 024 442 027 436 029 430 032

180 433 030 423 034 414 039 405 043 399 048

0 500 000 500 000 500 000 500 000 500 000

60 476 013 473 019 464 020 463 020 457 022

120 451 022 444 026 430 033 431 039 416 042

180 426 036 412 044 398 055 393 060 380 066 a Metal ion concentration 10ndash50 times 10

ndash4 M

Table 81 continued

Table 82 Concentration of RF (M times 105) and CDRF (M times 10

5) (02 M Phosphate

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 448 014 457 014 457 014 465 014 467 013

120 405 015 416 015 416 015 424 015 436 014

180 363 017 374 016 381 015 395 014 408 013

0 500 000 500 000 500 000 500 000 500 000

60 425 015 416 015 416 016 398 016 389 016

120 369 017 346 018 338 019 323 019 309 020

180 322 019 298 020 279 021 257 023 245 025

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 407 016 407 016 387 016

120 371 017 363 017 338 019 323 020 300 020

180 319 019 302 020 279 023 259 024 234 026

0 500 000 500 000 500 000 500 000 500 000

60 416 015 398 016 380 017 371 017 352 018

120 346 018 323 019 295 021 275 022 255 024

180 291 021 257 023 229 027 203 029 177 034

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 421 015 416 015 406 016

120 380 017 363 017 354 018 338 018 320 020

180 331 019 310 020 298 021 279 022 262 024

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 015 416 015 396 016

120 380 017 363 017 346 018 338 018 323 019

180 328 019 308 020 295 022 274 023 256 025

0 500 000 500 000 500 000 500 000 500 000

60 454 014 426 015 426 015 426 015 416 015

120 406 015 371 017 363 017 354 018 338 018

180 367 019 316 022 311 023 295 025 281 026

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 015 407 016 396 016

120 381 017 363 017 346 018 331 019 323 020

180 334 019 311 020 293 022 274 027 251 029

0 500 000 500 000 500 000 500 000 500 000

60 436 015 416 015 407 016 398 016 381 017

120 371 017 346 018 331 019 316 020 293 022

180 319 019 291 022 266 023 244 025 228 028

0 500 000 500 000 500 000 500 000 500 000

60 426 015 426 015 407 016 398 016 361 018

120 371 017 354 018 338 019 323 019 262 024

180 320 021 299 025 279 028 259 031 189 037

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 016 416 016 406 016

120 381 017 363 017 354 018 346 018 330 019

180 328 021 314 024 299 025 286 029 273 033

0 500 000 500 000 500 000 500 000 500 000

60 426 015 416 015 421 015 416 015 404 016

120 371 017 354 017 354 018 346 018 330 020

ndash4 M

Table 82 continued

5) (04 M Phosphate

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 416 011 421 010 428 009 436 009 447 007

120 347 027 354 026 369 021 389 018 402 015

180 292 034 303 030 319 028 343 026 359 021

0 500 000 500 000 500 000 500 000 500 000

60 403 018 372 026 375 026 358 030 347 033

120 325 024 282 037 276 038 251 045 244 046

180 252 032 216 042 194 044 171 053 165 055

0 500 000 500 000 500 000 500 000 500 000

60 393 019 381 020 375 021 358 024 347 026

120 307 028 289 031 276 033 254 038 244 041

180 237 037 215 039 200 041 181 044 170 048

0 500 000 500 000 500 000 500 000 500 000

60 375 016 347 022 334 024 319 027 295 022

120 272 030 246 033 219 035 195 036 182 035

180 200 040 167 045 143 048 122 051 103 061

0 500 000 500 000 500 000 500 000 500 000

60 393 011 381 015 384 015 375 016 364 018

120 319 025 298 029 289 031 276 033 263 036

180 251 033 233 036 221 038 209 041 197 043

0 500 000 500 000 500 000 500 000 500 000

60 393 011 381 014 384 014 375 017 364 021

120 317 022 298 025 289 027 276 031 263 035

180 251 029 229 032 223 034 207 037 194 039

0 500 000 500 000 500 000 500 000 500 000

60 393 014 384 016 384 016 375 018 364 020

120 303 022 298 023 289 025 276 027 263 029

180 241 031 229 033 221 035 203 037 191 039

0 500 000 500 000 500 000 500 000 500 000

60 393 013 384 015 384 015 367 017 345 018

120 315 019 298 021 289 023 272 026 237 029

180 255 025 225 029 215 033 198 035 169 039

0 500 000 500 000 500 000 500 000 500 000

60 393 011 375 018 367 019 347 021 337 025

120 302 022 282 026 26 030 242 035 231 038

180 237 033 207 036 188 038 169 041 155 044

0 500 000 500 000 500 000 500 000 500 000

60 393 014 375 019 367 020 347 022 337 024

120 302 019 282 025 266 029 242 032 231 034

180 234 027 213 031 171 041 171 043 159 047

0 500 000 500 000 500 000 500 000 500 000

60 393 013 384 014 367 019 363 019 350 022

120 309 019 295 021 272 026 263 027 251 031

180 242 026 226 029 207 032 195 034 183 036

0 500 000 500 000 500 000 500 000 500 000

60 393 016 381 019 375 021 358 022 347 024

120 315 027 289 033 276 034 254 038 244 040

ndash4 M

Table 83 continued

824 Fluorescence Characteristics of MetalndashFlavin Complexes

The complexation of metal ions with RF results in the quenching of RF

fluorescence This is due to the fact that metalndashRF complexation involves charge transfer

from metal d orbitals to RF π orbital in the excited state (Varnes et al 1971)

The quenching of RF fluorescence by different metal ions at pH 70 is shown in

Fig 84 and the loss of intensity in the fluorescence spectrum of RF (530 nm) in the

presence of increasing concentrations of divalent ions such as Fe2+

ions is shown in

Fig 85 The increase in fluorescence loss of RF at 5 times 10ndash4

M metal ion concentration is

in the order

2+lt Fe

3+ lt Ca

2+ +lt Fe

2+ lt Cd

2+ lt Cu

2+lt Mn

2+lt Pb

2+ lt Mg

2+lt Zn

2+lt Ag

Thus Ni2+

ions on interaction with RF produces the lowest loss in the

fluorescence intensity (37) and Ag+

ions produce the highest loss in fluorescence

intensity (224) of RF There is a gradual loss of RF fluorescence with an increase in

the metal ion concentration for all the metal ions studied This appears to be due to a

greater degree of metalndashRF complexation

825 Kinetic of Photolysis of MetalndashFlavin Complexes

The photochemistry of RF has been studied in detail (see Introduction) and its

modes of photolysis are well known (Heelis 1982 1991 Ahmad and Vaid 2006 Ahmad

et al 2008) (Fig 81) Metal ions are known to modify the redox reactivity of flavins

(Fukuzumi and Kojima 2008)

Fig 84 The percent decrease in fluorescence intensity of RF solutions (pH 70

0001 M phosphate buffer) in the presence of metal ions () Ni2+

ions (∆) Co2+

(loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

() Mg2+

ions () Zn2+

ions and () Fe3+

00 10 20 30 40 50 60

Metal ion concentration (M times 104)

Fig 85 Excitation spectrum of RF (5 times 10ndash5

M) (pH 70) (a) Fluorescence spectra

of RF pure RF (b1) RF + Fe2+

M) (b2)

RF + Fe2+

M) (b3)

However no work on the kinetics of photolysis of metalndashRF complexes has been

conducted to study the behaviour of these complexes on UV or visible irradiation and to

identify the photoproducts formed RF is known to undergo photolysis in aqueous

solution by an apparent firstndashorder kinetics (Ahmad et al 1980 2004a 2008 2010

2014 2016 Sheraz et al 2014)

In the present study the photolysis of 5 times 10ndash5

M RF solutions (pH 70) at low

(0001 M) and high (02ndash04 M) phosphate buffer concentrations has been carried out in

the presence of various metal ions to evaluate the kinetics of these reactions The various

rate constants for the photolysis of RF (kobs) and for the formation of LC (k1) and LF (k2)

(photoreduction pathway) and CDRF (k3) (photoaddition pathway) (Heelis 1982 1991

Ahmad and Vaid 2006) by parallel firstndashorder reactions have been determined by the

method described by Ahmad et al (2016) A typical set of firstndashorder plots for the loss of

RF concentration on photolysis as a function of the increasing concentration of metal

ions at low (0001 M) and high buffer concentrations (02ndash04 M) are shown in Fig 86ndash

818 and 819ndash842 respectively The greater loss of RF in the presence of increasing

concentrations of Fe2+

ions may be due to a change in the equilibria of RF and the metalndash

RF complexes and their greater susceptibility of photolysis

RF + Fe2+ RF-Fe2+

Significant enhancement of the electronndashtransfer reactivity of the singlet excited

state of flavins has been observed by complexation with metal ions (Fukuzumi et al

1985 Fukuzumi and Kojima 2008 Clarke et al 1979)

Fig 86 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH

70) in the presence Ag+

ions at different concentrations (M times 10ndash4

) (diams) 10

() 20 () 30 () 40 () 50

70) in the presence Fe2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

log co

70) in the presence Cu2+

) (diams) 10

() 20 () 30 () 40 () 50

70) in the presence Zn2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

70) in the presence Mg2+

) (diams) 10

() 20 () 30 () 40 () 50

70) in the presence Pb2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

70) in the presence Ni2+

) (diams) 10

() 20 () 30 () 40 () 50

70) in the presence Ca2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

70) in the presence Mn2+

) (diams) 10

() 20 () 30 () 40 () 50

70) in the presence Cd2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

70) in the presence Co2+

) (diams) 10

() 20 () 30 () 40 () 50

Fig 817 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)

in the presence Fe2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ag+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Cu2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

in the presence Zn2+

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Mg2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

in the presence Pb2+

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ni2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

in the presence Ca2+

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Mn2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

in the presence Cd2+

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Co2+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ag+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

This would suggest an increase in the photoreduction of RF (Heelis 1982 1991

Ahmad and Vaid 2006) and hence an increase in the rate of photolysis The apparent

firstndashorder rate constants (kobs) for the photolysis of RF in metalndashRF complexes along

with the rate constants for the formation of LC (k1) LF (k2) and CDRF (k3) at different

buffer concentrations are reported in Table 84ndash86 The values of kobs k1 k2 and k3 show

that the photolysis of RF and the formation of LC LF and CDRF are enhanced with an

increase in the metal ion concentration indicating that the metal ions promote the

photolysis reactions of RF as observed by earlier workers (Isaka 1955 Isaka and Ishida

1953 Sakai 1956 Rutter 1958 Varnes et al 1971) In order to develop a correlation

between the rate of photolysis and the fluorescence quenching of RF a plot of kobs versus

fluorescence loss of RF has been prepared as shown in Fig 843 It indicates an increase

in kobs of RF photolysis with an increase in the fluorescence loss of RF in the presence a

metal ion Thus the higher the fluorescence loss the higher the values of kobs due to the

greater complexation of RF and metal ions The photolysis of RF at low buffer

concentration (eg 0001 M) follows photoreduction pathway in aqueous solution

(Ahmad et al 2004a 2008 2014 Sheraz et al 2014 Ahmad and Vaid 2006) and at

high phosphate buffer concentration (eg 02ndash04 M) the photoaddition pathway (Heelis

1982 1991 Ahmad and Vaid 2006 Ahmad et al 2010 2016) (Fig 81) Therefore a

difference in the rate of photolysis of RF with a change in buffer concentration in the

presence of various metal ions could be expected

Table 84 Apparent Firstndashorder Rate Constants (kobs) for the Photolysis of RF in the

Presence of Various Metal Ions at pH 70 (0001 M Phosphate Buffer) for the formation

of LC (k1) LF (k2) and the SecondndashOrder Rate Constants for the Interaction of RF and

Metal Ions (kʹ)

Metal Ion Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

00 063 016 006

Ag+ 10 059 050 041 017

20 054 038 015

30 049 035 014

40 044 033 010

50 038 029 008

10 089 256 070 018

20 115 080 034

30 142 101 040

40 169 129 039

50 191 143 047

10 099 360 078 020

20 136 084 051

30 172 107 064

40 206 138 067

50 243 164 078

10 105 462 073 031

20 155 113 041

30 199 138 060

40 245 164 080

50 294 190 094

10 101 416 071 029

20 142 099 042

30 184 131 052

40 225 160 064

50 271 182 088

10 106 410 079 026

20 145 105 039

30 185 128 056

40 224 152 071

50 268 180 087

10 075 104 058 016

20 085 062 022

30 095 068 026

40 105 075 029

50 115 083 031

10 089 232 063 025

20 112 075 036

30 136 092 043

40 158 106 051

50 179 120 058

10 102 360 072 029

20 132 089 042

30 167 110 056

40 210 140 070

50 243 162 081

10 091 284 069 021

20 118 086 031

30 148 104 043

40 176 122 053

50 205 139 065

10 078 128 054 023

20 091 063 027

30 104 071 032

40 116 080 035

50 127 087 039

10 082 180 060 021

20 099 075 023

30 118 091 026

40 135 151 029

50 153 174 035

Table 84 continued

Presence of Various Metal Ions at pH 70 (02 M Phosphate Buffer) for the Formation of

LC (k1) LF (k2) CDRF (k3) and the SecondndashOrder Rate Constants for the Interaction of

RF and Metal Ions (kʹ )

Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

00 204 111 038 054 205

Ag+ 10 182 184 125 027 028 446

20 164 112 025 026 430

30 144 094 023 025 376

40 127 084 019 023 365

50 112 072 016 022 327

10 243 384 195 020 027 722

20 285 231 022 031 724

30 325 256 032 035 726

40 363 291 033 040 728

50 396 315 036 043 730

10 249 410 201 021 027 724

20 285 229 025 031 726

30 325 256 034 035 728

40 365 290 036 039 730

50 409 329 033 045 732

10 285 742 226 027 031 729

20 358 283 036 038 733

30 435 343 043 048 736

40 505 402 048 054 738

50 575 446 059 060 741

10 235 246 180 024 029 620

20 265 201 030 032 628

30 295 223 036 034 655

40 325 245 039 036 671

50 358 286 035 041 697

10 235 334 180 024 029 620

20 269 207 029 033 625

30 302 228 035 036 629

40 335 243 044 038 633

50 371 284 045 044 637

10 227 232 149 035 042 354

20 260 179 032 049 360

30 283 195 035 053 360

40 304 210 038 056 365

50 332 230 041 061 369

10 235 358 178 025 030 593

20 270 207 029 034 605

30 305 231 035 037 624

40 334 253 041 040 631

50 373 284 045 044 636

10 251 462 196 025 031 625

20 301 233 031 036 647

30 345 268 036 039 687

40 385 303 041 043 699

50 427 333 048 046 711

10 254 410 179 032 043 411

20 285 201 039 043 467

30 323 231 044 048 475

40 362 259 049 054 479

50 404 289 056 059 483

10 236 256 168 029 039 425

20 255 184 032 038 484

30 280 204 034 040 510

40 300 220 038 042 519

50 319 232 043 044 523

10 237 308 189 021 026 726

20 271 218 024 029 730

30 302 238 030 032 734

40 332 265 030 036 736

50 358 284 036 038 738

Table 85 continued

Presence of Various Metal Ions at pH 70 (04 M Phosphate Buffer) for the Formation

of LC (k1) LF (k2) CDRF (k3) and the SecondndashOrder Rate Constants for the

Interaction of RF and Metal Ions (kʹ)

Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

00 351 222 049 075 296

Ag+ 10 315 332 184 061 069 263

20 280 171 042 066 259

30 247 152 035 059 257

40 214 129 033 051 252

50 190 114 030 046 247

10 402 528 262 059 079 331

20 462 290 070 101 287

30 515 310 094 109 284

40 570 335 098 136 246

50 615 363 104 147 246

10 407 496 259 069 079 325

20 460 295 072 092 320

30 509 328 077 103 317

40 560 357 089 113 315

50 599 373 099 126 296

10 475 1048 302 075 096 314

20 580 359 106 115 310

30 681 414 128 137 302

40 784 475 151 158 299

50 875 505 173 196 257

10 390 348 257 058 073 352

20 425 275 066 082 335

30 458 296 071 090 328

40 490 315 075 099 318

50 525 335 082 107 313

10 386 348 273 050 061 447

20 427 301 057 068 442

30 458 321 060 075 428

40 490 336 068 084 400

50 525 355 077 091 390

10 387 508 254 058 073 347

20 424 273 069 081 337

30 494 317 080 096 330

40 545 347 089 107 324

50 605 380 104 119 319

10 389 600 271 057 060 451

20 426 287 070 067 428

30 494 327 080 085 384

40 545 359 089 095 377

50 651 432 103 116 370

10 415 600 282 057 075 376

20 475 318 071 085 374

30 535 363 074 098 370

40 605 405 090 110 366

50 651 423 109 117 361

10 413 570 287 060 065 441

20 470 320 072 077 415

30 530 337 091 101 333

40 590 370 102 116 318

50 636 392 110 132 296

10 395 414 273 059 061 447

20 438 296 069 071 416

30 479 321 076 081 396

40 524 350 084 089 393

50 558 369 093 095 388

10 405 468 260 055 083 313

20 455 290 072 093 310

30 505 322 077 104 309

40 548 346 086 115 300

50 585 363 093 128 283

Table 86 continued

Fig 843 A plot of kobs for the photolysis of RF versus fluorosecne loss in the

presence of different metal ions () Ni2+

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

ions () Mg2+

ions ()

ions () Fe3+

00 30 60 90

Fluorescence loss

bs times

For example the values of kobs for the photolysis of RF in the presence of Fe2+

ions (10ndash50 times 10ndash4

M) at 0001M buffer concentration (089ndash191 times 10ndash3

minndash1

) (Table

84) are lower than those obtained at 02 M buffer concentration (243ndash396 times 10ndash3

minndash1

(Table 85) and 04 M buffer concentration (402ndash615 times 10ndash3

minndash1

) (Table 86) The

bimolecular rate constants (kprime) for the interaction of Fe

2+ ions and RF in these reactions

are 256 384 and 528 times 10ndash3

Mndash1

minndash1

respectively These results indicate that the

metal ions not only accelerate the photolysis of RF but also influence the reaction

pathways by altering the ratio of the products formed by the photoreduction (LC) and

photoaddition (CDRF) pathways (Heelis 1982 1991 Ahmad and Vaid 2006) in the

presence of high buffer concentration This is evident from the values of the ratios of

k1k3 in the presence of Fe2+

ions at 02 M buffer concentration (72ndash73) and at 04 M

buffer concentration (33ndash25) It also shows that at the highest buffer concentration

(04 M) the formation of CDRF is increased with an increase in metal ion concentration

These observations suggest that the formation of the 5ndashmembered chelate ring (Fig 82)

in the metalndashRF complex may be affected by an increase in metal ion concentration at

high buffer concentration to influence the formation of the two photoproducts The

increase in metal ion concentration may alter the photoreduction pathway leading to the

formation of LC by k1 in favour of the photoaddition pathway leading to the formation of

CDRF by k3 and hence a change in k1k3 ratios with a change in buffer concentration A

similar pattern of product formation ratios (k1k3) has been observed in the presence of

other divalent ions (Cu2+

) and monovalent

(Ag+) and trivalent (Fe

3+) metal ions at high buffer concentrations (Table 85 and 86)

Thus all the metal ions studied behave in a similar manner to affect the product

formation by different pathways in the photolysis of RF at higher buffer concentration

The secondndashorder rate constants (kprime) for the interaction of metal ions with RF are in the

order Zn2+

gt Mg2+

gt Pb2+

gt Mn2+

gt Cu2+

gt Cd2+

gt Fe2+

gt Ca2+

gt Fe3+

gt Co2+

gt Ni2+

gt Ag+

This indicates that Zn2+

has the highest rate of interaction and Ag+ has the lowest rate of

interaction with RF The metal ion effect on the reaction is probably due to the

facilitation of the photoaddition pathway which originates from the excited singlet state

interaction of RF and HPO42ndash

ions (Schuman Jorns et al 1975) This would inhibit the

photoreduction pathway occurring through the excited triplet state of RF (Heelis 1991

Ahmad and Vaid 2006 Cairns and Metzler 1971)

826 Mode of Interaction of Metal Ions with RF

The present study shows that the divalent and trivalent metal ions promote the

photolysis reactions of RF in aqueous solution Earlier studies suggested that RF

catalyzes the photooxidation of Fe2+

ions and photoreduction of Fe3+

ions (Rutter 1958)

It was later suggested that metalndashflavin complexes involve extensive charge transfer from

metal d orbitals to flavin π orbitals and the excited states of flavins should interact much

more strongly than the ground state with metal ions (Varnes et al 1971) The mechanism

of photolysis reactions of RF in the absence of metal ions has been discussed in detail

(Heelis 1982 1951 Ahmad and Vaid 2006) The mode of interaction or complexation

of different metal ions with RF to enhance its degradation appears to be different It has

been shown that the monovalent metal ions (eg Ag+) form a 11 red complex with RF in

which the Ag+ atom binds to the flavin (isoalloxazine) ring (Weber 1950 Baarda and

Metzler 1961 Bamberg and Hemmerich 1961) The divalent ions (eg Fe2+

) bind to RF

in the N(5)ndashC(4a)ndashC(4)ndashO(4) site to form a planar fivendashmembered chelate ring (Kaim et

al 1999 Fukuzumi et al 1985 Fukuzumi and Kojima 2008) (Fig 82) Similarly the

trivalent ions (eg Fe3+

) also form a planar fivendashmembered chelate ring similar to that of

the divalent ions with RF (Fukuzumi et al 1985 Fukuzumi and Kojima 2008

Fukuzumi and Okhubo 2010) Thus all the divalent and trivalent metal ions enhance the

photolysis of RF through metalndashRF complexation

In view of the results obtained in this study indicating the role of metal ions as

promoters of photolysis of RF a scheme for the sequence of reactions involved may be

presented (Fig 844)

RF reacts with a metal ion eg Fe2+

ion to form a [RFhellipFe2+

] complex (Eq

(85)) This complex on absorption of a photon of light is promoted to the excited singlet

state [1RFhellipFe

2+] (Eq (86)) In this state charge transfer takes place resulting in the

formation of a loosely bound semireduced semiquinone radical [RFH] and an oxidized

] ion (Eq (87)) followed by their separation to give free [RFH] radicals and Fe3+

ions (Eq (88)) 2[RFH] radicals react to give a reduced RF molecule [RFprimeH2] with an

altered side chain (Eq (89)) The [RFprimeH2] molecules are oxidized by air to form FMF

and sidendashchain products (Eq (810)) FMF then undergoes hydrolysis to give LC LF and

sidendashchain products as the final photoproducts of RF (Eq (811)) The [1RFhellipFe

2+] state

in the presence of HPO42ndash

ions leads to the formation of a CDRF molecule and a Fe3+

(Eq (812))

RF + Fe2+ [RFFe2+]

metal-RF complex

[RFFe2+] [1RFFe2+]

excited singlet state complex

[1RFFe2+] [RFHFe3+]

[RFHFe3+] RFH

+ Fe3+

2RFH RFH2

RFH2 FMF + side-chain products

FMF LC + LF + side-chain products

[1RFFe2+] CDRF + Fe3+ HPO

H+ OH_

Fig 844 Scheme for the photolysis of RF in metalndashRF complex

The reaction scheme described for the photochemical interaction of Fe2+

ions and

RF (Eq (81)ndash(812)) may be considered analogous to that presented for the

photostabilization of RF by phosphatidylcholine (PC) in liposomes It involves the

formation of a photoinduced charge transfer complex between RF and PC (Ahmad et al

2015 Bhowmik and Sil 2004) and norfloxacin and PC (Ahmad et al 2016) as a basis of

the stabilization of these drugs in liposomes

CHAPTER IX

PHOTOCHEMICAL PREPARATION

CHARACTERIZATION AND FORMATION

KINETICS OF RIBOFLAVIN CONJUGATED

SILVER NANOPARTICLES

91 INTRODUCTION

Nanoparticles (NPs) are a rapidly growing field in nanotechnology due to their

size (nm) and unique characteristics which make them an ideal candidate for application

in physical chemical and biological systems (Nairn et al 2006 Noguchi et al 2011

Routh et al 2012 Arce et al 2014 Bala et al 2016 Foresti et al 2017) NPs exhibit a

particle size of less than 100 nm and possess versatile properties as compared to the bulk

material of a compound They need high pressure energy or temperature for their

formation They also require some toxic material for their stabilization which may lead to

adverse effects when subjected to biomedical and pharmaceutical applications (Goodsell

2004 Abbasi et al 2016 Rajavel et al 2017)

Different methods have been used for the preparation of silver (Ag) NPs ie

sequential injection method (Passos et al 2015) chemical reduction (Wei et al 2015)

photochemical reduction (Chen et al 2007 Frattini et al 2005) irradiationndashassisted

chemical reaction (Sotiriou et al 2010) electrochemical reduction (Abbasi et al 2016)

biosynthesis (Ramanathan et al 2013) lithography (Ahmed et al 2016) and physical

methods (Dang et al 2014 Tien et al 2008) The mechanism of formation of Ag NPs

(Hussain et al 2011) RF conjugated ZnO NPs (Bala et al 2016) and Cu NPs (Noguchi

et al 2011) has been described Ag NPs are of great importance due to their unique

features and different applications in the fields of drug delivery (Benyettou et al 2015)

food technology (Costa et al 2011 De Moura et al 2012) agriculture (Kim et al

2012) environmental technology (Benn and Westerhoff 2008) catalysis (Huang et al

2012) water purification (Das et al 2012) and textile industry (Ilic et al 2009

Montazer et al 2012)

Riboflavin (RF) (1) is a photosensitive vitamin (British Pharmacopoeia 2016)

and acts as an important precursor for the synthesis of flavin mononucleotide (FMN) and

flavin adenine dinucleotide (FAD) (Foraker et al 2003) It is widely used for the

treatment of neonatal jaundice (Ebbesen et al 2015) HIV induced infections (Leeansyah

et al 2015 Fernandez et al 2015) and keratoconus (Henriquez et al 2011 Farjadina

and Naderan 2015) In photodynamic therapy RF is used as a potential drug to kill tumor

tissues (Ionita et al 2003) and colorectal adenomas (Figueiredo et al 2008) RF along

with magnesium citrate and condashenzyme Q10 is effectively used for the prevention of

migraine (Gaul et al 2015) When exposed to light RF is rapidly degraded to form

different photoproducts (ie formylmethylflavin (FMF) (4) lumichromre (LC)

(5) lumiflavin (LF) (6) and carboxymethylflavin (CMF) (7)) (Smith and Metzler 1963

Cairns and Metzler 1971 Ahmad et al 2004 2014 2016) (Fig 91) Due to the

photosensitive nature of RF different attempts have been made for its stabilization using

liposomal preparations (Habib and Asker 1991 Loukas et al 1995ab Senndashverma et al

1995 Bhowmik and Sil 2004 Ahmad et al 2015) complexation with chemical agents

(Evstigneev et al 2005 Ahmad et al 2009 Sheraz et al 2014a) and cyclodextrins (CD)

(Loukas et al 1995ab Terekhova et al 2011ab) stabilizers (Asker and Habib 1990)

and borate (Ahmad et al 2008) and citrate buffers (Ahmad et al 2011)

RF is known to form complexes with Ag+ ions and other metal ions (Weber

1950 Wade and Fritiche 1973 Ahmad et al 2017) Different studies have been carried

out on the interaction of RF with Ag NPs (Voicescu et al 2013 Routh et al 2012

Mokashi et al 2014) photoactivation of RF by Ag NPs (Khaydukov et al 2016)

detection of RF by Ag NPs (Ma et al 2016) effect of Ag NPs on the photophysics of RF

(1)(4)

(5)(7) (6)

[O] neutral and alkaline pHacid neutral

and alkaline pH

Fig 91 Photodegradation pathway of RF

(Rivas Aiello et al 2016) preparation of RF conjugated Zn NPs (Bala et al 2016) and

Cu NPs (Noguchi et al 2003 2011) and adsorption of RF on the surface of silver (Liu et

al 2012 Akhond et al 2016) However there is a dearth of information on the effect of

some factors on the formation of RFndashAg NPs in these studies The object of present

investigation is to sprepare RFndashconjugated silver nanoparticles (Ag NPs) by

photoreduction their characterization by physical methods and the evaluation of the

effect of pH ionic strength concentration of Ag+ ions and irradiation source (visible

light UV light) on the formation kinetics of RFndashAg NPs

(Chapter 5)

921 Characterization of RFndashConjugated Ag NPs

9211 Optical studies

A colour change of the RFndashAg NPs solution (yellow green to brown) was

observed which indicated the formation of RFndashconjugated Ag NPs (Fig 92) This

change in colour was due to the reduction of Ag+ ions into Ag NPs (AbdelndashHafez et al

2016 Krupa et al 2016 Mosae Selvakumar et al 2016 Alzahrani et al 2017)

9212 Spectral characteristics of RFndashAg NPs

RF exhibits absorption maxima at 223 267 374 and 444 nm in aqueous solution

(British Pharmacopoeia 2016) Ag NPs absorb in the visible region with the appearance

of a surface Plasmon resonance (SPR) band depending on the size and shape of Ag NPs

Fig 92 Colour change for the formation of RFndashAg NPs from yellow green

to brown

(Haes and Van Duyne 2002 Lee et al 2008 Amendola et al 2010 Hou and Cronin

2013 Mogensen and Kneipp 2014) The absorption maxima of SPR band of Ag NPs

have been reported in the wavelength range of 408ndash422 nm (Chairam and Somsook

2008 Tai et al 2008 Chairam et al 2009)

In the present study the effect of photochemical interaction between RF and Ag+

ions and the formation of Ag NPs on changes in their spectral characteristics has been

investigated The absorption spectrum of RF and the changes occurring on the addition of

AgNO3 formation of Ag NPs and interaction of RF with Ag NPs during a period of 6 h

are shown in Fig 93 There is a significant change in the 374 and 444 nm bands of RF

which undergo bathochromic (red) and hypsochromic (blue) shift respectively to form

the SPR band of Ag NPs with a maximum at 422 nm Similar spectral shifts of RF

maxima to form a SPR band of Ag NPs (426 nm) have been observed by Zhang et al

(2011) and Mokashi et al (2014) These spectral changes have been attributed to the

interaction of RF and Ag NPs through the hydroxyl group or methyl groups (Mokashi et

al 2014) The spectra also show a gradual increase in the absorption at 267 nm

maximum of RF during the interaction with Ag NPs An increase in RF absorption in

250ndash300 nm region with an increase in Ag NPs concentration is probably due to greater

interaction between the two species (Mohashi et al 2014)

9213 Fluorescence characteristics of RF

RF is a highly fluorescent compound and emits fluorescence in the 520ndash530 nm

region (Weber 1950 Varnes et al 1972 Heelis et al 1981 Sikorska et al 2005

Ahmad and Vaid 2006 Arce et al 2014 Ahmad et al 2017)

Fig 93 Absorption spectra of RF and RFndashAg NPs

Its fluorescence is quenched by acid and alkali (Weber 1950) complexation with organic

compounds (Penzer and Radda 1967) and metal ions including Ag+ ions (Weber 1950

Wade and Fritchie 1973 Ahmad et al 2107) The fluorescence of aqueous solutions of

RF is also quenching by Ag NPs (Zhang et al 2011 Mokashi et al 2014 Rivas Aiello

et al 2016) Cu NPs (Noguchi et al 2011) and cinnamic alcohol chemisorbed on silica

NPs (Arce et al 2014)

The fluorescence quenching of RF by Ag NPs observed in this study is shown in

Fig 94 and a plot of fluorescence loss versus irradiation time is shown in Fig 95 The

loss of fluorescence intensity of RF at 525 nm is due to the interaction of RF and Ag NPs

and the total loss of fluorescence indicates complete conversion of RF to form the RFndashAg

NPs conjugates It has been suggested that the fluorescence quenching of RF by Ag NPs

is due to the fluorescence energy transfer (FRET) from RF (donor) to Ag NPs (acceptor)

on the adsorption of RF (Mokashi et al 2014) A photoinduced electron transfer from

excited RF to metal ions such as Cu2+

ions resulting in loss of fluorescence and copper

deposition has been reported (Morishita and Suzuki 1995 Noguchi et al 2003 2011)

Such photoinduced electron transfer reactions have been observed in the formation of Ag

colloids (Mennig et al 1992 Lei et al 2017) and Cu NPs (Giuffrida et al 2004)

9214 FTIR studies

FTIR studies have been carried out to confirm the structure of RF and to ascertain

the nature of interaction between RF and Ag NPs The FTIR spectra of RF and RFndashAg

NPs conjugates are shown in Fig 96 RF (Fig 96a) exhibits strong absorption peaks at

Fig 94 Excitation spectrum of RF (green colour) and Fluorescence spectra of RFndash

Ag NPs at different time 0 min (blue) 60 min (black) 120 min (pink) 180 min

(orange) 240 min (dark blue) 300 min (purple)

Fig 95 A plot of fluorescence loss versus time (h) for the formation of

RFndashAg NPs

0 1 2 3 4 5 6 7

Time (h)

Fig 96 FTIR spectrum of RF (a) and RFndashAg NPs (b)

1074 (ribose moiety) 1150 (CndashOH) 1550 (C=N) 1580 (C=C) 1650 (C=O) and 3370

cmndash1

(OHNH) These values are in agreement with the absorption peaks of RF reported

by Blout and Fields (1949) Fall and Petering (1956) Ahmad (1968) Moffat et al (2013)

and Akhond et al (2016) The IR spectrum of RFndashAg NPs (Fig 6b) conjugates shows an

intense absorption peak at 2920 cmndash1

(CndashH stretching) which may be due to chemical

interaction between RF and silver It has also been found that there is a shift in 1550 cmndash1

of RF peak to 1475 cmndash1

which may be due to the interaction of Ag with N(5) of RF IR

peaks at 15429 16509 and 17281 cmndash1

have been observed indicating the adsorption of

RF on the surface of AgFe3O4 NPs (Akhond et al 2016)

9215 Dynamic light scattering (DLS)

DLS has been used to determine the size of RFndashAg NPs The hydrodynamic radii

(Hd) of these NPs range from 579ndash722 nm RFndashAg NPs have been found to be

polydispersed with a polydispersity index of 275 to 290 (Fig 97) The mean

autocorrelation function (Fig 7A (a)) of RFndashAg NPs is good and indicates that the

particles are of nanoscale range It has been reported that if the particles are of larger size

their decay time is higher (Liu et al 2009) In the case of RFndashAg NPs the decay time has

been found to be 10ndash3

sec which indicates that the particles are decaying rapidly and are

in nanorange The mean radius distribution (Fig 97A (b)) of the particles has been found

to be less than 100 nm and the area under the curve indicates that the particles are less

than 100 nm in size The intensity of the peak shows that all the particles are in the 50ndash80

nm range The aggregation of RFndashAg NPs with time has been evaluated and it was found

that there is no aggregation between the particles with the passage of time

Fig 97 Dynamic light scattering measurements of RFndashAg NPs

The Hd of RFndashAg NPs remained the same with time and aggregation did not occur during

this period (Fig 97A (c)) The histograms in Fig 97B(a) also indicates that the RFndashAg

NPs are polydispersed in nature and the major particles are of 722 nm in size This is

evident from the histogram between the radius and the frequency of occurrence of the

particles The fluorescence in Fig 97B (b) is also complimentary to the Fig 97 (A (abc)

B(a)) showing that the particles are in the nanoscale range (579ndash722 nm) and are evenly

distributed

9216 Atomic force microscopy (AFM)

The morphological characteristics of RFndashAg NPs were studied by AFM and the

images obtained show the topographical organization of RFndashAg NPs at micrometer scale

with nanometer resolution in height (Fig 98ab) The prepared RFndashAg NPs are of

spherical shape and polydisperesed in nature AFM images show that the particles are of

bimodal distribution and the major particles are of nanoscale range with a size ranging

from 57 to 73 nm These results are complimentary to those obtained from DLS

measurements

922 Factors Affecting Particle Size of RFndashAg NPs

Different factors which affect the particles size of RFndashAg NPs are discussed

Fig 98 AFM micrograph (25 times 25 microm) of RFndashAg NPs

9221 pH

The Effect of pH (20ndash120) on the size (Hd) of RFndashAg NPs has been evaluated

At a lower pH the H+ ion concentration increases that result in an increased protonation at

the surface of NPs to form aggregates and thus an increase in the size of NPs It has been

reported that with an increase in pH the OHndash ion concentration increases which results in

the generation of negative sites at Ag NPs that do not allow the formation of aggregates

and thus the Hd of Ag NPs would be low (Badawy et al 2010) It has been found that at

acidic pH (20ndash60) a decrease in OHndash ion concentration leads to an increase in the

formation of aggregates of RFndashAg NPs that cause the settling of the particles due to an

increase in the Hd of RFndashAg NPs Whereas at alkaline pH (80ndash120) the Hd of RFndashAg

NPs decreases resulting in low aggregation and low settling of the particles On the

interaction of silver atoms (coordinately unsaturated) at the surface of NPs with a

nucleophile (OHndash ions) these are negatively charged This does not lead to the formation

of aggregates or increase in the particle size (Badawy et al 2010) The broadening of

absorption peaks of RFndashAg NPs at lower pH as compared to that of the higher pH

indicates the formation of aggregates which are due to an increase in the size of RFndashAg

NPs (Fig 99)

9222 Ionic strength

The effect of ionic strength (01ndash1000 mM) on the particle size has also been

evaluated and it has been found that with an increase in ionic strength the Hd of RFndashAg

NPs is also increased as evident from the broadening of the UVndashvis spectra (Fig 910) It

has previously been reported that with an increase in the ionic strength the Hd of the

Fig 99 Absorption spectra of RFndashAg NPs at different pH values 20 (black) 40

(red) 60 (blue) 80 (green) 100 (pink) 120 (light green)

Fig 910 Absorption spectra of RFndashAg NPs at different ionic strengths (mM) 01

(black) 10 (red) 50 (blue) 100 (light green) 500 (purple) 100 (green) 250 (dark

blue) 500 (maroon) 1000 (pink)

Ag NPs is also increased (Badawy et al 2010) The effect of ionic strength clearly shows

the broadening of the absorption spectra of RFndashAg NPs which is due to an increase in the

interaction of RFndashAg NPs with NaCl (250ndash1000 mM) It leads to greater aggregation and

settling of RFndashAg NPs due to an increase in the size of these particles The RFndashAg NPs

have been found to be stable at low ionic strength (01ndash100 mM) due to low or no

interaction between NPs and NaCl The particle size of these NPs is small as compared to

that observed in the presence of a higher concentration of NaCl due to aggregation This

is evident from the absorption spectra of RFndashAg NPs which have a broad peak at high

salt concentration (Fig 910) The sharpness of an absorption peak is an indication of the

decrease in the particle size of metal NPs (Kelly et al 2003)

923 Kinetics of Formation of RFndashAg NPs Conjugates

The rates of formation of RFndashAg NPs conjugates in the presence of UV and

visible light have been determined in the pH range of 80ndash105 and at different

concentrations of Ag+

ions (0002ndash001mM) It has been observed that the formation of

RFndashAg NPs follows a biphasic firstndashorder reaction This is probably due to the formation

of Ag NPs in the first phase (~ 30 min) and further reaction of RF with Ag NPs in the

second phase It has been reported (Noguchi et al 2011) in the case of RFndashCu 2+

interaction that the photoinduced electron transfer from RF to Cu2+

ions takes place only

during the initial stage of irradiation (~ 05 h) (which may be considered as the fast first

phase of the reaction) This is followed by a slow photoinduced electron transfer reaction

from the major photoproduct of RF (ie LC) to Cu2+

ions (this may be considered as the

slow second phase of the reaction) The biphasic formation of RFndashAg NPs in the present

case may also be explained on the basis of RFndashCu2+

ions reactions involving the

photoinduced electron transfer from RF to Ag+ ions (k1) and than from LC to Ag

+ ions

(k2) The photochemical formation of LC from RF is well established (Smith and Metzler

1963 Cairns and Metzler 1971 Ahmad et al 1990 2004a 2014 2016 2017)

However LC exhibits an absorption maximum at 356 nm (Koziol 1966) which would

not contribute to an increase in absorbance at the SPR band (422 nm) on interaction with

Ag NPs as observed in the case of RF Therefore the participation of LC in

photoinduced electron transfer to Ag+ ions in the second phase of the reaction is

questionable

An alternative explanation of the biphasic formation of RFndashAg NPs may be

considered The absorption spectrum of RF during the formation of Ag NPs indicates a

rapid increase in absorbance with a shift in the maximum of RF from 444 nm to 440 nm

(~ 30 min) The rapid absorbance changes during this period represent the first phase of

the kinetic plot (Fig 911) indicating the photoinduced electron transfer from RF to Ag+

ions to form RFndashAg NPs The reaction further goes on with an increase in absorbance

and the gradual shift of the maximum to the SPR band at 422 nm This absorbance

increase is slow and is almost constant at around 6 h irradiation This phase may indicate

the adsorption of the RF on Ag surface The adsorption process may be prolonged due to

the formation of RF multilayers on Ag surface through the involvement of C=O and NndashH

groups of the uracil ring of RF (Liu et al 2012) (Fig 912) IR peaks indicating the

adsorption of RF on the surface of AgFe3O4 have been observed (Akhond et al 2016)

The rate constants (k1 and k2) for these reactions at pH 80ndash105 and in the

presence of various concentrations of Ag+ ions (0002ndash001 mM) are reported in Table

91 and 92

Fig 911 A plot of log absorbance versus time for the formation of RF-Ag NPs

0 50 100 150 200 250 300 350 400

Time (min)

Fig 912 A scheme for the formation of Ag NPs (first phase) and the adsorption of

RF on the surface of Ag NPs (second phase)

Table 91 First-order Constants for the Photoinduced Electron Transfer Reaction

of RF and Ag+ ions (k1) and Adsorption of RF at Ag NPs Surface (k2) in UV and

UV light Visible light

k1times 103

k2 times 103

(min-1

k1times 103

(min-1

k2 times 103

(min-1

80 736 0060 521 0036

85 999 0088 843 0061

90 1285 0110 1122 0091

95 1523 0129 1324 0112

100 1740 0147 1480 0128

105 1822 0153 1524 0135

of RF and Ag+ ions (k1) and Adsorption of RF at Ag NPs (k2) in UV and visible light

ion Concentration

k1times 103

k2 times 103

(min-1

k1times 103

(min-1

k2 times 103

(min-1

0002 754 0012 355 0009

0004 892 0022 622 0020

0006 1279 0046 1088 0043

0008 1630 0072 1399 0052

001 1740 0079 1444 0054

The values of k1 and k2 indicate an increase as a function of pH (Fig 913 and

914) This is probably due to an increase in the OHndash ion concentration which is an

initiator in this reaction and thus facilitates the formation of RFndashAg NPs The plots of k1

and k2 versus Ag+ ions concentration are shown in Fig 915 and 916 which indicate that

there is a significant effect of Ag+ ions on the formation of RFndashAg NPs

The kinetic data indicate that the values of k1 and k2 for the formation of RFndashAg

NPs in the presence of UV light are greater than those obtained under visible light

(Table 91) This is due to the fact that UV light has a greater energy compare to that of

the visible light and hence causes a greater effect on the interaction of RF and Ag+ ions to

form the RFndashAg NPs conjugates

Biphasic reactions have been found to occur in chemical and photochemical

systems and may involve the formation of an intermediate species that controls the rate

determining step (Ahmad and Tollin 1981) Some examples of biphasic reactions include

the hydrolysis of pndashnitrophenyl acetate (AhmedndashOmer et al 2008) biphasic process for

the synthesis of clofibric acid and analogues (Bose et al 2005) biphasic conversion of

hydrophobic substrates by amine dehydrofuran (Au et al 2014) biphasic photolysis of

riboflavin (Sato et al 1984) and multiexponential decay kinetics of primary radical pair

in photosystem 2 reaction centers (Booth et al 1991)

924 Mode of Photochemical Interaction of RF and Ag+ Ions

A scheme for the photochemical interaction of RF and Ag+ ions to form Ag NPs

and photoproducts of RF is presented involving the following reactions

Fig 913 Plots of k1 () (left hand side) and k2 () (right hand side) versus pH for

the formation of RF-Ag NPs in UV light

750 800 850 900 950 1000 1050 1100

k1times

k2 times

the formation of RF-Ag NPs in visible light

750 800 850 900 950 1000 1050 1100

k1times

k2 times

Fig 915 Plots k1 () (left hand side) and k2 ()(right hand side) versus Ag+ ion

concentrations (mM) for the formation of RF-Ag NPs in UV light

0 0002 0004 0006 0008 001 0012

Ag+ ions concentration (mM)

k1times

k2 times

concentrations (mM) for the formation of RF-Ag NPs in visible light

0 0002 0004 0006 0008 001 0012

k1times

k2 times

RF1RFhv

3RFisc1RF

RF3RF + Ag+

+ Ag NP

0RFRF + H+

RF3RF + 0RF + RFH

0RF + RFH22RFH (cyclic intermediate)

+ side-chain productsRFH2

+ side-chain productsFMF LCH+ OH-

According to this scheme RF on light absorption is promoted to the excited

singlet state [1RF] (Eq (91)) and is then transformed to the excited triplet state [

(Eq (92)) by intersystem crossing (isc) The 3RF reacts with an Ag

+ ion to form RF

radical [RF] (Eq (93)) by excited state electron transfer to Ag

+ ions resulting in the

reduction of Ag+ ions to Ag NPs as observed by Noguchi et al (2011) in the case of the

photoreduction of Cu+ ions by RF to form Cu NPs The Ag

+ ions can also be reduced by

the excited triplet state of acetone (Mening et al 1992 Giuffrida et al 2004) The RF

radical [RF] accepts a proton and is converted to RF ground state [

0RF] Further

reactions of the photolysis of RF have been described previously (Heelis 1981 1982

Ahmad and Vaid 2006 Ahmad et al 2005 2013 2015) and are as follows

The RF triplet [3RF] on reaction with a ground state RF molecule [

0RF] lead to

the formation of an oxidized [RF] and a reduced semiquinone [RFH

] radical (Eq (95)

The RFH Radicals may react to yield a ground state

0RF molecule and a reduced cyclic

intermediate product [RFH2] (Eq (96) RFH2 is oxidized to give FMF and the sidendashchain

products of RF (Eq (97) FMF then undergoes acidbase hydrolysis to form LC LF and

sidendashchain products (Song et al 1965 Ahmad et al 1980) Thus the main role of RF in

the photochemical interaction with Ag+ ions is the photoinduced electron transfer to form

Ag NPs and its subsequent photodegradation to yield a number of products

CONCLUSIONS

The main conclusions of the present study the effect of various factors on the

photolysis of riboflavin (RF) in aqueousorganic solvents are as follows

1 Photoprodcuts of RF

The TLC studies have shown that RF photolysis in aqueous solution (pH 70)

leads to the formation of formylmethylflavin (FMF) lumichrome (LC) lumiflavin (LF)

and carboxymethylflavin (CMF) by photoredution pathway and cyclodehydroriboflavin

(CDRF) by photoaddition pathway CDRF is only formed in the presence of HPO42-

at a concentration exceeding 02 M above pH 60 FMF LC and CMF are only formed in

organic solvents at a rate slower than that of water All the above mentioned

photoproducts are formed in the presence of different divalent and trivalent metal ions at

a rate greater than that observed in the absence of metal ions as indicated by the

fluorescence intensity of the spots of these photoproducts

A multicomponent spectrometric method has been found to be most appropriate

for the assay of RF and its different photoproducts It involves the adjustment of the pH

of photolyzed solution to 20 and extracted of LC and LF by chloroform and their

determination (after evaporation and dissolution of the residue in pH 45 acetate buffer)

by two-component assay at 356 and 445 nm The aqueous phase is used for the

determination of RF and FMF as a two-component assay at 385 and 445 nm or of RF

FMF and CDRF as a three-component assay at 385 410 and 445 nm respectively The

method in all can determine 4 to 5 components in a photolyzed solution with a precision

of plusmn 5 This method gives a good molar balance of RF and photoproducts in photolysis

reactions and has previously be applied to the photolysis of RF in aqueous and organic

solvents

3 Kinetics of Photolysis of RF

RF undergoes photolysis by an apparent first-order kinetics to form FMF as an

intermediate and is further degraded to LC and LF by intramolecular photoreduction

pathway In the presence of HPO42-

ions (gt 02 M) RF is photolyzed by intramolecular

photoaddition pathway to form CDRF Both the intramolecular photoredcution and

photoaddition reactions occur simultaneously in the presence of various concentrations of

HPO42-

ions with a change in the rate to form the two major photoproducts LC and

CDRF by different pathways The kinetics of photolysis of RF is affected by the ionic

strength of the buffer The results imply the participation of a charged species in the rate

determining step of the reaction The Cl- ions may react with RF in the excited singlet

state to accelerate the photolysis of RF The log kobs against radicmicro1 + radicmicro and log kko

against radicmicro plots for the photolysis reactions are linear

4 Solvent Effect on RF Photolysis

The photolysis of RF is affected by the solvent characteristics and this may be

utilized for the photostablization of RF The photolysis of RF is a function of solvent

dielectric constant and the rate of photolysis has been found to increase with an increase

in solvent polarity Thus a decrease in solvent dielectric constant would tend to stabilize

RF The photolysis of RF has also been found to be affected by the viscosity of the

medium Thus an increase in solvent viscosity would lead to stabilization of RF The use

of appropriate cosolvents with water would be a best choice to achieve greater

stabilization of RF and similar drugs

5 Metal Ion Effect on RF Photolysis

The effect of a number of monovalent divalent and trivalent metal ions (Ag+

) has been studied on the

photolysis of RF at low (0001 M) and high (02-04 M) phosphate buffer concentration

Spectral and fluorescence measurements of RF solutions in the presence of metal ions

have shown a change in UV and visible spectra and loss of RF fluorescence indicating

the formation of RF-metal complexes The divalent and trivalent metal ions have been

found to accelerate the photolysis of RF whereas the monovalent ions (Ag+) inhibit the

photolysis of RF The acceleration in the rate of photolysis is probably due to electron

transfer from the metal ion to RF in the excited singlet state resulting in the

photoreduction and degradation of RF A relation has been observed between the values

of kobs for the photolysis of RF and the respective loss of fluorescence as an indication of

the degree of RF-metal complexation

6 Photochemical Preparation Characterization and Formation Kinetics of RF-Ag

The photoreduction of Ag+ ions in the presence of RF leads to the formation of

RFndashAg NPs These NPs exhibits a specific SPR band at 422 nm in the visible spectrum

and a strong band at 2900 cmndash1

in the FTIR spectrum due to interaction of RF and Ag

NPs The degree of fluorescence quenching of RF by Ag NPs indicates the magnitude of

the formation of RFndashAg NPs conjugates DLS studies have shown the Hd of these NPs in

the 579ndash722 nm range The mean autocorrelation function has confirmed that these NPs

are in the nanoscale range with a decay time of 10ndash3

s The pH and ionic strength have

been found to affect the particle size of RFndashAg NPs An increase in the particle size in

acidic medium leads to aggregation of NPs as well as broadening of the SPR band The

formation of RFndashAg NPs involves the initial conversion of Ag+ ions of Ag NPs by a fast

firstndashorder reaction and subsequently the interaction of RF and Ag NPs to form RFndashAg

NPs conjugates by a slow firstndashorder reaction An increase in pH leads to an increase in

the formation of RFndashAg NPs In UV light the formation of RF-Ag NPs is greater than

that observed in visible light

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AbdelndashHafez SI Nafady NA AbdelndashRahim IR Shaltout AM Mohamed MA

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Ahmad I Abbas SH Anwar Z Sheraz MA Ahmed S Arsalan A Bano R Stabilityndash

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Ahmad I Ahmed S Anwar Z Sheraz MA Sikorski M Photostability and

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Ahmad I Ahmed S Sheraz MA Vaid FH Ansari IA Effect of divalent anions on

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Ahmad I Ahmed S Sheraz MA Vaid FH Effect of borate buffer on the photolysis of

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Ahmad I Ahmed S Sheraz MA Vaid FHM Ansari IA Effect of divalent anions on

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Ahmad I Anwar Z Ahmed S Sheraz MA Bano R Hafeez A Solvent effect on the

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Ahmad I Anwar Z Ali SA Hasan KA Sheraz MA Ahmed S Ionic strength effects on

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Photobiol B Biol 2016157113ndash119

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Effect of acetate and carbonate buffers on the photolysis of riboflavin in

aqueous solution a kinetic study AAPS PharmSciTech 201415550ndash559

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study AAPS PharmSciTech 2014151588ndash1597

Ahmad I Bano R Musharraf SG Sheraz MA Ahmed S Tahir H ul Arfeen Q Bhatti

MS Shad Z Hussain SF Photodegradation of norfloxacin in aqueous and

organic solvents a kinetic study J Photochem Photobiol A Chem 20153021ndash

Ahmad I Bano R Sheraz MA Ahmed S Mirza T Ansari SA Photodegradation of

levofloxacin in aqueous and organic solvents a kinetic study Acta Pharm

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Ahmad I Beg AE Zoha SM Studies on degradation of riboflavin and related

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Ahmad I Cusanovich MA Tollin G Laser flash photolysis studies of electron transfer

between semiquinone and fully reduced free flavins and horse heart cytochrome

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cytochrome oxidase complex Biochemistry 1982213122ndash3128

Ahmad I Fasihullah Q Noor A Ansari IA Ali QN Photolysis of riboflavin in aqueous

solution A kinetic study Int J Pharm 2004a280199ndash208

Ahmad I Fasihullah Q Vaid FH A study of simultaneous photolysis and

photoaddition reactions of riboflavin in aqueous solution J Photochem

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Ahmad I Fasihullah Q Vaid FH Effect of phosphate buffer on photodegradation

reactions of riboflavin in aqueous solution J Photochem Photobiol B Biol

2005 78229ndash234

Ahmad I Fasihullah Q Vaid FHM Effect of light intensity and wavelengths on

photodegradation reactions of riboflavin in aqueous solution J Photochem

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Ahmad I Fasihullah Q Vaid FHM Photolysis of formylmethylflavin in aqueous and

organic solvents Photochem Photobiol Sci 2006b5680ndash685

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Ahmad I Fasihullah Q Effect of solvent on UV and visible spectra of

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viscosity on the photolysis of formylmethylflavin a kinetic study Aust J Chem

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and viscosity on the photolysis of formylmethylflavin A kinetic study Aust J

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Ahmad I Tollin G Solvent effect on flavin electron transfer reactions Biochemistry

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Ahmad I Vaid FHM Ahmed S Sheraz MA Hasan S Advances in biochemical

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Ajayi OA George BO Ipadeola T Clinical trial of riboflavin in sickle cell disease East

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Chen P Song L Liu Y Fang YE Synthesis of silver nanoparticles by γndashray irradiation

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Handbook for Pharmacist 2nd ed Wiley New York 1986 pp 38ndash41 99ndash100

Constable EC Homoleptic complexes of 2 2primendashbipyridine Adv Inorg Chem 1989341ndash

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Garcı NA Kinetic study of the riboflavinndashsensitised photooxygenation of two

hydroxyquinolines of biological interest J Photochem Photobiol B Biol

20037119ndash25

Dang MC Dang TM FribourgndashBlanc E Silver nanoparticles ink synthesis for

conductive patterns fabrication using inkjet printing technology Adv Nat Sci

Nanosci Nanotechnol 20146015003

Das BS Das DB Satpathy RN Patnaik JK Bose TK Riboflavin deficiency and

severity of malaria Eur J Clin Nutr 198842277ndash283

Das SK Khan MM Guha AK Das AR Mandal AB Silverndashnano biohybride material

synthesis characterization and application in water purification Bioresour

de Jesus MB Fraceto LF Martini MF Pickholz M Ferreira CV de Paula E

Non‐inclusion complexes between riboflavin and cyclodextrins J Pharm

De Moura MR Mattoso LH Zucolotto V Development of cellulosendashbased bactericidal

nanocomposites containing silver nanoparticles and their use as active food

packaging J Food Eng 2012109520ndash524

Delgado JN Remers WA Wilson and Gisvoldlsquos Textbook of Organic Medicinal and

Pharmaceutical Chemistry 10th ed LippincottndashRaven Philadelphia 2004 pp

899ndash 901 915

Deritter E Vitamins in pharmaceutical formulations J Pharm Sci 1982711073ndash1096

Dias DA Smith TA Ghiggino KP Scollary GR The role of light temperature and

wine bottle colour on pigment enhancement in white wine Food Chem

20121352934ndash2941

Dollery C Therapeutic Drugs Vol 2 Churchill Livingstone London 1999 pp R24ndash

Drexler KE Engines of creation the coming era of nanotechnology Anchor Press

Doubleday New York 1986

Drexler KE Nanosystems molecular machinery manufacturing and computation John

Wiley amp Sons New York 1992

Dutta P Seirafi J Halpin D Pinto J Rivlin R Acute ethanol exposure alters hepatic

glutathione metabolism in riboflavin deficiency Alcohol 19951243ndash47

Dutta P Disturbances in glutathione metabolism and resistance to malaria current

understanding and new concepts J Soc Pharm Chem 19932311ndash15

Dyke SF The Chemistry of Vitamins Interscience London 1965 Chap 3

Ebbesen F Madsen P Stoslashvring S Hundborg H Agati G Therapeutic effect of

turquoise versus blue light with equal irradiance in preterm infants with

jaundice Acta Paediatrica 200796837ndash841

Economou A Fielden PR Square wave adsorptive stripping voltammetry on mercury

film electrodes Anal Chim Acta 199327327ndash34

Eitenmiller RR Ye L Landen WO Jr Vitamin Analysis for the Health and Food

Sciences 2nd ed CRC Press Boca Raton FL 2008 Chap 7

Ellinger P Holden M Quenching effect of electrolytes on the fluorescence intensity of

riboflavin and thiochrome Biochem J 194438147ndash150

Emmett AD Luros GO Waterndashsoluble vitamins I Are the antineuritic and the

growthndashpromoting waterndashsoluble B vitamins the Same J Biol Chem

192043265ndash287

Enns K Burgess WH The photochemical oxidation of ethylenediaminetetraacetic acid

and methionine by ribolflavin J Am Chem Soc 1965875766ndash5770

Evstigneev MP Rozvadovskaya AO Hernandez Santiago AA Mukhina YV Veselkov

KA Rogova OV Davies DB Veselkov AN A 1H NMR study of the

association of caffeine with flavin mononucleotide in aqueous solutions Russian

J Phys Chem 200579573ndash578

Fall HH Petering HG Metabolic inhibitors 1 67-Dimethyl-9-

formylmethylisoalloxazine 67-dimethyl-9-(12-hydroxyethyl)-isoalloxazine and

derivatives J Am Chem Soc 195678377ndash381

Farjadnia M Naderan M Corneal crossndashlinking treatment of keratoconus Oman J

Ophthalmol 2015886

Farokhzad OC Langer R Impact of nanotechnology on drug delivery ACS Nano

2009316ndash20

Fernandez CS Amarasena T Kelleher AD Rossjohn J McCluskey J Godfrey DI Kent

SJ MAIT cells are depleted early but retain functional cytokine expression in

HIV infection Immunol Cell Biol 201593177ndash188

Ferrari M Cancer nanotechnology opportunities and challenges Nat Rev

Cancer20055161ndash71

Figueiredo JC Levine AJ Grau MV Midttun Oslash Ueland PM Ahnen DJ Barry EL

Tsang S Munroe D Ali I Haile RW Vitamins B2 B6 and B12 and risk of new

colorectal adenomas in a randomized trial of aspirin use and folic acid

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Florence AT Attwood D Physicochemical Principles of Pharmacy 4th ed

Pharmaceutical Press London 2006 pp 120ndash122

Foraker AB Khantwal CM Swaan PW Current perspectives on the cellular uptake and

trafficking of riboflavin Adv Drug Deliv Rev 2003551467ndash1483

Foresti ML Vaacutezquez A Boury B Applications of bacterial cellulose as precursor of

carbon and composites with metal oxide metal sulfide and metal nanoparticles

A review of recent advances Carbohydr Polym 2017157447ndash467

Fox JL Researchers discuss NIHs nanotechnology initiative Nature Biotechnol

200018821ndash825

Frattini A Pellegri N Nicastro D Sanctis O Preparation of amine coated silver

nanoparticles using triethylenetetraamine Mater Chem Phys 200594148ndash152

French RA Jacobson AR Kim B Isley SL Penn RL Baveye PC Influence of ionic

strength pH and cation valence on aggregation kinetics of titanium dioxide

nanoparticles Environ Sci Technol 2009431354ndash1359

Fritz BJ Kasai S Matsui K Photochemical properties of flavin derivatives Photochem

Photobiol 198745113ndash117

Frost A Pearson RG Kinetics and Mechanism 2nd Ed John Wiley New York 1964

pp 150ndash155 160ndash162

Fuguitt RE Hawkins JE Rate of the thermal isomerization of αndashpinene in the liquid

phase J Am Chem Soc 194769319ndash322

Fukamachi C Sakurai Y The photolytic formation of 6 7ndashdimethylflavinndash9ndashacetic

acid from riboflavin J Vitaminol 19551217ndash220

Fukumachi C Sakurai Y Vitamin B2 photolysis V The photolytic formation of 6 7-

dimethylflavin-9-acetic acid ester from riboflavin Vitamins (Kyoto)

19547939ndash943

Fukuzumi S Kojima T Control of redox reactivity of flavin and pterin coenzymes by

metal ion coordination and hydrogen bonding J Biol Inorg Chem 200813321ndash

Fukuzumi S Kuroda S Tanaka T Flavin analoguendashmetal ion complexes acting as

efficient photocatalysts in the oxidation of pndashmethylbenzyl alcohol by oxygen

under irradiation with visible light J Am Chem Soc 19851073020ndash3027

Fukuzumi S Ohkubo K Metal ionndashcoupled and decoupled electron transfer Coord

Chem Rev 2010254372ndash385

Fukuzumi S Tanaka T Flavins and deazaflavins In Fox MA Chanon M Eds

Photoinduced Electron Transfer Part C Elsevier Amsterdam 1988 pp 636ndash

Futterman S Rollins MH The catalytic isomerization of allndashtransndashretinal to 9ndashcisndash

retinal and 13ndashcisndashretinal J Biol Chem 19732487773ndash7779

Ganji V Kafai MR Frequent consumption of milk yogurt cold breakfast cereals

peppers and cruciferous vegetables and intakes of dietary folate and riboflavin

but not vitamins Bndash12 and Bndash6 are inversely associated with serum total

homocysteine concentrations in the US population Am J Clin Nutr

2004801500ndash1507

Garcia L Blazquez S SanndashAndres MP Vera SC Determination of thiamin riboflavin

and pyridoxine in pharmaceuticals by synchronous fluorescence spectrometry in

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Garland WT Fritchie CJ Metalloflavoprotein models the crystal structure of bis

(riboflavin) bis (cupric perchlorate) dodecahydrate J Biol Chem

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Garrett ER Kinetics and mechanisms in stability of drugs In Bean HS Beckett AH

Carless JE Eds Advances in Pharmaceutical Sciences Vol 2 Academic Press

London 1967 pp 57ndash58

Gaul C Diener HC Danesch U Improvement of migraine symptoms with a proprietary

supplement containing riboflavin magnesium and Q10 a randomized placebondash

controlled doublendashblind multicenter trial J Headache Pain 20151632

Ghanem AH Hassan ES Hamdi AA Stability of indomethacin solubilized system

Pharmazie 197934406ndash407

Ghasemi J Abbasi B Niazi A Nadaf E Mordai A Simultaneous spectrophotometric

multicomponent determination of folic acid thiamine riboflavin and pyridoxal

by using double divisorndashratio spectra derivativendashzero crossing method Anal lett

2004372609ndash2623

Ghasemi J Abbasi B Simultaneous spectrophotometric determination of group B

vitamins using parallel factor analysis PARAFAC J Chin Chem Soc

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Ghasemi J Vosough M Simultaneous spectrophotometric determination of folic acid

thiamin riboflavin and pyridoxal using partial leastndashsquares regression method

Spectrosc lett 200235153ndash169

Giuffrida S Condorelli GG Costanzo LL Fragalagrave IL Ventimiglia G Vecchio G

Photochemical mechanism of the formation of nanometerndashsized copper by UV

irradiation of ethanol bis (2 4ndashpentandionato) copper (II) solutions Chem

Mater 2004161260ndash1266

Gladys M Knappe WR Photochemie des (Iso) Alloxazins III Intramolekulare

Photodealkylierung von 10‐Alkylisoalloxazinen eine Modellreaktion fuumlr den

Riboflavin‐Photoabbau Chem Ber 19741073658ndash3673

Goodrich RP Edrich RA Li J Seghatchian J The Mirasoltrade PRT system for pathogen

reduction of platelets and plasma an overview of current status and future

trends Transfus Apher Sci 2006355ndash17

Goodsell DS Bionanotechnology lessons from nature John Wiley amp Sons USA 2004

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homocysteine as a risk factor for vascular disease The European Concerted

Action Project JAMA 19972771775ndash1781

Greulich S Kaim W Stange AF Stoll H Fiedler J Zališ S Cp Ir (dab)(dab= 1 4ndashBis

(2 6ndashdimethylphenyl)ndash1 4ndashdiazabutadiene) A coordinatively unsaturated sixndash

πndashelectron metallaheteroaromatic compound Inorg Chem 1996353998ndash

Gu HY Yu AM Chen HY Electrochemical behavior and simultaneous determination

of vitamin B2 B6 and C at electrochemically pretreated glassy carbon

electrode Anal lett 2001342361ndash2374

Guillory JK Poust RI Chemical kinetics and drug stability In Banker GS Rhodes CT

Eds Modern Pharmaceutics 4th ed Marcel Dekker New York 2002 pp

158ndash159

Guo J Lu Y Dong H HPLCndashMS analysis of the riboflavin crude product of

semisynthesis J Chromatogr Sci 200644552ndash556

Gutieacuterrez MI Fernaacutendez SM Massad WA Garciacutea NA Kinetic study on the

photostability of riboflavin in the presence of barbituric acid Redox Report

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fluorescence experiment for physical chemistry J Chem Educ 200582302ndash

Gwinn MR Vallyathan V Nanoparticles health effectsndashndashpros and cons Environ

Health Perspect 20061141818ndash1825

Habib MJ Asker AF Photostabilization of riboflavin by incorporation into liposomes J

Parenter Sci Technol 199145124ndash127

Haes AJ Van Duyne RP A nanoscale optical biosensor sensitivity and selectivity of an

approach based on the localized surface plasmon resonance spectroscopy of

triangular silver nanoparticles J Am Chem Soc 200212410596ndash10560

Haggi E Bertolotti S Garcıa NA Modelling the environmental degradation of water

contaminants Kinetics and mechanism of the riboflavinndashsensitisedndash

photooxidation of phenolic compounds Chemosphere 2004551501ndash1507

Halwer M The photochemistry of riboflavin and related compounds J Am Chem

Soc1951734870ndash4874

Hameed A Ali SA Khan AA Moin ST Khan KM Hashim J Basha FZ Malik MI

Solventndashfree click chemistry for tetrazole synthesis from 1 8ndashdiazabicyclo [54

0] undecndash7ndashene (DBU)ndashBased fluorinated ionic liquids their micellization and

density functional theory studies RSC Adv 2014464128ndash64137

Hashmi MH Assay of vitamins in pharmaceutical preparations Wiley New York

USA 1973 Chap 3

Hatchard CG Parker CA A new sensitive chemical actinometer II Potassium

ferrioxalate as a standard chemical actinometer Proc Roy Soc (Lond)

1956A235518ndash536

ferrioxalate as a standard chemical actinometer Proc Nat Acad Sci

1956235518ndash536

Hazzard JT Moench SJ Erman JE Satterlee JD Tollin G Kinetics of intracomplex

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noncovalent complexes of cytochrome c and cytochrome c peroxidase by free

flavin semiquinones Biochemistry 1988272002ndash2008

Hazzard JT Poulos TL Tollin G Kinetics of reduction by free flavin semiquinones of

the components of the cytochrome cndashcytochrome c peroxidase complex and

intracomplex electron transfer Biochemistry 1987262836ndash2848

Heelis PF Parsons BJ Phillips GO McKellar JF The flavin sensitised photooxidation

of ascorbic acid a continuous and flash photolysis study Photochem Photobiol

1981337ndash13

Heelis PF Phillips GO Ahmad I Rapson HDC The photodegradation of

formylmethylflavin A steady state and laser flash photolysis study

Photobiochem Photobiophys 19801125ndash130

Heelis PF The photochemistry of flavins In Muller F Ed Chemistry and

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171ndash 193

Heelis PF The photophysical and photochemical properties of flavins (isoalloxazines)

Chem Soc Rev 19821115ndash39

Heitele H Dynamic solvent effects on electron transfer reactions Angew Chem Int Ed

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Hemmerich P Veeger C Wood HC Progress in the chemistry and molecular biology of

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Hemmerich P The present status of flavin and flavocoenzyme chemistry In Fortschritte

der Chemie Organischer NaturstoffeProgress in the Chemistry of Organic

Natural Products Springer Vienna 1976pp 451ndash527

Hemmerich P The present status of flavin and flavoenzyme chemistry Fortschr Chem

Org Naturst 197633451ndash527

Henriquez MA Izquierdo Jr L Bernilla C Zakrzewski PA Mannis M

RiboflavinUltraviolet A corneal collagen crossndashlinking for the treatment of

keratoconus visual outcomes and Scheimpflug analysis Cornea 201130281ndash

Hiraku Y Ito K Hirakawa K Kawanishi S Photosensitized DNA damage and its

protection via a novel mechanism Photochem Photobiol 200783205ndash212

Hoffman-La Roche F Analytical Procedures for the Determination of Vitamins in

Multivitamin Preparations Hoffman-La Roche Basle 1970 pp 69ndash70

Hoitink MA Beijnen JH Bult A van der Houwen OA Nijholt J Underberg WJ

Degradation kinetics of gonadorelin in aqueous solution J Pharm Sci

1996851053ndash1059

Holmstrom B Oster G Riboflavin as an electron donor in photochemical reactions J

Am Chem Soc 1961831867ndash1871

Holmstrom B Mechanism of photoreduction of riboflavin Arkiv Kemi 1964a22329

Holmstrom B Spectral studies of the photobleaching of riboflavin phosphate Arkiv

Kemi 1964b 22281ndash301

Horikoshi S Serpone N Eds Microwaves in nanoparticle synthesis fundamentals and

applications John Wiley amp Sons USA 2013

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activities in relation to structure J Pharm Sci 196960503ndash532

Hou W Cronin SB A review of surface plasmon resonance‐enhanced photocatalysis

Adv Funct Mater 2013231612ndash1619

Hou W Wang E Liquid chromatography with series dualndashelectrode electrochemical

detection for riboflavin Analyst 1990115139ndash141

Huang R Choe E Min DB Kinetics for singlet oxygen formation by riboflavin

photosensitization and the reaction between riboflavin and singlet oxygen J

Food Sci 200469C726ndashC732

Huang R Kim HJ Min DB Photosensitizing effect of riboflavin lumiflavin and

lumichrome on the generation of volatiles in soy milk J Agric Food Chem

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Huang X Xiao Y Zhang W Lang M Inndashsitu formation of silver nanoparticles

stabilized by amphiphilic starndashshaped copolymer and their catalytic application

Appl Surf Sci 20122582655ndash2660

Hurley JK Hazzard JT Martiacutenez‐Juacutelvez M Medina M Goacutemez‐Moreno C Tollin G

Electrostatic forces involved in orienting Anabaena ferredoxin during binding to

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kinetic measurements and electrostatic surface potentials Protein Sci

199981614ndash1622

Hussain A Truelove J Effect of hydroxyl group substituents on pyran ring on

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197965235ndash266

Hussain E Fatima RA Ali IAF Naseem I Photoilluminated riboflavinriboflavinndashCu

(II) inactivates trypsin Cu (II) tilts the balance Indian J Biochem Biophys

200643312ndash318

Hussain JI Kumar S Hashmi AA Khan Z Silver nanoparticles preparation

characterization and kinetics Adv Mater Lett 20112188ndash194

Hussain W Effect of pH on the Photostability of Cyanocobalamin M Pharm thesis

University of Karachi 1987

Hustad S Ueland PM Schneede J Quantification of riboflavin flavin mononucleotide

and flavin adenine dinucleotide in human plasma by capillary electrophoresis

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Ikeda K Studies on decomposition and stabilization of drugs in solution IV Effect of

dielectric constant on the stabilization of barbiturate in alcohol-water mixtures

Chem Pharm Bull19608504ndash509

Ilić V Šaponjić Z Vodnik V Potkonjak B Jovančić P Nedeljković J Radetić M The

influence of silver content on antimicrobial activity and color of cotton fabrics

functionalized with Ag nanoparticles Carbohydr Polym 200978564ndash569

Insinska-Rak M Golczak A Sikorski M Photochemistry of riboflavin derivatives in

methanolic solutions J Phys Chem 20121161199ndash1207

Insinska-Rak M Sikorski M Riboflavin interactions with oxygen survey from the

photochemical perspective Chem Eur J 20142015280ndash15291

Ionita MA Ion RM Carstocea B Photochemical and photodynamic properties of

vitamin B2ndashriboflavin in liposomes Oftalmologia 20035531ndash36

Ioniţă MA Ion RM Carstocea B Photochemical and photodynamic properties of

vitamin B2ndashriboflavin and liposomes Oftalmologia 20025829ndash34

Isaka S Ishida S Photochemistry of riboflavin II Effect of divalent metallic ions upon

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1953190ndash94

Isaka S Photochemistry of riboflavin III Photondashoxidative activity of flavins and their

behavior in relation of cupric ions J Coll Arts Chiba Univ 19551263ndash266

Jabbar MA Salahuddin S Mannan RJ Mahmood AJ Electrochemical evidences for

the enhancement of heterogeneous electron transfer rates of riboflavin in the

presence of copper Dhaka Univ J Sci 201562147ndash152

Jiang J Chen DndashR Biswas P Synthesis of nanoparticles in a flame aerosol reactor with

independent and strict control of their size crystal phase and morphology

Nanotechnol 2007181ndash8

Johannsen M Gineveckow U Eckelt LFeussner A Waldofner N Scholz R Degar S

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magnetic nanoparticles presentation of a new interstitial technique Int J

Hyperthermia 200521637ndash647

Jortner J Rao CNR Nanostructures advanced materials Perspectives and directions

Pure Appl Chem 2002741491ndash1506

Jumaa M Carlson B Chimilio L Silchenko S Stella VJ Kinetics and mechanism of

degradation of epothilone‐D An experimental anticancer agent J Pharm Sci

2004932953ndash2961

Jung MY Kim SK Kim SY Riboflavinndashsensitized photooxidation of ascorbic acid

kinetics and amino acid effects Food Chem 199553397ndash403

Jung MY Oh YS Kim DK Kim HJ Min DB Photoinduced generation of 23ndash

butanedione from riboflavin J Agric Food Chem 200755170ndash174

Junqing Z Spectrofluorometric Determination of riboflavin in tablets of vitamin B_2

Nat Sci J Hainan Uni 19974014

Juris A Balzani V Barigelletti F Campagna S Belser PL Von Zelewsky A Ru (II)

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chemiluminescence Coord Chem Rev 19888485ndash277

Jusko WJ Levy G Absorption Protein binding and elimination of riboflavin In

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Kaikai P Thurnham DI The influence of riboflavin deficiency on Plasmodium berghei

infection in rats Transactions of the Royal Society of Tropical Medicine and

Hygiene 198377680ndash686

Kaim W Schwederski B Heilmann O Hornung FM Coordination compounds of

pteridine alloxazine and flavin ligands structures and properties Coord Chem

Rev 1999182323ndash342

Kamran S Asadi M Absalan G Adsorption of folic acid riboflavin and ascorbic acid

from aqueous samples by Fe3O4 magnetic nanoparticles using ionic liquid as

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Karrer P Schopp K Benz F Synthesis of flavins IV Helv Chim Acta 193518426ndash

Kelly KL Coronado E Zhao LL Schatz GC The optical properties of metal

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Kemlo JA Shepherd TM Quenching of excited singlet states by metal ions Chem

Phys Lett 197747158ndash162

Khattak SU Shaikh D Ahmad I Usmanghani K Sheraz MA Ahmed S

Photodegradation and stabilization of betamethasonendash17 valerate in

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201314177ndash182

Khaydukov EV Mironova KE Semchishen VA Generalova AN Nechaev AV

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Panchenko VY Riboflavin photoactivation by upconversion nanoparticles for

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Kim MJ Kim HJ Kim JM Kim B Han SH Cha GS Homogeneous assays for

riboflavin mediated by the interaction between enzymendashbiotin and avidinndash

riboflavin conjugates Anal Biochem 1995231400ndash406

Kim SW Jung JH Lamsal K Kim YS Min JS Lee YS Antifungal effects of silver

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20124053ndash58

Kim TY Kim DW Chung JY Shin SG Kim SC Heo DS Kim NK Bang YJ Phase I

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Cancer Res 2004103708ndash3716

Kim W Wang R Majumdar A Nanostructuring expands thermal limits Nano Today

2007240ndash47

King JM Min DB Riboflavin photosensitized singlet oxygen oxidation of vitamin D J

Food Sci 19986331ndash34

Knappe WR Hemmerich P Covalent intermediates in flavinndashsensitized

photodehydrogenation and photodecarboxylation Zeitschrift fuumlr

Naturforschung B 1972271032ndash1035

Knappe WR Hemmerich P Reduktive photoalkylierung des flavinkerns struktur und

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Knappe WR Photochemistry of (iso)alloxazines IV Dealkylation and decarboxylation

of shortndashchained isoalloxazinendash10ndashalkanoic acids Ber Dtsch Chem Ges

19751082422ndash2432

Knobloch E Hodr R Janda J Herzmann J Houdkova V Sectrofluorimetric

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Koppenol WH Effect of a molecular dipole on the ionic strength dependence of a

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Kosaka Y Egami K Tomita S Yanagi H Plasmonic circular dichroism using Au fine

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Kostenbauder HB DeLuca PP Kowarski CK Photobinding and photoreactivity of

riboflavin in the presence of macromolecules J Pharm Sci 1965541243ndash

Kozioł J Knobloch E The solvent effect on the fluorescence and light absorption of

riboflavin and lumiflavin Biochim Biophys Acta 1965102289ndash300

Koziol J Studies on flavins in organic solvents‐i spectral characteristics of riboflavin

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in the presence of oxygen Photochem Photobiol 1966a555ndash62

Kreuter J On the mechanism of termination in heterogeneous polymerization J

Polymer Sci Polym Lett Ed 198220543ndash545

Krupa AN Abigail ME Santhosh C Grace AN Vimala R Optimization of process

parameters for the microbial synthesis of silver nanoparticles using 3ndashlevel

BoxndashBehnken Design Ecol Eng 201687168ndash174

Kuhn R Reinemund KD Kaltschmitt H Strobele R Trischmann H Synthetisches 67ndash

dimethylndash9ndashdndashriboflavin Naturwiss 193523260

Kuhn R Wagner‐Jauregg T Uumlber das Reduktions‐Oxydations‐Verhalten und eine

Farbreaktion des Lacto‐flavins (Vitamin B2) Eur J Inorg Chem 193467361ndash

Kumar DR Manoj D Santhanalakshmi J Electrostatic fabrication of oleylamine

capped nickel oxide nanoparticles anchored multiwall carbon nanotube

nanocomposite A robust electrochemical determination of riboflavin at

nanomolar levels Anal Methods 201461011ndash1020

Kumar V Lockerbie O Keil SD Ruane PH Platz MS Martin CB Ravanat JL Cadet

J Goodrich RP Riboflavin and UV-light based pathogen reduction extent and

consequence of DNA damage at the molecular level Photochem Photobiol

20048015ndash21

Kurtin WE Latino MA Song PS A study of photochemistry of flavins in pyridine and

with a donor Photochem Photobiol 19676247ndash59

Lachman L DeLuca P Akers MJ Kinetic principles and stability testing in Lachman

L Liberman HA Kanig JL Eds The Theory and Practice of Industrial

Pharmacy 3rd ed Lea amp Febiger Philadelphia 1986 pp 769ndash770

Laidler KJ Chemical kinetics 3rd ed Harper amp Row New York 1987 p 183ndash195

197ndash206 279-280

Lallemand F Perottet P FeltndashBaeyens O Kloeti W Philippoz F Marfurt J Besseghir

K Gurny R A waterndashsoluble prodrug of cyclosporine A for ocular application

a stability study Eur J Pharm Sci 200526124ndash129

Lavanya N Radhakrishnan S Sekar C Navaneethan M Hayakawa Y Fabrication of

Cr doped SnO2 nanoparticles based biosensor for the selective determination of

riboflavin in pharmaceuticals Analyst 20131382061ndash2067

Lee KC Lin SJ Lin CH Tsai CS Lu YJ Size effect of Ag nanoparticles on surface

plasmon resonance Surf Coat Technol 20082025339ndash5342

Leeansyah E Svaumlrd J Dias J Buggert M Nystroumlm J Quigley MF Moll M

Soumlnnerborg A Nowak P Sandberg JK Arming of MAIT cell cytolytic

antimicrobial activity is induced by ILndash7 and defective in HIVndash1 infection

PLoS Pathog 201511e1005072

Lei G Gao PF Yang T Zhou J Zhang HZ Sun SS Gao MX Huang CZ

Photoinduced electron transfer process visualized on single silver nanoparticles

ACS Nano 2017112085ndash2093

Li K Simultaneous determination of nicotinamide pyridoxine hydrochloride thiamine

mononitrate and riboflavin in multivitamin with minerals tablets by

reversed‐phase ion‐pair high performance liquid chromatography Biomed

Chromatogr 200216504ndash507

Li YQ Huang XZ Xu JG Synchronous spectrofluorimetry for simultaneous

determination of riboflavin and pyridoxine and its application in multivitamin

preparations Acta Pharmaceutica Sinica 19921

Liu F Gu H Lin Y Qi Y Dong X Gao J Cai T Surface-enhanced Raman scattering

study of riboflavin on borohydride-reduced silver colloids Dependence of

concentration halide anions and pH values Spectrochim Acta Part A Mol

Biomol Spectrosc 201285111-119

Liu F Gu H Lin Y Qi Y Dong X Gao J Cai T Surfacendashenhanced Raman scattering

study of riboflavin on borohydridendashreduced silver colloids Dependence of

concentration halide anions and pH values Spectrochim Acta Mol Biomol

Spectrosc 201285111ndash119

Liu Z Ren G Zhang T Yang Z Action potential changes associated with the

inhibitory effects on voltagendashgated sodium current of hippocampal CA1

neurons by silver nanoparticles Toxicol 2009264179ndash184

Loacutepez-Leytoacuten TL Yusty ML Pintildeeiro MA Constant-wavelength synchronous

spectrofluorimetry for determination of riboflavin in anchovies Fresenius

journal of analytical chemistry 1998362341ndash343

Loukas Y 2 (kndashp) Fractional factorial design via fold over application to optimization

of novel multicomponent vesicular systems Analyst 19971221023ndash1028

Loukas YL Jayasekera P Gregoriadis G Characterization and Photoprotection Studies

of a Model gammandashCyclodextrinndashIncluded Photolabile Drug Entrapped in

Liposomes Incorporating Light Absorbers J Phys Chem 1995a9911035ndash

Loukas YL Jayasekera P Gregoriadis G Novel liposomendashbased multicomponent

systems for the protection of photolabile agents Int J Pharm 1995b11785ndash94

Loukas YL Vraka V Gregoriadis G Use of a nonlinear leastndashsquares model for the

kinetic determination of the stability constant of cyclodextrin inclusion

complexes Int J Pharm 1996144225ndash231

Loukas YL A PlackettndashBurnam screening design directs the efficient formulation of

multicomponent DRV liposomes J Pharm Biomed Anal 200126255ndash263

Ma Q Song J Zhang S Wang M Guo Y Dong C Colorimetric detection of riboflavin

by silver nanoparticles capped with βndashcyclodextrinndashgrafted citrate Colloids

Surf B Biointerfaces 201614866ndash72

Maafi M Maafi W Modeling and elucidation of the kinetics of multiple consecutive

photoreactions AB4 (4Φ) with Φndashorder kinetics Application to the

photodegradation of riboflavin J Pharm Sci 20161053537ndash3548

Macek TJ Stability problems with some vitamins in pharmaceuticals Am J Pharm

1960 132433ndash455

Mao YP Tao XL Lipsky PE Analysis of the stability and degradation products

triptolide J Pharm Pharmcol 2000523ndash12

Marcus AD Taraszka AJ A kinetic study of the specific hydrogen ion catalyzed

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19594877ndash84

Martens HJ Stability of water soluble vitamins in various infusion bags

Krankenhauspharmazie 198910359ndash361

Massey V Palmer G Ballou D Oxidases and Related Redox Systems In King TE

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Matos C Chaimovich H Lima JL Cuccovia IM Reis S Effect of liposomes on the

rate of alkaline hydrolysis of indomethacin and acemetacin J Pharm Sci

200190298ndash309

Mattivi FU Monetti AM Vrhovšek U Tonon DI AndreacutesndashLacueva C Highndash

performance liquid chromatographic determination of the riboflavin

concentration in white wines for predicting their resistance to light J

Chromatogr A 2000888121ndash127

McBride MM Metzler DE Photochemical degradation of flavins III Hydroxymethyl

and formylmethyl analogs of riboflavin Photochem Photobiol 19676113ndash

McCormick DB Two interconnected B vitamins riboflavin and pyridoxine Physiol

Rev 1989691170ndash1198

McDowell LR Riboflavin Vitamins in Animal and Human Nutrition Iowa State

University Press Iowa USA 2000 pp 311ndash346

Mennig M Spanhel J Schmidt H Betzholz S Photoinduced formation of silver

colloids in a borosilicate solndashgel system J NonndashCryst Solids 1992147326ndash

Merrill Jr AH Edmondson DE McCormick DB Formation and mode of action of

flavoproteins Annu Rev Nutr 19811281ndash317

Meyer TE Watkins JA Przysiecki CT Tollin G Cusanovich MA Electronndashtransfer

reactions of photoreduced flavin analogues with cndashtype cytochromes

quantitation of steric and electrostatic factors Biochemistry 1984234761ndash

Mielech K Simultaneous voltammetric determination of riboflavin and Lndashascorbic acid

in multivitamin pharmaceutical preparations J Trace Microprobe Tech

200321111ndash121

Min DB Boff JM Chemistry and reaction of singlet oxygen in foods Compr Rev Food

Sci Food Saf 2002158ndash72

Miranda A Caraballo I Millan M Stability study of flutamide in solid state and in

aqueous solution Drug Dev Ind Pharm 200228413ndash422

Miyazawa T Sato C Kaneda T Antioxidative effects of αndashtocopherol and riboflavinndash

butyrate in rats dosed with methyl linoleate hydroperoxide Agric Biol Chem

1983471577ndash1582

Miyazawa T Tsuchiya K Kaneda T Riboflavin tetrabutyrate an antioxidative

synergist of alphandashtocopherol as estimated by hepatic chemiluminescence

[Vitamin E] Nutr Rep Int 198429157ndash162

Moffat AC Osselton MD Widdop B Clarkelsquos Analysis of Drugs and Poison 3rd ed

Mogensen KB Kneipp K Sizendashdependent shifts of plasmon resonance in silver

nanoparticle films using controlled dissolution monitoring the onset of surface

screening effects J Phys Chem C 201411828075ndash28083

Mohamed AM Mohamed HA Mohamed NA Marwa ZR Chemometric methods for

the simultaneous determination of some waterndashsoluble vitamins J AOAC Int

201194467ndash 481

Mokashi VV Walekar LS Anbhule PV Lee SH Patil SR Kolekar GB Study of

energy transfer between riboflavin (vitamin B2) and AgNPs J Nanopart

Res 2014162291ndash2298

Montazer M Alimohammadi F Shamei A Rahimi MK Durable antibacterial and

crossndashlinking cotton with colloidal silver nanoparticles and butane

tetracarboxylic acid without yellowing Colloids Surf B Biointerfaces

201289196ndash202

Montessori V Press N Harris M Akagi L Montaner JSG Adverse effect of

antiretroviral therapy for HIV infection CMAJ 2004170229ndash238

Moore WM Baylor Jr C Photochemistry of riboflavine IV Photobleaching of some

nitrogenndash9 substituted isoalloxazines and flavines J Am Chem Soc

1969917170ndash7179

Moore WM Ireton RC The photochemistry of riboflavin V The photodegradation of

isoalloxazines in alcoholic solvents Photochem Photobiol 197725347ndash356

Moore WM Spence JT Raymond FA Colson SD Photochemistry of riboflavin I The

hydrogen transfer process in the anaerobic photobleaching of flavins J Am

Chem Soc1963853367ndash3372

Morishita S Suzuki KI Deposition of Copper Using Photoexcited Riboflavin Bull

Chem Soc Japan 1995682425ndash2428

Mortland MM Lawless JG Hartman H Frankel R Smectite interactions with

flavomononucleotide Clays Clay Min 198432279ndash282

Mortland MM Lawless JG Smectite interactions with riboflavin Clays Clay Min

198331435ndash439

Mosae Selvakumar P Antonyraj CA Babu R Dakhsinamurthy A Manikandan N

Palanivel A Green synthesis and antimicrobial activity of monodispersed silver

nanoparticles synthesized using lemon extract Synth React Inorg MetalndashOrg

NanondashMetal Chem 201646291ndash294

Munoz A Ortiz R Murcia MA Determination by HPLC of changes in riboflavin levels

in milk and nondairy imitation milk during refrigerated storage Food Chem

199449203ndash206

Murthy US Podder SK Adiga PR The interaction of riboflavin with a protein isolated

from hens egg white A spectrofluorimetric study Biochim Biophys Acta

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Nairn JJ Shapiro PJ Twamley B Pounds T Von Wandruszka R Fletcher TR

Williams M Wang C Norton MG Preparation of ultrafine chalcopyrite

nanoparticles via the photochemical decomposition of molecular singlendashsource

precursors Nano Lett 200661218ndash1223

Natera J Massad W Garciacutea NA The role of vitamin B6 as an antioxidant in the

presence of vitamin B2ndashphotogenerated reactive oxygen species A kinetic and

mechanistic study Photochem Photobiol Sci 201211938ndash945

Nath R Health and disease role of micronutrients and trace elements Recent advances

in the assessment of micronutrients and trace elements deficiency in humans 1st

ed APH publishers New Delhi 2000 Chap 6

Noguchi M Fukuda N Fujimura K Ishizuka K Uchida Y Matsui K Formation of

copper nanoparticles in silica doped with riboflavin by photoinduced electron

transfer J Japan Soc Colour Mater 200376459ndash462

Noguchi M Kazama H Katoh A Uchida Y Matsui K Photoinduced degradation of

fluorescence and formation of copper nanoparticles in solndashgel silica doped with

flavins J NonndashCryst Solids 20113572966ndash2969

OlsquoNeil MJ ed The Merck Index 13th ed Rohway NJ Merck and Co Inc 2013

Electronic Version

Ortega GR Diemling MJ Delgado JM Eds Vitamins and related compounds

Textbook of Organic Medicinal and Pharmaceutical Chemistry 2004 Chap

Oster G Bellin JS Holmstrom B Photochemistry of riboflavin Experientia

196218249ndash253

Oster G Fluorescence quenching by nucleic acids Trans Faraday Soc 195147660ndash

Owen WR Stewart PJ Kinetics and mechanism of chlorambucil hydrolysis J Pharm

Sci 197968992ndash996

Parker AJ Protic-dipolar aprotic solvent effects on rates of bimolecular reactions

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the automatic synthesis of silver nanoparticles in a greener way Talanta

201513345ndash51

Patil DT Bhattar SL Kolekar GB Patil SR Spectrofluorimetric studies of the

interaction between quinine sulfate and riboflavin J Solution Chem

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Peng Z HaiXia L SiDe YWenFengW Effect of pH and polarity on the excited states

of norfloxacin and its 4-N-acetyl derivative a steady state and time-resolved

study Sci China Chem 201457409ndash416

Penzer GR Radda GK Photochemistry of flavins Methods in Enzymol

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Penzer GR Radda GK The chemistry and biological function of isoalloxazines

(flavines) Quart Rev Chem Soc 19672143ndash65

Penzer GR The chemistry of flavins and flavoproteins Aerobic photochemistry

Biochem J 1970116733ndash743

Penzkofer A Tyagi A Kiermaier J Room temperature hydrolysis of lumiflavin in

alkaline aqueous solution J Photochem Photobiol A Chem 2011217369ndash375

PeacuterezndashRuiz T MartiacutenezndashLozano C Sanz A Tomaacutes V Photokinetic determination of

riboflavin and riboflavin 5primendashphosphate using flow injection analysis and

chemiluminescence detection Analyst 19941191825ndash1828

PeacuterezndashRuiz T MartinezndashLozano MC Tomaacutes V Determination of B2 vitamers in

pharmaceutical preparations foods and animal tissues by a photokinetic method

Analyst 1987112237ndash241

Perveen S Yasmin A Khan KM Quantitative simultaneous estimation of water soluble

vitamins riboflavin pyridoxine cyanocobalamin and folic acid in nutraceutical

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Picaud T Desbois A Interaction of glutathione reductase with heavy metal the binding

of Hg (II) or Cd (II) to the reduced enzyme affects both the redox dithiol pair

and the flavin Biochemistry 20064515829ndash15837

Powers HJ Riboflavin (vitamin B-2) and health Am J Clin Nutr 2003771352-1360

Pramar Y Gupta VD Preformulation studies of spironolactone effect of pH two buffer

species ionic strength and temperature on stability J Pharm Sci 199180551ndash

Radda GK Calvin M Chemical and photochemical reductions of flavin nucleotides and

analogues Biochemistry 19643384ndash393

Rajavel K Gomathi R Pandian R Rajendra Kumar RT In situ attachment and its

hydrophobicity of sizendashand shapendashcontrolled silver nanoparticles on fabric

surface for bioapplication Inorg NanondashMetal Chem 2017471196ndash1203

Ramanathan R Field MR OMullane AP Smooker PM Bhargava SK Bansal V

Aqueous phase synthesis of copper nanoparticles a link between heavy metal

resistance and nanoparticle synthesis ability in bacterial systems Nanoscale

201352300ndash2306

Rao CNR Cheetham AK Science and technology of nanomaterials current status and

features J Mat Chem 2001112887ndash2894

Rao CNR Kulkarni GU Thomas PJ Edwards PP Sizendashdependent chemistry

properties of nanocrystals Chem Eur J 2002828ndash35

Rashid I Potts D Riboflavin determination in milk J Food Sci 198045744ndash745

Reddy RYV Riboflavin photosensitized oxidation of Amino acids PhD thesis Ohio

State University Canada 2008

Reichardt C Solvent effects on chemical reactivity Pure Appl Chem 1982541867ndash

Reichardt C Solvents and Solvent Effects in Organic Chemistry 2nd ed New York

VCH Publishers 1988

Rexroad J Evans RK Middaugh CR Effect of pH and ionic strength on the physical

stability of adenovirus type 5 J Pharm Sci 200695237ndash247

RezaeindashZarchi S Saboury AA Javed A Barzegar A Ahmadian S Bayandorindash

Moghaddam A Nanondashcomposition of riboflavinndashnafion functional film and its

application in biosensing J Appl Electrochem 2013431175ndash 1183

Ribeiro DO Pinto DC Lima LM Volpato NM Cabral LM de Sousa VP Chemical

stability study of vitamins thiamine riboflavin pyridoxine and ascorbic acid in

parenteral nutrition for neonatal use Nutr J 20111047

Rivas Aiello MB Romero JJ Bertolotti SG Gonzalez MC Martire DO Effect of Silver

Nanoparticles on the Photophysics of Riboflavin Consequences on the ROS

Generation J Phys Chem C 201612021967ndash21975

Rivlin RS Dutta P Vitamin B2 (Riboflavin) Relevance to malaria and antioxidant

activity Nutr Today 19953062ndash67

Rivlin RS Pinto JT In Bowman B Russell R Eds Present Knowledge in Nutrition

8th ed ILSI Press Washington DC 2001 pp 1313ndash1332

Rivlin RS Riboflavin (vitamin B2) In Zempleni J Rucker RB McCormick DB Suttie

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Raton FL 2007 Chap 7

Rocha FRP Filho OF Reis BF A multicommuted flow system for sequential

spetcrophotometric determination of hydrosoluble vitamins in pharmaceutical

preparations Talanta 200357200ndash256

Rochette AD Silva E BirlouezndashAragon I Mancini M Edwards AM Morliegravere P

Riboflavin photodegradation and photosensitizing effects are highly dependent

on oxygen and ascorbate concentrations Photochem Photobiol 200072815ndash

Roe DA McCormick DB Lin RT Effects of riboflavin on boric acid toxicity J Pharm

Sci 1972611081ndash1085

Roseman TJ Sims B Stehle RG Stability of prostaglandins Am J Hosp Pharm

197330236ndash239

Roushani M Abdi Z Daneshfar A Salimi A Hydrogen peroxide sensor based on

riboflavin immobilized at the nickel oxide nanoparticlendashmodified glassy carbon

electrode J Appl Electrochem 2013431175ndash1183

Roushani M Shahdostndashfard F A novel ultrasensitive aptasensor based on silver

nanoparticles measured via enhanced voltammetric response of electrochemical

reduction of riboflavin as redox probe for cocaine detection Sensors Actuators

B Chem 2015207764ndash771

Routh P Layek RK Nandi AK Negative differential resistance and improved

optelectronic properties in Ag nanoparticlesndashdecorated grapheme oxidendash

riboflavin hybrids Carbon 2012503422ndash 3434

optoelectronic properties in Ag nanoparticlesndashdecorated graphene oxidendash

riboflavin hybrids Carbon 2012503422ndash3434

Royal Society Nanoscience and nanotechnologies opportunities and uncertainties

Rozen R Methylenetetrahydrofolate reductase a link between folate and riboflavin

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Rutter WJ The Interaction of Riboflavin FMN and FAD with various metal ions the

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Sakai K Fluorescence of riboflavin Nagoya J Med Sci 195618245ndash51

Sakai K On the influences of several metal ions upon photolysis of riboflavin Nagoya

J Med Sci 195518222ndash231

Salo JP Yli‐Kauhaluoma J Salomies H On the hydrolytic behavior of tinidazole

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Sandor PS Arfa J Ambrosini A Schoenen P Prophylactic treatment of migraine with

βndashblockers and riboflavin Differential effects on the intensity dependence of

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Sanvordeker DR Kostenbauder HB Mechanism for riboflavin enhancement of

bilirubin photodecomposition in vitro J Pharm Sci 197463404ndash408

Sato Y Chaki H Suzuki Y Biphasic photolysis of riboflavin III Effects of ionic

strength on the photolysis Chem Pharm Bull 1984321232ndash1235

Sato Y Yokoo M Takahashi S Takahashi T Biphasic photolysis of riboflavine with a

lowndashintensity light source Chem Pharm Bull 1982301803ndash1810

Schmid R Sapunov VN Non-formal kinetics in search of chemical reactions

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Schmidt WC Light-induced redox cycles of flavins in various alcoholacetic

acidmixtures Photochem Photobiol 198236699ndash703

Schoenen P Jaccuy J Lenaerts M Effectiveness of high dose riboflavin in migraine

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Schoumlllnhammer G Hemmerich P Nucleophilic addition at the photoexcited flavin

cation Synthesis and properties of 6‐and 9‐hydroxy‐flavocoenzyme

chromophores Eur J Biochem 197444561ndash577

SchoumlnlebenndashJanas A Kirsch P Mittl PR Schirmer RH KrauthndashSiegel RL Inhibition

of human glutathione reductase by 10ndasharylisoalloxazines Crystallographic

kinetic and electrochemical studies J Med Chem 1996391549ndash1554

Schuman Jorns M Schollnhammer G Hemmerich P Intramolecular addition of the

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SenndashVarma C Ghosh S Bhowmik BB Photondasheffect in phospholipid liposome

containing riboflavin Chem Phys Lipids 19957649ndash54

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Shah JJ Riboflavin In Augustin J Klein BP Becker D Venugopal PB Eds Method

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Sheraz MA Kazi SH Ahmed S Anwar Z Ahmad I Photo thermal and chemical

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Sheraz MA Kazi SH Ahmed S Mirza T Ahmad I Evstigneev MP Effect of

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Photobiol A Chem 2014a27317ndash22

Sheraz MA Kazi SH Ahmed S Qadeer K Khan MF Ahmad I Multicomponent

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Shimizu S Photolytic mechanism of riboflavin J Vitaminol 1955139ndash47

Shin CT Sciarrone BJ Discher CA Effect of certain additives on the photochemistry

of riboflavin J Pharm Sci 197059297ndash302

Sigel H Song B Liang G Halbach R Felder M Bastian M Acidndashbase and metal ionndash

binding properties of flavin mononucleotide (FMN2minus

) Is a dielectriclsquoeffect

responsible for the increased complex stability Inorg Chim Acta

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Sikorska E Khmelinskii I Komasa A Koput J Ferreira LF Herance JR Bourdelande

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photophysics of flavin related compounds riboflavin and isondash(6 7)ndashriboflavin

Chem Phys 2005314239ndash247

Sikorska E Koziolowa A Sikorski M Siemiarczuk A The solvent effect on the excited

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Sikorski E Worrall DR Bourdelande JI Sikroski M Photophysics of lumichrome and

its analogs Polish J Chem 20037765ndash73

Silva E Ruumlckert V Lissi E Abuin E Effects of pH and ionic micelles on the

riboflavinndashsensitized photoprocesses of tryptophan in aqueous solution J

Photochem Photobiol B Biol 19911157ndash68

Silva ED Ugarte R Andrade A Edwards AM Riboflavinndashsensitized photoprocesses of

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Singh S Gupta RI Dielectric constant effects on degradation of azothioprine in

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Sinha R Kim GJ Nie S Shin DM Nanotechnology in cancer therapeutics

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Smith EC Metzler DE The photochemical degradation of riboflavin J Am Chem

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Song PS Kurtin WE Photochemistry of the model phototropic system involving

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Song PS Metzler DE Photochemical degradation of FlavinsndashIV Studies of the

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Song PS Smith EC Metzler DE Photochemical degradation of flavins IV The

mechanism of alkaline hydrolysis of 67ndashdimethylndash9ndash

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Song PS Chemistry of flavins in their excited states In Kamin H Ed Flavins and

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Waterndashsoluble Vitamins Wiley New York 2000 Chap 7

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Speck WT Chen CC Rosenkranz HS In vitro studies of effects of light and riboflavin

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Spoerl E Wollensak G Dittert DD Seiler T Thermomechanical behavior of collagenndash

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Spoerl E Wollensak G Seiler T Increased resistance of crosslinked cornea against

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Stankovičova M Bezakova Ţ Beneš L Kinetics of hydrolysis of acetyl valeroyl and

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Stern KG Holiday ER Zur Konstitution des Photo‐flavins Versuche in der

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Strauss G Nickerson WJ Photochemical cleavage of water by riboflavin II Role of

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SuendashChu M Kristensen S Toslashnnesen HH Influence of lagndashtime between light exposure

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Suelter CH Metzler DE The oxidation of a reduced pyridine nucleotide analog by

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Sunkara G Navarre CB Kompella UB Influence of pH and temperature on kinetics of

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Svobodova S Hais J Kostir J The influence of pH and light on riboflavin solutions

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Sweetman SC ed Martindale The Complete Drug Reference 36th ed Pharmaceutical

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Tada H Decomposition reaction of hexamine by acid J Am Chem Soc 196082255ndash

Tai CY Wang YH Liu HS A green process for preparing silver nanoparticles using

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Tan KL Phototherapy for neonatal jaundice Acta Paediatr 199685277ndash279

Tang AM Graham NM Saah AJ Effects of micronutrient intake on survival in human

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Taniguchi M Harm T Effects of riboflavin and selenium deficiencies on glutathione and

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Nutr Sci Vitaminol 198329283ndash292

Tatsumi K Ichikawa H Wada S Flavinndashsensitized photooxidation of substituted phenols

in natural water J Contam Hydrol 19929207ndash219

Terekhova IV Koźbiał M Kumeev RS Alper GA Inclusion complex formation between

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Terekhova IV Tikhova MN Volkova TV Kumeev RS Perlovich GL Inclusion

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Theorell H Reindarstellung (Kristallistaion) des gelben Atmungsfermentes und die

reversible Spaltung desselben Biochem Z 1934272155ndash156

Thomas S Kumar R Sharma A Issarani R Nagori BP Stabilityndashindicating HPLC

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Tien DC Liao CY Huang JC Tseng KH Lung JK Tsung TT Kao WS Tsai TH

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Tonnesen HH Formulation and stability testing of photolabile drugs Int J Pharm

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TorresndashSequeiros RA GarciandashFalcon MS SimalndashGandara J Analysis of fluorescent

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Chromatographia 200153S236ndashS239

Traber R Vogelmann E Schreiner S Werner T Kramer HEA Reactivity of excited

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Photobiol 1981a3341ndash48

Treadwell GE Cairns WL Metzler DE Photochemical degradation of flavins V

Chromatographic studies of the products of photolysis of riboflavin J

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Uchegbu IF Pharmaceutical nanotechnology polymeric vesicles for drug and gene

delivery Expert Opin Drug Deliv 20063629ndash640

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United States Pharmacopoeia 29 United States Pharmacopoeial Convention Rockville

MD 2016 Electronic version

Vaish SP Tollin G Flash photolysis of flavins V Oxidation and disproportionation of

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Vaishnavi E Renganathan R Photochemical events during photosensitization of

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Valls F Sancho MT FernaacutendezndashMuintildeo MA Checa MA Determination of total

riboflavin in cooked sausages J Agric Food Chem 1999471067ndash1070

Van der Horst A Martens HJ De Goede PN Analysis of waterndashsoluble vitamins in

total parenteral nutrition solution by high pressure liquid chromatography

Pharmaceutisch Weekblad 198911169ndash174

Varnes AW Dodson RB Wehry EL Interactions of transitionndashmetal ions with

photoexcited states of flavins Fluorescence quenching studies J Am Chem

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Voicescu M Angelescu DG Ionescu S Teodorescu VS Spectroscopic analysis of the

riboflavinndashserum albumins interaction on silver nanoparticles J Nanopart Res

2013151555

Wade TD Fritchie CJ The Crystal Structure of a riboflavinndashmetal complex riboflavin

silver perchlorate hemihydrate J Biol Chem 19732482337ndash2343

Walsh C Flavin coenzymes at the crossroads of biological redox chemistry Acc Chem

Res 198013148ndash155

Wang Y Zhu PH Tian T Tang J Wang L Hu XY Synchronous fluorescence as a

rapid method for the simultaneous determination of folic acid and riboflavin in

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Warburg O Christian W A new oxidation enzyme and its absorption spectrum

Biochem Z 1932254438ndash458

Watkins JA Cusanovich MA Meyer TE Tollin G A ―parallel plate electrostatic

model for bimolecular rate constants applied to electron transfer proteins

Protein Sci 199432104ndash2114

Weber G Fluorescence of riboflavin and flavinndashadenine dinucleotide Biochem J

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Wei L Lu J Xu H Patel A Chen ZS Chen G Silver nanoparticles synthesis

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Witte AB Leistra AN Wong PT Bhatathi S Refior K Smith P Kaso O Sinniah K

Choi SK Atomic force microscopy probing of receptor naniparticle interactions

for riboflavin receptor targeted goldndashdendrimer nanocomposites J Phys Chem

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Wolf MM Schumann C Gross R Domratcheva T Diller R Ultrafast infrared

spectroscopy of riboflavin dynamics electronic structure and vibrational mode

analysis Phys Chem B 200811213424ndash13432

Wu GH He CY Application of artificial neural network to simultaneous

spectrofluorimetric determination of vitamin B1 B2 and B6 Guang Pu Xue Yu

Guang Pu Fen Xi 200323535ndash538

Wypych G Hand book of solvents 2nd ed Chem Tec Publishing Toronto 2001 pp

577ndash581

Xiaondashyan L Ruindashyong W Rundashxiu C Zhindashhong L Highly sensitive spectrofluorimetric

determination of riboflavin based on the generation of active oxygen coupled

with enzymatic reaction Wuhan Univ Nat Sci 20027361ndash364

Xie ZP Wand JW Xiao G LI L Determination of VB_2 by flow injection analysis

with chemiluminescence detection J Nanchang Univ 20052014

Xinhe LC Determination of thiamine riboflavin pyridoxideand niacinamide in the

vitamin chewable tabletby reversedndashphase ion pair HPLC J Suzhou Institute of

Urban Constr Environ Protect 19994013

Xu D Zhao HW Huang CZ Wu LP Pu WD Zheng J Zuo Y Sensitive and selective

detection of mercury (II) based on the aggregation of gold nanoparticles

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Yamada K Chen Z Rozen R Matthews RG Effects of common polymorphisms on the

properties of recombinant human methylenetetrahydrofolate reductase Proc Nat

Acad Sci 20019814853ndash14858

Yamaoka K Nakajima Y Terashima T Stability of vitamins in TPN mixture in a

doublendashchambered bag Jpn J Hosp Pharm 199521357ndash364

Yamato S Kawakami N Shimada K Ono M Idei N Itoh Y Onndashline automated highndash

performance liquid chromatographic determination of total riboflavin

phosphates using immobilized acid phosphatase as a prendashcolumn reactor J

Yang LH Xue Q Lu JG Determination of VitB_1 VitB_2 VitB_6 and sorbic acid in

alvityl syrup by HPLC Qilu Pharmaceutical Affairs 20106018

Yang PH Zhou ZJ Feng DX YE XY Studies on adsorptive cyclic voltammetry of

VB~ 2 on glassy carbon electrode JournalndashJinan Univ Nat Sci 20012293ndash97

Yantih N Widowati D Aryani T Validation of HPLC method for determination of

thiamine hydrochloride riboflavin nicotinamide and pyridoxine hydrochloride

in syrup preparation Can J Sci Indus Res 20112269ndash278

Yeh MK Degradation kinetics of neostigmine in solution Drug Dev Ind Pharm

2000261221ndash1226

Yoshioka S Stella VJ Stability of Drugs and Dosage Forms Kluwer AcademicPlenum

Publishers New York 2000 pp 99ndash104

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1975250946ndash951

Yuan LJ Chen DB Determination of vitamin B1 vitamin B2 in four vitamin glucose

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19966354ndash66

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soluble vitamins and vitaminndashlike compounds in infant formula by UPLCndash

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Zhang H Zhao J Liu H Wang H Liu R Liu J Application of poly (3ndash

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AAPS PharmSciTechAn Official Journal of the AmericanAssociation of Pharmaceutical Scientists e-ISSN 1530-9932 AAPS PharmSciTechDOI 101208s12249-015-0304-2

Solvent Effect on the Photolysis ofRiboflavin

Iqbal Ahmad Zubair Anwar SofiaAhmed Muhammad Ali Sheraz RaheelaBano amp Ambreen Hafeez

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Research Article

Solvent Effect on the Photolysis of Riboflavin

Iqbal Ahmad1 Zubair Anwar1 Sofia Ahmed1 Muhammad Ali Sheraz13 Raheela Bano1 and Ambreen Hafeez2

Received 8 December 2014 accepted 27 January 2015

Abstract The kinetics of photolysis of riboflavin (RF) in water (pH 70) and in organic solvents (aceto-nitrile methanol ethanol 1-propanol 1-butanol ethyl acetate) has been studied using a multicomponentspectrometric method for the assay of RF and its major photoproducts formylmethylflavin andlumichrome The apparent first-order rate constants (kobs) for the reaction range from 319 (ethyl acetate)to 461times10minus3 minminus1 (water) The values of kobs have been found to be a linear function of solvent dielectricconstant implying the participation of a dipolar intermediate along the reaction pathway The degradationof this intermediate is promoted by the polarity of the medium This indicates a greater stabilization of theexcited-triplet states of RF with an increase in solvent polarity to facilitate its reduction The rate constantsfor the reaction show a linear relation with the solvent acceptor number indicating the degree of solutendashsolvent interaction in different solvents It would depend on the electron-donating capacity of RFmolecule in organic solvents The values of kobs are inversely proportional to the viscosity of the mediumas a result of diffusion-controlled processes

KEY WORDS dielectric constant kinetics photolysis riboflavin solvent effect viscosity

INTRODUCTION

The influence of solvents on the rates of degradation ofdrugs is an important consideration for the formulation chem-ist The effects of dielectric constant and viscosity of themedium may be significant on the stability of pharmaceuticalformulations Theoretical basis of the effects of solvent on therates and mechanism of chemical reactions has been exten-sively dealt by many workers (1418212837475665) Theeffect of dielectric constant on the degradation kinetics andstabilization of chloramphenicol (40) barbiturates (31)methanamine (59) ampicillin (29) prostaglandin E2 (48)chlorambucil (43) 2-tetrahydropyranyl benzoate (30) indo-methacin (24) aspirin (16) phenoxybenzamine (2) azathio-prine (55) polypeptides (17) neostigmine (64) triprolidine(39) 10-methylisoalloxazine (12) formylmethylflavin (7)levofloxacin (6) and moxifloxacin (4) has been reportedThe viscosity of the medium may also affect the stability of adrug A linear relation has also been found between the rateconstant and the inverse of solvent viscosity for thephotodegradat ion of 10-methyl isoal loxazine (12) formylmethylflavin (9) levofloxacin (6) and moxifloxacin(4) in organic solvents

Some kinetic studies of the photolysis of riboflavin (RF)in carboxylic acids (3458) alcoholic solvents (32425057)

and pyridine (36) have been conducted However the methodused for the determination of RF is based on the measurementof absorbance at 445 nm without any consideration of theinterference caused by photoproducts formed during degra-dation Thus the kinetic data obtained may not be accurateand specific methods may be required for assay (1013)Studies on the photolysis of formylmethylflavin (FMF) amajor intermediate in the photolysis sequence of the RF inorganic solvents have been conducted (79) Solvent effects onflavin electron transfer reactions have been found to be sig-nificant (1251) The present work involves a detailed study ofthe kinetics of photolysis of RF in a wide range of organicsolvents using specific multicomponent spectrometric methodfor the assay of RF and photoproducts (101352) and todevelop correlations between the kinetic data and solventparameters such as dielectric constant and viscosity Theseconsiderations are important in the formulation of drugs withdifferent polar characters using cosolvents and those whoseoxidation is viscosity dependent to achieve their stabilization

RF lumichrome (LC) and lumiflavin (LF) were obtainedfrom Sigma Chemical Co St Louis MO USAFormylmethylflavin (FMF) and carboxymethylflavin (CMF)were synthesized by the previously reported methods (2223)All solvents and reagents were of analytical grade from Merckamp Co Whitehouse Station NJ USA

The methods of photolysis chromatography and assayare the same as previously described for FMF in organicsolvents (79) and in aqueous solution (8) These are brieflydescribed below

1 Baqai Institute of Pharmaceutical Sciences BaqaiMedical UniversityToll Plaza Super Highway Gadap Road Karachi 74600 Pakistan

2 Department of Biochemistry Dow International Medical College DowUniversity of Health Sciences Ojha Campus Karachi 74200 Pakistan

3 To whom correspondence should be addressed (e-mailali_sheraz80hotmailcom)

AAPS PharmSciTech ( 2015)DOI 101208s12249-015-0304-2

1530-9932150000-00010 2015 American Association of Pharmaceutical Scientists

Authors personal copy

Photolysis

A 3times10minus5 M solution of RF (100 ml) was prepared inwater (pH 70 0005 M phosphate buffer) and in organicsolvents in a volumetric flask (Pyrex) and immersed in awater bath maintained at 25plusmn1degC The solution was ex-posed to a Philips HPL-N 125 W high-pressure mercurylamp (emission bands at 405 and 435 nm the later bandoverlaps the 445 nm band of RF (13)) fixed at a distanceof 25 cm from the center of the flask for a period of 2ndash3 h depending upon the nature of the solvent usedSamples of photolyzed solution were withdrawn at a var-ious time intervals for thin-layer chromatography andspectrometric assay

pH Measurements

The pHmeasurements of solutions were performed on anElmetron pH meter (ModelmdashCP501 sensitivity plusmn001 pHunits Poland) using a combination pH electrode The elec-trode was automatically calibrated using phthalate (pH 4008)phosphate (pH 6865) and disodium tetraborate (pH 9180)buffer solutions

Thin-Layer Chromatography

The thin-layer chromatography (TLC) of the photo-lyzed solutions of RF in aqueous and organic solvents wascarried out on 250 μm cellulose plates using the followingsolvent systems (a) 1-butanolndashacetic acidndashwater (401050vv organic phase) and (b) 1-butanolndash1-propanolndashaceticacidndashwater (5030218 vv) (11) The compounds weredetected by their characteristic fluorescence on exposureto UV (365 nm) light RF LF FMF CMF (yellow green)LC (sky blue)

Spectrometric Assay

A 5-ml aliquot of the photolyzed solution of RF wasevaporated to dryness under reduced pressure at room tem-perature and the residue dissolved in 02 M KClndashHCl buffersolution (pH 20) The solution was extracted with 3times5 ml ofchloroform the chloroform was evaporated and the residuedissolved in 02 M acetate buffer solution (pH 45) The ab-sorption of this solution was measured at 356 nm to determinethe concentration of LC The aqueous phase (pH 20) wasused to determine the concentrations of RF and FMF indegradation solutions by a two-component spectrometric as-say at 385 and 445 nm according to the method of Ahmad andRapson (10)

Determination of Light Intensity

The intensity of the Philips HPL-N 125 W lamp wasdetermined using potassium ferrioxalate actinometry (25) as121plusmn010times1017 quanta sminus1

RESULTS

Photoproducts of RF

TLC of the photolyzed solutions of RF in organic solventsusing solvent systems (a) and (b) showed the presence of FMFand LC as the main photoproducts of this reaction CMF wasalso detected as a minor oxidation product of FMF in thesesolvents (79) These products were identified by comparisonof their fluorescence emission and Rf values with those of theauthentic compounds FMF and LC as the main photoprod-ucts of RF in organic solvents have previously been reported(7934) The formation of LC in organic solvents may takeplace through FMF as an intermediate in the photolysis of RFas observed in the case of aqueous solutions (7ndash10) The

Fig 1 Absorption spectra of RF photolyzed in methanol at 0 30 60 90 and 120 min

Ahmad et al

fluorescence intensity of the photoproducts on TLC plates isan indication of the extent of their formation in a particularsolvent during the irradiation period In aqueous solutions(pH 70) LF is also formed in addition to FMF and LC aspreviously observed (857)

Spectral Characteristics

RF exhibits absorption maxima in organic solvents in theregion of 440ndash450 344ndash358 and 270ndash271 nm (35) A typicalset of absorption spectra for the photolysis of RF in methanolis shown in Fig 1 There is a gradual loss of absorbancearound 445 nm with a shift of the peak at 358 to 350 nm withtime due to the formation of LC (λmax in methanol 339 nm)(54) the major photoproduct of RF in organic solvents LC isformed through the mediation of FMF an intermediate in thephotolysis of RF (57) FMF has an absorption spectrum sim-ilar to that of RF and therefore it could not be distinguishedfrom the absorption spectrum of RF in organic solvents

Assay of RF and Photoproducts

The photolyzed solutions of RF have been assayed at pH20 by extraction of LC with chloroform and its determinationat pH 45 at 356 nm The aqueous phase was used to deter-mine RF and FMF by a two-component assay at 385 and445 nm corresponding to the absorption maxima of thesecompounds The molar concentrations of RF and itsphotoproducts FMF and LC determined in a photolysisreaction (10) carried out in methanol are reported in Table IThe assay method shows uniformly increasing values of FMFand LC with an almost constant molar balance with time

indicating a good reproducibility of the method CMF a minoroxidation product of FMF in organic solvents (7) accountingto less than 1 (9) does not interfere with the assay method

Kinetics of Photolysis

The photolysis of RF in aqueous solution (3857) and inorganic solvents (3657) follows first-order kinetics A kineticplot for the photolysis of RF in methanol (Fig 2) shows thatLC is the final product in this reaction as observed by previousworkers (3242) The first-order rate constants (kobs) deter-mined for the photolysis reactions in organic solvents andwater range from 319 (ethyl acetate) to 461times10minus3 minminus1

(water) (correlation coefficients 0997ndash0999) (Table II) Thevalues of kobs increase with an increase in the dielectric con-stant showing the influence of solvent on the rate of reactionThe value for the photolysis of RF in aqueous solution (pH70 0005 M phosphate buffer) is also included for compari-son A plot of kobs for the photolysis of RF as a function ofsolvent dielectric constant is presented in Fig 3 It shows thatthe rate constants are linearly dependent upon the solventdielectric constant Similarly a linear relation has been foundbetween the values of kobs and the solvent acceptor numberindicating the degree of solutendashsolvent interaction (Fig 4) Inorder to observe the effect of viscosity on the rate of photol-ysis a plot of kobs versus inverse of viscosity was constructed(Fig 5) It showed a linear relation between the two valuesindicating the influence of solvent viscosity on the rate ofreaction These results are supported by the fact that a plotof dielectric constant versus inverse of viscosity of organicsolvents is linear However the values of kobs for RF in ethylacetate and water do not fit in the plot probably due todifferent behaviors of RF in acetate (compared to alcohols)and water (eg degree of hydrogen bonding)

DISCUSSION

Effect of Solvent

It is known that solvents could influence the degradationof drugs depending on the solutendashsolvent interaction Solventsmay alter the rate and mechanism of chemical reactions(11538444651) and thus play a significant role in the stabi-lization of pharmaceutical products (21) Pharmaceutical for-mulations of ionizable compounds such as RF may bestabilized by an alteration in the solvent characteristics A

0 30 60 90 120

on times

Time (min)

Fig 2 A kinetic plot for the photolysis of RF in methanol

Table I Concentrations of RF and Photoproducts in Methanol

Time(min)

RF(Mtimes105)

FMF(Mtimes105)

LC(Mtimes105)

Total(Mtimes105)

0 300 00 00 30030 255 036 015 30660 215 058 029 30290 201 071 032 304120 191 079 037 307

RF riboflavin FMF formylmethylflavin LC lumichrome

suppression of the ionization of a drug susceptible to degra-dation in water may be achieved by the addition of a cosolvent(eg alcohol) This would result in the destabilization of thepolar excited state and therefore a decrease in the rate ofreaction as observed in the case of many drugs (65) The useof organic solvents as cosolvent can have a photostabilizingeffect on the product as a result of a change in the polarity andviscosity of the medium (61) These considerations are impor-tant in the formulation of drugs with different polar charactersand those whose oxidation is viscosity dependent Theseaspects with respect to the photolysis of RF as a modelcompound used in the clinical treatment of neonatal jaundice(60) keratoconus (19) and HIV infection (41) would now beconsidered and correlations would be developed between thesolvent characteristics and the rate of reaction

Effect of Dielectric Constant

The rate of degradation reactions between ions and di-poles in solution depends on bulk properties of the solventsuch as the dielectric constant Any change in the dielectricconstant of a solvent can lead to variation in the energy ofactivation (ΔG) and hence in the rate constants (65) This canbe applied to the degradation of RF since its rate of photolysisis a linear function of dielectric constant This can be ex-plained on the basis of the participation of a polar intermedi-ate in the reaction pathway to facilitate the reaction (712)The rate of RF photolysis is affected by solvent polarity prob-ably due to changes in the conformation of the ribityl side

chain in different solvents (42) Quenching of flavin excited-triplet state [3FL] by oxygen during the reaction has beensuggested (733) and this may affect the rate of photolysisHowever under the present reaction conditions (ie solventsin equilibrium with the air) first-order plots are linear for RFsolutions photolyzed up to 30 and the values of kobs arerelative to these conditions The electron-donating capacity ofa molecule (eg fluoroquinolone RF) is affected by the na-ture of the solvent (545) and hence its rate of degradationThe acceptor number is a measure of the ability of solvents toshare electron pairs from suitable donors (4963) and thiscould affect the rate of photolysis The results obtained anddegradation behavior of RF in organic solvents suggest thatthe stability of such polar drugs can be improved by alterationof dielectric constant of the medium

Effect of Viscosity

The viscosity of the medium can also influence the rate ofdegradation particularly of an oxidizable drug The photolysis ofRF involves oxidation of the ribityl side chain (42) and thus maybe affected by the solvent viscosity The values of kobs for RF inethyl acetate and water do not follow the relation (Fig 5) prob-ably due to its different structural orientation (42) and degree ofhydrogen bonding (53) compared to those of the organic sol-vents The behavior of RF in organic solvents indicates that theviscosity of the medium suppresses the rate of photolysis prob-ably as a result of solute diffusion-controlled processes (1262) Ithas been observed that [3RF] quenching depends on solvent

00 100 200 300 400 500 600 700 800

times10

inndash1

Dieletric Constant

Fig 3 Plot of kobs for the photolysis of RF versus dielectric constant(letter x) ethyl acetate (black diamond) 1-butanol (black triangle) 1-propanol (black square) ethanol (black circle) methanol (cross sign)acetonitrile (asterisk) water

Table II Apparent First-Order Rate Constants for the Photolysis of Riboflavin (kobs) in Organic Solvents and Water

Solvents Acceptor number Dielectric constant (ϵ) (25degC) Inverse viscosity (mPasminus1) (25degC) kobstimes103 minminus1plusmnSD

Ethyl acetate 171 602 2268 319plusmn0141-Butanol 368 178 0387 328plusmn0131-Propanol 373 201 0514 334plusmn016Ethanol 371 243 0931 345plusmn015Methanol 413 326 1828 364plusmn017Acetonitrile 189 385 2898 381plusmn016Water 548 785 1123 461plusmn025

SD standard deviation

Ahmad et al

viscosity (12) that would affect the rate of reaction Similar effectsof viscosity have been observed on the photooxidative degrada-tion of formylmethylflavin (9) and fluoroquinolones (4ndash6)

Mode of Photolysis

The photochemistry of RF has widely been studied byseveral workers and the various modes of its photodegradationreactions (ie intramolecular and intermolecular photoreduc-tion photodealkylation and photoaddition) have been discussed(791320262751) The pathway of RF degradation in organicsolvents appears to be similar to that of the aqueous solutioninvolving intramolecular photoreduction followed by side-chaincleavage (13) However the rate of the reaction is solvent de-pendent due to the participation of a dipolar intermediate (12)

whose degradation is promoted by polar environment and sup-pressed by nonpolar media It has been observed by laser flashphotolysis that the reduction of [3FL] in organic solvents pro-ceeds through the mediation of the dipolar intermediate accord-ing to the following reaction (12)

3 FLthornAHrarr Fσndashhellip Hhellip Aσndashthorn rarrFLHbull thornAbull eth1THORN

The flavin semiquinone radical [FLH] undergoes fur-ther reactions to give the final products shown by Eqs (2)and (3)

2FLHbullrarrFLthorn FLH2 eth2THORN

The extent of the reaction to form radicals is controlled bythe degree of solutendashsolvent interaction The polar character ofthe reaction intermediate would determine the rate of reactionand the rate would be higher in solvents of greater polarityThus the solvent characteristics play an important role in deter-mining the rate of RF degradation An appropriate combinationof waterndashalcohol mixture would be a suitable medium for thestabilization of RF and drugs of similar character

CONCLUSION

Solvent characteristics are an important factor in the stabi-lization of pharmaceutical formulations The choice of a solventor cosolvent would depend on the chemical nature polar char-acter and the behavior of the drug in a particularmedium In thepresent study it has been demonstrated that solvent character-istics such as dielectric constant and viscosity may alter the rate

00 05 10 15 20 25 30

bstimes

103 (m

inndash1

Viscosity (mPa s)-1

Fig 5 Plot of kobs for the photolysis of RF versus inverse of viscositySymbols are as in Fig 3

00 100 200 300 400 500 600lnk o

bs times

Fig 4 Plot of lnkobs for the photolysis of RF versus acceptor numberSymbols are as in Fig 3

FLH2 degraded FL + side chain products eth3THORN

of degradation of a drug to achieve stabilization In the case ofRF it has been found that the rate of photolysis is linearlydependent on solvent polarity and is inversely dependent onsolvent viscosity This is reflected in the values of kobs obtainedfor the photolysis of RF in different solvents The value of kobs inwater (ϵ 785) is nearly one and half times that of ethyl acetate (ϵ60) indicating a prominent effect of dielectric constant on therate of reaction Similarly the value of kobs increases with adecrease in solvent viscosity Thus a change in the medium onthe basis of solvent characteristics could improve the stability ofa drug and prolong its shelf life A rational approach in thisdirection and the use of appropriate cosolvents with waterwould enable the formulator to achieve better stabilization ofa drug

REFERENCES

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2 Adams WP Kostenbauder HB Phenoxybenzamine stability inaqueous ethanolic solutions II Solvent effects on kinetics Int JPharm 198525313ndash27

3 Ahmad I Anwar Z Iqbal K Ali SA Mirza T Khurshid A et alEffect of acetate and carbonate buffers on the photolysis ofriboflavin in aqueous solution a kinetic study AAPSPharmSciTech 201415550ndash9

4 Ahmad I Bano R Musharraf SG Ahmed S Sheraz MA ArfeenQU et al Photodegradation of moxifloxacin in aqueous andorganic solvents a kinetic study AAPS PharmSciTech2014151588ndash97

5 Ahmad I Bano R Musharraf SG Sheraz MA Ahmed S TahirH et al Photodegradation of norfloxacin in aqueous and organicsolvents a kinetic study J Photochem Photobiol A Chem20153021ndash10

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7 Ahmad I Fas ihu l lah Q Vaid FHM Photo lys i s o fformylmethylflavin in aqueous and organic solvents PhotochemPhotobiol Sci 20065680ndash5

8 Ahmad I Fasiullah Q Noor A Ansari IA Ali QNM Photolysisof riboflavin in aqueous solution a kinetic study Int J Pharm2004280199ndash208

9 Ahmad I Mirza T Iqbal K Ahmed S Sheraz MA Vaid FHMEffect of pH buffer and viscosity on the photolysis offormylmethylflavin a kinetic study Aust J Chem 201366579ndash85

10 Ahmad I Rapson HDC Multicomponent spectrophotometricassay of riboflavin and photoproducts J Pharm Biomed Anal19908217ndash23

11 Ahmad I Rapson HDC Heelis PF Phillips GO Alkaline hydro-lysis of 7 8-dimethyl-10(formylmethyl)-isoalloxazine A kineticstudy J Org Chem 19804531ndash3

12 Ahmad I Tollin G Solvent effects on flavin electron transferreactions Biochemistry 1981205925ndash8

13 Ahmad I Vaid FHM Photochemistry of flavins in aqueous andorganic solvents In Silva E Edwards AM editors Flavins pho-tochemistry and photobiology Cambridge Royal Society ofChemistry 2006 p 13ndash40

14 Amis ES Hinton JF Solvent effects on chemical phenomenaNew York Academic 1973

15 Amis ES Hinton JF Solvent effect on chemical phenomena NewYork Academic 1973

16 Baker SK Niazi S Stability of aspirin in different media J PharmSci 1983721024ndash6

17 Brennan TV Clarke S Spontaneous degradation of polypeptidesat aspartyl and asparaginyl residues Effects of solvent dielectricProtein Sci 19932331ndash8

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19 Caporossi A Mazzotta C Baiocchi S Tomaso C Long-termresults of riboflavin ultraviolet a corneal collagen cross-linkingfor keratoconus in Italy the Siena eye cross study Am J Opthal2010149585ndash93

20 Choe E Huang R Min DB Chemical reactions and stability ofriboflavin in food J Food Sci 200570R28ndash36

21 Connors KA Amidon GL Stella VJ Chemical stability of phar-maceuticals a handbook for the pharmacist 2nd ed New YorkWiley 1986 p 38ndash41

22 Fall HH Petering HG Metabolic inhibitors 1 67-Dimethyl-9-formylmethylisoalloxazine 67-dimethyl-9-(12-hydroxyethyl)-iso-alloxazine and derivatives J Am Chem Soc 195678377ndash81

23 Fukumachi C Sakurai Y Vitamin B2 photolysis V The photo-lytic formation of 6 7-dimethylflavin-9-acetic acid ester fromriboflavin Vitamins (Kyoto) 19547939ndash43

24 Ghanem AH Hassan ES Hamdi AA Stability of indomethacinsolubilized system Pharmazie 197934406ndash7

25 Hatchard CG Parker CA A new sensitive chemical actinometerII Potassium ferrioxalate as a standard chemical actinometerProc Roy Soc (Lond) 1956A235518ndash36

26 Heelis PF The photophysical and photochemical properties offlavin (isoalloxazines) Chem Soc Rev 19821115ndash39

27 Heelis PF The photochemistry of flavins In Muller F editorChemistry and biochemistry of flavoenzymes Boca Raton CRCPress 1991 p 171ndash93

28 Heitele H Dynamic solvent effects on electron transfer reactionsAngew Chem Int Ed Engl 199332359ndash77

29 Hou JP Poole JW β-lactam antibiotics their physicochemicalproperties and biological activities in relation to structure JPharm Sci 196960503ndash32

30 Hussain A Truelove J Effect of hydroxyl group substituents onpyran ring on hydrolysis rate of benzoates 2-tetrahydropyranylbenzoate J Pharm Sci 197965235ndash66

31 Ikeda K Studies on decomposition and stabilization of drugs insolution IV Effect of dielectric constant on the stabilization ofbarbiturate in alcohol-water mixtures Chem Pharm Bull19608504ndash9

32 Insinska-Rak M Golczak A Sikorski M Photochemistry of ribo-flavin derivatives in methanolic solutions J Phys Chem20121161199ndash207

33 Insinska-RakM Sikorski M Riboflavin interactions with oxygen-survey from the photochemical perspective Chem Eur J20142015280ndash91

34 Koziol J Studies on flavins in organic solventsndashII Photodecom-position of riboflavin in the presence of oxygen PhotochemPhotobiol 1966555ndash62

35 Koziol J Studies on flavins in organic solventsndashI Spectral char-acteristics of riboflavin riboflavin tetrabutyrate and lumichromePhotochem Photobiol 1966541ndash54

36 Kurtin WE Latino MA Song PS A study of photochemistry offlavins in pyridine and with a donor Photochem Photobiol19676247ndash59

37 Laidler KJ Chemical kinetics 3rd ed New York Harper amp Row1987 p 183ndash95

39 Mao YP Tao XL Lipsky PE Analysis of the stability and deg-radation products triptolide J Pharm Pharmcol 2000523ndash12

40 Marcus AD Taraszka AJ A kinetic study of the specific hydro-gen ion catalyzed solvolysis of chloramphenicol in water-propylene glycol systems J Pharm Sci 19594877ndash84

41 Montessori V Press N Harris M Akagi L Montaner JSG Ad-verse effect of antiretroviral therapy for HIV infection CMAJ2004170229ndash38

42 Moore WM Ireton RC The photochemistry of riboflavin V Thephotodegradation of isoalloxazines in alcoholic solventsPhotochem Photobiol 197725347ndash56

43 Owen WR Stewart PJ Kinetics and mechanism of chlorambucilhydrolysis J Pharm Sci 197968992ndash6

44 Parker AJ Protic-dipolar aprotic solvent effects on rates of bi-molecular reactions Chem Rev 1969691ndash32

45 Peng Z HaiXia L SiDe Y WenFengW Effect of pH and polarityon the excited states of norfloxacin and its 4-N-acetyl derivative asteady state and time-resolved study Sci China Chem201457409ndash16

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46 Reichardt C Solvent effects on chemical reactivity Pure ApplChem 1982541867ndash84

47 Reichardt C Solvents and solvent effects in organic chemistry2nd ed New York VCH Publishers 1988

48 Roseman TJ Sims B Stehle RG Stability of prostaglandins AmJ Hosp Pharm 197330236ndash9

49 Schmid R Sapunov VN Non-formal kinetics in search of chem-ical reactions pathways (monograph in modern chemistry)Weinheim Verlag Chemie 1982 p 123ndash54

50 Schmidt WC Light-induced redox cycles of flavins in variousalcoholacetic acidmixtures PhotochemPhotobiol 198236699ndash703

51 Sheraz MA Kazi SH Ahmed S Anwar Z Ahmad I Photothermal and chemical degradation of riboflavin Beilstein J OrgChem 2014101999ndash2012

52 Sheraz MA Kazi SH Ahmed S Qadeer K Khan MF Multicom-ponent spectrometric analysis of riboflavin and photoproductsand their kinetic applications Cent Eur J Chem 201412635ndash42

53 Sikorska E Koziolowa A Sikorski M Siemiarczuk A The sol-vent effect on the excited state proton transfer of lumichrome JPhotochem Photobiol A Chem 20031575ndash14

54 Sikorski E Worrall DR Bourdelande JI Sikroski MPhotophysics of lumichrome and its analogs Polish J Chem20037765ndash73

55 Singh S Gupta RI Dielectric constant effects on degradation ofazothioprine in solution Int J Pharm 198846267ndash70

56 Sinko PJ Chemical kinetics and stability In Martinrsquos PhysicalPharmacy and Pharmaceutical Sciences 5th ed PhiladelphiaLippincott Williams amp Wilkins 2006 p 413ndash6

57 Song PS Metzler DE Photochemical degradation of FlavinsndashIVStudies of the anaerobic photolysis of riboflavin PhotochemPhotobiol 19676691ndash709

58 Szezesma V Koziol J Photolysis of flavin in carboxylic acids InOstrowski W editor Flavins and flavoproteins Physiochemicalproperties and functions Warsaw Polish Scientific Publishers1977 p 117ndash26

59 Tada H Decomposition reaction of hexamine by acid J AmChem Soc 196082255ndash63

60 Tan KL Phototherapy for neonatal jaundice Acta Paediatr199685277ndash9

61 Tonnesen HH Formulation and stability testing of photolabiledrugs Int J Pharm 20012251ndash14

62 Turro NJ Ramamurthy V Scaierno JC Modern molecular pho-tochemistry of organic molecules Sausalito University Science2010 p 469ndash74

63 Wypych G Hand book of solvents 2nd ed Toronto Chem TecPublishing 2001 p 577ndash81

64 Yeh MK Degradation kinetics of neostigmine in solution DrugDev Ind Pharm 2000261221ndash6

65 Yoshioka S Stella VJ Stability of drugs and dosage forms NewYork Kluwer AcademicPlenum Publishers 2000 p 102ndash4

Ionic strength effects on the photodegradation reactions of riboflavin inaqueous solution

Iqbal Ahmad a Zubair Anwar a Syed Abid Ali b Khwaja Ali Hasan b Muhammad Ali Sheraz a Sofia Ahmed a

a Baqai Institute of Pharmaceutical Sciences Baqai Medical University Toll Plaza Super Highway Gadap Road Karachi 74600 Pakistanb HEJ Research Institute of Chemistry University of Karachi Karachi 75270 Pakistan

a b s t r a c ta r t i c l e i n f o

Article historyReceived 18 September 2015Accepted 8 February 2016Available online 12 February 2016

A study of the effect of ionic strength on the photodegradation reactions (photoreduction and photoaddition) ofriboflavin (RF) in phosphate buffer (pH 70) has been carried out using a specific multicomponent spectrometricmethod It has been found that the rates of photodegradation reactions of RF are dependent upon the ionicstrength of the solutions at different buffer concentrations The apparent first-order rate constants (kobs) forthe photodegradation of riboflavin at ionic strengths of 01ndash05 (05 M phosphate) lie in the range of735ndash3032 times 10minus3 minminus1 Under these conditions the rate constants for the formation of the major productslumichrome (LC) by photoreduction pathway and cyclodehydroriboflavin (CDRF) by photoaddition pathwayare in the range of 380ndash1603 and 170ndash607 times 10minus3minminus1 respectively A linear relationship has been observedbetween log kobs and radicμ1 + radicμ A similar plot of log kko against radicμ yields a straight line with a value of ~+1 forZAZB showing the involvement of a charged species in the rate determining step NaCl appears to promote thephotodegradation reactions of RF probably by an excited state interaction The implications of ionic strengthon RF photodegradation by different pathways and flavinndashprotein interactions have been discussed

KeywordsRiboflavinPhotodegradation modePhotoproductsSpectrometric assayIonic strength effect

1 Introduction

The ionic strength of a solution can have a significant effect on therate of a chemical reaction and is known as the primary kinetic salteffect The relationship between the rate constant and the ionic strengthfor aqueous solution at 25 degC may be expressed by the BronstedndashBjerrum equation [12]

log k frac14 log ko thorn 102ZAZBradic μ eth1THORN

where ZA and ZB are the charges carried by the reacting species insolution μ the ionic strength k the rate constant of degradation andko the rate constant at infinite dilution A plot of log kko against radicμshould give a straight line of slope 102 ZAZB

Eq (1) is valid for ionic solutions up to μ = 001 At higher concen-trations (μ le 01) the BronstedndashBjerrum equation can be expressed as

log k frac14 log ko thorn 102ZAZBradic μ= 1thorn βradic μ

eth2THORN

In Eq (2) the value of β depends on the ionic diameter of thereacting species and is often approximated to unity

If the rate constants of a reaction are determined in the presence of aseries of different concentrations of the same electrolyte then a plot oflog k against radicμ is linear even in the case of solutions of high ionicstrength [3] The influence of ionic strength on the kinetics of drugdegradation and chemical reactions has been discussed by severalworkers [3ndash10] Ionic strength has been found to effect the aggregationkinetics of TiO2 [11] and the stability of Ag nanoparticles [12] The pri-mary salt effects on the rates and mechanism of chemical reactionshave been discussed [1314]

In drug degradation and stability studies the reactions are normallycarried out at a constant ionic strength tominimize its effect on the rateof reaction [15ndash20] However a large number of studies have beenconducted to evaluate the influence of ionic strength on the kinetics ofchemical [21ndash30] and photodegradation of drug substances [31] Theionic strength effects have important implications in photoinducedelectron transfer reactions and the binding ability of proteins to flavinspecies [32] Laser flash photolysis studies of the kinetics of electrontransfer between flavin semiquinone and fully reduced flavins andhorse rate cytochrome c have shown that the presence of a chargedphosphate group in the N-10 ribityl side chain leads to small ionicstrength effects on the rate constant whereas a charged group attachedto the dimethylbenzene ring produces a large ionic strength effect [33]Attempts have been made to describe the dependence of bimolecularrate constants on ionic strength for small molecules and protein interac-tions [33ndash38] A temperature dependent study of the effect of ionicstrength on the photolysis of riboflavin (RF) using a low intensity

Journal of Photochemistry amp Photobiology B Biology 157 (2016) 113ndash119

Corresponding authorE-mail address zubair_anahotmailcom (Z Anwar)

Contents lists available at ScienceDirect

Journal of Photochemistry amp Photobiology B Biology

j ourna l homepage wwwe lsev ie r com locate jphotob io l

lamp has been conducted In higher ionic strength phosphate buffer(031 M) an initial faster photolysis phase is observed that is followedby a slower second phase and vice versa in lower ionic strength buffer(005M) [39] In the presence of higher concentration (N01M) of diva-lent phosphate anions (HPO4

2minus) and pH values above 60 the normalcourse of RF photolysis (photoreduction) involving 10-dealkylation toform formylmethylflavin (FMF) lumiflavin (LF) and lumichrome (LC)[40] is shifted to photoaddition to yield cyclodehydroriboflavin (CDRF)[4142] The present study involves the evaluation of ionic strengtheffects on the photodegradation of RF with a change in the mode ofreaction at higher buffer concentrations These effects may significantlyinfluence the rates and mechanism of RF degradation reactions flavinndashprotein interactions and the kinetics of electron transfer reactions Thestudy of ionic strength effects is also necessary since the single andmul-tivitamin parenteral and total parenteral nutrition (TPN) preparationscontaining RF are isotonic and the amount of NaCl present (09 wv)may influence the stability of RF on photodegradation The effects ofionic strength on a change in the mode of photodegradation of RFneed to be investigated Some related work on the effect of factorssuch as pH [40] and buffer [4344] on the photodegradation of RF hasbeen reported

2 Materials and Methods

Riboflavin (RF) lumiflavin (LF) and lumichrome (LC) were obtainedfrom Sigma Chemical Co St Louis MO Formylmethylflavin (FMF) andcyclodehydroriboflavin (CDRF) were prepared by the methods of Falland Petering [45] and Schuman Jorns et al [41] respectively Thefollowing buffer system was used throughout (01ndash05 M) KH2PO4ndashK2HPO4 pH 70 the ionic strength was adjusted in the range 01ndash05 Mwith NaCl

21 Photodegradation

A 10minus4 M aqueous solution of RF (100 ml) at pH 70 (01ndash05 Mphosphate buffer) with varying ionic strength (01ndash05 at each bufferconcentration) was prepared in a Pyrex flask and placed in a waterbathmaintained at 25plusmn 1 degC The solution was irradiated with a PhilipsHPLN 125 W high pressure mercury vapor fluorescent lamp (emissionat 405 and 435 nm) fixed horizontally at a distance of 25 cm from thecenter of the flask The solution was continuously stirred by bubblinga stream of air in the flask Samples of the photolysed solution wereused at various intervals for spectrometric assay

22 Assay Method

The assay of RF in the photodegraded solutions was performed by afive component spectrometric method to avoid any interference of itsnormal photolysis (FMF LF LC) and photoaddition (CDRF) productsThe absorption spectra of RF and photoproducts the scheme of analysisand the details of the method have been reported [2046] The RSD ofthe method is within plusmn5

23 Light Intensity Measurements

Potassium ferrioxalate actinometry [47] was used to determine theintensity of the Philips HPLN 125 W high pressure mercury vaporfluorescent lamp (112 plusmn 011 times 1017 quanta sminus1) The lamp emits at405 436 545 and 578 nm and only the 405 and 436 nm bands areabsorbed by RF This amount to about 54 of the photon energy avail-able for absorption by RF on the basis of the spectral power distributionof the lamp

24 Fluorescence Measurements

The fluorescence intensity of RF solutions in the presence and ab-sence of NaCl was measured at room temperature at about (~25 degC)on a Spectromax 5 flourimeter (Molecular Devices USA) in the endpoint mode using λex = 374 nm and λem = 520 nm [48] The fluores-cence intensity was recorded in relative fluorescence units using apure 005 mM RF solution (pH 70) as a standard

3 Results and Discussion

31 Preliminary Considerations

The Philips HPLN 125 W high pressure mercury fluorescent lampemits in the visible region at 405 and 435 nm the latter band partiallyoverlaps the absorption maximum of RF at 445 nm [46] Therefore thelamp is suitable for the photolysis of RF and has been used in earlierstudies [4042ndash444950]

An important consideration in kinetic studies is the use of a specificassay procedure to determine the desired compound in the presence ofdegradation products Themulticomponent spectrometricmethod usedin this study is capable of simultaneous determination of RF and itsphotoreduction and photoaddition products with reasonable accuracy[20] It has previously been applied to the assay of these compoundsduring the kinetic studies of photodegradation of RF [2043444950]Such an analysis cannot be carried out rapidly by the HPLC methodThe assay of RF and photoproducts in a typical reaction carried out atpH 70 with an ionic strength of 05 is reported in Table 1 A goodmolar balance is obtained during the reaction indicating the accuracyof the method

The absorption spectra of RF determined during a photolysis reac-tion at pH 70with zero and 05 ionic strengths show a gradual decreasein absorbance at themaximumat 445 [2046] indicating a greater loss ofRF and an increase in absorbance around 356 nm [20] at 05 ionicstrength (Fig 1) There does not appear to be any drastic change inthe shape of the spectra in the presence of a high ionic strength of thesolution However at this ionic strength the magnitude of spectralchanges is affected for instance there is a greater decrease inabsorbance at 445 nm and a greater increase in absorbance at 356 nmcompared to that at zero ionic strength This supports the view that anincrease in ionic strength leads to an increase in the rate of photolysisreactions

A large number of studies have been conducted on the photo-degradation of RF under different conditions [2039ndash4246] It has beenestablished that the photolysis of RF in aqueous solution follows first-order kinetics [4042445152] In this study the effect of ionic strengthon the photodegradation of RF under different conditions has beenstudied Considering the photodegradation of RF as parallel first-order re-actions leading to the formation of LC (k1) and LF (k2) as final products by

Table 1Concentrations of RF and photoproducts (pH 70) at 05 M ionic strength

Time(min)

RF(M times 105)

CDRF(M times 105)

FMF(M times 105)

LC(M times 105)

LF(M times 105)

Total(M times 105)

0 500 00 00 00 00 50030 210 062 082 124 026 50460 078 088 110 179 043 49890 034 094 120 188 055 491120 013 099 122 198 074 506

114 I Ahmad et al Journal of Photochemistry amp Photobiology B Biology 157 (2016) 113ndash119

photoreduction and CDRF (k3) by photoaddition pathways the values ofthe rate constants k1 and k2 can be calculated as previously reported[2049] These reactions can be expressed as follows

The mathematical treatment to determine k1 k2 and k3 for these re-actions is given by Frost and Pearson [13] Using the concentrationvalues of RF LC LF and CDRF and RF0 for the initial concentration

ndashdRF=dt frac14 k1RFthorn k2RFthorn k3RF frac14 k1 thorn k2 thorn k3eth THORNRF frac14 kobsRF eth3THORN

kobs frac14 k1 thorn k2 thorn k3 eth4THORN

andln RF0=RFeth THORN frac14 kobst eth5THORN

RF frac14 RF0eminuskt eth6THORN

Similarly

dLC=dt frac14 k1RF0eminuskt eth7THORN

LC frac14 minusRF0eminuskt

kobsthorn constant eth8THORN

Fig 1 Absorption spectra of the photolysed solutions of RF (5 times 10minus5 M) at pH 70 (a) at zero and (b) at 05 ionic strength

115I Ahmad et al Journal of Photochemistry amp Photobiology B Biology 157 (2016) 113ndash119

LC frac14 LC0 thorn k1RF0kobs

1minuseminuskt

eth9THORN

LF frac14 LF0 thorn k2RF0kobs

1minuseminuskt

eth10THORN

CDRF frac14 CDRF0 thorn k3RFkobs

1minuseminuskt

eth11THORN

If LC= LF=CDRF=0 the equation simplifies and its is readily seenthat

LF=FC frac14 k2=k1 CDRF=LC frac14 k3=k1 eth12THORN

LC LF CDRF frac14 k1 k2 k3 eth13THORN

The products are in constant ratio to each other independent of timeand initial concentration of the reactant The method has been appliedto the determination of rate constants for all the three primary process-es in the pure liquid-phase pyrolysis of α-pinene [53]

The values of k1 k2 and k3 determined as a function of ionic strengthat different phosphate buffer concentrations along with k1k3 ratios arereported in Table 2 The values of k1 show a greater increase comparedto those of k3 with an increase in ionic strength at a constant buffer con-centration It has been observed that a change in k1k3 ratios in favor ofk1 occurs with a change in ionic strength This indicates that the ionicstrength has a greater effect on k1 (photoreduction pathway) leadingto the formation of LC The mechanism of promotion of the rate ofphotoaddition reactions (k3) of RF by Clminus ions is not clear

The values of apparent first-order rate constants (kobs) (Table 2) forthe overall photodegradation of RF in reactions carried out at a phos-phate buffer concentration of 01 M (photoreduction pathway) [40] in-dicate the effect of ionic strength on this particular reaction Howeverthe photodegradation reactions carried out at phosphate buffer concen-trations above 01 M involve both photoreduction and photoadditionpathways the latter due to buffer effect [4142] Under these conditionsthe values of kobs for RF would not distinguish the ionic strength effects

on the rates of the two distinct reactions where as the individual rateconstants (k1 k2 for photoreduction pathway and k3 for photoadditionpathway) would indicate the effect of ionic strength on these reactionsThe values of rate constants are relative and have been observed undercontrolled conditions of light intensity and other factors

RF exhibits an intense yellow green fluorescence at 520 nm in aque-ous solution [48] that vanishes in strongly acidic and alkaline solutionsdue to ionization [54] In order to observe the effect of NaCl on the fluo-rescence intensity of RF fluorescence measurements were made on5 times 10minus5 M RF solutions (pH 70) at different ionic strengths at constantbuffer concentrations (Fig 2) These results indicate that at a 0001 Mbuffer concentration there is a 334 to 422 loss of florescence at 01to 05 M ionic strength With an increase in buffer concentration (01ndash05 M) there is a gradual increase in the loss of florescence reaching avalue of 271 to 332 at 01 to 05 M ionic strength respectively in05 M buffer concentration Since phosphate buffer also quenches theflorescence of RF [42] a combined effect of buffer and NaCl is being ob-served at each buffer concentration with an increase in ionic strengthThis is in agreement with a previous observation that NaCl (01 M)quenches the fluorescence of RF solutions [55] Since the kinetic resultsshow an increase in ratewith an increase in ionic strength at each bufferconcentration the loss of florescence cannot be attributed exclusively tothe excited singlet state quenching and some interaction between RFand NaCl may be stipulated This could be analogous to the excited sin-glet state quenching of RF by complexation with HPO4

2minus ions leading tothe formation of CDRF by the photoaddition pathway [41] On the basesof the kinetic results it can be suggested that a similar mechanism mayoperate between RF and NaCl as explained below

In the present case RF on the absorption of light is promoted to theexcited singlet state [1RF] (14) [1RF] could react with Clminus ions to forman excited state complex (exciplex) as suggested for the exited state re-actions of organic compounds [56] (15) and observed in the case of[1RF-HPO4

2minus] complex leading to the formation of CDRF [20] In bothcases RF complexation with Clminus ions observed in the present study or

Table 2Apparent first-order rate constants (kobs) for the photodegradation of riboflavin in the presence of phosphate buffer (pH 70) at different ionic strengths (01ndash05 M) for the formation oflumichrome (k1) lumiflavin (k2) and cyclodehydroriboflavin (k3)

Buffer concentration(M)

Ionic strength(M)

kobs times 103

(minminus1)k0 times 103

minminus1)k2 times 103

(minminus1)k1k3

01 01 201 079 145 055 ndash ndash02 301 210 090 ndash ndash03 396 261 134 ndash ndash04 490 321 168 ndash ndash05 625 416 208 ndash ndash

02 01 276 085 139 063 072 19302 485 284 070 144 19703 715 407 102 198 20504 978 535 177 255 20905 1190 684 201 321 213

03 01 445 120 224 109 111 20102 825 425 151 185 22903 1185 632 240 265 23804 1505 835 253 345 24205 1860 1042 296 521 248

04 01 525 135 259 127 121 21402 1150 501 282 226 22103 1571 756 370 325 23204 2030 1115 487 466 23905 2491 1279 561 522 245

05 01 735 141 380 166 170 22202 1250 660 285 277 23803 1891 991 478 402 24604 2421 1220 615 482 25305 3032 1603 638 607 264

with HPO42minus ions [42] results in the quenching of fluorescence involving

the [1RF] state as well as an acceleration of the photodegradation pro-cess The role of Clminus ions appears to be analogous to that of theHPO4

2minus ions in promoting the rate of degradation of RF This wouldlead to the formation of the photoproducts of RF (eg LC) (16)

RFrarrhv 1RF

eth14THORN

1RF thorn NaC1rarr RFhelliphellipC11minus

exciplexthorn Nathorn eth15THORN

1RFhelliphellipC1minus rarrPhotoproducts eth16THORN

Clminus appears to form a non-fluorescent complex with the groundstate RFmolecule by static quenching as suggested in the case of quinine[57] Thus the role of Clminus ions in the photodegradation of RF is topromote the degradation of RF by different pathways

In order to correlate the rate constants for the photodegradation ofRF by photoreduction (LC LF) and photoaddition (CDRF) pathwayswith ionic strength the log values of rate constants (kobs) were plottedagainst radicμ1 + radicμ (Eq (2)) which yielded straight lines indicating alinear relationship Extrapolation to zero ionic strength yielded thevalue for k0 the rate constant for the photodegradation of RF at zeroionic strength (Fig 3) Further plots of log k1k0 and k3k0 against radicμ(Eq (1)) gave straight lines with a positive slope of 102 ZAZB (Fig 4)

shown for a typical photodegradation reaction of RF at 05M buffer con-centration (ionic strength 01ndash05 M) The rate constant k2 for the for-mation of LF by photoreduction pathway is a minor reaction and hasbeen neglected The number of unit charges ZAZB can be calculatedfrom the slope of the plots

ZAZB frac14 105=102 frac14 103 frac14 thorn1 for k1eth THORN

ZAZB frac14 082=102 frac14 081 frac14 thorn080 ethfor k3 THORN

The values of ZAZB (+1) for photoreduction suggest that a chargedspecies is involved in the rate determining step of the reaction (k1) Ithas been earlier suggested by flash photolysis experiments that theflavin triplet reduction takes place via a dipolar intermediate [58] asfollows

eth22THORN

Thedegree towhich this intermediate proceeds to form the productswould be affected by the interaction with NaCl at a particular ionicstrength The higher the ionic strength the greater the interactionleading to degradation and hence an increase in the rate of reaction Apositive slope of the reaction indicates an increase in the rate of reactionbetween similarly charged species as a result of an increase in the ionicstrength of the solution The degradation of RF by the photoadditionpathway also involves the participation of a charged species in theform of a [1RFndashHPO4

2minus] complex Although Eq (1) is essentially truefor dilute solutions an effect due to ionic strength is in fact observedat higher concentrations [3] as found in the present case Since thevalue of ZAZB for the photoaddition reaction (k3) is 080 This value isnot an integer suggesting a complex mode of reaction between RFbuffer species and Clminus ions

It has been suggested [41] that the photoaddition pathway is notaffected by ionic strength These authors studied the analytical photo-chemistry of RF by absorbance changes at the λmax at 445 nm Theiranalytical datamay not be reliable due to the fact that all the photoprod-ucts of RF absorb at this wavelength and an accurate assay of RF is notpossible Thus any kinetic data obtained may not represent the truerate constants for the reactions involved The present study involves aspecific analytical method to determine RF accurately in the presenceof various photoproducts and therefore the rate constants derivedfrom such analytical data would be reliable as reported in severalprevious studies [2043444950]

Fig 2 Plots of fluorescence intensity of RF solutions (pH 70) versus ionic strength at 0001ndash05 M buffer concentration

The effect of ionic strength has also been observed in studies carriedout on the photolysis of RF and related reactions under conditionsdifferent from those of the present work These include the biphasicphotolysis of RF in the ionic strength range of 003ndash046 M usingphosphate buffer (pH 74) [39] the photolysis of RF in the presence ofmagnesium perchlorate at pH 70 [41] and the alkaline hydrolysis of67-dimethyl-9-formylmethylisoalloxazine (an intermediate in thephotolysis of RF) under various conditions of ionic strength and pH[52] Ionic strength effects play a significant role in studies involvingflavinndashprotein interactions A charged phosphate group attached tothe dimethylbenzene ring of flavins has been found to produce a largeionic strength effect on the rate of interaction [33] The kinetics ofelectron transfer reactions and the binding ability of flavins to proteinsare dependent upon the ionic strength due to electrostatic interactions[33ndash355960] and may be significantly influenced at large values ofionic strength

4 Conclusion

The photodegradation pathways of RF in aqueous solution (photore-duction and photoaddition) are significantly influenced by ionic strengthThe log k against radicμ1+ radicμ and the log kko against radicμ plots for the reac-tions are linear A charged species (ZAZB=+1) appears to be involved inthe rate determining step of these reactions Clminus ionsmay reactwith RF inthe excited state to promote the photodegradation reactions The ionicstrength effects on drug degradation rates and flavinndashprotein interactionscould be considerable Therefore the control of ionic strength is necessaryin kinetic studies to avoid such effects

References

[1] JN Bronsted Die Bedeutung des Aktivitaumlts Begriffs fuumlr die chemische Reaktionsgeschwindigkeit Z Phys Chem 102 (1922) 169ndash207

[2] N Bjerrum Zur theorie der chemischen reaktionsgeshwindigkeit Z Phys Chem108 (1924) 82ndash100

[3] AT Florence D Attwood Physicochemical Principles of Pharmacy fourth edPharmaceutical Press London 2006 120ndash122

[4] L Lachman P DeLuca MJ Akers Kinetic principles and stability testing in LLachman HA Liberman JL Kanig (Eds) The Theory and Practice of IndustrialPharmacy third edLea amp Febiger Philadelphia 1986 pp 769ndash770

[5] JT Carstensen Kinetics pH profiles in JT Carstensen CT Rhodes (Eds) DrugStability Principles and Practices third edMarcel Dekker New York 2000(pp 58ndash60 65ndash67)

[6] JK Guillory RI Poust Chemical kinetics and drug stability in GS Banker CTRhodes (Eds) Modern Pharmaceutics fourth edMarcel Dekker New York 2002pp 158ndash159

[7] PJ Sinko Martins Physical Pharmacy and Pharmaceutical Sciences fifth edLippincott Williams amp Wilkins Baltimore 2006 414ndash415

[8] S Yoshioka VJ Stella Stability of Drugs and Dosage Forms Kluwer AcademicPlenum Publishers New York 2000 99ndash102

[9] KJ Laidler Chemical Kinetics third ed Harper amp Row New York 1987 197ndash206[10] WH Koppenol Effect of a molecular dipole on the ionic strength dependence of a

bimolecular rate constant Biophys J 29 (1980) 493ndash508[11] RA French AR Jacobson B Kim SL Isley RL Penn PC Baveye Influence of ionic

strength pH and cation valence on aggregation kinetics of titanium dioxidenanoparticles Environ Sci Technol 43 (2009) 1354ndash1359

[12] M El Badawy TP Luxton RG Silva KG Scheckel MT Suidan TM TolaymatImpact of environmental conditions (pH ionic strength and electrolyte type) onthe surface charge and aggregation of silver nanoparticles suspensions EnvironSci Technol 44 (2010) 1260ndash1266

[13] A Frost RG Pearson Kinetics and Mechanism second ed John Wiley New York1964 (pp 150ndash155 160ndash162)

[14] G Corsaro Salt and solvent effects on reaction mechanism J Chem Educ 54 (1977)483ndash484

[15] G Sankara CB Navarre UB Kompella Influence of pH and temperature on kineticsof ceftiofur degradation in aqueous solution J Pharm Pharmacol 51 (1999)249ndash255

[16] M Stankovicova Z Bezakova L Benes Kinetics of hydrolysis of acetyl veleroyl andnicotinoyl acyl derivatives of stobadine Life Sci 65 (1999) 2007ndash2010

[17] MK Yeh Degradation kinetics of neostigmine in solution Drug Dev Ind Pharm 26(2000) 1221ndash1226

[18] R Chadha N Kashid DV Jain Kinetics of degradation of diclofenac sodium inaqueous solution determined by a calorimetric method Pharmazie 58 (2003)631ndash635

[19] M Jumaa B Carlson L Chimilio S Silchenko VJ Stella Kinetics and mechanism ofdegradation of epothilone an experimental anticancer drug J Pharm Sci 93 (2004)2953ndash2961

[20] I Ahmad Q Fasihullah FHM Vaid A study of simultaneous photolysis andphotoaddition reactions of riboflavin in aqueous solution J Photochem PhotobiolB Biol 75 (2004) 13ndash20

[21] Y Pramar VD Gupta Preformulation studies of spironolactone effect of pH twobuffer species ionic strength and temperature on stability J Pharm Sci 80 (1991)551ndash553

[22] MA Hoitink JH Beijnen A Bult OAGJ van der Houwen J Nijholt WJNUnderberg Degradation kinetics of gonadorelin in aqueous solution J Pharm Sci85 (2000) 1053ndash1059

[23] JA Zang J Pawelchak Effect of pH ionic strength and oxygen burden on thechemical stability of EPCcholesterol liposomes under accelerated conditions Part1 lipid hydrolysis Eur J Pharm Biopharm 50 (2000) 357ndash364

[24] C Matos H Chaimovich JLFC Lima IM Cuccovia S Reis Effect of liposomes onthe rate of alkaline hydrolysis of indomethacin and acemetacin J Pharm Sci 90(2001) 298ndash309

[25] A Miranda I Caraballo M Millan Stability study of flutamide in solid state and inaqueous solution Drug Dev Ind Pharm 28 (2002) 413ndash422

[26] G Alibrandi S Coppolino S DAliberti P Ficarre N Micali A Villari Variable-ionicstrength kinetic experiments in drug stability studies J Pharm Sci 92 (2003)1730ndash1733

[27] J-PK Salo J Yli-Kauhaluoma H Salomies On the hydrolytic behaviour oftinidazole metronidazole and ornidazole J Pharm Sci 92 (2003) 739ndash746

[28] GG Aloisi A Barbafina M Canton F DallAcqua F Elisei L Facciolo L Latterini GViola Photophysical and photobiological behaviour of antimalarial drugs in aqueoussolution Photochem Photobiol 79 (2004) 248ndash258

[29] F Lallemand P Perotter O Felt-BaeyensW Kloeti F Philippoz J Marfurt K BesseghirR Gurny A water-soluble prodrug of cyclosporine A for ocular application a stabilitystudy Eur J Pharm Sci 26 (2005) 124ndash129

[30] J Rexroad RK Evans CR Middough Effect of pH and ionic strength on the physicalstability of adenovirous type 5 J Pharm Sci 95 (2006) 237ndash247

[31] SR Khattak D Shaikh I Ahmad K Usmanghani MA Sheraz S AhmedPhotodegradation and stabilization of betamethasone-17 valerate in aqueousorganicsolvents and topical formulations AAPS PharmSci Tech 14 (2012) 177ndash182

[32] S Fukuzumi T Tanaka Flavins and deazaflavins in MA Fox M Chanon(Eds) Photoinduced Electron Transfer Part C Elsevier Amsterdam 1988pp 636ndash688

[33] I Ahmad MA Cusanovich G Tollin Laser flash photolysis studies of electrontransfer between semiquinone and fully reduced free flavins and horse heartcytochrome c Proc Natl Acad Sci U S A 78 (1981) 6724ndash6728

[34] I Ahmad MA Cusanovich G Tollin Laser flash photolysis studies of electrontransfer between semiquinone and fully reduced free flavins and the cytochromecndashcytochrome oxidase complex Biochemistry 21 (1982) 3122ndash3128

[35] JT Hazzard TL Poulos G Tollin Kinetics of reduction of free flavin semiquinone ofthe components of the cytochrome cndashcytochrome c peroxidase complex andintracomplex electron transfer Biochemistry 26 (1987) 2836ndash2848

[36] JT Hazzard SJ Moench JE Erman JD Satterlee G Tollin Kinetics of intracomplexelectron transfer and the reduction of the components of covalent and noncovalentcomplexes of cytochrome c and cytochrome c peroxidase by free flavin semiquinoneBiochemistry 27 (1988) 2002ndash2008

[37] JA Watkins MA Cusanovich TE Meyer G Tollin A ldquoparallel platerdquo electrostaticmodel for bimolecular rate constants applied to electron transfer proteins ProteinSci 3 (1994) 2104ndash2114

[38] D Zhong AH Zewail Femtosecond dynamics of flavoproteins charge separationand recombination in riboflavin (vitamin B2)-binding protein and in glucoseoxidase enzyme Proc Natl Acad Sci U S A 98 (2001) 11867ndash11872

[39] Y Sato H Chaki Y Suzuki Biphasic photolysis of riboflavin III Effects of ionicstrength on the photolysis Chem Pharm Bull (Jpn) 32 (1984) 1232ndash1235

[40] I Ahmad Q Fasihullah A Noor IA Ansari QNM Ali Photolysis of riboflavin inaqueous solution a kinetic study Int J Pharm 280 (2004) 199ndash208

[41] M Schuman Jorns G Schollnhammer P Hammerich Intramolecular addition of theriboflavin side chain Anion-catalysed neutral photochemistry Eur J Biochem 57(1975) 35ndash48

[42] I Ahmad Q Fasihullah FHM Vaid Effect of phosphate buffer on photodegradationreactions of riboflavin in aqueous solution J Photochem Photobiol B Biol 78(2005) 229ndash234

[43] I Ahmad T Mirza K Iqbal S Ahmed MA Sheraz FHM Vaid Effect of pH bufferand viscosity on the photolysis of formylmethylflavin a kinetic study Aust JChem 66 (2013) 579ndash585

[44] I Ahmad Z Anwar K Iqbal SA Ali T Mirza A Khurshid A Khurshid A ArsalanEffect of acetate and carbonate buffers on the photolysis of riboflavin in aqueoussolution a kinetic study AAPS PharmSci Tech 15 (2015) 550ndash559

[45] HH Fall HG Petering Metabolic inhibitors 1 67-Dimethyl-9formylmethylisoalloxazine 67-dimethyl-9-(2-hydroxyethyl) isoalloxazineand derivatives J Am Chem Soc 78 (1956) 377ndash381

[46] I Ahmad HDC Rapson Multicomponent spectrophotometric assay of riboflavinand photoproducts J Pharm Biomed Anal 8 (1990) 217ndash223

[47] CG Hatchard CA Parker A new sensitive chemical actinometer II Potassiumferrioxalate as a standard chemical actinometer Proc Roy Soc (Lond) A 235(1956) 518ndash536

[48] United States Pharmacopeia 30National Formulary 25 United States PharmacopeialConvention Inc Rockville MD 2007 Electronic version

[49] I Ahmad S Ahmed MA Sheraz FH Vaid IA Ansari Effect of divalent anions onphotodegradation kinetics and pathways of riboflavin in aqueous solution Int JPharm 390 (2010) 174ndash182

[50] I Ahmad S Ahmed MA Sheraz M Aminuddin FHM Vaid Effect of caffeinecomplexation on the photolysis of riboflavin in aqueous solution a kinetic studyChem Pharm Bull (Japan) 57 (2009) 1363ndash1370

[51] I Ahmad Z Anwar S Ahmed MA Sheraz R Bano A Hafeez Solvent effect on thephotolysis of riboflavin AAPS PharmSciTech 16 (2015) 1122ndash1128

[52] P-S Song EC Smith DE Metzler Photochemical degradation of flavins II Themechanism of alkaline hydrolysis of 67-dimethyl-9-formylmethylisoalloxazineJ Am Chem Soc 87 (1965) 4181ndash4184

[53] RE Fuguitt JE Hawkins Rate of thermal isomerization of α-pinene in the liquidphase J Am Chem Soc 69 (1947) 319ndash322

[54] G Weber Fluorescence of riboflavin and flavin-adenine dinucleotide Biochem J 47(1950) 114ndash121

[55] P Ellinger M Holden Quenching effect of electrolytes on the fluorescence intensityof riboflavin and thiochrome Biochem J 38 (1944) 147ndash150

[56] N J Turro V Ramamurthy J C Scaiano Modern Molecular Photochemistry ofOrganic Molecules University Science Books Sausalito CA (pp 253-254 458-461)

[57] JH Gutow Halide (Clminus) quenching of quinine sulfate fluorescence a time-resolvedfluorescence experiment for physical chemistry J Chem Edu 82 (2005) 302ndash305

[58] I Ahmad G Tollin Solvent effects on flavin electron transfer reactions Biochemistry20 (1981) 5925ndash5928

[59] TE Meyer JA Watkins CT Przysiecki G Tollin MA Cusanovich Electron-transferreactions of photoreduced flavin analogues with c-type cytochromes quantitationof steric and electrostatic factors Biochemistry 23 (1984) 4761ndash4767

[60] JK Hurley JT Hazzard M MartinezndashJulvez M Medina C GomezndashMoreno GTollin Electrostatic forces involved in orienting Anabaena ferredoxin during bindingto Anabaena ferredoxin NADP+ reductase site-specific mutagenesis transient ki-netic measurements and electrostatic surface potentials Protein Sci 8 (1999)1614ndash1622

Journal of Photochemistry and Photobiology B Biology

Available online 29 May 2017

In Press Accepted Manuscript mdash Note to users

Metal ion mediated photolysis reactions of riboflavin A kinetic study

Iqbal Ahmada

Zubair Anwara

Sofia Ahmeda

Muhammad Ali Sheraza

Saif-ur-Rehman Khattakb Show more

httpsdoiorg101016jjphotobiol201705033 Get rights and content

Highlights

Metal ion complexation alters the redox reactivity of riboflavin (RF) on photolysis

Photolysis of RF complexes is enhanced by electron transfer to RF in excited state

Reactivity of metal ion on RF photolysis is affected by phosphate concentration

Metal ions influence the kinetics of photoreduction and photoaddition pathways of RF

The study could throw light on the redox reactivity of RF in biological systems

Abstract

The effect of metal ion complexation on the photolysis of riboflavin (RF) using various metal ions (Ag+ Ni2 + Co2 + Fe2 + Ca2 + Cd2 + Cu2 + Mn2 + Pb2 + Mg2 + Zn2 + Fe3 +) has been studied Ultraviolet and visible spectral and fluorimetric evidence has been obtained to confirm the formation of metal-RF complexes The kinetics of photolysis of RF in metal-RF complexes at pH 70 has been evaluated The apparent first-order rate constant (kobs) for the photolysis of RF and the formation of lumichrome (LC) and lumiflavin (LF) (0001 M phosphate buffer) and LC LF and cyclodehydroriboflavin (CDRF) (02ndash04 M phosphate buffer) have been determined The values of kobs indicate that the rate of photolysis of RF is promoted by divalent and trivalent metal ions The second-order rate constants (kprime) for the interaction of metal ions with RF are in the order

Zn2 + gt Mg2 + gt Pb2 + gt Mn2 + gt Cu2 + gt Cd2 + gt Fe2 + gt Ca2 + gt Fe3 + gt Co2 + gt Ni2 + gt Ag+ In phosphate buffer (02ndash04 M) an increase in the metal ion concentration leads to a decrease in the formation of LC compared to that of CDRF by different pathways The photoproducts of RF have been identified and RF and the photoproducts have simultaneously been assayed by a multicomponent spectrometric method The mode of photolysis of RF in metal-RF complexes has been discussed

Graphical Abstract

BIODATA

Qualifications

Pharm D Baqai Medical University Karachi 2011

M Phil Baqai Medical University Karachi 2013

R Ph Pharmacy Council of Pakistan

Impact Factor 17001

Publications

Chapters

1 Anwar Z Khurshi Aq Khurshid Ad Ahmed S Baig QEN Ahmad I

Nanoparticles Physicochemical Properties Characterization Methods of

Preparation and Applications In Bartul A Trenor J Eds Advances in

Nanotechnology Nova Science Publishers USA 2017 (In Press)

2 Zuberi SA Sheraz MA Ahmed S Anwar Z Ali SA Ahmad I

Nanosponges Characteristics Methods of Preparation and Applications In

Bartul A Trenor J Eds Advances in Nanotechnology Nova Science

Publishers USA 2017 (In Press)

3 Ahmad I Bano R Sheraz MA Ahmed S Qadeer K Anwar Z Analytical

Methods for the Determination of Fluoroquinolones in Pharmaceutical

Preparations Biological Fluids and Degraded Solutions In Berhardt LV

Ed Advances in Medicine and Biology Nova Science Publishers USA

2017 (In Press)

Reviews

4 Arsalan A Anwar Z Ahmad I Shad Z Ahmed S Cronobacter sakazakii An

emerging contaminant in Pediatric infant milk formula Int Res J Pharm

2013417ndash22

5 Arsalan A Anwar Z Ahmad I Saba A Baqar S Naqvi S Microbes in

pediatric infant formula Annals Food Sci Technol 20131490ndash99

6 Anwar Z Arsalan A Khurshid Ad Khurshid Aq Ahmad I Helicobacter

pylori A major causative organism of peptic ulcer and its eradication J

Baqai Med Univ 20131441ndash49

7 Khurshid Aq Khurshid Ad Anwar Z Arsalan A Ahmad I Influenza virus

Infections and their treatment J Baqai Med Univ 20131465ndash71

8 Khurshid Ad Khurshid Aq Anwar Z Arsalan A Ahmad I (2013) The

prospects of vitamin c in cancer therapy J Baqai Med Univ 20131451ndash58

9 Arsalan A Naqvi SBS Ali SI Anwar Z Contamination of microorganisms

in pediatric infant formula marketed in Karachi Annals Food Sci Technol

201314 318ndash326

10 Sheraz MA Kazi SH Ahmed S Anwar Z Ahmad I Photo thermal and

chemical degradation of riboflavin Beilstein J Org Chem 2014101999ndash

11 Arsalan A Alam M Naqvi SB Ahmad I Anwar Z Oxygen as a facilitator

in the reduction of surgical site infections Sri Lanka J Surgery 201431

12 Gul W Anwar Z Qadeer K Perveen S Ahmad I Method of analysis of

riboflavin (vitamin B2) A review J Pharma Pharma Sci 20140210ndash21

13 Khurshid Ad Anwar Z Khurshid Aq Ahmad I Ascorbic acid Clinical use

and method of analysis Baqai J Health Sci 20151615ndash19

vitamin K A review J Pharma Pharma Sci 2015114ndash21

15 Gul W Anwar Z Khurshid A Khurshid A Ahmad I Ascorbic acid method

of analysis J Pharma Pharma Sci 201531ndash18

16 Anwar Z Baig QEN Khurshid Ad Khurshid Aq Ahmad I Peptic ulcer

diseases Pathogenesis and diagnosis Baqai J Health Sci 20151821ndash24

17 Shaikh S Anwar Z Mirza T Khurshid A Khurshid A Ahmad I Total

parenteral nutrition (TPN) Role of riboflavin (vitamin B2) and

cyanocobalamin (vitamin B12) Baqai J Health Sci 20151831ndash47

18 Baig QEN Anwar Z Ahmad I Qadeer K Silicosisndasha major occupational

threat Baqai J Health Sci 2015186ndash10

19 Mirza T Anwar Z Shaikh S Ahmad I Photochemical reactions of

formylmethylflavin and riboflavin Baqai J Health Sci 201518 30ndash34

20 Baig QEN Bano R Arsalan A Anwar Z Ahmad I Anaylsis of amino acids

by high performance liquid chromatography Baqai J Health Sci

20161951ndash57

21 Ahmad I Ahmed S Anwar Z Sheraz MA Sikorski M Photostability and

Research Papers

22 Ahmad I Anwar Z Iqbal K Ali SA Mirza T Khurshid A Khurshid A

Arsalan A Effect of acetate and carbonate buffers on the photolysis of

riboflavin in aqueous solution a kinetic study AAPS PharmSciTech

201415550ndash559

23 Anwar Z Mirza T Khurshid Ad Khurshid Aq Ahmad I (2014)

Photodegradation of Riboflavin In acetate buffer Baqai J Health Sci

2014153ndash7

24 Ahmad I Abbas SH Anwar Z Sheraz MA Ahmed S Arsalan A Bano R

Stabilityndashindicating photochemical method for the assay of riboflavin

lumichrome method J Chem 20152015

25 Ahmad I Arsalan A Ali SA Sheraz MA Ahmed S Anwar Z Munir I Shah

MR Formulation and stabilization of riboflavin in liposomal preparations J

26 Ahmad I Anwar Z Ahmed S Sheraz MA Bano R Hafeez A Solvent effect

on the photolysis of riboflavin AAPS PharmSciTech 2015161122ndash1128

27 Ahmad I Ahmed S Sheraz MA Anwar Z Qadeer K Noor A Evstigneev

MP Effect of Nicotinamide on the Photolysis of Riboflavin in Aqueous

Solution Scientia Pharmaceutica 201584289ndash304

28 Ahmad I Anwar Z Ali SA Hasan KA Sheraz MA Ahmed S Ionic strength

effects on the photodegradation reactions of riboflavin in aqueous solution J

29 Ahmad I Anwar Z Ahmed S Sheraz MA Khattak SUR Metal ion

mediated photolysis reactions of riboflavin A kinetic study J Photochem

Photobiol B Biol 2017 (In Press)

30 Ahmad I Anwar Z Ali SA Shah R Farid MA Ahmed S Photochemical

preparation characterization and formation kinetics of riboflavin conjugated

silver nanoparticles (Under Preparation)

01Front-Pagespdf

02Chapter-Ipdf

03Chapter-IIpdf

04Chapter-IIIpdf

05Chapter-IVpdf

06OBJECT-OF-PRESENT-INVESTIGATIONpdf

07Chapter-Vpdf

08Chapter-VIpdf

09Chapter-VIIpdf

10Chapter-VIIIpdf

11Chapter-IXpdf

12CONCLUSIONSpdf

13Referencespdf

14Solvent Effect on the Photolysis of Riboflavinpdf


Abstract

INTRODUCTION


Photolysis

pH Measurements


Spectrometric Assay


RESULTS

Photoproducts of RF




DISCUSSION

Effect of Solvent


Effect of Viscosity

Mode of Photolysis

Conclusion

References

15Ionic-Strength-Effect (1)pdf

Ionic strength effects on the photodegradation reactions of riboflavin in aqueous solution

1 Introduction


21 Photodegradation

22 Assay Method









4 Conclusion

References

16Metalpdf

17BIODATApdf

AUTHORS DECLARATION

I Zubair Anwar hereby state that my PhD thesis titled ldquoEffect of Solvent Ionic

Strength and Metal Ions on the Photolysis of Riboflavin and its Nanoparticlesrdquo is my own

work and has not been submitted previously by me for taking any degree from Baqai

Medical University or anywhere else in the countryworld

At any time if my statement is found to be incorrect even after my Graduation the

university has the right to withdraw my PhD degree

Name of Student Zubair Anwar

referred cited

Name Zubair Anwar

_____________________________

ABSTRACT

minminus1

ions (Ag+ Ni

3+) has been

2+ gt Mg

gt Mn2+

gt Cu2+

gt Cd2+

gt Fe2+

gt Ca2+

gt Fe3+

gt Co2+

gt Ni2+

2920 cmndash1

ACKNOWLEDGEMENTS

(Quran 20114)

institution

and my niece

Anushay Zeeshan

CONTENTS

Chapter Page

ABSTRACT vi

ACKNOWLEDGEMENTS ix

I INTRODUCTION

11 INTRODUCTION 2

15 CLINICAL USES 8

31 INTRODUCTION 38

3472 pH effect 60

41 INTODUCTION 71

422 Stabilizer 74

424 Biosensor 76

51 MATERIALS 86

52 REAGENTS 88

53 METHODS 89

and Photoproducts

61 INTRODUCTION 106

AQUEOUS SOLUTION

71 INTRODUCTION 135

81 INTRODUCTION 165

Solutions

91 INTRODUCTION 217

9221 pH 232

CONCLUSIONS 248

REFERENCES 252

and 120 min

RF () FMF () LC ()

(5 times 10ndash5

M) in water (pH 70)

acetonitrile

methanol

ethanol

1ndashpropanol

1ndashbutanol

ethyl acetate

M () 005 M (times) 01 M () 02 M (∆) 03 M () 04 M ()

buffer

M) (pH 70) (____

) in the

ions (b) Zn2+

ions and (c) Cu2+

() Ni2+

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

ions () Mg2+

() Zn2+

ions and () Fe3+

M) (pH 70) (a)

3 M) (b2) RF + Fe

2+ ions (2 times 10

ndash3 M) (b3)

ions at different

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams)

ions () Pb2+

ions () Mg2+

ions () Zn2+

ions () Fe3+

green to brown

300 min (purple)

RFndashAg NPs

green)

(pink)

UV light

visible light

cmminus1

) of RF and

Photoproducts

(pH 70)

Phosphate buffer

5) (0001

5) (02

5) (04

CHAPTER I

11 INTRODUCTION

12 BIOCHEMICAL ROLE

CH2O P

Thyroid Harmone

Empirical formula

C17H20N4O6

Molar mass 3764

pKa 19 102 (20o)

Redox potential

Water 33ndash606

1070 cmndash1

15 CLINICAL USES

Kafai 2004)

17 THERAPEUTIC USES

et al 1996)

18 PHARMACOKINETICS

RF (Nath 2000)

Chapters in Books

Reviews

(1998)

Physiochemical Data

CHAPTER II

RELATED COMPOUNDS

mg Lndash1

009 microg mLndash1

045 microg mLndash1

et al 2006)

Vosough 2002)

wavelength

(Hustad et al 1999)

4 times 10ndash2

secndash1

mol Lndash1

(Li et al 1992)

10 microg Lndash1

(Garcia et al 2001)

(Zhang et al 2009)

et al 2009)

al 2008)

mobile phase

500 nmol mlndash1

05 ml minndash1

of RF in RF

0007ndash01

15ndash45

microg mlndash1

10 ml minndash1

(Valls et al 1999)

) as the mobile

with LOD

of 05 ng Lndash1

50 times 10ndash8

mol Lndash1

to 2 times 10ndash4

mol Lndash1

15 times 10ndash6

15 times 10ndash4

mol Lndash1

with a LOD of

10 times 10ndash8

mol Lndash1

(Yang et al 2001)

to 3 times 10ndash6

mol Lndash1

25 ENZYMATIC ASSAYS

10 times 10ndash10

mol Lndash1

CHAPTER III

31 INTRODUCTION

1980 2004ab 2005 2006ab 2008 2009 2010ab 2011 2013 2014 2015ab 2016ab

SO42ndash

) RF undergoes

are discussed below

(8) (5)

(7) (6)

r phot

oadditi

341 Photoreduction

discussed below

and Metzler 1971)

Fl hv 1Fl

FlH + R1Fl + RH

Fl- + RH+1Flo + RH

Fl hv 1Fl

R + CO2RCOO-

radical addition

HFl + HFl

H2Flred + Flox

ProdcutsR

RFlredHHFl + R

1FloFl hv

1Fl oFlic

1Fl 3Flisc

3Fl oFl+ heat

(Eq (316))

oFlred + O2

oFl + H2O2

and Knappe 1974)

pH 70hv

(1) (5)

SO42ndash

344 Photooxidation

RFhv 1RF

3RF1RF isc

3RF RF + 3O2

and Min 2006)

ndash1s

ndash1 and

ndash1s

8 and 821 times 10

ndash1s

8 and 527times10

ndash1s

ndash1 It has

ndash is

CndashRF and 14

Cndash

(Reddy 2008)

qsndash1

3472 pH effect

et al 2007)

2004a)

3473 Buffer effect

2ndash species

(02 M ge)

tartarte succinate

as follows

RF [1RF] LC

hv H2PO4-

[3RF][1RF] RFH2

RFHPO4

RF-HPO42- hv [1RF]

complex

0 ∆cS

Jesus et al 2012)

et al 2004)

et al 2015a)

1991 Loukas 2001)

CHAPTER IV

41 INTODUCTION

1ndash100 nm ndash

American Society of

(ASTM)

1ndash100 nm

Health (NIOSH)

nanoscale

nanoscale range

Arbeitsschutz und

nanoscale range

nanostructure or a

nanosubstance

421 Photosenstizer

state 13

C and 29

422 Stabilizer

ions This

K+) (Xu et al

424 Biosensor

to 10times 10ndash4

molcm2 and

Mndash1

sndash1

(Zhang et al 2011)

Ag+ etc)

CHAPTER V

51 MATERIALS

24-dione Merck

C17H20N4O6 Mr 3764

C13H12N4O2 Mr 2563

C12H10N4O2 Mr 2423

C14H12N4O3 Mr 2843

C14H12N4O4 Mr 3003

al 1980)

C17H18O6N4 Mr 3744

Metal Salts

follows

CoSO47H2O (999)

52 REAGENTS

53 METHODS

change

(Merck)

al 1980)

(Ahmad et al1980)

532 pH Measurements

to 400 cm-1

A (1 1 cm) at 444 nm = 328

arc lamp

A 10minus4

A 5 times 10ndash5

M) were

Photoproducts

385 nm

photolysis)

at 445 385 and 410 nm

photolysis)

356 nm

cmminus1

following equation

A1 = 1a1 1C (51)

A1 = 1ε1 1C (52)

A1 = 1ε1 1C + 2ε1 2C (53a)

A2 = 1ε2 1C + 2ε2 2C (53b)

and 3C

A1 = 1ε1 1C + 2ε1 2C + 3ε1 3C (55a)

A2 = 1ε2 1C + 2ε2 2C + 3ε2 3C (55b)

A3 = 1ε3 1C + 2ε3 2C + 3ε3 3C (55c)

λ1 A1 1 ε 1 1C + 2 ε 1 2C + 3 ε 1 3C

λ2 A2 1 ε 2 1C + 2 ε 2 2C + 3 ε 2 3C

λ3 A3 1 ε 3 1C + 2 ε 3 2C + 3 ε 3 3C

A1 1ε1 2ε1 3ε1 1C

A2 = 1ε2 2ε2 3ε2 = 2C

A3 1ε3 2ε3 3ε3 3C

(AM) (ASM) (CM)

A1 2ε1 3ε1 1ε 1 2ε 1 3ε1

1C = A2 2ε2 3ε2 1ε2 2ε2 3ε2

A3 2ε3 3ε3 1ε3 2ε3 3ε3

1 ε 1 A1 3 ε 1

1ε 1 2ε 1 3ε1

2C = 1 ε 2 A2 3 ε 2 1ε2 2ε2 3ε2

1 ε 3 A3 3 ε 3 1ε3 2ε3 3ε3

1ε1 2ε1 A1

1ε 1 2 ε 1 3 ε 1

3C = 1ε2 2ε2 A2 1 ε 2 2 ε 2 3 ε 2

1ε3 2ε3 A3 1 ε 3 2 ε 3 3 ε 3

A1 2ε2 3ε2

2ε3 3ε3

ndash 2 ε 1

A2 3ε 2

A3 3ε3

+ 3 ε 1

A2 2ε2

A3 2ε3

ASM expanded

CHAPTER VI

RIBOFLAVIN

61 INTRODUCTION

53 (Chapter 5)

045 Non-

(Ahmad et al 1980)

and 120 min

250 300 400 500 600

Wavelength (nm)

method

(Mtimes 105)

0 300 000 000 000 300

30 263 028 008 004 305

60 229 060 012 007 308

90 197 078 023 009 309

120 173 086 030 012 311

(Mtimes 105)

0 300 000 000 300

30 269 023 012 304

60 239 040 021 308

90 213 058 031 304

120 194 066 045 311

(Mtimes 105)

0 300 00 00 300

30 255 036 015 306

60 215 058 029 308

90 201 071 032 306

120 191 079 037 312

(Mtimes 105)

0 300 000 000 300

30 273 017 014 306

60 245 032 024 310

90 223 042 036 308

120 199 049 052 306

(Mtimes 105)

0 300 000 000 300

30 268 020 015 305

60 245 031 028 307

90 223 040 039 304

120 202 049 050 302

(Mtimes 105)

0 300 000 000 300

30 269 022 012 303

60 245 037 022 304

90 222 052 031 307

120 204 060 039 309

(Mtimes 105)

0 300 000 000 300

30 275 017 011 308

60 251 031 023 309

90 227 037 039 306

120 208 046 050 304

method

minminus1

(water)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times10

Time (min)

M) in water

(pH 70)

acetonitrile

0 20 40 60 80 100 120 140

Time (min)

times10

0 20 40 60 80 100 120 140

Time (min)

M times

methanol

ethanol

0 20 40 60 80 100 120 140

Time (min)

times10

0 20 40 60 80 100 120 140

M times

Time (min)

1ndashpropanol

1ndashbutanol

0 20 40 60 80 100 120 140

times10

Time (min)

0 20 40 60 80 100 120 140

Time (min)

M times

ethyl acetate

0 20 40 60 80 100 120 140

times10

Time (min)

Solvents Dielectric

constant (isin)

(25 oC)

Acceptor

Number

Inverse

viscosity

(mPasndash1

(25 oC)

kobs times 103 min

ndash1

plusmnSDa

Ethanol 243 371 0931 345plusmn015

Water 785 548 1123 461plusmn025

() water

00 100 200 300 400 500 600 700 800

Dielectric constant

s times

() water

00 100 200 300 400 500 600 -70

s times

10 15 20 25 05 30 00

Viscosity (mPa s)-1

times 1

00 05 10 15 20 25 30 35

Viscosity (mPas)-1

the medium

3FL + AH (F

σndash+) FLH (61)

CHAPTER VII

71 INTRODUCTION

B radicmicro (71)

where ZA and Z

Corsaro 1977)

reported

53 (Chapter 5)

(M times105)

0 500 00 00 00 500

30 451 019 021 010 501

60 398 039 045 019 506

90 373 053 059 022 507

120 340 064 071 027 508

(M times105)

0 500 00 00 00 500

30 446 020 022 016 504

60 386 044 049 021 508

90 332 069 073 029 509

120 309 076 081 035 501

(M times105)

0 500 00 00 00 500

30 435 020 031 016 502

60 381 039 052 029 505

90 331 065 071 035 508

120 288 078 089 046 501

(M times105)

0 500 00 00 00 500

30 417 031 035 020 503

60 361 054 058 031 504

90 308 069 082 043 507

120 269 081 099 052 508

(M times105)

0 500 00 00 00 500

30 404 032 044 022 502

60 336 056 075 036 505

90 290 068 097 047 507

120 245 079 118 059 501

(M times 105)

0 500 00 00 00 00 500

30 435 015 016 039 008 513

60 378 026 028 048 020 508

90 329 035 046 071 030 511

120 280 048 060 092 042 522

(M times 105)

0 500 00 00 00 00 500

30 416 024 036 057 006 539

60 353 040 059 075 016 543

90 293 079 081 134 028 615

120 251 089 091 175 034 640

(M times 105)

0 500 00 00 00 00 500

30 386 023 032 059 006 500

60 307 040 056 083 014 511

90 239 059 069 119 021 516

120 194 064 081 131 033 503

(M times 105)

0 500 00 00 00 00 500

30 369 030 036 062 009 506

60 280 045 060 093 023 501

90 217 060 073 122 033 509

120 153 071 089 145 048 506

(M times 105)

0 500 00 00 00 00 500

30 338 036 046 074 009 503

60 238 055 081 112 014 510

90 164 064 116 131 027 502

120 119 073 126 149 037 504

(M times 105)

0 500 00 00 00 00 500

30 398 016 031 045 010 510

60 327 031 055 066 022 508

90 267 042 065 085 041 503

120 224 050 076 101 049 506

(M times 105)

0 500 00 00 00 00 500

30 367 027 037 056 013 504

60 286 047 051 096 020 511

90 221 059 069 120 031 513

120 178 057 082 139 044 509

(M times 105)

0 500 00 00 00 00 500

30 354 024 049 059 014 504

60 236 049 069 108 038 508

90 168 068 076 139 049 503

120 108 078 096 158 060 509

(M times 105)

0 500 00 00 00 00 500

30 295 040 051 100 015 506

60 160 056 108 143 033 505

90 097 069 121 168 045 502

120 076 075 132 177 051 506

(M times 105)

0 500 00 00 00 00 500

30 282 046 060 106 006 511

60 145 076 088 154 037 505

90 079 091 104 175 051 509

120 052 100 110 200 057 507

(M times 105)

0 500 00 00 00 00 500

30 397 029 026 035 017 504

60 309 036 049 076 037 507

90 239 048 061 105 051 504

120 180 067 075 126 062 508

(M times 105)

0 500 00 00 00 00 500

30 361 029 042 047 023 508

60 256 048 056 095 047 512

90 183 061 077 118 063 502

120 127 073 095 145 071 514

(M times 105)

0 500 00 00 00 00 500

30 314 032 050 075 035 506

60 195 055 090 113 050 513

90 130 070 108 133 062 508

120 075 085 130 145 071 506

(M times 105)

0 500 00 00 00 00 500

30 292 042 052 079 039 504

60 148 069 083 135 066 511

90 078 093 103 155 076 509

120 042 103 114 163 084 506

(M times 105)

0 500 00 00 00 00 500

30 217 049 070 113 055 504

60 113 060 096 157 074 509

90 057 073 106 178 086 511

120 024 082 117 187 093 506

(M times 105)

0 500 00 00 00 00 500

30 425 013 028 027 009 502

60 338 032 041 065 024 509

90 251 045 074 091 043 514

120 157 066 085 135 059 512

(M times 105)

0 500 00 00 00 00 500

30 313 041 046 085 019 506

60 214 056 068 115 047 509

90 140 072 085 150 057 506

120 099 081 096 164 067 507

(M times 105)

0 500 00 00 00 00 500

30 298 037 062 075 030 506

60 179 061 079 125 056 511

90 099 076 097 155 075 502

120 049 088 108 169 087 508

(M times 105)

0 500 00 00 00 00 500

30 249 049 068 099 036 501

60 099 071 118 145 067 509

90 049 082 128 167 077 506

120 023 088 137 178 086 512

(M times 105)

0 500 00 00 00 00 500

30 210 062 086 126 026 508

60 078 088 112 179 049 506

90 034 094 120 190 069 509

120 013 099 132 201 080 511

photoproducts

been studied

M) at pH 70

kobs= k1+ k2+ k3 (74)

RF = RF0 endashkt

Similarly

) (79)

) (710)

) (711)

k2 RF0 kobs

k3 RF0 kobs

ndash RF0 endashkt

k1 RF0

is not clear

other factors

5times10minus5

Buffer

Concentration

Strength

kobs times 103

(minndash1

k0 times 103

(minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

02 01 276 085 139 063 072 193

02 485 284 070 144 197

03 715 407 102 198 205

04 978 535 177 255 209

05 1190 684 201 321 213

03 01 445 120 224 109 111 201

02 825 425 151 185 229

03 1185 632 240 265 238

04 1505 835 253 345 242

05 1860 1042 296 521 248

04 01 525 135 259 127 121 214

02 1150 501 282 226 221

03 1571 756 370 325 232

04 2030 1115 487 466 239

05 2491 1279 561 522 245

05 01 735 141 380 166 170 222

02 1250 660 285 277 238

03 1891 991 478 402 246

04 2421 1220 615 482 253

05 3032 1603 638 607 264

() 005 M (times) 01 M () 02 M (∆) 03 M () 04 M () 05 M

0 01 02 03 04 05 06

Ionic Strength (M)

ions leading to the

of [1RFndashHPO4

2minus

RF [1RF] (714)

[1RF] + NaCl [

1RFhelliphellipCl

ndash] + Na

+ (715)

[1RF helliphellipCl

pathways

of the plots

B = 105 102 = 103 = ~ + 1 (for k1)

B = 161 102 = 157 = ~ + 160 (for k3)

exciplex

000 010 020 030 040

radicμ1 + radicμ

000 010 020 030 040 050 060 070 080

radicμ

] (717)

+H ndashH+

CHAPTER VIII

81 INTRODUCTION

) (Mortland

additi

photoreduction(1)

(4) (5)

H+ OH-

and Cd2+

and Zn2+

(Kaim et al 1999)

promoted by Fe3+

and Cu2+

(inhibited by

(1) (81)

Rearrangment

4a5 44a

and Zn2+

+ RH M(nndash1)+

+ H+ + R

˙ (81)

+ H+ + RʹO2

˙ (82)

reaction

(Chapter 5)

and Cu2+

11 RFndashFe2+

RF + 2Fe2+ +2HRFH2 + 2Fe3+

M) (pH 70) (____

ions (b) Zn2+

ions and (c) Cu2+

and Cu2+

ions appears to

complex formation

presence of Zn2+

and Cu2+

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 485 008 489 006 486 007 489 006 490 005

120 470 014 477 012 472 015 478 014 479 012

180 447 026 454 023 458 020 466 019 468 018

0 500 000 500 000 500 000 500 000 500 000

60 472 015 463 017 457 019 450 019 446 022

120 442 032 431 037 421 035 413 038 398 046

180 416 045 398 049 384 052 371 059 355 066

0 500 000 500 000 500 000 500 000 500 000

60 474 014 461 018 453 022 442 026 432 030

120 450 027 424 032 413 036 393 047 373 055

180 418 039 389 044 365 055 346 065 324 076

0 500 000 500 000 500 000 500 000 500 000

60 472 014 462 017 442 025 429 030 421 033

120 444 024 423 033 395 044 375 052 354 061

180 414 039 385 052 352 067 322 075 295 086

0 500 000 500 000 500 000 500 000 500 000

60 474 015 465 018 464 019 459 021 441 028

120 450 024 436 029 430 031 415 036 389 048

180 427 036 407 045 393 051 358 062 339 068

0 500 000 500 000 500 000 500 000 500 000

60 474 015 459 019 450 022 440 026 427 032

120 450 026 422 038 403 044 386 051 363 065

180 417 041 381 056 355 066 338 071 309 081

0 500 000 500 000 500 000 500 000 500 000

60 489 006 475 013 472 014 472 014 467 016

120 465 016 449 024 446 026 443 027 437 029

180 437 032 427 036 419 039 414 041 408 045

0 500 000 500 000 500 000 500 000 500 000

60 475 014 467 017 463 018 457 021 451 023

120 449 024 434 030 429 033 411 040 406 042

180 426 035 407 040 390 047 374 054 363 060

0 500 000 500 000 500 000 500 000 500 000

60 472 015 463 019 453 022 442 025 433 030

120 443 029 428 035 412 042 390 051 373 057

180 416 039 394 047 371 057 342 068 322 076

0 500 000 500 000 500 000 500 000 500 000

60 474 014 473 016 463 019 457 021 441 028

120 447 027 444 028 428 034 416 039 390 051

180 429 036 411 042 391 051 375 057 346 068

0 500 000 500 000 500 000 500 000 500 000

60 478 011 476 013 472 015 466 017 464 019

120 454 022 450 024 442 027 436 029 430 032

180 433 030 423 034 414 039 405 043 399 048

0 500 000 500 000 500 000 500 000 500 000

60 476 013 473 019 464 020 463 020 457 022

120 451 022 444 026 430 033 431 039 416 042

ndash4 M

Table 81 continued

5) (02 M Phosphate

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 448 014 457 014 457 014 465 014 467 013

120 405 015 416 015 416 015 424 015 436 014

180 363 017 374 016 381 015 395 014 408 013

0 500 000 500 000 500 000 500 000 500 000

60 425 015 416 015 416 016 398 016 389 016

120 369 017 346 018 338 019 323 019 309 020

180 322 019 298 020 279 021 257 023 245 025

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 407 016 407 016 387 016

120 371 017 363 017 338 019 323 020 300 020

180 319 019 302 020 279 023 259 024 234 026

0 500 000 500 000 500 000 500 000 500 000

60 416 015 398 016 380 017 371 017 352 018

120 346 018 323 019 295 021 275 022 255 024

180 291 021 257 023 229 027 203 029 177 034

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 421 015 416 015 406 016

120 380 017 363 017 354 018 338 018 320 020

180 331 019 310 020 298 021 279 022 262 024

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 015 416 015 396 016

120 380 017 363 017 346 018 338 018 323 019

180 328 019 308 020 295 022 274 023 256 025

0 500 000 500 000 500 000 500 000 500 000

60 454 014 426 015 426 015 426 015 416 015

120 406 015 371 017 363 017 354 018 338 018

180 367 019 316 022 311 023 295 025 281 026

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 015 407 016 396 016

120 381 017 363 017 346 018 331 019 323 020

180 334 019 311 020 293 022 274 027 251 029

0 500 000 500 000 500 000 500 000 500 000

60 436 015 416 015 407 016 398 016 381 017

120 371 017 346 018 331 019 316 020 293 022

180 319 019 291 022 266 023 244 025 228 028

0 500 000 500 000 500 000 500 000 500 000

60 426 015 426 015 407 016 398 016 361 018

120 371 017 354 018 338 019 323 019 262 024

180 320 021 299 025 279 028 259 031 189 037

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 016 416 016 406 016

120 381 017 363 017 354 018 346 018 330 019

180 328 021 314 024 299 025 286 029 273 033

0 500 000 500 000 500 000 500 000 500 000

60 426 015 416 015 421 015 416 015 404 016

120 371 017 354 017 354 018 346 018 330 020

ndash4 M

Table 82 continued

5) (04 M Phosphate

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 416 011 421 010 428 009 436 009 447 007

120 347 027 354 026 369 021 389 018 402 015

180 292 034 303 030 319 028 343 026 359 021

0 500 000 500 000 500 000 500 000 500 000

60 403 018 372 026 375 026 358 030 347 033

120 325 024 282 037 276 038 251 045 244 046

180 252 032 216 042 194 044 171 053 165 055

0 500 000 500 000 500 000 500 000 500 000

60 393 019 381 020 375 021 358 024 347 026

120 307 028 289 031 276 033 254 038 244 041

180 237 037 215 039 200 041 181 044 170 048

0 500 000 500 000 500 000 500 000 500 000

60 375 016 347 022 334 024 319 027 295 022

120 272 030 246 033 219 035 195 036 182 035

180 200 040 167 045 143 048 122 051 103 061

0 500 000 500 000 500 000 500 000 500 000

60 393 011 381 015 384 015 375 016 364 018

120 319 025 298 029 289 031 276 033 263 036

180 251 033 233 036 221 038 209 041 197 043

0 500 000 500 000 500 000 500 000 500 000

60 393 011 381 014 384 014 375 017 364 021

120 317 022 298 025 289 027 276 031 263 035

180 251 029 229 032 223 034 207 037 194 039

0 500 000 500 000 500 000 500 000 500 000

60 393 014 384 016 384 016 375 018 364 020

120 303 022 298 023 289 025 276 027 263 029

180 241 031 229 033 221 035 203 037 191 039

0 500 000 500 000 500 000 500 000 500 000

60 393 013 384 015 384 015 367 017 345 018

120 315 019 298 021 289 023 272 026 237 029

180 255 025 225 029 215 033 198 035 169 039

0 500 000 500 000 500 000 500 000 500 000

60 393 011 375 018 367 019 347 021 337 025

120 302 022 282 026 26 030 242 035 231 038

180 237 033 207 036 188 038 169 041 155 044

0 500 000 500 000 500 000 500 000 500 000

60 393 014 375 019 367 020 347 022 337 024

120 302 019 282 025 266 029 242 032 231 034

180 234 027 213 031 171 041 171 043 159 047

0 500 000 500 000 500 000 500 000 500 000

60 393 013 384 014 367 019 363 019 350 022

120 309 019 295 021 272 026 263 027 251 031

180 242 026 226 029 207 032 195 034 183 036

0 500 000 500 000 500 000 500 000 500 000

60 393 016 381 019 375 021 358 022 347 024

120 315 027 289 033 276 034 254 038 244 040

ndash4 M

Table 83 continued

ions is shown in

in the order

2+lt Fe

3+ lt Ca

2+ +lt Fe

2+ lt Cd

2+ lt Cu

2+lt Mn

2+lt Pb

2+ lt Mg

2+lt Zn

2+lt Ag

Thus Ni2+

ions (∆) Co2+

(loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

() Mg2+

ions () Zn2+

ions and () Fe3+

00 10 20 30 40 50 60

M) (b2)

RF + Fe2+

M) (b3)

2014 2016 Sheraz et al 2014)

RF + Fe2+ RF-Fe2+

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

log co

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ag+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ag+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

Metal Ions (kʹ)

Metal Ion Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

00 063 016 006

Ag+ 10 059 050 041 017

20 054 038 015

30 049 035 014

40 044 033 010

50 038 029 008

10 089 256 070 018

20 115 080 034

30 142 101 040

40 169 129 039

50 191 143 047

10 099 360 078 020

20 136 084 051

30 172 107 064

40 206 138 067

50 243 164 078

10 105 462 073 031

20 155 113 041

30 199 138 060

40 245 164 080

50 294 190 094

10 101 416 071 029

20 142 099 042

30 184 131 052

40 225 160 064

50 271 182 088

10 106 410 079 026

20 145 105 039

30 185 128 056

40 224 152 071

50 268 180 087

10 075 104 058 016

20 085 062 022

30 095 068 026

40 105 075 029

50 115 083 031

10 089 232 063 025

20 112 075 036

30 136 092 043

40 158 106 051

50 179 120 058

10 102 360 072 029

20 132 089 042

30 167 110 056

40 210 140 070

50 243 162 081

10 091 284 069 021

20 118 086 031

30 148 104 043

40 176 122 053

50 205 139 065

10 078 128 054 023

20 091 063 027

30 104 071 032

40 116 080 035

50 127 087 039

10 082 180 060 021

20 099 075 023

30 118 091 026

40 135 151 029

50 153 174 035

Table 84 continued

Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

00 204 111 038 054 205

Ag+ 10 182 184 125 027 028 446

20 164 112 025 026 430

30 144 094 023 025 376

40 127 084 019 023 365

50 112 072 016 022 327

10 243 384 195 020 027 722

20 285 231 022 031 724

30 325 256 032 035 726

40 363 291 033 040 728

50 396 315 036 043 730

10 249 410 201 021 027 724

20 285 229 025 031 726

30 325 256 034 035 728

40 365 290 036 039 730

50 409 329 033 045 732

10 285 742 226 027 031 729

20 358 283 036 038 733

30 435 343 043 048 736

40 505 402 048 054 738

50 575 446 059 060 741

10 235 246 180 024 029 620

20 265 201 030 032 628

30 295 223 036 034 655

40 325 245 039 036 671

50 358 286 035 041 697

10 235 334 180 024 029 620

20 269 207 029 033 625

30 302 228 035 036 629

40 335 243 044 038 633

50 371 284 045 044 637

10 227 232 149 035 042 354

20 260 179 032 049 360

30 283 195 035 053 360

40 304 210 038 056 365

50 332 230 041 061 369

10 235 358 178 025 030 593

20 270 207 029 034 605

30 305 231 035 037 624

40 334 253 041 040 631

50 373 284 045 044 636

10 251 462 196 025 031 625

20 301 233 031 036 647

30 345 268 036 039 687

40 385 303 041 043 699

50 427 333 048 046 711

10 254 410 179 032 043 411

20 285 201 039 043 467

30 323 231 044 048 475

40 362 259 049 054 479

50 404 289 056 059 483

10 236 256 168 029 039 425

20 255 184 032 038 484

30 280 204 034 040 510

40 300 220 038 042 519

50 319 232 043 044 523

10 237 308 189 021 026 726

20 271 218 024 029 730

30 302 238 030 032 734

40 332 265 030 036 736

50 358 284 036 038 738

Table 85 continued

Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

00 351 222 049 075 296

Ag+ 10 315 332 184 061 069 263

20 280 171 042 066 259

30 247 152 035 059 257

40 214 129 033 051 252

50 190 114 030 046 247

10 402 528 262 059 079 331

20 462 290 070 101 287

30 515 310 094 109 284

40 570 335 098 136 246

50 615 363 104 147 246

10 407 496 259 069 079 325

20 460 295 072 092 320

30 509 328 077 103 317

40 560 357 089 113 315

50 599 373 099 126 296

10 475 1048 302 075 096 314

20 580 359 106 115 310

30 681 414 128 137 302

40 784 475 151 158 299

50 875 505 173 196 257

10 390 348 257 058 073 352

20 425 275 066 082 335

30 458 296 071 090 328

40 490 315 075 099 318

50 525 335 082 107 313

10 386 348 273 050 061 447

20 427 301 057 068 442

30 458 321 060 075 428

40 490 336 068 084 400

50 525 355 077 091 390

10 387 508 254 058 073 347

20 424 273 069 081 337

30 494 317 080 096 330

40 545 347 089 107 324

50 605 380 104 119 319

10 389 600 271 057 060 451

20 426 287 070 067 428

30 494 327 080 085 384

40 545 359 089 095 377

50 651 432 103 116 370

10 415 600 282 057 075 376

20 475 318 071 085 374

30 535 363 074 098 370

40 605 405 090 110 366

50 651 423 109 117 361

10 413 570 287 060 065 441

20 470 320 072 077 415

30 530 337 091 101 333

40 590 370 102 116 318

50 636 392 110 132 296

10 395 414 273 059 061 447

20 438 296 069 071 416

30 479 321 076 081 396

40 524 350 084 089 393

50 558 369 093 095 388

10 405 468 260 055 083 313

20 455 290 072 093 310

30 505 322 077 104 309

40 548 346 086 115 300

50 585 363 093 128 283

Table 86 continued

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

ions () Mg2+

ions ()

ions () Fe3+

00 30 60 90

Fluorescence loss

bs times

minndash1

) (Table

minndash1

) (Table 86) The

Mndash1

minndash1

) and monovalent

order Zn2+

gt Mg2+

gt Pb2+

gt Mn2+

gt Cu2+

gt Cd2+

gt Fe2+

gt Ca2+

gt Fe3+

gt Co2+

gt Ni2+

gt Ag+

ions (Rutter 1958)

) bind to RF

presented (Fig 844)

] complex (Eq

state [1RFhellipFe

2+] state

(Eq (812))

RF + Fe2+ [RFFe2+]

metal-RF complex

[RFFe2+] [1RFFe2+]

[1RFFe2+] [RFHFe3+]

[RFHFe3+] RFH

+ Fe3+

2RFH RFH2

H+ OH_

ions and

CHAPTER IX

91 INTRODUCTION

(1)(4)

(5)(7) (6)

and alkaline pH

(Chapter 5)

to brown

9214 FTIR studies

RFndashAg NPs

0 1 2 3 4 5 6 7

Time (h)

cmndash1

distributed

measurements

9221 pH

NPs (Fig 99)

9222 Ionic strength

+ ions

questionable

91 and 92

0 50 100 150 200 250 300 350 400

Time (min)

k1times 103

k2 times 103

(min-1

k1times 103

(min-1

k2 times 103

(min-1

80 736 0060 521 0036

85 999 0088 843 0061

90 1285 0110 1122 0091

95 1523 0129 1324 0112

100 1740 0147 1480 0128

105 1822 0153 1524 0135

ion Concentration

k1times 103

k2 times 103

(min-1

k1times 103

(min-1

k2 times 103

(min-1

0002 754 0012 355 0009

0004 892 0022 622 0020

0006 1279 0046 1088 0043

0008 1630 0072 1399 0052

001 1740 0079 1444 0054

750 800 850 900 950 1000 1050 1100

k1times

k2 times

750 800 850 900 950 1000 1050 1100

k1times

k2 times

0 0002 0004 0006 0008 001 0012

k1times

k2 times

0 0002 0004 0006 0008 001 0012

k1times

k2 times

RF1RFhv

3RFisc1RF

RF3RF + Ag+

+ Ag NP

0RFRF + H+

RF3RF + 0RF + RFH

+ ion to form RF

0RF] Further

0RF] lead to

] radical (Eq (95)

CONCLUSIONS

solvents

HPO42-

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1953190ndash94

2004932953ndash2961

Rev 1999182323ndash342

Chem B 2003107668-677

201314177ndash182

20124053ndash58

2007240ndash47

19751082422ndash2432

198029493ndash507

20048015ndash21

197ndash206 279-280

1960 132433ndash455

19594877ndash84

200190298ndash309

Rev 1989691170ndash1198

200321111ndash121

1983471577ndash1582

201194467ndash 481

Res 2014162291ndash2298

201289196ndash202

1969917170ndash7179

198331435ndash439

199449203ndash206

197643469ndash81

Electronic Version

196218249ndash253

Sci 197968992ndash996

201513345ndash51

201140211ndash223

197118B479ndash506

201352300ndash2306

VCH Publishers 1988

Sci 1972611081ndash1085

197330236ndash239

1982 pp 123ndash154

19755735ndash48

1995240313ndash322

20031575ndash14

2006 pp 413ndash416

Soc1963853285ndash3288

200863545ndash546

200964428ndash435

2011b69167ndash172

19771071269ndash1276

20012251ndash14

254 458ndash461 469ndash474

1979271039ndash1042

2013151555

Res 198013148ndash155

195047114ndash121

B 20141162872ndash2882

577ndash581

2000261221ndash1226

1975250946ndash951

19966354ndash66

2014159282ndash286

Research Article

INTRODUCTION

Photolysis

pH Measurements

Spectrometric Assay

RESULTS

Photoproducts of RF

Ahmad et al

DISCUSSION

Effect of Solvent

0 30 60 90 120

on times

Time (min)

Time(min)

RF(Mtimes105)

FMF(Mtimes105)

LC(Mtimes105)

Total(Mtimes105)

0 300 00 00 30030 255 036 015 30660 215 058 029 30290 201 071 032 304120 191 079 037 307

Effect of Viscosity

00 100 200 300 400 500 600 700 800

times10

inndash1

Dieletric Constant

Ahmad et al

Mode of Photolysis

CONCLUSION

00 05 10 15 20 25 30

bstimes

103 (m

inndash1

Viscosity (mPa s)-1

00 100 200 300 400 500 600lnk o

bs times

REFERENCES

Ahmad et al

1 Introduction

eth2THORN

21 Photodegradation

22 Assay Method

Time(min)

RF(M times 105)

CDRF(M times 105)

FMF(M times 105)

LC(M times 105)

LF(M times 105)

Total(M times 105)

0 500 00 00 00 00 50030 210 062 082 124 026 50460 078 088 110 179 043 49890 034 094 120 188 055 491120 013 099 122 198 074 506

Similarly

1minuseminuskt

eth9THORN

1minuseminuskt

eth10THORN

1minuseminuskt

eth11THORN

Ionic strength(M)

kobs times 103

(minminus1)k1k3

02 01 276 085 139 063 072 19302 485 284 070 144 19703 715 407 102 198 20504 978 535 177 255 20905 1190 684 201 321 213

03 01 445 120 224 109 111 20102 825 425 151 185 22903 1185 632 240 265 23804 1505 835 253 345 24205 1860 1042 296 521 248

04 01 525 135 259 127 121 21402 1150 501 282 226 22103 1571 756 370 325 23204 2030 1115 487 466 23905 2491 1279 561 522 245

05 01 735 141 380 166 170 22202 1250 660 285 277 23803 1891 991 478 402 24604 2421 1220 615 482 25305 3032 1603 638 607 264

RFrarrhv 1RF

eth14THORN

eth22THORN

4 Conclusion

References

Iqbal Ahmada

Zubair Anwara

Sofia Ahmeda

Highlights

Abstract

Graphical Abstract

BIODATA

Qualifications

Impact Factor 17001

Publications

Chapters

2017 (In Press)

Reviews

2013417ndash22

201314 318ndash326

20161951ndash57

Research Papers

201415550ndash559

2014153ndash7

01Front-Pagespdf

02Chapter-Ipdf

03Chapter-IIpdf

04Chapter-IIIpdf

05Chapter-IVpdf

07Chapter-Vpdf

08Chapter-VIpdf

09Chapter-VIIpdf

10Chapter-VIIIpdf

11Chapter-IXpdf

12CONCLUSIONSpdf

13Referencespdf


Abstract

INTRODUCTION


Photolysis

pH Measurements


Spectrometric Assay


RESULTS

Photoproducts of RF




DISCUSSION

Effect of Solvent


Effect of Viscosity

Mode of Photolysis

Conclusion

References



1 Introduction


21 Photodegradation

22 Assay Method









4 Conclusion

References

16Metalpdf

17BIODATApdf

referred cited

Name Zubair Anwar

_____________________________

ABSTRACT

minminus1

ions (Ag+ Ni

3+) has been

2+ gt Mg

gt Mn2+

gt Cu2+

gt Cd2+

gt Fe2+

gt Ca2+

gt Fe3+

gt Co2+

gt Ni2+

2920 cmndash1

ACKNOWLEDGEMENTS

(Quran 20114)

institution

and my niece

Anushay Zeeshan

CONTENTS

Chapter Page

ABSTRACT vi

ACKNOWLEDGEMENTS ix

I INTRODUCTION

11 INTRODUCTION 2

15 CLINICAL USES 8

31 INTRODUCTION 38

3472 pH effect 60

41 INTODUCTION 71

422 Stabilizer 74

424 Biosensor 76

51 MATERIALS 86

52 REAGENTS 88

53 METHODS 89

and Photoproducts

61 INTRODUCTION 106

AQUEOUS SOLUTION

71 INTRODUCTION 135

81 INTRODUCTION 165

Solutions

91 INTRODUCTION 217

9221 pH 232

CONCLUSIONS 248

REFERENCES 252

and 120 min

RF () FMF () LC ()

(5 times 10ndash5

M) in water (pH 70)

acetonitrile

methanol

ethanol

1ndashpropanol

1ndashbutanol

ethyl acetate

M () 005 M (times) 01 M () 02 M (∆) 03 M () 04 M ()

buffer

M) (pH 70) (____

) in the

ions (b) Zn2+

ions and (c) Cu2+

() Ni2+

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

ions () Mg2+

() Zn2+

ions and () Fe3+

M) (pH 70) (a)

3 M) (b2) RF + Fe

2+ ions (2 times 10

ndash3 M) (b3)

ions at different

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams)

ions () Pb2+

ions () Mg2+

ions () Zn2+

ions () Fe3+

green to brown

300 min (purple)

RFndashAg NPs

green)

(pink)

UV light

visible light

cmminus1

) of RF and

Photoproducts

(pH 70)

Phosphate buffer

5) (0001

5) (02

5) (04

CHAPTER I

11 INTRODUCTION

12 BIOCHEMICAL ROLE

CH2O P

Thyroid Harmone

Empirical formula

C17H20N4O6

Molar mass 3764

pKa 19 102 (20o)

Redox potential

Water 33ndash606

1070 cmndash1

15 CLINICAL USES

Kafai 2004)

17 THERAPEUTIC USES

et al 1996)

18 PHARMACOKINETICS

RF (Nath 2000)

Chapters in Books

Reviews

(1998)

Physiochemical Data

CHAPTER II

RELATED COMPOUNDS

mg Lndash1

009 microg mLndash1

045 microg mLndash1

et al 2006)

Vosough 2002)

wavelength

(Hustad et al 1999)

4 times 10ndash2

secndash1

mol Lndash1

(Li et al 1992)

10 microg Lndash1

(Garcia et al 2001)

(Zhang et al 2009)

et al 2009)

al 2008)

mobile phase

500 nmol mlndash1

05 ml minndash1

of RF in RF

0007ndash01

15ndash45

microg mlndash1

10 ml minndash1

(Valls et al 1999)

) as the mobile

with LOD

of 05 ng Lndash1

50 times 10ndash8

mol Lndash1

to 2 times 10ndash4

mol Lndash1

15 times 10ndash6

15 times 10ndash4

mol Lndash1

with a LOD of

10 times 10ndash8

mol Lndash1

(Yang et al 2001)

to 3 times 10ndash6

mol Lndash1

25 ENZYMATIC ASSAYS

10 times 10ndash10

mol Lndash1

CHAPTER III

31 INTRODUCTION

1980 2004ab 2005 2006ab 2008 2009 2010ab 2011 2013 2014 2015ab 2016ab

SO42ndash

) RF undergoes

are discussed below

(8) (5)

(7) (6)

r phot

oadditi

341 Photoreduction

discussed below

and Metzler 1971)

Fl hv 1Fl

FlH + R1Fl + RH

Fl- + RH+1Flo + RH

Fl hv 1Fl

R + CO2RCOO-

radical addition

HFl + HFl

H2Flred + Flox

ProdcutsR

RFlredHHFl + R

1FloFl hv

1Fl oFlic

1Fl 3Flisc

3Fl oFl+ heat

(Eq (316))

oFlred + O2

oFl + H2O2

and Knappe 1974)

pH 70hv

(1) (5)

SO42ndash

344 Photooxidation

RFhv 1RF

3RF1RF isc

3RF RF + 3O2

and Min 2006)

ndash1s

ndash1 and

ndash1s

8 and 821 times 10

ndash1s

8 and 527times10

ndash1s

ndash1 It has

ndash is

CndashRF and 14

Cndash

(Reddy 2008)

qsndash1

3472 pH effect

et al 2007)

2004a)

3473 Buffer effect

2ndash species

(02 M ge)

tartarte succinate

as follows

RF [1RF] LC

hv H2PO4-

[3RF][1RF] RFH2

RFHPO4

RF-HPO42- hv [1RF]

complex

0 ∆cS

Jesus et al 2012)

et al 2004)

et al 2015a)

1991 Loukas 2001)

CHAPTER IV

41 INTODUCTION

1ndash100 nm ndash

American Society of

(ASTM)

1ndash100 nm

Health (NIOSH)

nanoscale

nanoscale range

Arbeitsschutz und

nanoscale range

nanostructure or a

nanosubstance

421 Photosenstizer

state 13

C and 29

422 Stabilizer

ions This

K+) (Xu et al

424 Biosensor

to 10times 10ndash4

molcm2 and

Mndash1

sndash1

(Zhang et al 2011)

Ag+ etc)

CHAPTER V

51 MATERIALS

24-dione Merck

C17H20N4O6 Mr 3764

C13H12N4O2 Mr 2563

C12H10N4O2 Mr 2423

C14H12N4O3 Mr 2843

C14H12N4O4 Mr 3003

al 1980)

C17H18O6N4 Mr 3744

Metal Salts

follows

CoSO47H2O (999)

52 REAGENTS

53 METHODS

change

(Merck)

al 1980)

(Ahmad et al1980)

532 pH Measurements

to 400 cm-1

A (1 1 cm) at 444 nm = 328

arc lamp

A 10minus4

A 5 times 10ndash5

M) were

Photoproducts

385 nm

photolysis)

at 445 385 and 410 nm

photolysis)

356 nm

cmminus1

following equation

A1 = 1a1 1C (51)

A1 = 1ε1 1C (52)

A1 = 1ε1 1C + 2ε1 2C (53a)

A2 = 1ε2 1C + 2ε2 2C (53b)

and 3C

A1 = 1ε1 1C + 2ε1 2C + 3ε1 3C (55a)

A2 = 1ε2 1C + 2ε2 2C + 3ε2 3C (55b)

A3 = 1ε3 1C + 2ε3 2C + 3ε3 3C (55c)

λ1 A1 1 ε 1 1C + 2 ε 1 2C + 3 ε 1 3C

λ2 A2 1 ε 2 1C + 2 ε 2 2C + 3 ε 2 3C

λ3 A3 1 ε 3 1C + 2 ε 3 2C + 3 ε 3 3C

A1 1ε1 2ε1 3ε1 1C

A2 = 1ε2 2ε2 3ε2 = 2C

A3 1ε3 2ε3 3ε3 3C

(AM) (ASM) (CM)

A1 2ε1 3ε1 1ε 1 2ε 1 3ε1

1C = A2 2ε2 3ε2 1ε2 2ε2 3ε2

A3 2ε3 3ε3 1ε3 2ε3 3ε3

1 ε 1 A1 3 ε 1

1ε 1 2ε 1 3ε1

2C = 1 ε 2 A2 3 ε 2 1ε2 2ε2 3ε2

1 ε 3 A3 3 ε 3 1ε3 2ε3 3ε3

1ε1 2ε1 A1

1ε 1 2 ε 1 3 ε 1

3C = 1ε2 2ε2 A2 1 ε 2 2 ε 2 3 ε 2

1ε3 2ε3 A3 1 ε 3 2 ε 3 3 ε 3

A1 2ε2 3ε2

2ε3 3ε3

ndash 2 ε 1

A2 3ε 2

A3 3ε3

+ 3 ε 1

A2 2ε2

A3 2ε3

ASM expanded

CHAPTER VI

RIBOFLAVIN

61 INTRODUCTION

53 (Chapter 5)

045 Non-

(Ahmad et al 1980)

and 120 min

250 300 400 500 600

Wavelength (nm)

method

(Mtimes 105)

0 300 000 000 000 300

30 263 028 008 004 305

60 229 060 012 007 308

90 197 078 023 009 309

120 173 086 030 012 311

(Mtimes 105)

0 300 000 000 300

30 269 023 012 304

60 239 040 021 308

90 213 058 031 304

120 194 066 045 311

(Mtimes 105)

0 300 00 00 300

30 255 036 015 306

60 215 058 029 308

90 201 071 032 306

120 191 079 037 312

(Mtimes 105)

0 300 000 000 300

30 273 017 014 306

60 245 032 024 310

90 223 042 036 308

120 199 049 052 306

(Mtimes 105)

0 300 000 000 300

30 268 020 015 305

60 245 031 028 307

90 223 040 039 304

120 202 049 050 302

(Mtimes 105)

0 300 000 000 300

30 269 022 012 303

60 245 037 022 304

90 222 052 031 307

120 204 060 039 309

(Mtimes 105)

0 300 000 000 300

30 275 017 011 308

60 251 031 023 309

90 227 037 039 306

120 208 046 050 304

method

minminus1

(water)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times10

Time (min)

M) in water

(pH 70)

acetonitrile

0 20 40 60 80 100 120 140

Time (min)

times10

0 20 40 60 80 100 120 140

Time (min)

M times

methanol

ethanol

0 20 40 60 80 100 120 140

Time (min)

times10

0 20 40 60 80 100 120 140

M times

Time (min)

1ndashpropanol

1ndashbutanol

0 20 40 60 80 100 120 140

times10

Time (min)

0 20 40 60 80 100 120 140

Time (min)

M times

ethyl acetate

0 20 40 60 80 100 120 140

times10

Time (min)

Solvents Dielectric

constant (isin)

(25 oC)

Acceptor

Number

Inverse

viscosity

(mPasndash1

(25 oC)

kobs times 103 min

ndash1

plusmnSDa

Ethanol 243 371 0931 345plusmn015

Water 785 548 1123 461plusmn025

() water

00 100 200 300 400 500 600 700 800

Dielectric constant

s times

() water

00 100 200 300 400 500 600 -70

s times

10 15 20 25 05 30 00

Viscosity (mPa s)-1

times 1

00 05 10 15 20 25 30 35

Viscosity (mPas)-1

the medium

3FL + AH (F

σndash+) FLH (61)

CHAPTER VII

71 INTRODUCTION

B radicmicro (71)

where ZA and Z

Corsaro 1977)

reported

53 (Chapter 5)

(M times105)

0 500 00 00 00 500

30 451 019 021 010 501

60 398 039 045 019 506

90 373 053 059 022 507

120 340 064 071 027 508

(M times105)

0 500 00 00 00 500

30 446 020 022 016 504

60 386 044 049 021 508

90 332 069 073 029 509

120 309 076 081 035 501

(M times105)

0 500 00 00 00 500

30 435 020 031 016 502

60 381 039 052 029 505

90 331 065 071 035 508

120 288 078 089 046 501

(M times105)

0 500 00 00 00 500

30 417 031 035 020 503

60 361 054 058 031 504

90 308 069 082 043 507

120 269 081 099 052 508

(M times105)

0 500 00 00 00 500

30 404 032 044 022 502

60 336 056 075 036 505

90 290 068 097 047 507

120 245 079 118 059 501

(M times 105)

0 500 00 00 00 00 500

30 435 015 016 039 008 513

60 378 026 028 048 020 508

90 329 035 046 071 030 511

120 280 048 060 092 042 522

(M times 105)

0 500 00 00 00 00 500

30 416 024 036 057 006 539

60 353 040 059 075 016 543

90 293 079 081 134 028 615

120 251 089 091 175 034 640

(M times 105)

0 500 00 00 00 00 500

30 386 023 032 059 006 500

60 307 040 056 083 014 511

90 239 059 069 119 021 516

120 194 064 081 131 033 503

(M times 105)

0 500 00 00 00 00 500

30 369 030 036 062 009 506

60 280 045 060 093 023 501

90 217 060 073 122 033 509

120 153 071 089 145 048 506

(M times 105)

0 500 00 00 00 00 500

30 338 036 046 074 009 503

60 238 055 081 112 014 510

90 164 064 116 131 027 502

120 119 073 126 149 037 504

(M times 105)

0 500 00 00 00 00 500

30 398 016 031 045 010 510

60 327 031 055 066 022 508

90 267 042 065 085 041 503

120 224 050 076 101 049 506

(M times 105)

0 500 00 00 00 00 500

30 367 027 037 056 013 504

60 286 047 051 096 020 511

90 221 059 069 120 031 513

120 178 057 082 139 044 509

(M times 105)

0 500 00 00 00 00 500

30 354 024 049 059 014 504

60 236 049 069 108 038 508

90 168 068 076 139 049 503

120 108 078 096 158 060 509

(M times 105)

0 500 00 00 00 00 500

30 295 040 051 100 015 506

60 160 056 108 143 033 505

90 097 069 121 168 045 502

120 076 075 132 177 051 506

(M times 105)

0 500 00 00 00 00 500

30 282 046 060 106 006 511

60 145 076 088 154 037 505

90 079 091 104 175 051 509

120 052 100 110 200 057 507

(M times 105)

0 500 00 00 00 00 500

30 397 029 026 035 017 504

60 309 036 049 076 037 507

90 239 048 061 105 051 504

120 180 067 075 126 062 508

(M times 105)

0 500 00 00 00 00 500

30 361 029 042 047 023 508

60 256 048 056 095 047 512

90 183 061 077 118 063 502

120 127 073 095 145 071 514

(M times 105)

0 500 00 00 00 00 500

30 314 032 050 075 035 506

60 195 055 090 113 050 513

90 130 070 108 133 062 508

120 075 085 130 145 071 506

(M times 105)

0 500 00 00 00 00 500

30 292 042 052 079 039 504

60 148 069 083 135 066 511

90 078 093 103 155 076 509

120 042 103 114 163 084 506

(M times 105)

0 500 00 00 00 00 500

30 217 049 070 113 055 504

60 113 060 096 157 074 509

90 057 073 106 178 086 511

120 024 082 117 187 093 506

(M times 105)

0 500 00 00 00 00 500

30 425 013 028 027 009 502

60 338 032 041 065 024 509

90 251 045 074 091 043 514

120 157 066 085 135 059 512

(M times 105)

0 500 00 00 00 00 500

30 313 041 046 085 019 506

60 214 056 068 115 047 509

90 140 072 085 150 057 506

120 099 081 096 164 067 507

(M times 105)

0 500 00 00 00 00 500

30 298 037 062 075 030 506

60 179 061 079 125 056 511

90 099 076 097 155 075 502

120 049 088 108 169 087 508

(M times 105)

0 500 00 00 00 00 500

30 249 049 068 099 036 501

60 099 071 118 145 067 509

90 049 082 128 167 077 506

120 023 088 137 178 086 512

(M times 105)

0 500 00 00 00 00 500

30 210 062 086 126 026 508

60 078 088 112 179 049 506

90 034 094 120 190 069 509

120 013 099 132 201 080 511

photoproducts

been studied

M) at pH 70

kobs= k1+ k2+ k3 (74)

RF = RF0 endashkt

Similarly

) (79)

) (710)

) (711)

k2 RF0 kobs

k3 RF0 kobs

ndash RF0 endashkt

k1 RF0

is not clear

other factors

5times10minus5

Buffer

Concentration

Strength

kobs times 103

(minndash1

k0 times 103

(minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

02 01 276 085 139 063 072 193

02 485 284 070 144 197

03 715 407 102 198 205

04 978 535 177 255 209

05 1190 684 201 321 213

03 01 445 120 224 109 111 201

02 825 425 151 185 229

03 1185 632 240 265 238

04 1505 835 253 345 242

05 1860 1042 296 521 248

04 01 525 135 259 127 121 214

02 1150 501 282 226 221

03 1571 756 370 325 232

04 2030 1115 487 466 239

05 2491 1279 561 522 245

05 01 735 141 380 166 170 222

02 1250 660 285 277 238

03 1891 991 478 402 246

04 2421 1220 615 482 253

05 3032 1603 638 607 264

() 005 M (times) 01 M () 02 M (∆) 03 M () 04 M () 05 M

0 01 02 03 04 05 06

Ionic Strength (M)

ions leading to the

of [1RFndashHPO4

2minus

RF [1RF] (714)

[1RF] + NaCl [

1RFhelliphellipCl

ndash] + Na

+ (715)

[1RF helliphellipCl

pathways

of the plots

B = 105 102 = 103 = ~ + 1 (for k1)

B = 161 102 = 157 = ~ + 160 (for k3)

exciplex

000 010 020 030 040

radicμ1 + radicμ

000 010 020 030 040 050 060 070 080

radicμ

] (717)

+H ndashH+

CHAPTER VIII

81 INTRODUCTION

) (Mortland

additi

photoreduction(1)

(4) (5)

H+ OH-

and Cd2+

and Zn2+

(Kaim et al 1999)

promoted by Fe3+

and Cu2+

(inhibited by

(1) (81)

Rearrangment

4a5 44a

and Zn2+

+ RH M(nndash1)+

+ H+ + R

˙ (81)

+ H+ + RʹO2

˙ (82)

reaction

(Chapter 5)

and Cu2+

11 RFndashFe2+

RF + 2Fe2+ +2HRFH2 + 2Fe3+

M) (pH 70) (____

ions (b) Zn2+

ions and (c) Cu2+

and Cu2+

ions appears to

complex formation

presence of Zn2+

and Cu2+

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 485 008 489 006 486 007 489 006 490 005

120 470 014 477 012 472 015 478 014 479 012

180 447 026 454 023 458 020 466 019 468 018

0 500 000 500 000 500 000 500 000 500 000

60 472 015 463 017 457 019 450 019 446 022

120 442 032 431 037 421 035 413 038 398 046

180 416 045 398 049 384 052 371 059 355 066

0 500 000 500 000 500 000 500 000 500 000

60 474 014 461 018 453 022 442 026 432 030

120 450 027 424 032 413 036 393 047 373 055

180 418 039 389 044 365 055 346 065 324 076

0 500 000 500 000 500 000 500 000 500 000

60 472 014 462 017 442 025 429 030 421 033

120 444 024 423 033 395 044 375 052 354 061

180 414 039 385 052 352 067 322 075 295 086

0 500 000 500 000 500 000 500 000 500 000

60 474 015 465 018 464 019 459 021 441 028

120 450 024 436 029 430 031 415 036 389 048

180 427 036 407 045 393 051 358 062 339 068

0 500 000 500 000 500 000 500 000 500 000

60 474 015 459 019 450 022 440 026 427 032

120 450 026 422 038 403 044 386 051 363 065

180 417 041 381 056 355 066 338 071 309 081

0 500 000 500 000 500 000 500 000 500 000

60 489 006 475 013 472 014 472 014 467 016

120 465 016 449 024 446 026 443 027 437 029

180 437 032 427 036 419 039 414 041 408 045

0 500 000 500 000 500 000 500 000 500 000

60 475 014 467 017 463 018 457 021 451 023

120 449 024 434 030 429 033 411 040 406 042

180 426 035 407 040 390 047 374 054 363 060

0 500 000 500 000 500 000 500 000 500 000

60 472 015 463 019 453 022 442 025 433 030

120 443 029 428 035 412 042 390 051 373 057

180 416 039 394 047 371 057 342 068 322 076

0 500 000 500 000 500 000 500 000 500 000

60 474 014 473 016 463 019 457 021 441 028

120 447 027 444 028 428 034 416 039 390 051

180 429 036 411 042 391 051 375 057 346 068

0 500 000 500 000 500 000 500 000 500 000

60 478 011 476 013 472 015 466 017 464 019

120 454 022 450 024 442 027 436 029 430 032

180 433 030 423 034 414 039 405 043 399 048

0 500 000 500 000 500 000 500 000 500 000

60 476 013 473 019 464 020 463 020 457 022

120 451 022 444 026 430 033 431 039 416 042

ndash4 M

Table 81 continued

5) (02 M Phosphate

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 448 014 457 014 457 014 465 014 467 013

120 405 015 416 015 416 015 424 015 436 014

180 363 017 374 016 381 015 395 014 408 013

0 500 000 500 000 500 000 500 000 500 000

60 425 015 416 015 416 016 398 016 389 016

120 369 017 346 018 338 019 323 019 309 020

180 322 019 298 020 279 021 257 023 245 025

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 407 016 407 016 387 016

120 371 017 363 017 338 019 323 020 300 020

180 319 019 302 020 279 023 259 024 234 026

0 500 000 500 000 500 000 500 000 500 000

60 416 015 398 016 380 017 371 017 352 018

120 346 018 323 019 295 021 275 022 255 024

180 291 021 257 023 229 027 203 029 177 034

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 421 015 416 015 406 016

120 380 017 363 017 354 018 338 018 320 020

180 331 019 310 020 298 021 279 022 262 024

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 015 416 015 396 016

120 380 017 363 017 346 018 338 018 323 019

180 328 019 308 020 295 022 274 023 256 025

0 500 000 500 000 500 000 500 000 500 000

60 454 014 426 015 426 015 426 015 416 015

120 406 015 371 017 363 017 354 018 338 018

180 367 019 316 022 311 023 295 025 281 026

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 015 407 016 396 016

120 381 017 363 017 346 018 331 019 323 020

180 334 019 311 020 293 022 274 027 251 029

0 500 000 500 000 500 000 500 000 500 000

60 436 015 416 015 407 016 398 016 381 017

120 371 017 346 018 331 019 316 020 293 022

180 319 019 291 022 266 023 244 025 228 028

0 500 000 500 000 500 000 500 000 500 000

60 426 015 426 015 407 016 398 016 361 018

120 371 017 354 018 338 019 323 019 262 024

180 320 021 299 025 279 028 259 031 189 037

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 016 416 016 406 016

120 381 017 363 017 354 018 346 018 330 019

180 328 021 314 024 299 025 286 029 273 033

0 500 000 500 000 500 000 500 000 500 000

60 426 015 416 015 421 015 416 015 404 016

120 371 017 354 017 354 018 346 018 330 020

ndash4 M

Table 82 continued

5) (04 M Phosphate

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 416 011 421 010 428 009 436 009 447 007

120 347 027 354 026 369 021 389 018 402 015

180 292 034 303 030 319 028 343 026 359 021

0 500 000 500 000 500 000 500 000 500 000

60 403 018 372 026 375 026 358 030 347 033

120 325 024 282 037 276 038 251 045 244 046

180 252 032 216 042 194 044 171 053 165 055

0 500 000 500 000 500 000 500 000 500 000

60 393 019 381 020 375 021 358 024 347 026

120 307 028 289 031 276 033 254 038 244 041

180 237 037 215 039 200 041 181 044 170 048

0 500 000 500 000 500 000 500 000 500 000

60 375 016 347 022 334 024 319 027 295 022

120 272 030 246 033 219 035 195 036 182 035

180 200 040 167 045 143 048 122 051 103 061

0 500 000 500 000 500 000 500 000 500 000

60 393 011 381 015 384 015 375 016 364 018

120 319 025 298 029 289 031 276 033 263 036

180 251 033 233 036 221 038 209 041 197 043

0 500 000 500 000 500 000 500 000 500 000

60 393 011 381 014 384 014 375 017 364 021

120 317 022 298 025 289 027 276 031 263 035

180 251 029 229 032 223 034 207 037 194 039

0 500 000 500 000 500 000 500 000 500 000

60 393 014 384 016 384 016 375 018 364 020

120 303 022 298 023 289 025 276 027 263 029

180 241 031 229 033 221 035 203 037 191 039

0 500 000 500 000 500 000 500 000 500 000

60 393 013 384 015 384 015 367 017 345 018

120 315 019 298 021 289 023 272 026 237 029

180 255 025 225 029 215 033 198 035 169 039

0 500 000 500 000 500 000 500 000 500 000

60 393 011 375 018 367 019 347 021 337 025

120 302 022 282 026 26 030 242 035 231 038

180 237 033 207 036 188 038 169 041 155 044

0 500 000 500 000 500 000 500 000 500 000

60 393 014 375 019 367 020 347 022 337 024

120 302 019 282 025 266 029 242 032 231 034

180 234 027 213 031 171 041 171 043 159 047

0 500 000 500 000 500 000 500 000 500 000

60 393 013 384 014 367 019 363 019 350 022

120 309 019 295 021 272 026 263 027 251 031

180 242 026 226 029 207 032 195 034 183 036

0 500 000 500 000 500 000 500 000 500 000

60 393 016 381 019 375 021 358 022 347 024

120 315 027 289 033 276 034 254 038 244 040

ndash4 M

Table 83 continued

ions is shown in

in the order

2+lt Fe

3+ lt Ca

2+ +lt Fe

2+ lt Cd

2+ lt Cu

2+lt Mn

2+lt Pb

2+ lt Mg

2+lt Zn

2+lt Ag

Thus Ni2+

ions (∆) Co2+

(loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

() Mg2+

ions () Zn2+

ions and () Fe3+

00 10 20 30 40 50 60

M) (b2)

RF + Fe2+

M) (b3)

2014 2016 Sheraz et al 2014)

RF + Fe2+ RF-Fe2+

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

log co

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ag+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ag+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

Metal Ions (kʹ)

Metal Ion Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

00 063 016 006

Ag+ 10 059 050 041 017

20 054 038 015

30 049 035 014

40 044 033 010

50 038 029 008

10 089 256 070 018

20 115 080 034

30 142 101 040

40 169 129 039

50 191 143 047

10 099 360 078 020

20 136 084 051

30 172 107 064

40 206 138 067

50 243 164 078

10 105 462 073 031

20 155 113 041

30 199 138 060

40 245 164 080

50 294 190 094

10 101 416 071 029

20 142 099 042

30 184 131 052

40 225 160 064

50 271 182 088

10 106 410 079 026

20 145 105 039

30 185 128 056

40 224 152 071

50 268 180 087

10 075 104 058 016

20 085 062 022

30 095 068 026

40 105 075 029

50 115 083 031

10 089 232 063 025

20 112 075 036

30 136 092 043

40 158 106 051

50 179 120 058

10 102 360 072 029

20 132 089 042

30 167 110 056

40 210 140 070

50 243 162 081

10 091 284 069 021

20 118 086 031

30 148 104 043

40 176 122 053

50 205 139 065

10 078 128 054 023

20 091 063 027

30 104 071 032

40 116 080 035

50 127 087 039

10 082 180 060 021

20 099 075 023

30 118 091 026

40 135 151 029

50 153 174 035

Table 84 continued

Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

00 204 111 038 054 205

Ag+ 10 182 184 125 027 028 446

20 164 112 025 026 430

30 144 094 023 025 376

40 127 084 019 023 365

50 112 072 016 022 327

10 243 384 195 020 027 722

20 285 231 022 031 724

30 325 256 032 035 726

40 363 291 033 040 728

50 396 315 036 043 730

10 249 410 201 021 027 724

20 285 229 025 031 726

30 325 256 034 035 728

40 365 290 036 039 730

50 409 329 033 045 732

10 285 742 226 027 031 729

20 358 283 036 038 733

30 435 343 043 048 736

40 505 402 048 054 738

50 575 446 059 060 741

10 235 246 180 024 029 620

20 265 201 030 032 628

30 295 223 036 034 655

40 325 245 039 036 671

50 358 286 035 041 697

10 235 334 180 024 029 620

20 269 207 029 033 625

30 302 228 035 036 629

40 335 243 044 038 633

50 371 284 045 044 637

10 227 232 149 035 042 354

20 260 179 032 049 360

30 283 195 035 053 360

40 304 210 038 056 365

50 332 230 041 061 369

10 235 358 178 025 030 593

20 270 207 029 034 605

30 305 231 035 037 624

40 334 253 041 040 631

50 373 284 045 044 636

10 251 462 196 025 031 625

20 301 233 031 036 647

30 345 268 036 039 687

40 385 303 041 043 699

50 427 333 048 046 711

10 254 410 179 032 043 411

20 285 201 039 043 467

30 323 231 044 048 475

40 362 259 049 054 479

50 404 289 056 059 483

10 236 256 168 029 039 425

20 255 184 032 038 484

30 280 204 034 040 510

40 300 220 038 042 519

50 319 232 043 044 523

10 237 308 189 021 026 726

20 271 218 024 029 730

30 302 238 030 032 734

40 332 265 030 036 736

50 358 284 036 038 738

Table 85 continued

Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

00 351 222 049 075 296

Ag+ 10 315 332 184 061 069 263

20 280 171 042 066 259

30 247 152 035 059 257

40 214 129 033 051 252

50 190 114 030 046 247

10 402 528 262 059 079 331

20 462 290 070 101 287

30 515 310 094 109 284

40 570 335 098 136 246

50 615 363 104 147 246

10 407 496 259 069 079 325

20 460 295 072 092 320

30 509 328 077 103 317

40 560 357 089 113 315

50 599 373 099 126 296

10 475 1048 302 075 096 314

20 580 359 106 115 310

30 681 414 128 137 302

40 784 475 151 158 299

50 875 505 173 196 257

10 390 348 257 058 073 352

20 425 275 066 082 335

30 458 296 071 090 328

40 490 315 075 099 318

50 525 335 082 107 313

10 386 348 273 050 061 447

20 427 301 057 068 442

30 458 321 060 075 428

40 490 336 068 084 400

50 525 355 077 091 390

10 387 508 254 058 073 347

20 424 273 069 081 337

30 494 317 080 096 330

40 545 347 089 107 324

50 605 380 104 119 319

10 389 600 271 057 060 451

20 426 287 070 067 428

30 494 327 080 085 384

40 545 359 089 095 377

50 651 432 103 116 370

10 415 600 282 057 075 376

20 475 318 071 085 374

30 535 363 074 098 370

40 605 405 090 110 366

50 651 423 109 117 361

10 413 570 287 060 065 441

20 470 320 072 077 415

30 530 337 091 101 333

40 590 370 102 116 318

50 636 392 110 132 296

10 395 414 273 059 061 447

20 438 296 069 071 416

30 479 321 076 081 396

40 524 350 084 089 393

50 558 369 093 095 388

10 405 468 260 055 083 313

20 455 290 072 093 310

30 505 322 077 104 309

40 548 346 086 115 300

50 585 363 093 128 283

Table 86 continued

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

ions () Mg2+

ions ()

ions () Fe3+

00 30 60 90

Fluorescence loss

bs times

minndash1

) (Table

minndash1

) (Table 86) The

Mndash1

minndash1

) and monovalent

order Zn2+

gt Mg2+

gt Pb2+

gt Mn2+

gt Cu2+

gt Cd2+

gt Fe2+

gt Ca2+

gt Fe3+

gt Co2+

gt Ni2+

gt Ag+

ions (Rutter 1958)

) bind to RF

presented (Fig 844)

] complex (Eq

state [1RFhellipFe

2+] state

(Eq (812))

RF + Fe2+ [RFFe2+]

metal-RF complex

[RFFe2+] [1RFFe2+]

[1RFFe2+] [RFHFe3+]

[RFHFe3+] RFH

+ Fe3+

2RFH RFH2

H+ OH_

ions and

CHAPTER IX

91 INTRODUCTION

(1)(4)

(5)(7) (6)

and alkaline pH

(Chapter 5)

to brown

9214 FTIR studies

RFndashAg NPs

0 1 2 3 4 5 6 7

Time (h)

cmndash1

distributed

measurements

9221 pH

NPs (Fig 99)

9222 Ionic strength

+ ions

questionable

91 and 92

0 50 100 150 200 250 300 350 400

Time (min)

k1times 103

k2 times 103

(min-1

k1times 103

(min-1

k2 times 103

(min-1

80 736 0060 521 0036

85 999 0088 843 0061

90 1285 0110 1122 0091

95 1523 0129 1324 0112

100 1740 0147 1480 0128

105 1822 0153 1524 0135

ion Concentration

k1times 103

k2 times 103

(min-1

k1times 103

(min-1

k2 times 103

(min-1

0002 754 0012 355 0009

0004 892 0022 622 0020

0006 1279 0046 1088 0043

0008 1630 0072 1399 0052

001 1740 0079 1444 0054

750 800 850 900 950 1000 1050 1100

k1times

k2 times

750 800 850 900 950 1000 1050 1100

k1times

k2 times

0 0002 0004 0006 0008 001 0012

k1times

k2 times

0 0002 0004 0006 0008 001 0012

k1times

k2 times

RF1RFhv

3RFisc1RF

RF3RF + Ag+

+ Ag NP

0RFRF + H+

RF3RF + 0RF + RFH

+ ion to form RF

0RF] Further

0RF] lead to

] radical (Eq (95)

CONCLUSIONS

solvents

HPO42-

REFERENCES

Sci 2011a111ndash15

201363221ndash227

2005 78229ndash234

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201366579ndash585

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1981a205925ndash5928

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Sci 2010b118ndash21

2003921730ndash1733

201411815348ndash15355

51071133ndash138

20051278ndash283

1961441001ndash1111

2003 601ndash10

B 201537237ndash7245

19948196ndash198

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Chem 2006546016ndash6020

version

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199532286

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20121352934ndash2941

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Ophthalmol 2015886

2009316ndash20

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19742492228ndash2234

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200611153ndash158

Soc1951734870ndash4874

USA 1973 Chap 3

1956A235518ndash536

1956235518ndash536

1981337ndash13

171ndash 193

1996851053ndash1059

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199981614ndash1622

197965235ndash266

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Rev 1999182323ndash342

Chem B 2003107668-677

201314177ndash182

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2007240ndash47

19751082422ndash2432

198029493ndash507

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197ndash206 279-280

1960 132433ndash455

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Rev 1989691170ndash1198

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Res 2014162291ndash2298

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1969917170ndash7179

198331435ndash439

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Electronic Version

196218249ndash253

Sci 197968992ndash996

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197118B479ndash506

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VCH Publishers 1988

Sci 1972611081ndash1085

197330236ndash239

1982 pp 123ndash154

19755735ndash48

1995240313ndash322

20031575ndash14

2006 pp 413ndash416

Soc1963853285ndash3288

200863545ndash546

200964428ndash435

2011b69167ndash172

19771071269ndash1276

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254 458ndash461 469ndash474

1979271039ndash1042

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Res 198013148ndash155

195047114ndash121

B 20141162872ndash2882

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2014159282ndash286

Research Article

INTRODUCTION

Photolysis

pH Measurements

Spectrometric Assay

RESULTS

Photoproducts of RF

Ahmad et al

DISCUSSION

Effect of Solvent

0 30 60 90 120

on times

Time (min)

Time(min)

RF(Mtimes105)

FMF(Mtimes105)

LC(Mtimes105)

Total(Mtimes105)

0 300 00 00 30030 255 036 015 30660 215 058 029 30290 201 071 032 304120 191 079 037 307

Effect of Viscosity

00 100 200 300 400 500 600 700 800

times10

inndash1

Dieletric Constant

Ahmad et al

Mode of Photolysis

CONCLUSION

00 05 10 15 20 25 30

bstimes

103 (m

inndash1

Viscosity (mPa s)-1

00 100 200 300 400 500 600lnk o

bs times

REFERENCES

Ahmad et al

1 Introduction

eth2THORN

21 Photodegradation

22 Assay Method

Time(min)

RF(M times 105)

CDRF(M times 105)

FMF(M times 105)

LC(M times 105)

LF(M times 105)

Total(M times 105)

0 500 00 00 00 00 50030 210 062 082 124 026 50460 078 088 110 179 043 49890 034 094 120 188 055 491120 013 099 122 198 074 506

Similarly

1minuseminuskt

eth9THORN

1minuseminuskt

eth10THORN

1minuseminuskt

eth11THORN

Ionic strength(M)

kobs times 103

(minminus1)k1k3

02 01 276 085 139 063 072 19302 485 284 070 144 19703 715 407 102 198 20504 978 535 177 255 20905 1190 684 201 321 213

03 01 445 120 224 109 111 20102 825 425 151 185 22903 1185 632 240 265 23804 1505 835 253 345 24205 1860 1042 296 521 248

04 01 525 135 259 127 121 21402 1150 501 282 226 22103 1571 756 370 325 23204 2030 1115 487 466 23905 2491 1279 561 522 245

05 01 735 141 380 166 170 22202 1250 660 285 277 23803 1891 991 478 402 24604 2421 1220 615 482 25305 3032 1603 638 607 264

RFrarrhv 1RF

eth14THORN

eth22THORN

4 Conclusion

References

Iqbal Ahmada

Zubair Anwara

Sofia Ahmeda

Highlights

Abstract

Graphical Abstract

BIODATA

Qualifications

Impact Factor 17001

Publications

Chapters

2017 (In Press)

Reviews

2013417ndash22

201314 318ndash326

20161951ndash57

Research Papers

201415550ndash559

2014153ndash7

01Front-Pagespdf

02Chapter-Ipdf

03Chapter-IIpdf

04Chapter-IIIpdf

05Chapter-IVpdf

07Chapter-Vpdf

08Chapter-VIpdf

09Chapter-VIIpdf

10Chapter-VIIIpdf

11Chapter-IXpdf

12CONCLUSIONSpdf

13Referencespdf


Abstract

INTRODUCTION


Photolysis

pH Measurements


Spectrometric Assay


RESULTS

Photoproducts of RF




DISCUSSION

Effect of Solvent


Effect of Viscosity

Mode of Photolysis

Conclusion

References



1 Introduction


21 Photodegradation

22 Assay Method









4 Conclusion

References

16Metalpdf

17BIODATApdf

referred cited

Name Zubair Anwar

_____________________________

ABSTRACT

minminus1

ions (Ag+ Ni

3+) has been

2+ gt Mg

gt Mn2+

gt Cu2+

gt Cd2+

gt Fe2+

gt Ca2+

gt Fe3+

gt Co2+

gt Ni2+

2920 cmndash1

ACKNOWLEDGEMENTS

(Quran 20114)

institution

and my niece

Anushay Zeeshan

CONTENTS

Chapter Page

ABSTRACT vi

ACKNOWLEDGEMENTS ix

I INTRODUCTION

11 INTRODUCTION 2

15 CLINICAL USES 8

31 INTRODUCTION 38

3472 pH effect 60

41 INTODUCTION 71

422 Stabilizer 74

424 Biosensor 76

51 MATERIALS 86

52 REAGENTS 88

53 METHODS 89

and Photoproducts

61 INTRODUCTION 106

AQUEOUS SOLUTION

71 INTRODUCTION 135

81 INTRODUCTION 165

Solutions

91 INTRODUCTION 217

9221 pH 232

CONCLUSIONS 248

REFERENCES 252

and 120 min

RF () FMF () LC ()

(5 times 10ndash5

M) in water (pH 70)

acetonitrile

methanol

ethanol

1ndashpropanol

1ndashbutanol

ethyl acetate

M () 005 M (times) 01 M () 02 M (∆) 03 M () 04 M ()

buffer

M) (pH 70) (____

) in the

ions (b) Zn2+

ions and (c) Cu2+

() Ni2+

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

ions () Mg2+

() Zn2+

ions and () Fe3+

M) (pH 70) (a)

3 M) (b2) RF + Fe

2+ ions (2 times 10

ndash3 M) (b3)

ions at different

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams)

ions () Pb2+

ions () Mg2+

ions () Zn2+

ions () Fe3+

green to brown

300 min (purple)

RFndashAg NPs

green)

(pink)

UV light

visible light

cmminus1

) of RF and

Photoproducts

(pH 70)

Phosphate buffer

5) (0001

5) (02

5) (04

CHAPTER I

11 INTRODUCTION

12 BIOCHEMICAL ROLE

CH2O P

Thyroid Harmone

Empirical formula

C17H20N4O6

Molar mass 3764

pKa 19 102 (20o)

Redox potential

Water 33ndash606

1070 cmndash1

15 CLINICAL USES

Kafai 2004)

17 THERAPEUTIC USES

et al 1996)

18 PHARMACOKINETICS

RF (Nath 2000)

Chapters in Books

Reviews

(1998)

Physiochemical Data

CHAPTER II

RELATED COMPOUNDS

mg Lndash1

009 microg mLndash1

045 microg mLndash1

et al 2006)

Vosough 2002)

wavelength

(Hustad et al 1999)

4 times 10ndash2

secndash1

mol Lndash1

(Li et al 1992)

10 microg Lndash1

(Garcia et al 2001)

(Zhang et al 2009)

et al 2009)

al 2008)

mobile phase

500 nmol mlndash1

05 ml minndash1

of RF in RF

0007ndash01

15ndash45

microg mlndash1

10 ml minndash1

(Valls et al 1999)

) as the mobile

with LOD

of 05 ng Lndash1

50 times 10ndash8

mol Lndash1

to 2 times 10ndash4

mol Lndash1

15 times 10ndash6

15 times 10ndash4

mol Lndash1

with a LOD of

10 times 10ndash8

mol Lndash1

(Yang et al 2001)

to 3 times 10ndash6

mol Lndash1

25 ENZYMATIC ASSAYS

10 times 10ndash10

mol Lndash1

CHAPTER III

31 INTRODUCTION

1980 2004ab 2005 2006ab 2008 2009 2010ab 2011 2013 2014 2015ab 2016ab

SO42ndash

) RF undergoes

are discussed below

(8) (5)

(7) (6)

r phot

oadditi

341 Photoreduction

discussed below

and Metzler 1971)

Fl hv 1Fl

FlH + R1Fl + RH

Fl- + RH+1Flo + RH

Fl hv 1Fl

R + CO2RCOO-

radical addition

HFl + HFl

H2Flred + Flox

ProdcutsR

RFlredHHFl + R

1FloFl hv

1Fl oFlic

1Fl 3Flisc

3Fl oFl+ heat

(Eq (316))

oFlred + O2

oFl + H2O2

and Knappe 1974)

pH 70hv

(1) (5)

SO42ndash

344 Photooxidation

RFhv 1RF

3RF1RF isc

3RF RF + 3O2

and Min 2006)

ndash1s

ndash1 and

ndash1s

8 and 821 times 10

ndash1s

8 and 527times10

ndash1s

ndash1 It has

ndash is

CndashRF and 14

Cndash

(Reddy 2008)

qsndash1

3472 pH effect

et al 2007)

2004a)

3473 Buffer effect

2ndash species

(02 M ge)

tartarte succinate

as follows

RF [1RF] LC

hv H2PO4-

[3RF][1RF] RFH2

RFHPO4

RF-HPO42- hv [1RF]

complex

0 ∆cS

Jesus et al 2012)

et al 2004)

et al 2015a)

1991 Loukas 2001)

CHAPTER IV

41 INTODUCTION

1ndash100 nm ndash

American Society of

(ASTM)

1ndash100 nm

Health (NIOSH)

nanoscale

nanoscale range

Arbeitsschutz und

nanoscale range

nanostructure or a

nanosubstance

421 Photosenstizer

state 13

C and 29

422 Stabilizer

ions This

K+) (Xu et al

424 Biosensor

to 10times 10ndash4

molcm2 and

Mndash1

sndash1

(Zhang et al 2011)

Ag+ etc)

CHAPTER V

51 MATERIALS

24-dione Merck

C17H20N4O6 Mr 3764

C13H12N4O2 Mr 2563

C12H10N4O2 Mr 2423

C14H12N4O3 Mr 2843

C14H12N4O4 Mr 3003

al 1980)

C17H18O6N4 Mr 3744

Metal Salts

follows

CoSO47H2O (999)

52 REAGENTS

53 METHODS

change

(Merck)

al 1980)

(Ahmad et al1980)

532 pH Measurements

to 400 cm-1

A (1 1 cm) at 444 nm = 328

arc lamp

A 10minus4

A 5 times 10ndash5

M) were

Photoproducts

385 nm

photolysis)

at 445 385 and 410 nm

photolysis)

356 nm

cmminus1

following equation

A1 = 1a1 1C (51)

A1 = 1ε1 1C (52)

A1 = 1ε1 1C + 2ε1 2C (53a)

A2 = 1ε2 1C + 2ε2 2C (53b)

and 3C

A1 = 1ε1 1C + 2ε1 2C + 3ε1 3C (55a)

A2 = 1ε2 1C + 2ε2 2C + 3ε2 3C (55b)

A3 = 1ε3 1C + 2ε3 2C + 3ε3 3C (55c)

λ1 A1 1 ε 1 1C + 2 ε 1 2C + 3 ε 1 3C

λ2 A2 1 ε 2 1C + 2 ε 2 2C + 3 ε 2 3C

λ3 A3 1 ε 3 1C + 2 ε 3 2C + 3 ε 3 3C

A1 1ε1 2ε1 3ε1 1C

A2 = 1ε2 2ε2 3ε2 = 2C

A3 1ε3 2ε3 3ε3 3C

(AM) (ASM) (CM)

A1 2ε1 3ε1 1ε 1 2ε 1 3ε1

1C = A2 2ε2 3ε2 1ε2 2ε2 3ε2

A3 2ε3 3ε3 1ε3 2ε3 3ε3

1 ε 1 A1 3 ε 1

1ε 1 2ε 1 3ε1

2C = 1 ε 2 A2 3 ε 2 1ε2 2ε2 3ε2

1 ε 3 A3 3 ε 3 1ε3 2ε3 3ε3

1ε1 2ε1 A1

1ε 1 2 ε 1 3 ε 1

3C = 1ε2 2ε2 A2 1 ε 2 2 ε 2 3 ε 2

1ε3 2ε3 A3 1 ε 3 2 ε 3 3 ε 3

A1 2ε2 3ε2

2ε3 3ε3

ndash 2 ε 1

A2 3ε 2

A3 3ε3

+ 3 ε 1

A2 2ε2

A3 2ε3

ASM expanded

CHAPTER VI

RIBOFLAVIN

61 INTRODUCTION

53 (Chapter 5)

045 Non-

(Ahmad et al 1980)

and 120 min

250 300 400 500 600

Wavelength (nm)

method

(Mtimes 105)

0 300 000 000 000 300

30 263 028 008 004 305

60 229 060 012 007 308

90 197 078 023 009 309

120 173 086 030 012 311

(Mtimes 105)

0 300 000 000 300

30 269 023 012 304

60 239 040 021 308

90 213 058 031 304

120 194 066 045 311

(Mtimes 105)

0 300 00 00 300

30 255 036 015 306

60 215 058 029 308

90 201 071 032 306

120 191 079 037 312

(Mtimes 105)

0 300 000 000 300

30 273 017 014 306

60 245 032 024 310

90 223 042 036 308

120 199 049 052 306

(Mtimes 105)

0 300 000 000 300

30 268 020 015 305

60 245 031 028 307

90 223 040 039 304

120 202 049 050 302

(Mtimes 105)

0 300 000 000 300

30 269 022 012 303

60 245 037 022 304

90 222 052 031 307

120 204 060 039 309

(Mtimes 105)

0 300 000 000 300

30 275 017 011 308

60 251 031 023 309

90 227 037 039 306

120 208 046 050 304

method

minminus1

(water)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times1

Time (min)

0 30 60 90 120

times10

Time (min)

RF () FMF () LC ()

0 30 60 90 120

times10

Time (min)

M) in water

(pH 70)

acetonitrile

0 20 40 60 80 100 120 140

Time (min)

times10

0 20 40 60 80 100 120 140

Time (min)

M times

methanol

ethanol

0 20 40 60 80 100 120 140

Time (min)

times10

0 20 40 60 80 100 120 140

M times

Time (min)

1ndashpropanol

1ndashbutanol

0 20 40 60 80 100 120 140

times10

Time (min)

0 20 40 60 80 100 120 140

Time (min)

M times

ethyl acetate

0 20 40 60 80 100 120 140

times10

Time (min)

Solvents Dielectric

constant (isin)

(25 oC)

Acceptor

Number

Inverse

viscosity

(mPasndash1

(25 oC)

kobs times 103 min

ndash1

plusmnSDa

Ethanol 243 371 0931 345plusmn015

Water 785 548 1123 461plusmn025

() water

00 100 200 300 400 500 600 700 800

Dielectric constant

s times

() water

00 100 200 300 400 500 600 -70

s times

10 15 20 25 05 30 00

Viscosity (mPa s)-1

times 1

00 05 10 15 20 25 30 35

Viscosity (mPas)-1

the medium

3FL + AH (F

σndash+) FLH (61)

CHAPTER VII

71 INTRODUCTION

B radicmicro (71)

where ZA and Z

Corsaro 1977)

reported

53 (Chapter 5)

(M times105)

0 500 00 00 00 500

30 451 019 021 010 501

60 398 039 045 019 506

90 373 053 059 022 507

120 340 064 071 027 508

(M times105)

0 500 00 00 00 500

30 446 020 022 016 504

60 386 044 049 021 508

90 332 069 073 029 509

120 309 076 081 035 501

(M times105)

0 500 00 00 00 500

30 435 020 031 016 502

60 381 039 052 029 505

90 331 065 071 035 508

120 288 078 089 046 501

(M times105)

0 500 00 00 00 500

30 417 031 035 020 503

60 361 054 058 031 504

90 308 069 082 043 507

120 269 081 099 052 508

(M times105)

0 500 00 00 00 500

30 404 032 044 022 502

60 336 056 075 036 505

90 290 068 097 047 507

120 245 079 118 059 501

(M times 105)

0 500 00 00 00 00 500

30 435 015 016 039 008 513

60 378 026 028 048 020 508

90 329 035 046 071 030 511

120 280 048 060 092 042 522

(M times 105)

0 500 00 00 00 00 500

30 416 024 036 057 006 539

60 353 040 059 075 016 543

90 293 079 081 134 028 615

120 251 089 091 175 034 640

(M times 105)

0 500 00 00 00 00 500

30 386 023 032 059 006 500

60 307 040 056 083 014 511

90 239 059 069 119 021 516

120 194 064 081 131 033 503

(M times 105)

0 500 00 00 00 00 500

30 369 030 036 062 009 506

60 280 045 060 093 023 501

90 217 060 073 122 033 509

120 153 071 089 145 048 506

(M times 105)

0 500 00 00 00 00 500

30 338 036 046 074 009 503

60 238 055 081 112 014 510

90 164 064 116 131 027 502

120 119 073 126 149 037 504

(M times 105)

0 500 00 00 00 00 500

30 398 016 031 045 010 510

60 327 031 055 066 022 508

90 267 042 065 085 041 503

120 224 050 076 101 049 506

(M times 105)

0 500 00 00 00 00 500

30 367 027 037 056 013 504

60 286 047 051 096 020 511

90 221 059 069 120 031 513

120 178 057 082 139 044 509

(M times 105)

0 500 00 00 00 00 500

30 354 024 049 059 014 504

60 236 049 069 108 038 508

90 168 068 076 139 049 503

120 108 078 096 158 060 509

(M times 105)

0 500 00 00 00 00 500

30 295 040 051 100 015 506

60 160 056 108 143 033 505

90 097 069 121 168 045 502

120 076 075 132 177 051 506

(M times 105)

0 500 00 00 00 00 500

30 282 046 060 106 006 511

60 145 076 088 154 037 505

90 079 091 104 175 051 509

120 052 100 110 200 057 507

(M times 105)

0 500 00 00 00 00 500

30 397 029 026 035 017 504

60 309 036 049 076 037 507

90 239 048 061 105 051 504

120 180 067 075 126 062 508

(M times 105)

0 500 00 00 00 00 500

30 361 029 042 047 023 508

60 256 048 056 095 047 512

90 183 061 077 118 063 502

120 127 073 095 145 071 514

(M times 105)

0 500 00 00 00 00 500

30 314 032 050 075 035 506

60 195 055 090 113 050 513

90 130 070 108 133 062 508

120 075 085 130 145 071 506

(M times 105)

0 500 00 00 00 00 500

30 292 042 052 079 039 504

60 148 069 083 135 066 511

90 078 093 103 155 076 509

120 042 103 114 163 084 506

(M times 105)

0 500 00 00 00 00 500

30 217 049 070 113 055 504

60 113 060 096 157 074 509

90 057 073 106 178 086 511

120 024 082 117 187 093 506

(M times 105)

0 500 00 00 00 00 500

30 425 013 028 027 009 502

60 338 032 041 065 024 509

90 251 045 074 091 043 514

120 157 066 085 135 059 512

(M times 105)

0 500 00 00 00 00 500

30 313 041 046 085 019 506

60 214 056 068 115 047 509

90 140 072 085 150 057 506

120 099 081 096 164 067 507

(M times 105)

0 500 00 00 00 00 500

30 298 037 062 075 030 506

60 179 061 079 125 056 511

90 099 076 097 155 075 502

120 049 088 108 169 087 508

(M times 105)

0 500 00 00 00 00 500

30 249 049 068 099 036 501

60 099 071 118 145 067 509

90 049 082 128 167 077 506

120 023 088 137 178 086 512

(M times 105)

0 500 00 00 00 00 500

30 210 062 086 126 026 508

60 078 088 112 179 049 506

90 034 094 120 190 069 509

120 013 099 132 201 080 511

photoproducts

been studied

M) at pH 70

kobs= k1+ k2+ k3 (74)

RF = RF0 endashkt

Similarly

) (79)

) (710)

) (711)

k2 RF0 kobs

k3 RF0 kobs

ndash RF0 endashkt

k1 RF0

is not clear

other factors

5times10minus5

Buffer

Concentration

Strength

kobs times 103

(minndash1

k0 times 103

(minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

02 01 276 085 139 063 072 193

02 485 284 070 144 197

03 715 407 102 198 205

04 978 535 177 255 209

05 1190 684 201 321 213

03 01 445 120 224 109 111 201

02 825 425 151 185 229

03 1185 632 240 265 238

04 1505 835 253 345 242

05 1860 1042 296 521 248

04 01 525 135 259 127 121 214

02 1150 501 282 226 221

03 1571 756 370 325 232

04 2030 1115 487 466 239

05 2491 1279 561 522 245

05 01 735 141 380 166 170 222

02 1250 660 285 277 238

03 1891 991 478 402 246

04 2421 1220 615 482 253

05 3032 1603 638 607 264

() 005 M (times) 01 M () 02 M (∆) 03 M () 04 M () 05 M

0 01 02 03 04 05 06

Ionic Strength (M)

ions leading to the

of [1RFndashHPO4

2minus

RF [1RF] (714)

[1RF] + NaCl [

1RFhelliphellipCl

ndash] + Na

+ (715)

[1RF helliphellipCl

pathways

of the plots

B = 105 102 = 103 = ~ + 1 (for k1)

B = 161 102 = 157 = ~ + 160 (for k3)

exciplex

000 010 020 030 040

radicμ1 + radicμ

000 010 020 030 040 050 060 070 080

radicμ

] (717)

+H ndashH+

CHAPTER VIII

81 INTRODUCTION

) (Mortland

additi

photoreduction(1)

(4) (5)

H+ OH-

and Cd2+

and Zn2+

(Kaim et al 1999)

promoted by Fe3+

and Cu2+

(inhibited by

(1) (81)

Rearrangment

4a5 44a

and Zn2+

+ RH M(nndash1)+

+ H+ + R

˙ (81)

+ H+ + RʹO2

˙ (82)

reaction

(Chapter 5)

and Cu2+

11 RFndashFe2+

RF + 2Fe2+ +2HRFH2 + 2Fe3+

M) (pH 70) (____

ions (b) Zn2+

ions and (c) Cu2+

and Cu2+

ions appears to

complex formation

presence of Zn2+

and Cu2+

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 485 008 489 006 486 007 489 006 490 005

120 470 014 477 012 472 015 478 014 479 012

180 447 026 454 023 458 020 466 019 468 018

0 500 000 500 000 500 000 500 000 500 000

60 472 015 463 017 457 019 450 019 446 022

120 442 032 431 037 421 035 413 038 398 046

180 416 045 398 049 384 052 371 059 355 066

0 500 000 500 000 500 000 500 000 500 000

60 474 014 461 018 453 022 442 026 432 030

120 450 027 424 032 413 036 393 047 373 055

180 418 039 389 044 365 055 346 065 324 076

0 500 000 500 000 500 000 500 000 500 000

60 472 014 462 017 442 025 429 030 421 033

120 444 024 423 033 395 044 375 052 354 061

180 414 039 385 052 352 067 322 075 295 086

0 500 000 500 000 500 000 500 000 500 000

60 474 015 465 018 464 019 459 021 441 028

120 450 024 436 029 430 031 415 036 389 048

180 427 036 407 045 393 051 358 062 339 068

0 500 000 500 000 500 000 500 000 500 000

60 474 015 459 019 450 022 440 026 427 032

120 450 026 422 038 403 044 386 051 363 065

180 417 041 381 056 355 066 338 071 309 081

0 500 000 500 000 500 000 500 000 500 000

60 489 006 475 013 472 014 472 014 467 016

120 465 016 449 024 446 026 443 027 437 029

180 437 032 427 036 419 039 414 041 408 045

0 500 000 500 000 500 000 500 000 500 000

60 475 014 467 017 463 018 457 021 451 023

120 449 024 434 030 429 033 411 040 406 042

180 426 035 407 040 390 047 374 054 363 060

0 500 000 500 000 500 000 500 000 500 000

60 472 015 463 019 453 022 442 025 433 030

120 443 029 428 035 412 042 390 051 373 057

180 416 039 394 047 371 057 342 068 322 076

0 500 000 500 000 500 000 500 000 500 000

60 474 014 473 016 463 019 457 021 441 028

120 447 027 444 028 428 034 416 039 390 051

180 429 036 411 042 391 051 375 057 346 068

0 500 000 500 000 500 000 500 000 500 000

60 478 011 476 013 472 015 466 017 464 019

120 454 022 450 024 442 027 436 029 430 032

180 433 030 423 034 414 039 405 043 399 048

0 500 000 500 000 500 000 500 000 500 000

60 476 013 473 019 464 020 463 020 457 022

120 451 022 444 026 430 033 431 039 416 042

ndash4 M

Table 81 continued

5) (02 M Phosphate

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 448 014 457 014 457 014 465 014 467 013

120 405 015 416 015 416 015 424 015 436 014

180 363 017 374 016 381 015 395 014 408 013

0 500 000 500 000 500 000 500 000 500 000

60 425 015 416 015 416 016 398 016 389 016

120 369 017 346 018 338 019 323 019 309 020

180 322 019 298 020 279 021 257 023 245 025

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 407 016 407 016 387 016

120 371 017 363 017 338 019 323 020 300 020

180 319 019 302 020 279 023 259 024 234 026

0 500 000 500 000 500 000 500 000 500 000

60 416 015 398 016 380 017 371 017 352 018

120 346 018 323 019 295 021 275 022 255 024

180 291 021 257 023 229 027 203 029 177 034

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 421 015 416 015 406 016

120 380 017 363 017 354 018 338 018 320 020

180 331 019 310 020 298 021 279 022 262 024

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 015 416 015 396 016

120 380 017 363 017 346 018 338 018 323 019

180 328 019 308 020 295 022 274 023 256 025

0 500 000 500 000 500 000 500 000 500 000

60 454 014 426 015 426 015 426 015 416 015

120 406 015 371 017 363 017 354 018 338 018

180 367 019 316 022 311 023 295 025 281 026

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 015 407 016 396 016

120 381 017 363 017 346 018 331 019 323 020

180 334 019 311 020 293 022 274 027 251 029

0 500 000 500 000 500 000 500 000 500 000

60 436 015 416 015 407 016 398 016 381 017

120 371 017 346 018 331 019 316 020 293 022

180 319 019 291 022 266 023 244 025 228 028

0 500 000 500 000 500 000 500 000 500 000

60 426 015 426 015 407 016 398 016 361 018

120 371 017 354 018 338 019 323 019 262 024

180 320 021 299 025 279 028 259 031 189 037

0 500 000 500 000 500 000 500 000 500 000

60 436 015 426 015 416 016 416 016 406 016

120 381 017 363 017 354 018 346 018 330 019

180 328 021 314 024 299 025 286 029 273 033

0 500 000 500 000 500 000 500 000 500 000

60 426 015 416 015 421 015 416 015 404 016

120 371 017 354 017 354 018 346 018 330 020

ndash4 M

Table 82 continued

5) (04 M Phosphate

20a 30

0 500 000 500 000 500 000 500 000 500 000

60 416 011 421 010 428 009 436 009 447 007

120 347 027 354 026 369 021 389 018 402 015

180 292 034 303 030 319 028 343 026 359 021

0 500 000 500 000 500 000 500 000 500 000

60 403 018 372 026 375 026 358 030 347 033

120 325 024 282 037 276 038 251 045 244 046

180 252 032 216 042 194 044 171 053 165 055

0 500 000 500 000 500 000 500 000 500 000

60 393 019 381 020 375 021 358 024 347 026

120 307 028 289 031 276 033 254 038 244 041

180 237 037 215 039 200 041 181 044 170 048

0 500 000 500 000 500 000 500 000 500 000

60 375 016 347 022 334 024 319 027 295 022

120 272 030 246 033 219 035 195 036 182 035

180 200 040 167 045 143 048 122 051 103 061

0 500 000 500 000 500 000 500 000 500 000

60 393 011 381 015 384 015 375 016 364 018

120 319 025 298 029 289 031 276 033 263 036

180 251 033 233 036 221 038 209 041 197 043

0 500 000 500 000 500 000 500 000 500 000

60 393 011 381 014 384 014 375 017 364 021

120 317 022 298 025 289 027 276 031 263 035

180 251 029 229 032 223 034 207 037 194 039

0 500 000 500 000 500 000 500 000 500 000

60 393 014 384 016 384 016 375 018 364 020

120 303 022 298 023 289 025 276 027 263 029

180 241 031 229 033 221 035 203 037 191 039

0 500 000 500 000 500 000 500 000 500 000

60 393 013 384 015 384 015 367 017 345 018

120 315 019 298 021 289 023 272 026 237 029

180 255 025 225 029 215 033 198 035 169 039

0 500 000 500 000 500 000 500 000 500 000

60 393 011 375 018 367 019 347 021 337 025

120 302 022 282 026 26 030 242 035 231 038

180 237 033 207 036 188 038 169 041 155 044

0 500 000 500 000 500 000 500 000 500 000

60 393 014 375 019 367 020 347 022 337 024

120 302 019 282 025 266 029 242 032 231 034

180 234 027 213 031 171 041 171 043 159 047

0 500 000 500 000 500 000 500 000 500 000

60 393 013 384 014 367 019 363 019 350 022

120 309 019 295 021 272 026 263 027 251 031

180 242 026 226 029 207 032 195 034 183 036

0 500 000 500 000 500 000 500 000 500 000

60 393 016 381 019 375 021 358 022 347 024

120 315 027 289 033 276 034 254 038 244 040

ndash4 M

Table 83 continued

ions is shown in

in the order

2+lt Fe

3+ lt Ca

2+ +lt Fe

2+ lt Cd

2+ lt Cu

2+lt Mn

2+lt Pb

2+ lt Mg

2+lt Zn

2+lt Ag

Thus Ni2+

ions (∆) Co2+

(loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

() Mg2+

ions () Zn2+

ions and () Fe3+

00 10 20 30 40 50 60

M) (b2)

RF + Fe2+

M) (b3)

2014 2016 Sheraz et al 2014)

RF + Fe2+ RF-Fe2+

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

log co

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ag+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

in the presence Ag+

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

0 50 100 150 200

Time (min)

) (diams) 10

() 20 () 30 () 40 () 50

0 50 100 150 200

Time (min)

Metal Ions (kʹ)

Metal Ion Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

00 063 016 006

Ag+ 10 059 050 041 017

20 054 038 015

30 049 035 014

40 044 033 010

50 038 029 008

10 089 256 070 018

20 115 080 034

30 142 101 040

40 169 129 039

50 191 143 047

10 099 360 078 020

20 136 084 051

30 172 107 064

40 206 138 067

50 243 164 078

10 105 462 073 031

20 155 113 041

30 199 138 060

40 245 164 080

50 294 190 094

10 101 416 071 029

20 142 099 042

30 184 131 052

40 225 160 064

50 271 182 088

10 106 410 079 026

20 145 105 039

30 185 128 056

40 224 152 071

50 268 180 087

10 075 104 058 016

20 085 062 022

30 095 068 026

40 105 075 029

50 115 083 031

10 089 232 063 025

20 112 075 036

30 136 092 043

40 158 106 051

50 179 120 058

10 102 360 072 029

20 132 089 042

30 167 110 056

40 210 140 070

50 243 162 081

10 091 284 069 021

20 118 086 031

30 148 104 043

40 176 122 053

50 205 139 065

10 078 128 054 023

20 091 063 027

30 104 071 032

40 116 080 035

50 127 087 039

10 082 180 060 021

20 099 075 023

30 118 091 026

40 135 151 029

50 153 174 035

Table 84 continued

Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash1

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

00 204 111 038 054 205

Ag+ 10 182 184 125 027 028 446

20 164 112 025 026 430

30 144 094 023 025 376

40 127 084 019 023 365

50 112 072 016 022 327

10 243 384 195 020 027 722

20 285 231 022 031 724

30 325 256 032 035 726

40 363 291 033 040 728

50 396 315 036 043 730

10 249 410 201 021 027 724

20 285 229 025 031 726

30 325 256 034 035 728

40 365 290 036 039 730

50 409 329 033 045 732

10 285 742 226 027 031 729

20 358 283 036 038 733

30 435 343 043 048 736

40 505 402 048 054 738

50 575 446 059 060 741

10 235 246 180 024 029 620

20 265 201 030 032 628

30 295 223 036 034 655

40 325 245 039 036 671

50 358 286 035 041 697

10 235 334 180 024 029 620

20 269 207 029 033 625

30 302 228 035 036 629

40 335 243 044 038 633

50 371 284 045 044 637

10 227 232 149 035 042 354

20 260 179 032 049 360

30 283 195 035 053 360

40 304 210 038 056 365

50 332 230 041 061 369

10 235 358 178 025 030 593

20 270 207 029 034 605

30 305 231 035 037 624

40 334 253 041 040 631

50 373 284 045 044 636

10 251 462 196 025 031 625

20 301 233 031 036 647

30 345 268 036 039 687

40 385 303 041 043 699

50 427 333 048 046 711

10 254 410 179 032 043 411

20 285 201 039 043 467

30 323 231 044 048 475

40 362 259 049 054 479

50 404 289 056 059 483

10 236 256 168 029 039 425

20 255 184 032 038 484

30 280 204 034 040 510

40 300 220 038 042 519

50 319 232 043 044 523

10 237 308 189 021 026 726

20 271 218 024 029 730

30 302 238 030 032 734

40 332 265 030 036 736

50 358 284 036 038 738

Table 85 continued

Metal ion

concentration

(Mtimes104)

kobs times 103

(minndash1

kʹ times 10

(Mndash1

minndash

k1 times 103

(minndash1

k2 times 103

(minndash1

k3 times 103

(minndash1

00 351 222 049 075 296

Ag+ 10 315 332 184 061 069 263

20 280 171 042 066 259

30 247 152 035 059 257

40 214 129 033 051 252

50 190 114 030 046 247

10 402 528 262 059 079 331

20 462 290 070 101 287

30 515 310 094 109 284

40 570 335 098 136 246

50 615 363 104 147 246

10 407 496 259 069 079 325

20 460 295 072 092 320

30 509 328 077 103 317

40 560 357 089 113 315

50 599 373 099 126 296

10 475 1048 302 075 096 314

20 580 359 106 115 310

30 681 414 128 137 302

40 784 475 151 158 299

50 875 505 173 196 257

10 390 348 257 058 073 352

20 425 275 066 082 335

30 458 296 071 090 328

40 490 315 075 099 318

50 525 335 082 107 313

10 386 348 273 050 061 447

20 427 301 057 068 442

30 458 321 060 075 428

40 490 336 068 084 400

50 525 355 077 091 390

10 387 508 254 058 073 347

20 424 273 069 081 337

30 494 317 080 096 330

40 545 347 089 107 324

50 605 380 104 119 319

10 389 600 271 057 060 451

20 426 287 070 067 428

30 494 327 080 085 384

40 545 359 089 095 377

50 651 432 103 116 370

10 415 600 282 057 075 376

20 475 318 071 085 374

30 535 363 074 098 370

40 605 405 090 110 366

50 651 423 109 117 361

10 413 570 287 060 065 441

20 470 320 072 077 415

30 530 337 091 101 333

40 590 370 102 116 318

50 636 392 110 132 296

10 395 414 273 059 061 447

20 438 296 069 071 416

30 479 321 076 081 396

40 524 350 084 089 393

50 558 369 093 095 388

10 405 468 260 055 083 313

20 455 290 072 093 310

30 505 322 077 104 309

40 548 346 086 115 300

50 585 363 093 128 283

Table 86 continued

ions (∆) Co2+

ions (loz) Ca2+

ions (+) Fe2+

ions () Cd2+

ions (ndash) Cu2+

ions (diams) Mn2+

ions () Pb2+

ions () Mg2+

ions ()

ions () Fe3+

00 30 60 90

Fluorescence loss

bs times

minndash1

) (Table

minndash1

) (Table 86) The

Mndash1

minndash1

) and monovalent

order Zn2+

gt Mg2+

gt Pb2+

gt Mn2+

gt Cu2+

gt Cd2+

gt Fe2+

gt Ca2+

gt Fe3+

gt Co2+

gt Ni2+

gt Ag+

ions (Rutter 1958)

) bind to RF

presented (Fig 844)

] complex (Eq

state [1RFhellipFe

2+] state

(Eq (812))

RF + Fe2+ [RFFe2+]

metal-RF complex

[RFFe2+] [1RFFe2+]

[1RFFe2+] [RFHFe3+]

[RFHFe3+] RFH

+ Fe3+

2RFH RFH2

H+ OH_

ions and

CHAPTER IX

91 INTRODUCTION

(1)(4)

(5)(7) (6)

and alkaline pH

(Chapter 5)

to brown

9214 FTIR studies

RFndashAg NPs

0 1 2 3 4 5 6 7

Time (h)

cmndash1

distributed

measurements

9221 pH

NPs (Fig 99)

9222 Ionic strength

+ ions

questionable

91 and 92

0 50 100 150 200 250 300 350 400

Time (min)

k1times 103

k2 times 103

(min-1

k1times 103

(min-1

k2 times 103

(min-1

80 736 0060 521 0036

85 999 0088 843 0061

90 1285 0110 1122 0091

95 1523 0129 1324 0112

100 1740 0147 1480 0128

105 1822 0153 1524 0135

ion Concentration

k1times 103

k2 times 103

(min-1

k1times 103

(min-1

k2 times 103

(min-1

0002 754 0012 355 0009

0004 892 0022 622 0020

0006 1279 0046 1088 0043

0008 1630 0072 1399 0052

001 1740 0079 1444 0054

750 800 850 900 950 1000 1050 1100

k1times

k2 times

750 800 850 900 950 1000 1050 1100

k1times

k2 times

0 0002 0004 0006 0008 001 0012

k1times

k2 times

0 0002 0004 0006 0008 001 0012

k1times

k2 times

RF1RFhv

3RFisc1RF

RF3RF + Ag+

+ Ag NP

0RFRF + H+

RF3RF + 0RF + RFH

+ ion to form RF

0RF] Further

0RF] lead to

] radical (Eq (95)

CONCLUSIONS

solvents

HPO42-

REFERENCES

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Sci 1972611081ndash1085

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Res 198013148ndash155

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Research Article

INTRODUCTION

Photolysis

pH Measurements

Spectrometric Assay

RESULTS

Photoproducts of RF

Ahmad et al

DISCUSSION

Effect of Solvent

0 30 60 90 120

on times

Time (min)

Time(min)

RF(Mtimes105)

FMF(Mtimes105)

LC(Mtimes105)

Total(Mtimes105)

0 300 00 00 30030 255 036 015 30660 215 058 029 30290 201 071 032 304120 191 079 037 307

Effect of Viscosity

00 100 200 300 400 500 600 700 800

times10

inndash1

Dieletric Constant

Ahmad et al

Mode of Photolysis

CONCLUSION

00 05 10 15 20 25 30

bstimes

103 (m

inndash1

Viscosity (mPa s)-1

00 100 200 300 400 500 600lnk o

bs times

REFERENCES

Ahmad et al

1 Introduction

eth2THORN

21 Photodegradation

22 Assay Method

Time(min)

RF(M times 105)

CDRF(M times 105)

FMF(M times 105)

LC(M times 105)

LF(M times 105)

Total(M times 105)

0 500 00 00 00 00 50030 210 062 082 124 026 50460 078 088 110 179 043 49890 034 094 120 188 055 491120 013 099 122 198 074 506

Similarly

1minuseminuskt

eth9THORN

1minuseminuskt

eth10THORN

1minuseminuskt

eth11THORN

Ionic strength(M)

kobs times 103

(minminus1)k1k3

02 01 276 085 139 063 072 19302 485 284 070 144 19703 715 407 102 198 20504 978 535 177 255 20905 1190 684 201 321 213

03 01 445 120 224 109 111 20102 825 425 151 185 22903 1185 632 240 265 23804 1505 835 253 345 24205 1860 1042 296 521 248

04 01 525 135 259 127 121 21402 1150 501 282 226 22103 1571 756 370 325 23204 2030 1115 487 466 23905 2491 1279 561 522 245

05 01 735 141 380 166 170 22202 1250 660 285 277 23803 1891 991 478 402 24604 2421 1220 615 482 25305 3032 1603 638 607 264

RFrarrhv 1RF

eth14THORN

eth22THORN

4 Conclusion

References

Iqbal Ahmada

Zubair Anwara

Sofia Ahmeda

Highlights

Abstract

Graphical Abstract

BIODATA

Qualifications

Impact Factor 17001

Publications

Chapters

2017 (In Press)

Reviews

2013417ndash22

201314 318ndash326

20161951ndash57

Research Papers

201415550ndash559

2014153ndash7

01Front-Pagespdf

02Chapter-Ipdf

03Chapter-IIpdf

04Chapter-IIIpdf

05Chapter-IVpdf

07Chapter-Vpdf

08Chapter-VIpdf

09Chapter-VIIpdf

10Chapter-VIIIpdf

11Chapter-IXpdf

12CONCLUSIONSpdf

13Referencespdf


Abstract

INTRODUCTION


Photolysis

pH Measurements


Spectrometric Assay


RESULTS

Photoproducts of RF




DISCUSSION

Effect of Solvent


Effect of Viscosity

Mode of Photolysis

Conclusion

References



1 Introduction


21 Photodegradation

22 Assay Method









4 Conclusion

References

16Metalpdf

17BIODATApdf

Documents

EFFECT OF SOLVENT, IONIC STRENGTH AND