STUDIES ON ION - EXCHANGE MATERIALS, CHROMATOGRAPHIC AND PAPER

ELECTROPHORETIC SEPARATION OF SUBSTANCES

ABSTRACT

THESIS SUBMITTED FOR THE DEGREE OF

JBoctor of $l|tlo)Biop{ip IN

BY

^oofta ?ii8(mant

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA) 1994

T4660

A B S T R A C T

There are five chapters in this thesis. The first

chapter consists of a detailed and uptodate literature of

the subject. Applications of three component Ion-exchange

materials receiving increasing attention due to their resis­

tance to heat and radiations. They are used for high temper­

ature separation of cationic and anionic species in radio­

active waste and in pharmaceutical analysis. Chelating

resins have also drawn attention due to their high stability

and complex forming attitude with different metal ions.

Their utility can apply in the selective separation of heavy

metals from industrial waste and contaminated water. Use of

other chromatographic techniques such as coloumn, thin layer

and paper chromatography determination and separation of

organic and inorganic components have also paved the way of

analysis. Inorganic ion-exchanger itself or mixed with

conventional adsorbents e.g. silica gel as thin layer have

been exploited for the analysis of biological, pharmaceuti­

cal and industrial samples. Thin layer chromatography is a

simple, rapid, versatile and low cost method which is appli­

cable to the analysis of a variety of multicomponent

mixtures except volatile or reactive substances. Paper

electrophoresis is also a suitable frequently used conven­

tional technique for the analysis of charged species present

in electrolyte solution.

The second chapter of this thesis comprise of the

electrophoretic behaviour of 21 cimino acids on whatman No.l

paper strips in citrate-phosphate buffer system of varying

pH. The role of sodium dodecyl sulphate (SDS) with citrate

phosphate buffer on the mobility of amino acids has been

critically observed. It has been found that the presence of

SDS in citrate phosphate buffer enhances the separation

possibilities significantly. On the basis of difference in

the movement of amino acids a number of important qualita­

tive and quantitative separations have been achieved in

these solvent systems the practical utility of the method

has been demonstrated by achieving quantitative separation

and determination of amino acids constituents of

commercially available standard drugs namely Santevini

(plus) and Astymin-3.

The third chapter include(the synthesis of samples of

zirconium(IV) iodophosphate under varying conditions at pH-

1.0. The most chemically and thermally stable sample is

prepared by adding a mixture of aqueous solution of O.lM

potassium iodate and O.lM potassium dihydrogen orthophos-

phate to aqueous solution of O.lM zirconium oxychloride. Its

ion-exchange capacity for Na is 1.78 meq/dry gm exchanger.

The material has been characterized on the basis of chemical

composition, FTIR, TGA and DTA. / The effect of heating on

the exchanger at different temperature on the exchange capa­

city has also been studied. The sorption behaviour of impor­

tant metal ions in HCl-DMSO systems has been studied. On

the basis of this study a number of analytically important

metal ion separations have been achieved on the column of

zirconium(IV) iodophosphate. Practical utility of these

separations have been reflected in the determination and

separation of metal ion contents in antacid drug sample.

In the fourth chapter I thin layer chromatographic

studies of amino acids on thin layers of stannic-arsenate

silica gel have been reported. The ion -exchange and adsorp­

tion takes place simultaneously is an added advantagte to

the technique.' Amino acids have been chosen as they play a

vital role in controlling the physiological activity in

human body. The study has shown that the degree of retention

of different amino acids varies with the nature of develop­

ing solvent. It has been observed that pH also plays a

prominent role in the movement of amino acids. Thus it has

been possible to achieve a good number of important separa­

tions in citrate phosphate buffer of varying pH. Phenylal­

anine has been successfully separated from other amino acids

in n-Butanol-1,4-Dioxane-potassium chloride (5:4:1) system.

Besides this a number of binary and ternary separations have

also been achieved in different solvent system.

The last chapter describes [the modification of anion

exchange resin Amberlite IRA-400 by interaction with

eriochrome black-T. Distribution coeffecients of metal ions

in water and formic acid have been determined. ) It has been

found that the selectivity of the material is enhanced due

to the presence of chelating group in the resin material.

Besides a number of important and analytically difficult

2+ bmary separations, selective separation of Hg from a

synthetic fmixture of other heavy metal ions have been

successfully achieved on small columns of this material.

T^/t60

STUDIES ON ION - EXCHANGE MATERIALS. CHROMATOGRAPHIC AND PAPER

ELECTROPHORETIC SEPARATION OF SUBSTANCES

THESIS SUBMITTED FOR THE DEGREE OF

Bottor of ^liilofiioplip IN

Cfiemifl^trp

BY

^oofta QSieimant

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH ( INDIA) 1994

(Qe nnj

Jyappa and f4^ntnti

Dr. Syed Ashfaq Nabi M.Sc. M.Phil., Ph.D. (Alig.)

DEPARTMENT OF CHEMISTRY AUGARH MUSLIM VMIERSITY AUiiARH - 202002 (V.P.) INDIA Phone: 405515

50- iZ '"^^^

CERTIFICATE

Certified that the work embodied in this thesis

entitled "Studies on lon-Excbange Materials, Cbromato-

grapbic and paper Electrophoretic Separation of

Substances" is the result of the original research work

of Miss Soofia Usmani carried out under my supervision

and is suitable for submission for the award of the

Degree of Doctor of Pbilosophy in Chemistry, of Aligarh

Muslim University.

^ '

(Dr.Syed Ashfaq Nabi)

ACKNOWLEDGEMENT

It is my first privilege to acknowledge with deep

sense of gratitude and humble submission the enthusiastic

support and never failing encouragement, extended by my

supervisor Dr.Syed Asbfaq Nabi, who made it possible for me

to bring the thesis in hand to a glorious furnish. I really

feel that without bis valuable suggestions this attempt

could not have take place.

The necessary research facilities provided to me by

Prof. Nurul Islam, Chairman, Department of Chemistry are

also thankfully acknowledged. I wish to express my sincere

thanks to Prof.5.Z. Qureshi, for bis help and moral support.

The invaluable help and much fruitful suggestions rendered

to me by Dr.Nafisur Rahman are thankfully acknowledged. I

wish to thank my lab colleagues Mr.Irshad A. Qureshi,

Rasbeed M.A. Jamhaur, R. Kbayer and Muzaffar A. Andrabi for

their help and friendly attitude.

My special thanks to my dear friends Ghazia,

Dr.Shabjahan, Namita and Farba for their whole hearted

cooperation and encouragement throughout the work.

I shall be failing in my duties if I forget here my

loving parents whose dedication to the fulfilment of this

task whose material and ideological support and whose energ­

ising encouragement paved this way for me to carry on this

work all through the time it has taken.

contd.

I shall be ever indebted to my brother Ayaz and sister

Fouzia for their encouragement and love. My thanks also

goes to my Mamu, Mr.Ismail Husain and Auntie Dr.Tayyaba

Husain for their encouragement and affection throughout the

tenure of this work.

Financial sup port provided by MAAS, Aligarb is also

thankfully acknowledged.

In the last but not the least I express my hearty

gratitude to Almighty Allah who led me to this path of

success.

l^^ ( SOOFIA USMANI )

C O N T E N T S

CHAPTERS PAGE Nos.

CHAPTER -

CHAPTER - II

CHAPTER - III

Introduction

References

1

37

Paper Electrophoretic studies of ff -Amino Acids in Citrate Phosphate Buffer and Sodium Dodecyl Sulphate Mixed Systems. 56

Synthesis, Characterization and Ana­lytical Application Of Ion-Exchange Material: Zirconium(IV) lodophos-phate. 76

36

55

75

101

CHAPTER - IV Thin Layer Chromatographic Separa­tions of a -Amino Acids on Stannic-Arsenate — Silica-gel Mixed Layers. 102 116

CHAPTER - Sorption Studies of Metal Ions on Modified Anion Exchange Resin, Selective Separation of Mercuryill) from Other Heavy Metals. 117 13U

**********

LIST OF PUBLICATIONS

1. Paper Electrophoretic studies of ot-Amino Acids in

Citrate Phosphate Buffer and Sodium Dodecyl Sulphate

Mixed Systems.

J. Planar Chromatogr.- Mod. TLC (In Press)

2. Sorption Studies of Metal Ions on Modified Anion

Exchange Resint Selective, Separation Of Mercury. (II)

from other Heavy Metals.

J. Indian Chem. Soc. (In Press)

3. Selective separation of Mercury from other Heavy Metal

Ions on Bromophenol Blue Sorbed Anion Exchange Resin.

Indian J. Chem. (In press).

4. Synthesis Ion-Exchange Properties and Analytical

Applications of a Novel Tin(IV) Sulphosalicylate

Exchanger.

Indian J. Chem. (In Press)

CHAPTER -1

INTRODUCTION

INTRODDCTIOH

There are various methods in analytical chemistry that

are employed for separation. Today ion exchange is recognised

as one of the most versatile and standard analytical tool

and is widely used in inorganic, organic and biochemical

separations. It offers many advantages over the classical

methods of separation such as precipitation, distillation and

filtration. The technique can be applied to both micro as

well as macro analysis and may serve even for the routine

analysis.

The development of ion-exchangers and sorbents can be

divided into several periods (1).

1. The period upto year 1850 was the period of first experi­

mental observation and information, principle of ion-

exchange had not yet been discovered.

2. The period from 1850-1905 is characterized by discovery

of the principle of ion-exchange and the experiment in

the technical utilization of ion exchanger.

3. 1905-1935, characterized by the use of inorganic ion-

exchange sorbents and modified natural organic materials.

4. The period of 1935-1940 was the period of synthetic

organic ion-exchangers. Inorganic ion-exchange sorbents

were almost completely eliminated from all applications.

5. The period from the mid 1940s to the present

characterized both by the continued rapid development of

artificial organic ion-exchangers and by renaissance in

inorganic ion-exchange sorbents and their practical

applications.

Though the use of solid adsorbents to improve water

quality has been recorded since ancient times. In 16 23

Francis Bacon described a method for removing salt from sea

water, Hales also recommended that sea water be desalinated

by filtration through stone water. In 1790 Loitz purified

sugar beet juice by passing it through charcoal.

The first information regarding the discovery of ion-

exchange principle was found in the 19th century, when some

soil chemist found, that soil and specially clay retain

disssolved fertilizers particles, Gazzeri (1819). In 1826

Sprengel stated that the humus frees certain acids from soil.

Fuchs (1833) pointed out that the action of lime frees

potassium and sodium from some clays. By the middle of 19th

century sufficient experimental information had been

collected on the as yet unformulated"ion-exchange" principle.

In the middle of 1845 Thompson and Spence carried out

studies of the behaviour of ammonia salts in soils and found

that ammonium sulfate converted in the calcium sulfate (2).

During 1850-1855 the agrochemist Way (3) published a number

of papers dealing with the behaviour of soils in the presence

of various cations. He demonstrated the following mechanism

to be one of the ion-exchange involving the complex silicates

present in the soil. As described by Way the process observed

by Thompson could be formulated.

Ca - Soil + ()viH4)2S04 ^^ >> NH4 - Soil + CaS04

The work of Eichorn (1850) (4) was very important and

demonstrated that exchange process in soils are reversible.

Harm (1896) and Rumpler (1903) (5) proposed the use of nat­

ural and artificial aluminosilicates to purify beet syrup.

Cans (6) developed the basis for the synthesis and technical

application of inorganic cation exchangers in the begining of

the 20th century. He termed the cation exchangers based on

aluminosilicates "permutitis" and felt that these

ion-exchangers would find broad applications. Nonetheless in

1917, Folin and Bell ,(7) developed an analytical method based

on his material for the determination of ammonia in urine

using zeolites.

During the period between 1930s and 1940s inorganic

ion-exchagne sorbents were replaced in all fields by new

organic high molecular weight ion-exchangers.

The rebirth of inorganic ion-exchange sorbents began

in 1950. A great impulse for their renewed use came from the

field of nuclear research. Common organic ion-exchange resins

were found to be inadequate under high temparature and in

highly acidic media. They also decompose in the presence of

ionizing radiations. Therefore organic exchangers replaced by

inorganic ion-exchangers. Pioneering work was carried out in

this field by the research team at the Ook Ridge National

Laboratory led by Kraus and by the English team led by

Araphlett (8,9,10). In the last decade intense research has

continued on the synthesis of crystalline ion-exchange

materials, elucidation of their structure and correlation

with their physical, chemical and ion-exchange properties.

Great work has been done in this field by Clearfield et.al

(11,12). Alberti (13,14) and Walton (15,16,17,18) have also

worked on different aspects of synthetic inorganic ion-

exchangers. In India Qureshi and co workers have developed a

large number of such inorganic materials and studied their

ion-exchange behaviour during last fifteen years; few

improtant references are (19-24).

Synthetic organic ion-exchanges can be classified on

the basis of exchangeable species (cation, anion, ampholyte

and multifuncutional types) funcutional groups (strong, weak

and acid/base type) skeleton types (polymers, copolymers and

polycondensates) and the rigidity of the polymeric structure

(microporous gel, macroporous, isoporous).

On the basis of chemical characteristic of the ion

exchanging species inorganic ion-exchanger can be classified

in to following main groups as propsoed by Vesely et al.

(25).

1. Hydrous oxide

2. Acidic salts of multivalent metals

3. Salts of heteropolyacids

4. Insoluble ferrocyanides

5. Alumino silicates.

Hydrous oxides groups include bivalent, trivalent

tetravalent, pentavalent and hexavalent atoms. Hydrous oxide

1 2 -f2 +2 of Be , Mg , Zn and their mixtures with Fe(OH)T or

Al (OH)^. Hydrous Beo acts as cation and anion exchanging

material. A mixture of Mg(0H)2 with Fe(0H)2 or AKOH)^

exhibits interesting sorption properties and has been

successfully used to adsorb radio nuclides in trace

concentration. Hydrous oxide of trivalent metals includes

pseudomorphic iron hydroxide, hydrous Alumina, Qi -A1„0-,,

QLr AlOOH and A1(0H)_. These sorbents used in thin layer

chromatography. Hydrous oxides of tetravalent metals includes

oxides of Si02, Sn02, '^^'^2' '^^^2' ^^^2 ^^^ y.nO^. They act

either as cation exchagner in alkaline solutions or as anion

exchanger in acid solutions. The hydrous manganese dioxide

with rather unusual selectivity sequence for alkali metals

has been described by Tsuji (26). Inuoe and Coworkers (27)

have studied the isotopic exchange rate of sodium ions

between the hydrous tin(IV) oxide in the Na form and aqueous

solution of sodium salt.

A wide rage of compound of acidic salts of polyvalent

metals has been, described as ion-exchangers. The renaissence

of the practical application of these substances is closely

connected with the development of nuclear science and also

with the modern, more exact approach to the understanding and

elucidation of sorption processes. The mechanism of

ion-exchange on the acidic salts of polyvalent metals is also

a very complex process The work of Clearfield et.al 1982

(28) who carried out structural studies and studies of the

phase conversion during ion-exchange to elucidate the

sorption mechanism on these materials and correlated the

degree of crystallinity with the sorption properties. For

example difference in the structure, composition and exchange

behaviour of amorphous zirconium phosphate can result from

different preparation conditions. It has been found that it

is possible to obtain an almost continous increase in the

crystallinity until true crystals of «6-zirconium phosphate

are obtained. The sorption process on amorphous zirconium

phosphate is a function of the loading. The exchangeable

sites are not energetically equivalent. The incoming ions

initially occupy the most favourable sites, so that the

alkali ions are more or less hydrated when they enter the

cavities. As the exchange process proceeds smaller and

smaller cavities are occupied and the less strongly bonded

water is forced out by the larger cations. The process is

accompanied by an increase in the interlayer spacing. Two

cations must approach each other closely as they occupy the

same cavity, lattice disordering occure and is reflected in

the release of phosphate in the solution. Among metals

studied have been zirconium. Thorium, Titanium, Cerium (IV)

tin (IV) etc. and anions employed include phsophate,

arsenate, antimonate, vanadate silicate etc. These salts

acting as cation exchangers are gel like or micro

crystalline materials and possess usually a high chemical,

temparature and radiation statiblity (29,30,31). The cation

exchange properties arise from the presence of readily

exchangeable hydrogen ions, associated with the anionic

groups present in the salts.

Zirconium phosphate is the most t^xtensively studied

inorganic ion exchange material. It shows amorphous (30,31,

32) semicrystalline (33,34) and crystalline (35). Clearfield

and others (33,35,36,37,38,39) have tried to solve the

structure of Ct-zirconium phosphate. Stereiko (40) has given

the formula of H form and salt form of zirconium phosphate

as -

Zr(OH)x(HP04)2-Zx.YH20 (X = 0-2)

Zr(OH)x[(MP04)ni(HP04)j^]2_zr.YH20

respectively.

A few analytically important applcations of zirconium

phosphate exchanger have been cited below.

1. Purification of reactor Coolants (41)

2. Decontamination of D_0 (42)

3. Decontamination of radio active waste water (43)

137 4. Cs form and reprocessing solution (44)

+4 5. Pu from irradicated Uranium (45)

The parent acids of these salt are the

12-heteropolyacids with the general formula H XY,„0.„.nH„0

where m=3,4 or 5, X can be phosphorous, arsenic, silicon,

germaniiim and boron and Y is the number of different elements

such as molybdenum, tungston and vanadium. Much of subsequent

investigation of the ion exchange properties of these salts

have been carried out by Smith and Robt(46,47). Three component

ion-exchangers show superiority over simple salts mainly in

three aspects. They are more thermally and cheir.ically stable

and more selective (49). Qureshi and Qureshi (48) have

presented a review on the applications of ion-exchange

methods in radiochemical separations which is needed in

activationanalysis, waste processing, fuel processing or

reactor coolant water purifications.

When metal salt solutions are mixed with H^[Fe(CN)^],

Na.[Fe(CN^] solutions (50) precipitates with various composi­

tion are formed, depending on the acidity, order of mixing

and the initial ratio of the reacting components. Pure

compounds are mixtures with limiting compositions

M^[Fe(CN)g] (M''"=Ag), M2[Fe(CN)g] M^"^=Zn,Cd,Cu,Co,Ni,Mn ) , M^

M^'*'[Fe(CK)g] (M'''M "*'=CsZn,HCu, KCo,KNi, HMn), l^^M^[Fe{Cli) ^] 2

(M'^M^''"=HZn,NaZn,KZn,HCo) , M"''M- ' [Fe(CN) g ] (M"'"M- "*"=NaFe,KFe ,RbFe, 2+

CsFe) and M^M [Fe(CN) g ] (M'^M^=RbNi ,KNi ) with various amounts

of water of crystallization have been reported.

Insoluble ferrocyanides have found various

applications in analytical chemistry and in technological

practice because of their highly selective ion-excharige

p::operties and satisfactory chemical and mechanical

properties.

Ion-exchange in aluminosilicate is a very vast topic.

They can be divided into three main groups air rphous

substances, two dimensional layered aluminosilicates as

synthetic analogue of clay minerals, and three dimensional

10

structures (zeolite). The greatest attention will be paid to

synthetic zeolites, as their molecular and ion-seive

properties are useful for analytical applications.

Synthetic aluminosilicates can be approximately

related to their naturally occuring analogues. The most

important of these compounds are listed in the following

table.

Zeolite Idealized unit cell Free diameter Total exchangi composition of the largest capacity(meg/

channel (if) gm)of water species

ZK

ZK

2.4-6.1

2.8

3.5

3.7-4.2

3.8

4 . 1

Heulandite- : Ca4[!:A102)3(5102)23•24H20

Ana lc i t e Na^g[(AIO2)^g ^ ^^2^32 ^•I6H2O

Linde-T Na3 IIAIO2) Q (Si02) 23 ] • 27H2O

CJiabazite Ca2 [fU02) 4 (Si02) 3 ].I3H2O

Na3o[(Al02)3o(Si02)gg].98H20

Nag [ (AIO2) 9 (Si02) 15 ] . 27H2O

ClinoptilQlLte( Ca ,Na2 ,K2) 3 [ (AIO2) g (Si02) 3Q ] . 24H2O 4 .1 -6 .2

P h i l l i p s i t e (K,Na)^Q[ (AIO2)3^0(8102)22]-2OH2O 4 .2 -4 .4

Mordenite Nag [ (AIO2) 3 (Si02) ^Q ] . 24H2O 6.7

Linde X Nag^C (AIO2)35(8102)^Q^] .256H2O 7.4

Linde Y Nag2[ (^°2^52^^^°2^140^ '^^^^2° "^'^

Linde A Ngg[ (Al02)gg(Si02)gg].216H20 4 . 1

AT p r e s e n t a l a r g e number of i n o r g a n i c i o n - e x c h a n g e r s

h a v e b e e n s y n t h e s i z e d . V a r i o u s s y n t h e t i c i n o r g a n i c i o n -

e x c h a n g e r s , t h e i r c o m p o s i t i o n , i o n - e x c h a n g e r s c a p a c i t y a r e

3.45

4.95

3.43

4.95

4.57

5.50

2.64

4.67

2.62

6.34

4.10

7.04

11

listed in Table- 1.1. It has been found that mixed salts or

three component ion-exchanger possess ion-exchange proper­

ties different from simple salts or two components ion-

exchangers they show superiority over simple salts mainly in

three aspects. They are thermally and chemically stable,

secondly they are more selective in natures (50), and

thirdly their ion-exchange capacities, are higher (51-53) as

compared to two component ion exchanger (54,55).

Limitations of zeolites and clay is their stability

in acids led to the discovery of some ion-exchange materials.

In 1931 Kullgren (104) observed that sulfide cellulase work

as an ion exchanger for the deterroination of Cu. In 1935

Adam and Holmes (105) found ion-exchange property in crushed

phonograph.

The discovery of ion exchange resins with three

dimensional network obtained by polymerization of organic

monomers has resulted a break through in separation Science.

The polymeric ion exchange resins which are commercially

available based mainly on cross-linked polymeric compounds.

A list of commercial ion exchange resins is given in Table

1.2. The use of weak acid cation exchange resin for the

removal of permanent hardness and inversion of sugar with

cation exchange resin are pertinent examples.

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19

The interesting discovery led the inventors to the

synthesis of organic ion exchange resins which had much

better properties. These resins were stable towards acids

and easy to handle. The structure can be varied as desired,

therefore, the difficulties observed with zeolite and clays

were removed by introduction of resins.

Phenolic resins have been used for a number of analy­

tical applications, but the more modern styrene- divinylben-

zene resins are for several reasons more attractive to the

analytical chemist. These resins are produced by sulfona-

tion of a co-polymer of styrene with divinylbenzene. In

this copolymer the divinylbenzene forms cross-linkages

between the chains of polymerized styrene (Fig. 1.1).

^5»!

-CH-CH -CH-CH2- CH-CH,

n - CH-CH2 - CH - Ch2-

-CH-CH„-CH-CH^- CH-CH,

^6«5 n

- CH -CH,, - CH -CH„-

-CH-CH2-CH-CH2-

C6H5

CH-CH,

^6 H5

- CH -CH2- CH -CH--

Ms -Vi

-CH-CH2-

Fig. 1.1 Chemical Structure of a styrene-divinylbenezene resin

20

Since then these organic ion exchangers have been

used both in laboratories and industries for separations,

recoveries of metals, deionization of water, concentration

of electrolytes and elucidating the mechanism of a great

many reactions (106). The application of these ion-exchange

resins progressed so rapidly that the theory lagged behind

and could not follow the experiments.

The applications of organic ion exchangers are also

limited under certain conditions i.e. they are unstable in

aqueous systems at high temperatures and in the presence of

the ionizing radiations.

The first polymerization type of ion-exchanger was

produced by D'Alelio (107) in 1945 and the first ion-

exchange membarane were produced by Juda and Mcrae (108).

The polymeric ion-exchangers which are commercially avail­

able are based mainly on crosslinked polystyrene (109).

Anion exchange resin (110) of strongly basic type are

much more useful than weakly basic resins, it can be used

for anion exchange and chromatographic work in acid neutral

or alkaline medium, extremely important is the uptake and

chromatographic separation of complex metal ions, uranium

sulfate complex. Resin of strongly basic type have much

21

more resistant to oxidation e.g. chlorine as compared with

weakly basic resins of phenolic type( 111} . In long term

tests on resins used for water deionization it has been

found that the resin will last much longer if the water is

deairated before the ion exchange (112). According to

Lindsey and D'Amico (113), the resins are insoluble in all

common solvents including aliphatic and aromatic hydro­

carbons. Strongly basic resins are yellow in colour.

In recent years ion exchange method have applied in

various fields such as ion exchange treatment of sea water

(114). In water demineralization (115,116) traced metal in

drinking water (117) in environmental analysis (118). Desa­

lination of water (119), removal of heavy metals from river

+ 2 +2 water (120} determination of Ca and Mg (121)Cl2and N^

determination in water (122) for the production of water

used in pharmaceutical purposes (123), treatment of waste

+2 water containing Hg (124) for separation of heavy metals

on chelating resins (125). In separation preconcentration of

Cr(VI) from Cr(III) (126) for the chromatography of biopoly-

mer (127). Separation of amino acids (128) and phenolic

compounds (129). In clinical and pharmaceutical process

(130) extraction of uranium (131).

Chelating ion exchangers show a definite selectivity

towards certain ions or group of ions. Thus the chelating

22

ion exchangers may provide a convenient technique for the

analytical concentrations of many of the more interesting

trace elements from neutral waters and collection of toxic

elements from industrial waste waters. The selectivity of

most complexing agents resides predominantly in their

ability to form chelates with certain cations. Therefore

the developments of complexing ion exchangers have taken

place. In this connection Brajter (132) recommended

exchange method with highly selective resins. These chela­

ting resins prepared by immobilization of chelating agents

on various support (133,134). In the recent years number of

papers related to the study of loaded resin in different

field of separation analysis have been published few

references are given here (135-:144).

Chromatography is an analytical or separatory proce­

dure based upon the adsorption experiments described by

Tswett in 1903 and 1906 (145) when he was attempting to

separate colored leaf pigments on a small column packed with

calcium carbonate. Tswett had not only resolved a multi

component mixture into its constitutents, but had also

isolated the constituents without chemical change or altera­

tion, so that they could be utilized for investigation of

their properties. In this way he provided scientists with a

remarkably useful tools for the investigation of chemical

substances. So Chromatography is a method of analysis in

which the flow of solvent or gas promotes the separation of

substances by differential migration from a narrow initial

23

zone in a porous sorptive medium. Gas chromatography and

solution chromatography are the major subdivision of chroma­

tography. Solution chromatography can be divided again into

column chromatography, thin layer chromatography and paper

chromatography.

Martin and Synge in 1941 (146,147) developed the

technique of liquid-liquid partition chromatography. They

used a stationary liquid phase spread over the surface of

the adsorbents and immiscible with the mobile phase. The

sample component partitioned themselves between the two

liquid phases according to their solubilities. In the early

days of column chromatography, reliable identification of

small quantities of separated substances was difficult which

led to the development of paper chromatography. In this

"planar" technique separations are achieved on sheets of

filter paper mainly through partition. This gave birth to

thin layer chromatography in which separations are carried

out on thin layers of adsorbent supported on plate of glass

or some other rigid materials TLC gained the popularity

after the classic work by stahl in 1958. The most recently

developed chromatographic technique is gas chromatography

was first described by Martin and James in 1952, and widely

used of all chromatographic techniques, particularly for

mixture of gases or for volatile liquid and solids (148).

Liquid Chromatography refers to any chromatographic

technique in which the moving phase is a liquid. Traditional

24

column chromatography, thin layer and paper chromatography

and modern liquid chromatography are each example of liquid

chromatography. The difference between these older and

modern involves improvements in equipments, materials,

techniques and the applciation of theory. It offers major

advantages in convenience, accuracy, speed and ability to

carry out difficult separations. Modern liquid

chromatography has been called high performance or high

pressure liquid chromatography (HPLC). The advantages of

HPLC first come to the attention of a wide audience in early

1969, when a session were organized as part of the Fifth

International Symposium on Advances in Chromatography.

However it had its begining in the late 1950s, with the

introduction of automated amino acids analysis by Spackman,

Stein and Moore (149 ). This followed by the work of C D .Scott

of Oak Ridge on high pressure ion exchange chromatography

and the introduction of get permition chromatography by J.C.

Moore (150). In modern LC closed, reusable columns are

employed, so that hundreds of individuals separations can be

carried out on a given column. Detection and quantitation

are achieved with continuous detectors of various types.

These yield a final chromatogram without intervention by the

operator (151) .

Ion-exchange chromatography was the first of the

various liquid chromatography method to be used udner modern

25

LC conditions. Automated high resolution ion-exchange

chromatography dates from the early 1960s with the

introduction of routine amino acids analysis. Basically the

same technique was later extended to the analysis of

literally hundreds of different compound in physiological

fluid such as urine and serum. The renaissance on LC that

began in the late 1960s features ion-exchange chromatography

applications fromthe very start (152,153) for various reason

however lEC has remained less popular than the other LC

methods and more recently the new technique of ion-pair

chromatography has begun to displace lEC in some of its

traditional areas of application.

Ion-pair chromatography as adapted to modern LC is of

comparatively recent origin being first applied in the mid

1970s. However its use in classicle liquid chromatography

and liquid-liquid extraction is considerably older. The

rapid acceptance of ion pair chromatography as a new high

performance liquid chromatography owes much work by Schill

et.al. (154 ), and to its unique advantages. The current

popularity of ion-pair chromatography arises mainly from the

limitations of ion exchange chromatography and from the

difficulty in handling certain samples by the other liquid

chromatographic methods. IPC can carried out either in

normal-pahse or reverse phase modes each of which has its

own advantages.

26

The newest of four liquid chromatographic methods,

Size exclusion liquid chromatography is also referred to as

gel chromatography, gel filtration and gel permition

chromatography is the easiest of the LC methods to

udnerstand and used as the most predectable. Despite its

simplicity, this powerful technique has been applied to a

broad variety of sample types to solve widely different

separation problems. SEC is the preferred method

forseparating higher molecular weight components (MW >200)

particularly those that are nonionic. Macromolecules such as

proteins and nucleic acid are best separated by SEC. It can

be used to separate simple mixtures conveniently and

rapidly, when the components of mixture have sufficient

difference in molecular weight. SEC is a powerful method for

the initial exploratory separation of unknown samples,

quickly providing an overall picture of sample composition

with a minimum of separation development. It separates

molecules according to their effective size in solution. It

is commonly divided into the techniques of gel filtration

chromatography using aqueous solvents, and gel permeation

chromatography using organic solvents for applciation to

water soluble and organic soluble samples respectivley. Size

exclusion chromatography can be carried out with either

rigid or nonrigid column packings. Rigid packings are

required for high pressure liquid chromatography, but some

27

samples are better separated on the non-rigid gels. A book

on modern size exclusion chromatography treats its detail

including the newer aspects of gel filtration chromatography

(155).

Planar Chromatography has found wide spread use in

forensic chemistry identification of drug sample and

analysis of ink in suspected forgery cases. A tiny amount of

ink can be taken from the document and spotted on paper or

thin-layer plate. The R ; values and colors for ink

components are compared with reference ink values or with a

sample from the suspects pen. Plant extract and other

biochemical sample can also be identified with planar

chromatography.

The begining of inorganic chromatography may be

attributed to the work of Runge (1850) on paper

chromatography (156 ) Beyerinck (1889) on thin layers of

gelatin (157) and Schwab (1937) on alumina column. Among

chromatographic techniques, thin layer chromatography is a

simple, rapid, versatile and low cost method which is

applicable to the analysis of a variety of multicomponent

mixtures except volatile or reactive substances.

TLC always been recognized as a practicle and

effective technique for purifying materials before their

analysis with sophisticated instruments. The stationary

28

phase in normal TLC is an active solid termed as the

sorbents whereas a liquid containing a single solvent or a

mixture of solvent is used as mobile phase. A suitabely

closed vessel containing mobile phase and a plate (glass or

plastic) coated with a suitable sorbents (silica, alumina,

cellulose, polyamide or ion-exchanger) are all that required

to perform qualitative and quantitative separations. TLC has

certain advantages over paper chromatography and gas

chromatography, for example resulting separations were much

better than in classicle liquid chromatography, and required

much less time, it can handle several samples

simultaneously, and has a higher sample loading capacity.

Moreover, corrosive reagents and acids can be sprayed on

thin layer chromatoplates without any adverse effect.

Recently an improved version of TLC have been

introduced (158) and referred to high performance-thin layer

chromatography. It has been implied that this new technique

will displace modern LC from many of its present

applications. This seems to us an overoptimistic assessment

of the potential of HP-TLC. However TLC itself has proved

to be a complementry technqiue that can be used effectively

in conjunction with LC and any improvement inTLC will only

increase the value of TLC in these applications.

The history of TLC has been nicely reviewed by

Kirchner (159) Hefteiann (160) stahl (161) and Pek lick (162).

29

The discovery of TLC is usually ascribed to Izmailov and

Schraiber (163) who utilized thin layer of alumina on glass

plates for chromatographic separations. After pioneering

work of Kirchner (164) and Stahl (165,166). TLC become

popular as separation techniques (124,125) for sample not

amendable to GC. TLC technique has been applied since years

in the analysis of organic and inorganic substances and to

the analysis of biological pharmaceutical and environmental

samples (167 ) .

In thin layer chromatography variety of coating

materials are available as stationary phase such as silica

gel, cellulose powder, alumina and activated charcoal.

Ion-exchange resin in particle size of 40-80um are also

suitable for preparing thin layer plates. The presence of an

plate is advantageous particularly for the studies of ionic

species. The ion-exchange property of the adsorbent plays a

more prominent role than its simply adsorption behaviour

Sherma and Fried (168) in their review have maintained the

analytical capabilities of synthetic inorganic

ion-exchangers as adsorbents in TLC. For the sake of

convenience inorganic ion-exchangers have been classified in

to four catagories, and all of them have found their use in

thin layer Chromatography.

30

1. Thin layer of hydrated oxide

2. Thin layer of isoluble metal salts of polybasic

acids.

3. Thin layers of metal Ferrocyanide

4. Thin layer of heteropoly acid salts

1. • Zabin and Rollins (169) used for the first time

zirconium oxide for the separation of metal ions.

Berger et.al (170,171) also used for the study of

ferrocyanide, ferricynide and sulfocyanide. Kirchner

et.al. (172) separated terpenes, Sen and Coworkers

. 2+ separated Ei from 25 ternay and 12 quaternary

mixtures. Sen also chromatographed some anions on

stannie oxide (173,174) layers. Rathore et.al. (175 )

have isolated citric acid from 10 carboxylic acid on

Calcium sulfate, ammonium molybdate, zinc oxide and

titanium oxide thin paltes. Grace (176) also used

titanium oxide for the chromatography of ortho-para and

meta amino phenols. Cremer and Seidl (177 ) prepared

indium oxide plates and studied the movement of various

anions.

2. Zirconium phsophate got prefrence as a medium for TLC.

Zabin and Rollins (169 ) studied cations, Keonig and

Demiel(l78), Alberti (179) Keonig and Gray (180) studied

the movement of cation on the layer of zirconiumhypo-

phosphate, titanium phosphate and Cerium phosphate

31

layers respectively. Srivastava et.al. (181) used Cerium

molybdate for the study of cations. Qureshi et.al. (182)

prepared stannic tungstate layers for the study of 15

binary metal 'ions. Studies of phenolic compounds have

been performed by Nabi et.al. (183). Chromatography of

57 metal ions on stannic arsenate layers was performed by

Husain and associates (184,185) Qureshi et.al. (186)

reported 2 0 binary separations of metal ions on

non-refluxed stannic arsenate layers. They have also

chromatographed 34 metal ions on stannic arsenate layers

(187 ). Sharma et.al (188) have recorded the movement of

47 metal cations on stannic arsenate layers.

3. Thin layer of zince f errocyanide were prepared by Fog

and Wood (189) who chromatographed 16 samples of

sulfonamides Kawamura and Co-workers (ISO ) have analysed

various combinations of alkali metals.

4. Analytical applications of this group is not so old-

Lesigang et.al. chromatographed alkali metals on thin

layers of ammonium phosphododeca molybdate, arsenododeca

molybdate germano dodecamolybdate and oxinium and

pyridium germanododeca molybdate (191,192). Lepri and

Desideri (193) have presented exhaustive explorations of

ammonium molybdophosphate and ammonium tungstophosphate

layers. Pyridium tungstoarsenate thin layers were

32

successfully utilized for the separations of amino

acids and metal ions (194,195). Varshney and asso­

ciates have performed TLC of alkaline earth transi­

tion metals and amino acids on tin (IV) arsenosili-

cate and arsenophosphate layers (196, 197).

The selectivity of the ion exchange material depends

upon its nature as well as on the medium of exchange.

Present work deals with the qualitative and quantitative

separations of amino acids on stannic arsenate thin layers.

There are some references related to the separation of amino

acids on TLC (198-201).

Electrophoresis may be considered as one of the

branch of chromatography and defined as the migration of

particles under the influence of an electric field. Elec­

trophoresis probably the oldest form of migratory analysis

is certainly the most gentle one towards the substances

it separates. It was first used in 1909 to describe

the the movement of colloidal particles in an electric

field (202). Kendell (203) in the period (1923-1928) who

fully explored its analytical possibilities, separated rare

earths and also alkaloids. The first full fledged method

for protein electrophoresis was developed by Tiselius (204).

33

There are three different types of electrophorectic

systems (205 ) .

1. Moving boundry

2. Zone electrophoresis

3. Steady state

The original work of Tiselius in 1930s was with

moving boundry, and from this technique others have been

derived.

Moving boundry electrophoresis was used quite widely

between 1935 and the 1950s but it is replaced by zone

electrophoresis techniques, in which the components of

mixture separate completely from one another during

electrophoresis, forming discrete zones. There are a number

of ways of stabilising the separated zones, including the

use of supporting media, density gradients and free zone

technqieus. In steady state electrophoresis after procedding

for a certain length of time, a steady state is attained in

which the widths and positions of the zones of the separated

components do not change with time.

There are many types of support media that used in

zone electrophoresis. The use of filter paper was introduced

in 1950s and followed by the use of cellulose acetate

membrane in 1957. The introduction of gels as a support

J4

media has also been used in zone electrophorresis. Gel is a

three dimensional polymeric network with a random structure.

Starch gels used in the electrophoresis of proteins. Many

workers used polyacrylamide gels as a supporting medium. The

voltage plays a very important role in the electrophoresis.

It may be noted that the substance of low molecular weight

separate better with high potential gradients and substance

of high molecular weight with low potential gradient.

Methods have been developed for the analysis of

hydrolysates of peptides using high-voltage electrophoresis

(206 ) . A rather large effect due to the formation of

ion-pair_ has been observed repeatedly in paper

electrophoresis, Chakraborty (207 ) Blasius and Bilal (208)

and Mazzel ( 209) did the work on ion pair fcrmation in

electrophoresis for the separation ofmetal complexes.

Extensive studies have been done on the separation of high

polymer on electrophoresis (210, 211). However Kiso et.al

(212) and many others have done the studies on low molecular

weight substances. Amino acids separated by the high

voltage paper electrophoresis (213), electrophoresis have

also been applied in separation of low molecular weight

substances for example amino acid and peptides (214). In

India Mohsin et.al. done the metal ion separation on the

paper strips impregnated with inorganic ion exchangers by

35

electrophoresis (-215,216). Though Gray et.al. separated the

dansyl derivatives of amino acids by electrophoresis( 217 ).

In 1955 Smithies published a paper on the separation

of human serum in starch gel (218)- Gel electrophoresis has

been used principally in protein chemistry for the

investigation of human and animal sera. Maizel (219) et.al

reported the separations of proteins by polyacrylamide

electrophoresis in the presence of the anionic detergent

sodium dodecyl sulfate is dependent on the molecular weight

of their polypeptide chains. Proteins and peptides of chain

have been studied by polyacrylamide gel electrophoresis in

the presence of sodium dodecyl sulfate (220).

Capillary electrophoresis (CE) was initially reported

by Jorgenson and Lukacs in 1981 (221). Capillary column

(10-100 um) filled with buffer solution and a voltage is

then applied across the column. Number of papers have been

publised related to capillary electrophoresis in recent

years (222-226).

CE in linear polymer solution, derivatized

cellulose, or linear polyacrylamide is finding increasing

acceptance for the rapid and efficient separations of many

kind of biopolymers. Pulsed field capillary gel

electropharesis (PFCGE) is now emerging as a promising

technique to improve nucleic acid separation as well as

36

separation of other biopolymers (227).

A review by Monnig and Kennedy (228) described the

contribution in the development of this technique. Texts by

Kuhn and Hoffstettes-Kuhn (229) and Jandick and Bonn (230)

also provide a useful introduction to capillary

electrophoresis technqieus. Capillary electrophoresis

continues to have a growing influence on olignucleotides

analysis. Kohen and Coworkers (231) reviewed capillary gel

electrophoresis of DNA and proteins. It is also applicable

in the field of pharmecautical analysis. Seventeen basic

drugs of different classess were separated in 11 minute by

capillary zone electrophorsis, the analysis of most of these

components in urine and plasma also demonstrated (232).

37

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Anal., 612 (1993) 172.

CHAPTER - / /

Paper Electrophoretic Studies of

CL -Amino Acids in Citrate Phosphate Buffer and

Sodium Dodecyl Sulphate Mixed Systems

56

INTRODUCTION

Attempts have been made for the separation of amino

acids using different techniques such as ion-exchange Column

Chromatography (1), Ion-exchange paper Chromatography (2),

Ion-exchange paper electrophoresis (3-5) and ion-exchange

thin layer Chromatography (6). Among different techniques

paper electrophoresis has been most widely used for the

separation of species. Electrophoresis on ion-exchange

papers suffer from the following major limitations:

i) Preparation of exchange papers involves expensive

chemicals and at the same time tedious and time

consuming job,

ii) The ion exchanger generated on the papers may

not be of uniform composition.

iii) The spots on the strips are usually distorted.

iv) The development time is relatively higher.

I have therefore utilized the conventional Whatman

papers using simple electrolyte systems for the quantitative

separation of amino acids. It has been observed that the

57

use of miceller reagents often improves the separation

potentialities of eluting solvents in many instances as they

form ion pair with certain species. The capability of SDS

as a miceller reagent has already established for the sepa­

ration of certain pairs of amino acids using HPLC techniques

(7). These facts have prompted us to choose SDS mixed

solvents systems to enhance its utility further in the sepa­

ration of amino acids by using electrophoretic techniques.

The method has specifically developed for the estimation of

amino acids in commercially available drugs.

58

EXPERIMENTAL

Apparatus :

Hindustan Power Trenix Inc electrophoresis chamber

was used. The unit was powered by power supply model EPH-

510. The power supply is a 500 V, 100 itiA unit. Electropho­

resis was carried out using the enclosed strip method. A

micropippete was used to put a spot of known amount for

quantitative separations. Bausch and Lomb spectronic 20

spectrophotometer was used for colorimetric studies.

Reagents and Chemicals :

Sodium dodecyl sulphate (SDS) and amino acids were

obtained from Loba Chemie (India). All other chemicals and

reagents were of A.R. grade. Drug samples, Santevini plus

(Sandoz India Ltd.) and Astymin-3 (Tablets India Ltd.) were

purchased locally.

Test Solution :

0.8% aqueous solutions of amino acids were prepared

in double distilled water.

Background electrolyte solutions :

citrate phosphate buffer of pH 2.6, 4.0, 5.0, 6.0 and

7.0 were prepared as usual. In each buffer solution differ­

ent amount of SDS were mixed so as to give a concentration

of ImM, 5mM, lOmM and 50mM.

59

Ninhydrin Reagent :

0.2% ninhydrin solution in n-butanol saturated with

water was used for the detection of amino acids.

Procedure :

Amino acids were spotted on the Whatman No.l paper

strips (30 X 5 cm) with the help of thin glass capillaries.

Electrophoresis chamber was filled with the desired back­

ground electrolyte. The strips were placed in the chamber

and electrophorised for 1 hr. at 300v/20 mA. After develop­

ment the strips were dried in an oven at 60 °C and the

position of each amino acid was detected by ninhydrin as

usual.

Quantitative Separations :

Mixtures of amino acids were prepared by mixing equal

volume of each. The mixtures were then spotted with the help

of micropippete on the centre of the paper strip and elec­

trophorised in the usual way. A pilot electrophoretogram

was run under the same experimental conditions to locate the

actual position of spots on the experimental strips. After

electrophoresis, the spots on the pilot paper strip were

detected by spraying ninhydrin. The pilot electrophoreto­

gram was compared with the experimental strips to find out

the position of amino acids. The same portions of the paper

strips were then cut out. The amino acids present in these

portions were extracted with ethanol and determined spectre-

60

photometrically using ninhydrin as a chromogenic agent (8).

Results are reported in Table- 2.7

Drug samples of Astymin-3 (injection) and Santevini

plus (syrup) containing amino acids were taken for the

analysis. A known amount of drug sample was applied on the

centre of the strip with the help of a micropippete and

electrophorised in the usual way. The resuls are reported

in Table- 2.8

61

RESULTS AND DISCUSSION

Paper electrophoretic studies of -amino acids in

mixed SDS-citrate phosphate buffer systems (Tables 2.1-2.5)

have revealed many interesting results. It has been obser­

ved that the presence of SDS in citrate phosphate buffer

significantly differentiate the behaviour of these amino

acids. This is due to the fact that most of the amino acids

form ion pairs with SDS with the exception of few namely

serine aspartic acid, gluamic acid, threonine, glycine,

cystine and alanine (7). This differentiating ability of

SDS toward amino acids offers amazing potentials for diffi­

cult and important separations of these acids. The experi­

ments performed in buffer solution of pH 2.6 show that the

movement of basic amino acids is relatively higher as

compared to acidic and neutral amino acids and the direction

is towards cathode. It is apparent from Table-2.1 that on

addition of 1 mmole of SDS to this buffer system, movement

of amino acids is retarted. Further addition of SDS to the

buffer system movement of arginine and histidine is reversed

i.e. they move towards anode. On further addition of 10

mmole SDS to the system all the basic amino acids i.e. 1-

arginine-HCl, 1-histidine, HCl, 1-lysine-HCl and 1-Ornithine

HCl and neutral amino acids namely Phenylalanine, tryptophan

and nor-leucine are directed towards anode. It is interest-

62

TABLE - 2.1

ELECTROPHORETIC MOVEMENT(cm) OF AMINO ACIDS ON

WHATMAN No.l PAPER IN CITRATE-PHOSPHATE BUFFER OF

pH 2.6 AND SDS MIXED SYSTEMS

Cora]

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

pound

DL-alanine

DL-2amino n-butyric acid

L-arginine HCl

DL-aspartic acid

Cysteine

Cystine

L-Glutamic acid

Glycine

L-Histidine HCl

L-leucine

DL-isoleucine

DL-nor-leucine

L-lysine HCl

DL-Methionine

L-Ornithine HCl

DL-Phenylalanine

DL-Serine

DL-Threonine

DL-Tryptophan

L-Tyrosin

DL-Valine

Buffer

PH 2.6

3.3

3.8

9.0

1.5

2.8

1.5

3.7

7.3

7.5

3.9

4.4

2.0

6.9

2.3

6.7

2.8

4.1

6.5

4.8

1.5

4.5

Buffer

ImM

5.3

4.0

6.9

3.5

-

-

2.0

6.3

6.3

-

3.3

3.1

6.2

4.3

5.5

3.3

4.4

3.8

4.0

4.3

3.9

pH 2.6- SDS-Systems 5mM

9.0

5.2

-5.8

5.0

1.5

0.0

7.3

9.3

-2.3

0.8

2.1

1.3

5.3

5.0

1.5

0.0

8.8

8.0

0.8

1.5

3.1

lOmM

6.0

2.3

-4.1

1.8

2.0

0.0

4.7

5.0

-3.1

0.0

1.3

-1.3

2.4

0.0

0.0

-1.0

3.4

3.0

-1.0

0.0

0.0

Mixed

50mM

2.4

1.5

-5.9

3.5

1.3

4.0

2.0

5.0

-4.7

5.9

-5.0

-5.5

-5.0

0.0

-2.5

-4.5

4.8

5.2

-3.8

0.0

-2.0

-ve & +ve signs indicate movements towards anode and cathode

respectively.

63

ing to note that other neutral amino acids move towards

cathode. This peculiar behaviour may be attributed to the

fact that in the case of basic amino acids such as arginine,

histidine, lysine and ornithine, the mean charge in the low

pH range is greater than unity whereas in other amino acids

the mean charge in the same pH range is usually less than

unity (9). A comparison of movement of basic amino acids in

pure buffer system (pH-2.6) and SDS mixed buffer system show

that the movement is reversed from cathode to anode. It is

probably due to the interaction of positively charged basic

amino acids with that of negatively charge hydrophilic

region of SDS molecule (10) causing net negative charge on

the species. It is also clear from Table-1 that the move­

ment of basic amino acid is retarted with increasing concen­

trations of SDS in the system. Thus the presence of SDS

plays an important role in the separation of basic amino

acids (Table- 2.6). The behaviour of basic amino acids in

buffer of pH 4.0 is interesting. They all move appreciably

towards cathode and follow the order 1-Ornithine HCl > 1-

lysine-HCl > 1-histidine-HCl > 1-arginine-HCl. It clearly

indicates that the movement depend on the molecular weight

of basic amino acids. Greater the molecular weight the

smaller distance amino acid traverses (Fig. 2.1). However,

on increasing the concentration of SDS the movement of basic

amino acids is retarted as a result of formation of heavier

64

TABLE - 2.2

ELECTROPHORETIC MOVEMENT (cm) OF AMINO ACIDS ON

WHATMAN NO.l PAPER IN CITRATE -

PHOSPHATE BUFFER OF pH 4.0 AND SOS MIXED SYSTEMS

Com]

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

pound

DL-alanine

DL-2 amino-n-butyric acid

L-arginine-HCl

DL-aspartic acid

Cysteine

Cystine

L-glutamicac id

Glycine

L-Histidine HCl

L-Leucine

DL-isoleucine

DL-nor-leucine

L-lysine HCl

DL-methionine

L-ornithine HCl

DL-phenylalanine

DL-serine

DL-threonine

DL-tryptophan

L-tyrosin

DL-valine

Buffer

pH 4.0

1.0

2.0

5.5

0.0

1.9

0.0

0.0

6.0

5.8

4.0

3.0

3.5

6.5

3.5

7.6

3.9

4.8

5.3

2.5

-5.5

2.4

Buffer pH 4.0-SDS systems

5mM SDS lOmM SDS

3.4

2.2

2.1

1.5

1.1

1.3

2.5

5.6

5.0

1.5

1.3

3.9

4.0

2.1

4.7

3.4

2.1

2.5

1.3

2.3

3.1

3.9

2.8

1.8

1.6

4.5

-

1.0

4.0

3.4

3.1

5.5

5.9

3.4

3.0

3.9

3.5

3.5

5.6

1.8

2.5

3.3

mixed

50mMSDS

4.1

3.9

1.4

1.8

3.9

-

2.8

2.3

2.4

3.4

1.9

1.4

2.3

3.6

3.5

2.3

2.8

2.2

0.5

1.4

3.6

65

lO-Or

E o *-* ^ • r f

c a> E Ut > o 2

ftO

6-0

•

o Q. O

(J

UJ

m 150 150

Molecular Weight

180

Fig. 2.1 Electrophoretic Movement vs. Molecular Weight of Basic Amino Acids At Different pH Values (0,2.6; ,4.0; ,5.0; o,6.0; X, 7.0) .

66

TABLE - 2.3

ELECTROPHORETIC MOVEMENT (cm) OF AMINO ACIDS ON

WHATMAN NO.l PAPER IN CITRATE-PHOSPHATE BUFFER OF

pH 5.0 AND SDS MIXED SYSTEMS

Coitij

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

pound

DL-alanine

DL-2 amino-n butyric acid

L-arginine-HCl

DL-aspartic acid

Cysteine

Cystine

L-glutamic acid

Glycine

L-Histidine HCl

L-leucine

DL-isoleucine

DL-nor-leucine

L-lysine HCl

DL-methionine

L-ornithine HCl

DL-phenylalanine

DL-serine

DL-threonine

DL-tryptophan

L-tyrosin

DL-valine

Buffer

pH 5.0

0.0

1.0

3.4

6.8

1.4

1.4

2.3

5.4

1.8

1.8

4.5

0.7

1.8

0.0

2.4

2.0

0.0

1.0

1.9

1.5

2.0

Buffer pH 5.0 - SDS systems

5mM SDS lOmM SDS

2.6

4.2

4.5

-2.8

2.7

1.6

-1.6

4.2

3.4

3.3

2.3

3.6

4.5

3.4

3.9

1.5

2.8

2.9

4.3

1.6

3.6

2.2

1.5

5.5

-2.3

2.3

1.9

0.0

2.0

3.7

2.2

2.3

2.7

4.5

3.1

4.2

2.4

4.2

4.1

2.8

3.4

2.7

mixed

5GmMSDS

2.1

3.8

6.0

-2.0

5.0

-

0.0

1.0

-

-

-

-

-

2.6

4.5

2.1

3.3

4.0

2.5

-

3.0

67

molecule of these amino acids with SDS. While movement of

neutral amino acids generally increases. On the other hand,

reverse trend has been observed at pH-5.0. In the case of

basic amino acids movement has been found to increase with

the increase in molecular weight. In buffer system of pH

6.0 acidic amino acids move towards anode and movement is

affected by molecular weight. But the basic amino acids

have the same behaviour as observed in buffer of pH 5.0.

The movement of acidic amino acid i.e. aspartic acid

and glutamic acid is also appreciably affected by pH of the

buffer system. Both the amino acids move towards cathode

when the pH of electrolyte is below 6.0 except at pH 4.0

where the movement becomes zero. This may be owing to the

fact that aspartic acid and glutamic acid exist as uncharged

species at this pH. However, the movement is reversed when

the pH of buffer system is increased to 6.0 and onwards

showing the behaviour of these amino acids as negatively

charged species.

The results of these studies (Tables 2.1-2.5) suggest

that a number of binary, ternary and multinary separations

are possible by varying the pH and SDS concentrations in the

buffer system. A very effective separation of phenylalanine

arginine, methionine cysteine and Ornithine from each other

has been achieved by enhancing the SDS concentration to 10

mmole in pH 2.6. In the same background electrolyte the

68

TABLE - 2.4

ELECTROPHORETIC MOVEMENT (cm) OF AMINO ACIDS ON

WHATMAN NO.l PAPER IN CITRATE - PHOSPHATE BUFFER OF

pH 6.0 AND SDS MIXED SYSTEMS

Com]

_1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

pound

:DL-alanine

DL-2 amino-n butyric acid

L-arginine-HCl

DL-aspartic acid

Cysteine

Cystine

L-glutamic acid

Glycine

L-Histidine HCl

L-Leucine

DL-isoleucine

DL-nor-leucine

L-lysine HCl

DL-methionine

L-ornithine HCl

DL-phenylalanine

DL-serine

DL-threonine

DL-tryptophan

L tyrosin

DL-valine

Buffer

pH 6.0

0.8

1.5

4.0

-2.2

2.7

5.6

-2.3

1.1

3.8

2.0

1.0

2.3

3.8

0.9

2.4

1.8

1.2

2.1

1.0

1.0

0.0

Buffer

ImM SDS

1.6

3.3

2.8

-1.5

1.9

3.5

-2.0

1.3

3.6

2.0

2.4

2.0

4.6

1.0

3.5

.0

1.5

2.2

1.2

1.3

2.5

pH 6.0- SDS systems 5mM SDS

3.3

2.1

2.8

3.8

2.0

-

1.3

4.3

3.5

4.5

2.0

4.8

4.8

2.0

5.0

1.4

2.0

2.5

2.0

1.4

2.3

lOmM SDS

2.0

2.2

2.5

2.3

2.0

0.0

1.2

5.5

3.5

2.9

1.0

1.4

4.9

2.5

6.0

0.0

3.0

2.7

2.3

1.6

2.0

mixed

50mM SDS

4.0

6.0

2.8

0.0

4.0

4.8

1.1

8.0

3.5

2.8

4.5

3.3

5.0

3.1

6.3

3.3

3.4

3.8

1.1

2.0

4.5

69

TABLE - 2.5

ELECTROPHORETIC MOVEMENT (cm) OF AMINO ACIDS ON

WHATMAN NO.l PAPER IN CITRATE - PHOSPHATE BUFFER OF

pH 7.0 AND SDS MIXED SYSTEMS.

Compound

1. DL-alanine

2. DL-2 amino-n butyric acid

3. L-arginine-HCl

4. DL-aspartic acid

5. Cysteine

6. Cystine

7. L-glutamic acid

8. Glycine

9. L-Histidine HCl

10. L-Leucine

11. DL-ISO-leucine

12. DL-nor-leucine

13. L-lysine HCl

14. DL-methionine

15. L-ornithine HCl

16. DL-phenylalanine

17. DL-serine

18. DL-threonine

19. DL-tryptophan

20. L-tyrosin

21. DL-valine

Buffer

pH 7.0

4.8

5.0

5.5

-1.0

2.5

-2.8

-1.5

5.0

2.8

4.0

3.3

5.5

2.5

1.3

2.4

2.0

1.3

1.0

2.8

0.5

1.5

Buffer

imM SDS

2.3

2.0

4.8

0.0

2.1

1.3

0.0

0.0

2.4

2.6

1.3

0.0

5.5

1.8

2.0

3.1

1.0

0.0

0.8

4.5

1.3

pH 7.0 - SDS systems

lUmM SDS

1.4

1.6

1.9

1.3

1.2

-

-1.5

2.0

0.8

1.0

0.8

2.6

2.3

1.3

3.0

2.1

2.9

1.5

0.6

2.6

1.2

mixed

5UmM SDS

1.0

1.3

0.0

4.5

0.0

-

-1.0

1.1

0.0

0.0

0.0

1.2

1.8

1.3

1.8

0.0

1.0

2.1

0.0

0.5

1.1

70

separation of histidine and arginine has been achieved from

a mixture containing cystine, tyrosine, methionine, valine

and leucine as they remain at the point of application

(Table- 2.6).

A very efficient selective qualitative separation of

tyrosine has been achieved from a synthetic mixture of 21

neutral, basic and acidic amino acids at pH-4.0, as it is

the only amino acid which moves towards the cathode.

Aspartic acid and glutamic acid both belonging to

monoaminodicarboxhlic acid type have been separated from

each other in buffer pH 5.0 containing 10 mmole SDS (Table-

2.6). Separation of acidic amino acids from the mixture of

neutral and basic amino acids have been achieved at pH-6.0.

Cystine has been selectively separated from a synthetic

mixture of all the neutral and basic amino acids in buffer

system of pH 7.0 (Table- 2.6).

The utility of these separations has been practically

demonstrated in the quantitative separation of amino acids

constituents in commercially available drug samples namely

Santevini plus and Astymin-3 (Table- 2.8). Threonine has

been separated quantitatively from other amino acids consti­

tuents of Astymin-3 injection. Lysin and methionine consti­

tuents of Santevini plus has also been separated and deter­

mined.

71

TABLE - 2 . 6

QUALITATIVE SEPARATION OF AMINO ACIDS

ACHIEVED EXPERIMENTALLY

B a c k g r o u n d E l e c t r o l y t e S e p a r a t i o n a c h i e v e d

A. pH 2.6 with 5 mM SDS (I) Histidine -(4.0-2.0) from cystine ( o.o ) and alanine (7.0-9.0)

(II) Arginine -(6.5-4.0) from ornithine (0-3.0) and glycine (8.0-11.0)

(III) Arginine -(6.5-4.0) from ornithine (0-3.0) and lysine (5.3)

(IV) Cysteine - (3.5-0) Serine (7.0-8.0)

(V) DL-nor leccine-(2.5-0.0) from gluta­mic acid (5.5-7.0)

(VI) Iso-leucine -(2.5-0.0) from aspartic acid (4.1-6.5)

B. pH-2.6 with lOmM SDS (I) Phenylalanine -(1.3-0.0), Arginine -(3.2-8.5) Methionine (0.0), cysteine (1.0-2.5), and ornithine (3,5-5.5)

(II) DL-nor leucine -(3.5-0.5), Aspartic acid (1.5-3.0) and glycine (3.5-4.5)

(III) Arginine -(8.5-4.2) leucine (0.0) Histidine (4.0-6.5)

(IV) Histidine (4.0-6.5) and arginire -(8.5-4.2) from cystine (0.0), tyro-sin (0.0) Methionine (0.0) Valine (0.0) and L-leucine (0.0)

C. pH 2.6 with 50mM SDS (I) Glutamic acid (2.1-3.6) and L-leucine -(6.0-4.5)

(II) Ornithine -(5.9-4.2) Serine (5.0-5.8)

Table Contd 2

72

D. Buffer pH'-4.0

(III) Iso-leucine -(5.7-4.0) DL-2 Amino-n Butyric acid (0-1.4)

(IV) Valine -(5.0-2.0), phenylalanine -(8.0-6.5) Methionine (0.0), Alanine (4.5-6.5) DL-2 Amino-n- Butyric acid (1.5-3.5)

(V) Methionine (0.0), Nor-leucine -(7.9-1.9) DL-2 Amino-n-Butyric acid (1.5-3.5)

(I) Tyrosin -(3.0-2.0) with all others amino acid (1.0-3.0)

E. pH-5.0 with 5mM SDS

F. pH-6.0 with lOmM SDS

(I) Arginine (5.0-8.0) Aspartic acid -(3.6-0.0)

(II) Tryptophan (2.5-6.0) glutamic acid -(3.2-0-0)

(I) Glutamic acid -(3.0-0.0) Alanine (1.0-3.0)

(II) Glutamic acid -(3.0-0.0) DL-nor Leuc­ine (0.2-2.5) and ornithine (3.5-6.5)

(III) Cystine (0.0), Histidine (1.0-4.0)

G. Buffer pH-7.0 (I) Cystine -(4.0-1.5) with other neutral and basic amino acid (1.0-6.0)

73

TABLE - 2.7

QUANTITATIVE SEPARATION OF AMINO ACIDS IN SYNTHETIC MIXTURES,

Separation Achieved Amount taken ug

50.0 50.0

50.0 50.0

50.0 50.0

50.0 50.0

Amount found ug

49.0 49.5

49.8 48.6

49.6 49.4

49.2 49.8

% error

2.0 1.0

0.4 2.8

0.8 1.2

1.6 0.4

Background electrolyte

pH2.6+50mM 1.

2.

3.

4.

Glycine Histidine

Threonine Leucine

Threonine Lysine

Ornithine Serine

5. Histidine from alanine, 50.0 glutamic acid, 2-amino-n butyric acid,glycine methionine and threonine

49.2 1.6

Lysine from alanine 50.0 aspartic acid,glycine methionine and threonine

49.3 1.4

7. Aspartic acid glycine

8. Aspartic acid threonine

50.0 50.0

50.0 50.0

49.1 49.2

49.3 49.7

1.8 1.6

1.4 0.6

pH5.0+50mM SDS

74

TABLE - 2.8

DETERMINATION OF AMINO ACIDS IN COMMERCIALLY AVAILABLE DRUGS

Commercial namfi of the drug

Background electrolyte

Labelled amount of amino acid mg/ml

Amount applied ug

** Amount %Erro; found ug

Santevini Plus (Syrup)

Astymin-3

Buffer pH2.6 DL-methio 38.7 38.0 -1.8 + 50mM SDS nine 1.93

L-Lysine-Hcl 13.3 133.3 131.2 -1.6

Buffer pH2.6 L-threonine 54.0 53.5 -0.9 + 50mM SDS 5.4

Astymin-3 : Each ml contains L-arginine HCL 8.0mg, L-histidine HCL, 4.0mg, L-lsoleucine 5.5mg, L-leucine 12.3mg, L-lysine HCL 22.3, L-methionine 7.1mg, L-phenylalanine 8.7mg, L-threonine 5.4mg, L-tryptophan 1.8mg, L-valine S.lmg, glycine lO.Omg and sorbitol 50mg.

* * Average of three determinations,

75

REFERENCES

1. P.B. Hamilton, Advan. Chromatog., 2 (1966) 3.

2. C.S. Knight, Advan. Chromatog., 4 (1967) 100.

3. E. Selegney, J.C. Fenyo, G.Broun, F. Matray and C.D.E

Bernerdy, J. Chromatogr., 47 (1970) 552.

4. A.K. Mishra, J. Planar Chromatogr-Mod. TLC, 4 (1991)

237.

5. A.K. Mishra, D.K. Mishra and V.K. Maheshwari, J.Liq.

Chromatogr., 14 (1991) 1469.

6. R.L. Munier and C. Thommegay, Bull. Soc. Chin. France

No.9 (1967) 3171.

7. N. Seller and B. Knodgen, J. Chromatogr., 341( 1985 ) 11

8. S. Moore and W.H. Stein, J. Biol. Chem.176 (1948) 367

9. Y. Kiso and E. Falk, J. Chromatogr., 59 (1971) 401-

404.

10. P.H. Holworthy, A.T. Florence, C.B. Macfurlane,

"Solubilization by surface-active agents" Champan and

Hall Ltd., 11, Newfetter lane, LONDON ECL, (1968).

CHAPTER - / / /

Synthesis, Characterization and Analytical Applica­tion of Ion-Exchange Material: Zirconium (Il\) lodophophate

76

INTRODUCTION

Revived interest in the development of new inorganic

ion-exchange material is owing to the specific nature

towards certain ionic species which is important for solving

complicated problem in diverse fields. The inorganic ion-

exchangers based on three components have been found to show

relatively increased ion exchange capacity and selectivity

(1-5). However, only a few studies have been reported on

iodophosphate of tin (IV), zirconium(IV) and Fe(III) (6,7).

Zirconium(IV) phosphate has been extensively studied and its

ion-exchange characteristic is now well established.

Studies on zirconium(IV) iodophosphate has been taken with a

view to enhance its exchange characteristic in a systematic

way and to explore its separation potentialities in the

field of pharmaceutical analysis.

EXPERIMENTAL

Reagents:

Zirconium(IV) oxychloride (Loba chemie). Sodium

iodate(BDH), potassium dihydrogen orthophosphate and all

other chemicals used were of A.R. grade.

Apparatus :

Toshniwal (India) single electrode pH meter and a

Bausch and Lomb Spectronic-20 spectrophotometer were used

77

for pH and absorbance measurements respectively.

Synthesis :

The material was synthesized by adding zirconium( IV)

oxychloride gradually into sodium iodate and dihydrogen

orthophosphate solution in the ratio and concentration given

in Table- 3.1 and shaking the mixtures intermittently keeping

the pH constant throughout the mixing. The gelatinous

precipitate so formed was agitated at 80°C for 8 hours using

a magnetic stirrer. The supernatant liquid was removed and

washed with distilled water and finally filtered under

suction. The product was completely dried at 40+2°C in an

oven and subsequently treated with distilled water in order

to get the material in granular form. To convert the

material in H form it was kept for 24 hrs. in 1.0 M HNO.

solution. Excess acid was removed by washing with distilled

water and finally dried in an oven at 40_+2°C.

Ion Exchange Capacity :

The ion-exchange capacity was determined by the

column process. The slurry of 0.50 gm of the exchanger in H"*"

form was transferred to a glass column fitted with glass

wool at the bottom. It was washed with distilled water to

remove any excess of acid remained sticking on the particles.

To elute H"*" ions 250.0 ml of 1.0 M solution of different

uni- and bi-valent salts were passed through the column

78

maintaining the flow rate 8-9 drops/min. The amount of H

in the total effluent was determined titrimetrically using

NaOH as a titrant. The results are given in Table- 3.2

pH-titrations :

pH titrations were performed using batch procedure.

0.5 gm exchanger in H form was equiliberated with a 50 ml.

equimolar mixture of either (NaCl and NaOH) or (KC1& KOH)

for 24 hours. The pH of each solution was determined and

plotted against meq. of OH ions (Fig.'3.1).

Chemical Stability :

A 0.5 gm of exchange material (sample C) was equili­

brated with 50 ml of the solution of interest for 24 hrs. at

room temperature (30+4Oc) Zirconium, iodate and phosphate

released in the solution were determined spectrophotometri-

cally using Alizarin red-S(8), Pyragallol( 9) and phosphovan-

domolybdate (10) as colouring reagents respectively. The

results are given in Table- 3.4.

Chemical Composition :

For the determination of chemical composition of

sample C, 0.10 gm of exchanger was dissolved in approxi­

mately 10 ml concentrated sulphuric acid. Then the solution

was diluted to 100 ml with distilled water. The amount of

zirconium, iodate and phosphate were determined spectro-

79

photometrically (8,9,10) as described earlier.

Infrared spectriim :

Infrared spectra of zirconium(IV) iodophosphate in H

form were obtained using KBr disc on a Nicolet Fourier

transform spectrometer. The spectra have been shown in

figure 3.4 .

Thermal treatment :

Thermogravimetric analysis of sample C in H form was

performed at heating rate of 10°C min~ . To examine the

effect of heat on the properties of zirconium(IV) iodophos­

phate sample C was heated at different temperatures in a

muffle furnace for one hour. ( Figure-3.3).

Distribution Coefficient :

The distribution coefficients of metal ions in

various concentration of HCl and HCL:DMSO were determined.

A 0.5 gm of exchanger in H form was treated with 25.0 ml

erlenmeyer flask. The mixture was shaken intermittantly for

8 hours. The amount of metal ion species left in the

solution was then determined by titrating against the

standard solution of 0.002M disodium salt of EDTA. The K^ d

values were calculated according to the formula. The

results are given in Table 3.5.

j _ mmoles of metal species/qram of exchanger d mmoles of metal species/ml of the total

volume of the solution

80

Sepaxation of Metal Ions :

Quantitative separations of metal ions were achieved

in a glass column using 2.0 gm of exchanger in H form. A

metal ion mixture was poured on the top of the column. The

flow rate of the effluent was maintained at 1ml min~

throughout the elution process. The results are given in

Table- 3.6.

2+ 3+ Determination of Mg and Al in Antacid drug Samples:

One tablet or 5 ml of antacid suspension was dissol­

ved in 10 ml concentrated HCl and diluted to 250 ml with

DMW. One ml of this solution was evaporated to almost

dryness and the residue was taken in 1 ml of DMW. It was

loaded in a column having 2.0 gm of exchanger in H form.

The results are given in Table- 3.7.

81

RESULTS AND DISCUSSION

It is apparent from Table- 3.1 that the ion exchange

capacity of zirconium(IV) iodophosphate (1.75 meq/g ) was

found to be higher as compared to that of zirconium iodate

(0.56 meq/g) and zirconium phosphate (0.60 meq./g ) prepared

under identical codntions. It was also observed that ion-

exchange capacity increased significantly to a greater

extent fo'r samples prepared at elevated temperatures.

Improvement in exchange capacity and chemical stability was

observed for sample-C of exchanger prepared by agitating the

gelatinous precipitate in mother liquor at 80°C for 8 hrs

using a magnetic stirrer. Sample C was therefore selected

for further studies. The data on exchange capacity (Table

3.2) show that capacity decreases as the hydrated radii of

the alkali metals as well as alkaline earth increases as

expected. Ion exchange capacity of Zr(IV) iodophosphate is

also affected by drying temperature. As the drying tempera­

ture of the material increases exchange capacity decreases

(Table-3.3). A comparision of the exchange capacity of

different materials dried at 500°C show the sequence-

Zirconium iodophosphate > Zirconium Selenophosphate( 11)

> Tin Iodophosphate(12) > Zirconium iodomolybdate( 13).

Moreover, ZriP also appears to be most thermally stable as

it retains considerable ion exchange capacity (0.66 meq/g)

82

w E-t < K en o K

o Q o

s D M

Z o u « H ISl

O

w w t - l

w a, o

w

o

M

w E EH 2 >H CO

J«i

u (0

E (U

Oi

0) cr >i c VJ (0 •« x: >i O -P 1

^ Q) cn-l-

X -H en c Q) U \ (0 1 to crx: c a (u 0 (0 e M U —

(U 1 O >i C W VJ (0 T) '0 M (0 (0 (U )-l <U £1 0) a -p 04 4-1 M-l < 0 to

(U O 1 C -H (0 U 5-1 (U Q) (0 V -P <u a (0 a 4-1 a4- i -H < 0 a

o X 0)

+J ra

en C

•H

(0 z u 0 M-l

u 0 o ^

in •H cn

4-1

c w

o tn c o

• H 4-1 - H TD C O

U

cn c o >

•H -H \ X 4J >

•H (0 \ ^ U >

O

O

u o tS3

rH •

o. o E 2 (0 w

1 (0 4

0) D. E 0) 4J

E 0 0 Pi

0) u 3 4-1

QJ 4-1 -H x: s

1

rH Q) cn 0) 4-1 -H X

s

01 P 0 c

•H 4J

u M o 0 O

U-J <X)

O CP4J •H C (C +J -H <u V4 in C 4 V4 cn-H x : n3 4-1

U 0

> 4 - l

-d (U X 3

i H

14-1

• 10 ^ x: Q) -sr

M o

144

-0 <D • X 10 D U

rH X U-( QJ O

S 10 CO tf CM

o i n

IT) o

« in

in U3 in

• • •• • • ••

rH r-l

o o

i-H rH

• • •• CN CM

l - l rH

• • • o o o

o o

o o

o o

o o o

m u D W fe

83

TABLE - 3 . 2

ION-EXCHANGE CAPACITY ( m e q / g . DRY EXCHANGER) OF ZIRCONIUM

(IV) lODOPHOSPHATE (SAMPLE C) FOR VARIOUS METAL IONS AT pH

6 . 5 .

S .No . M e t a l i o n H y d r a t e d I o n - e x c h a n g e c a p a c i t y ( m e q / g - d r y e x c h a n g e r )

1 . L i 1 0 . 0 1 .37

2 . Na"^ 7 . 9 1 .70

3 . K"^ 5 . 3 1 .78

4 . Mg^"^ 1 0 . 8 1 .25

5 . Ca^"^ 9 . 6 1 .56

6 . Sr^"^ 9 . 4 1 .60

7 .

Metal ion

Li+

Na+

K^

Mg2+

Ca2+

Sr2+

Ba2+

Hydrated ionic radius

A°

10.0

7.9

5.3

10.8

9.6

9.4

8.8

84

TABLE - 3.3

EFFECT OF HEATING ON ION-EXCHANGE CAPACITY OF

VARIOUS INORGANIC ION-EXCHANGERS

Heating Ion-exchange capacity Temperature (meq/g-dry exchanger)

40

100

150

200

300

400

500

600

ZIP TIP ZIM ZSP

1 . 7 5

1 . 6 0

1 . 6 0

1 . 4 0

1 . 0 0

0 . 9 2

0 . 8 0

0 . 6 6

1 . 6 0

1 . 4 4

1 . 3 3

0 . 9 9

0 . 7 2

0 . 6 6

0 . 4 4

0 . 3 8

1 .54

1 .30

-

1 .00

0 . 7 8

0 . 6 6

0 . 4 2

0 . 1 6

1 . 5 1

1.4

-

1 .38

1 .18

0 . 5 4

0 . 5 0

0 . 2 0

ZIP = zirconium(IV) iodophosphate (Sample C),

TIP = tin(IV) iodophosphate,

ZIM = zirconium(IV) iodomolybdate,

ZSP = zirconium!IV) selenophosphate

85

even at 600°C. It is apparent from the Table-?'.4 that the

material (sample-C) is quite stable in moderate concentra­

tion of mineral acids like HCL, HNO^ and H-SO. and organic

acids e.g. HCOOH, CH^COOH but dissolved completely in 2M

NaOH solution.

FigureG Dshows the titration curves for the K"*" H^

+ + and N H exchange processes. The titration process

occurs in one stage suggesting its behaviour as a monofunc-

tional weak with pKa value 2.87. The ion exchange capaci-

+ -t-ties calculated from the titration curves for K and Na are

found to be 1.78 meq/gm and 1.70 meq/gm at pH 6, and 3.50

meq/gm and 3.4 meq/gm at pH 10.5 respectively. These values

are in good agreement with those obtained theoretically

(1.73 meq/gm at pH-6.5 and 3.46 meq/gm at pH 10.5). These

values have been calculated on the basis of three exchange­

able hydrogen at pH 6.5 and six exchangeable hydrogen at pH

10.5 per mole formulae.

The pyrolysis studies of the material reveals

interesting results. The DTA curve (Fig.3.3) exhibit two

endothermic with T max at 50° and 780°C and one exothermic

peak wit T-max at 280°C corresponding to three distinct

weight losses. The external water molecule is lost in the

region 40-130°C and the corresponding weight loss is 12.5%

as evident from TGA curve. The exothermic peak occurs

86

Na"

1-0 _ 2-0 3<3 A-0 me^ Of OhT^dded/O-Sg e x c h a n g e r

F i g u r e - 3 . 1 pH- T i t r a t i o n Curve of Zr (IV) l o d o p h o s p h a t e i n H Form.

87

ZIP

100 200 300 ^00

Drying temperature "b

500

Figure-3.2 Exchange Capacity as a Function of Temparazure of various Inorganic Ion-Exchanger

88

during heating from 130°-520°C with a weight loss of 10^

group byvolatilization. The disappearance of iodate peak in

i.r. spectrum of this material dried at 52 0°C also supports

this conclusion. Further loss in weight beyond 520°C which

_2 continioes upto 630°C may be due to condensation of HPO.

group into pyrophosphate. The infrared absorption spectra

of zirconium(IV) iodophosphate in H form are shown in fig. 3.4

The strong and broad band in the region 3600-3100 cm may

be assigned to interstitial water molecule and -OH

groups(14). Another strong and sharp peak with maximum at

162 0 cm" may be due to HOH bending. A very weak band

appears in the range 2460-2300 cm that may be due to

-2 HPO. group. The spectrum also shows strong bands m the

regions 1120-920 cm" and 780-680 cm" indicating the -2

presence of HPO. and 10^ groups respectively (15) . A very

strong and sharp peak occurs at 1370 cm which is assigned

to >P=0 (17). The Zr-0 stretching vibration also falls in

the region 1120-920 cm" (16). Peaks appearing at 600, 550

and 500 cm may also be due to the HPO. and 10^ groups(15).

The i.r. spectrum of the material dried at 520°c shows the

absence of peak in the region 780-680 cm confirming the

elimination of iodate group.

On the basis of chemical composition, ion exchange

capacity, i.r. and thermogravimetric analysis a tentative

formula for zirconium(IV) iodophosphate may be suggested as

89

Weight (Vo)

0

9t Q.

E

0) 4-1

x; tn O

j : : a o -o o

IS]

0

0) > u a u

EH Q I < o

n •

n I U D

O O o 3 • a

r O o o

I

520'C

400 C

001

liOOO 3 540 3680

F i g u r e - 3 . 4

780 320

Infrared Spectra of Zr(IV) lodophosphate

2620 2160 1700,, 12^0 WAVENUMBER CCm )

91

(ZrO)^ (OH) (lO^) (H0P^)g.nH20- The weight loss (12.5%)

upto 130°C is due to the removal of external water molecules

The number of water molecules per mole of the material as

calculated from Alberti's equation (18) is found to be 12.0.

•"•^ X 100 = % weight loss. M+18n

The ion exchange capacity calculated on the basis of

proposed formula is 1.73 meq/g dry exchanger and is in

agreement with the experimental value at pH 6.5. The weight

loss upto 520°C due to elimination of H_0 and I0-. groups

corresponds to 22.5% (Fig.3.3) which is close to the theore­

tical value 23.5%..

It is apparent from the Table 3.5 that the distribu­

tion coefficient of metal ions decreases with the increase

in concentration of hydrochloric acid as expected. However,

in mixed DMSO-HCl systerr.sthe K, value increases with increa­

sing percentage of DMSO. This effect is more prominantly

observed in the case of transition metals and inner transi-

3+ 2+ 3+ 4+ 4+ 3 + tion metals e.g. Al / Pb , Fe , Zr , Th and La have

exceptionally high K, value. The large variation in K,

values for different metal ions may be due to difference in

the stability constant of the complex formed with DMSO.

These findings are in good agreement with the studies of

Janauer et.al (19,20). Due to high dielectric constant and

excellent solvating properties, DMSO plays a very important

92

TABLE - 3 . 4

CHEMICAL STABILITY OF ZIRCONIUM( IV) lODOPHOSPHATE (SAMPLE C)

ON DIFFERENT SOLUTIONS

Solution

H^O

l.OM

2.0M

2.0M

2.0M

2.0M

2.0M

2.0M

HNO3

HNO^

H2SO4

HCl

CH^COOH

HCOOH

NaOH

Zr(V)

released mg/50 ml

0.00

0.00

0.35

1.38

1.20

0.00

0.08

Complete

IO3

released mg/50 ml

0.00

0.00

0.85

1.24

1.12

0.00

0.11

dissolution

-r released mg/50 ml

0.00

0.00

1.08

1.15

0.98

0.00

0.18

93

TABLE - 3 . 5

DISTRIBUTION COEFFICIENT OF METAL IONS ON lODOPHOSPHATE

OF z r ( I V ) IN DIFFERENT SOLVENT SYSTEMS.

M e t a l 0.05M O.lM 5% DMSO 20%DMSO 30%DMSO 50%DMSO

I o n s . HCl HCl

Mg2+

Cd^^

Zn

Ba2+

Ca+2

Al+3

Co+2

Cr+3

Cu2+

Hg2 +

Pb2+

Fe3+

Zr4+

Th^+

La3+

Sr2+

Mn2+

Ni2+

56.25

2.27

4.16

25.0

18.0

22400

125.0

33.0

33.0

18.0

650.0

212.0

22400

337.0

24900

21.62

25.0

38.0

13.6

12.5

25.0

18.0

5.5

22400

7.14

-

4.10

5.5

181.0

25.0

22400

1650.0

24900

12.5

4.80

25.0

56.20

7.14

25.0

32.0

31.9

22400

125

25.0

56.2

97.9

1025.0

316.0

22400

22400

24900

25.0

25.0

25.0

66.0

25.0

31.0

104.0

58.0

22400

150

50.0

70.4

97.9

22400

1150.0

22400

22400

24900

-

33.0

25.0

-

150.0

31.0

181.0

69.0

22400

462

50.0

87.5

493.75

22400

22400

22400

22400

24900

-

100

25.0

-

181.25

38.0

221.0

236.50

22400

650

150

134.3

6910.0

22400

22400

22400

22400

24900

-

150

25.0

94

role in reflecting different behaviour of metal ions towards

ion-exchange materials. On the basis of difference in K,

values a number of binary and ternary separations have been

achieved (Table 3.6, fig. 3.5). The practical utility of

these separations has been demonstrated in the analysis of

drug samples of antacids. The results obtained are in good

agreement with those of the labelled values (3.7). The

separation of metals by selective elution from a cation

exchange resin with hydrochloric acid is a well acceptable

technique. The formation of non-dissolved chloro compound

is most favourable with the transition metals (21) which are

strongly held by the cation exchange are conveniently eluted

2+ 2+ with IM HCl; Mg and Ca with low K, values are eluted

with lower concentration of HCl.

95

TABLE - 3.6

SEPARATION OF METAL IONS ACHIEVED ON THE

COLUMN OF Zr (IV) lODOPHOSPHATE.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10

Separations achieved

Ca+2

Mg+2

.. +2 Zn Mg+2

Mg^2

A1^3

Mg^2

Fe

Sr^2

Ba^2

Cu^2

v+2 Ni

o +2 Ca

Al- 3

Cd

Al+2

^ +2 Ca Mg^2

A1^3

. Cu+2

Co- 2

Fe^3

Amount loaded mg

0.184

0.120

0.253

0.120

0.120

0.135

0.120

0.196

0.408

0.515

0.252

0.236

0.184

0.135

0.165

0.135

0.184

0.120

0.135

0.252

0.289

0.196

%of metal ions eluted

98%

98%

100%

98%

98%

96%

98%

99.4%

98.8%

100%

99%

98%

98%

96%

98.6%

96.0%

98%

98%

96%

99%

100%

99.4%

Volume of effluent in ml.

20 ml

40 ml

30 ml

40 ml

40 ml

100 ml

40 ml

90 ml

50 ml

40 ml

50 ml

50 ml

20 ml

100 ml

40 ml

100 ml

20 ml

40 ml

100 ml

50 ml

70 ml

90 ml

Fluent used

.05M HCl

.20M HCl

.05M HCl

.20M HCl

.20M HCl

l.OM HCl

.20M HCl

IM HCl

.05M HCl

.20M HCl

.05M HCl

.2M HCl

.05M HCl

l.OM HCl

.05M HCl

l.OM HCl

.05M HCl

.20M HCl

l.OM HCl

.05M HCl

.2 0M HCl

IM HCl

96

-0-2MHCI H 1-OM HCl-

10 20 30 40 SO 60 70 | 0 90 100 no f^O 130 UO ISO

Volumt of t f f l u t n t ( mIO

10 20 30 40 50 60 70 60 0

Volumt of »ftlutn« (ml )

'° 2 0 3 0 4 0 5 0 60 70 9 T 9

2+ ,-,3+ ,^^ ^_2+ „_2 + Figure-3.5 Separation of (a) Mg^'^-Al (b) Ca " -Mg (c) Zn2+ - Mg2+.

97

o - o

o 01

0

-7

£ ^ c

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W O I X O ' Z V i Q 3 /o sa|OUJUj W^OlvQ.f VIQ3 >o saioiuuj

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o k E

1-

1-

2

0

O'Q

0

0'

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2

0 To 20 30 ^0 50 60 70 80 90 100 Ho 120 130 UO 150 160

Volume of ef f luent ( ml)

F i g \ i r e - 3 . 5 S e p a r a t i o n of (g) Ca^'^-Al'^'*" (h) Cd^'^'-Al^'*',

99

V i a 3 i o S3|oujuj W Oix 3. V i a 3 >o sa|oai ai

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101

REFERENCES

1. M.L. Berardelli, P. Galli, A.L. Ginestra, M.A.

Massucci and K.G. Varshney, J. Chem. Soc, Dalton

Trans., 1737 (1985).

2. K.G. Varshney and A.A. Khan, J. Inorg. Nucl. Chem.,

41, 241 (1979).

3. S.A. Nabi, R.A.K. Rao and A.R. Siddiqi, Z. Anal.

Chem., 311, 505 (1982) .

4. M. Qureshi and R.C. Kaushik, Anal. Chem., 49(1), 165

(1977).

5. M. Qureshi, R. Kumar, V. Sharma and T. Khan, J. Chro-

matogr. 11^, 175 (1976).

6. S.A. Nabi, R.A.K. Rao and W.A. Siddiqi, J. Liq. Chro-

matogr. 6, 777 (1983) .

7. S.A. Nabi and W.A. Siddiqi, J. Liq. Chromatogr, , 8 ,

1159 (1985).

8. F.D. Snell and C.T. Snell, "Colorimetric Methods of

Analysis". D. Van Nostrand, Princeton, New Jersey

(1959) Vol. IIA, p.335.

9. F.D. Snell and C.T. Snell, "Colorimetric Methods of

Analysis", D. Van Nostrand, Princeton, New Jersey,

(1949) Vol.11 p.741.

10. Vogel's Text Book of Quantitative Inorganic Analysis,

Fourth ELBS edition Longman. Revised by J. Bassett,

R.C.Denney, G.H. Jeffery and J.Mendham (1986),pp.756.

lOlB

11. S.Z. Qureshi and N. Rahman, Bull. Chem. Soc. Japan,

^ , 2627 (1987).

12. S.A. Nabi, R.A.K. Rao and W.A. Siddiqui, J. Liq.

Chromatogr., 6, 777 (1983).

13. S.Z. Qureshi and N. Rahman, Bull. Soc. Chim., France,

959 (1987).

14. M. Davis, Infrared Spectroscopy and molecular struc­

ture Elsevier Publishing Co., Amesterdam (1963), p.

318.

15. G. Socrates, "Infrared characteristic group frequen­

cies", John Wiley & Sons, Ltd., New York (1980), p.

145.

16. W. Weltner, Jr. and D. McLeod, Jr, J. Phys. Chem.,69,

3488 (1965).

17. K. Nakamoto, 'Infrared spectra of Inorganic and

Coordination Compounds', John Wiley, New York, p.16 9

(1963).

18. G. Alberti, E. Torracca and E. Conte, J. Inorg.Nucl.

Chem. , , 607 (1966) .

19. G.E. Janauer, Mikrochim Acta, 1111 (1968).

20. G.E. Janauer, H.E. Vanwart and J.T. Carrano, Anal.

Chem., 42 (1970).

21. Ilga Birze, Leland W. Marple and Harvey Diehl,

Talanta, 15, 1441 (1968).

CHAPTER-IV

Thin Layer Chromatographic Separations of a^ -Amino Acids on

Stannicarsenate - Silica gel Mixed Layers

102

INTRODUCTION

Thin layer of inorganic ion exchange material has

been widely used for the separation of metal ions (1-3). Use

of these materials for the separation of organic compounds

has received little attention. Nabi et al. successfully

utilized pure cation exchanger stannic tungstate layer for

typical separations of amino acids and phenols (4,5). Lepri

et al. used mixture of ammonium tungstophosphate and

silanized silica gel for the study of amino acids (6). A few

studies have been reported on inorganic ion-exchange

material mixed with silica gel (7). The present work is

therefore undertaken to explore the potentiality of the

inorganic exchange layers mixed with silica gel for the

separation of organic compounds. <?C -Amino acids have been

chosen as they play important role in controlling the

physiological activity in human body. The study has shown

that degree of retention for different amino acids varies

with the nature of developing solvent.

EXPERIMENTAL

Reagents and Chemicals:-

Stannic chloride pentahydrated (Loba Chemie , India,).

Sodium arsenate dibasic (J.T. Baker Chemical Co. USA), Amino

103

Acids kit (Loba Chemie,India). Silica gel (E.Merck,(India)

Ltd.) All other reagent used were of A.R. grade.

Apparatus : -

Bausch and Lomb spectronic-20 spectrophotometer was

used for spectrophotometric studies.

Detectors :-

A ninhydrin 0.25% in acetone-water (9:1) solution

was used for the detection of amino acids.

Preparation of ion-excheuige material and thin layer plates:-

Stannic arsenate was prepared by mixing O.IM solution

of sodium arsenate with O.IM solution of stannic chloride in

1:1 ratio at pH-1.0 and digesting the resulting precipitate

at room temparature for 24 hours. The precipitate was

filtered under suction and completely dried in an oven at

40+_4 C. The material so obtained was cracked in deminera-

lized water and then placed in l.OM HNO^ for 24 hours to con­

vert it in H form. It was washed with demineralized water

to remove excess acid and finally dried at 40°C.

For the preparation of thin layer plates the granules

of stannic arsenate was well powdered in a mortar and mixed

with silica gel G in 1:1 ratio. The slurry of this mixed

product was prepared by adding 50ml of water per 10 gm of

104

material and spread over glass plate with the help of

applicator to obtain a uniform thin layer of 0.2mm. The

plates were then dried in an oven at 60 C.

Procedure:-

Approximately 0.05ml. of amino acid solutions were

applied with the help of glass capillary on the plates.

After drying the spots , the plates were developed in

various solvent systems and solvent were allowed to ascend

upto 15.0cm from the point of application in all cases.

Quantitative Separations:-

0.01ml mixture of amino acids containing 50ug each

were spotted with the help of a micropippete at the point of

application on the plate. The plates were developed in the

usual way. Pilot Chromatograms were run under similar

conditions to ascertain the actual position of the spots on

the experimental plates. After the development the spots on

plates were detected using the ninhydrin reagent. The same

portion of the experimental plates were then scratched out

and the amino acids present in these portions were extracted

with small portions of the absolute alcohol. Total 5.0ml

being used for complete elution in each case. Amino acids

were then determined spectrophotometrically using ninhydrin

reagent (8) .

105

RESULTS AND DISCUSSION

Result of these studies reveal that stannic arsenate-

silica gel-G layer offers potentials for the separation of

amino acids. The behaviour of amino acids towards adsorbent

is altered due to the presence of ion-exchange material.

Thus in addition to simple adsorption ion-exchange also

plays important role in differential migration of amino

acids. A number of diverse organic solvents have been tried

for the study of amino acids but results were not

Satisfactory due to distorted spots. It has been observed in

general amino acids remain at the point of application in

solvent of low dielectric constant such as

ethylmethylketone, n-butanol, formic acid, acetone, alcohol,

propionic acid, benzene etc. showing that these compounds

have little interaction or no interaction at all with the

mobile phase. However, the addition of little quantity of

potassium chloride or sodium hydroxide in n-butanol and

l,4,Dioxane gives interesting results and reflects many fine

separations. In solvent system KCl-n-BuoH (3:2) and KCl-1,4,

Dioxane (3:2). R^ values of almost all amino acids show

similar behaviour. The systems having different ratios of

n-BuoH-KCl-1,4,Dioxane the R^ values of arginine, cysteine,

cystine, histidine, glycine, lysine and ornithine are in

general low, while nor leucine, valine, phenylalanine having

106

higher values. Thus a number of important binary and ternary

separation have been actually achieved. It has been observed

that increase in the quantity of dioxane in the developing

solvent decrease the movement. (Table-4.1).

Similarly addition of potassium chloride to the

acetone enhances the movement of amino acid except a few

cases. Valine, serine, 3(3,4-Dihydroxy phenyl)-DL-Alanine

(Dopa), hydroxy proline and phenylalanine in

acetone-potassiunichloride (4:1) move uptil the front, while

the R^ values of acidic and basic amino acids are in the

range of (0.43-0.66) as apparent from table-4.2. As the

ratio of potassium chloride increases the movement of almost

ell amino acids increases except alanine, 2-amino-n-butyric

acid and cysteine. Similar behaviour has been observed on

increasing the acetic acid composition in the same solvent

system only with the exception of Iso-leucine, cysteine and

cystine which show the reverse trend. On the basis of this

difference in behaviour a number of binary separations of

neutral and basic amino acids have been actually achieved

(Table-4.5).

It is also clear from the table-4.3 that the movement

of amino acids is not observed even in acetone-sodium

hydroxide (3:2) mixed system. However interesting results

have been reflected on decreasing the concentration of

TABLE - 4.1

R^ VALUES OF AMINO ACIDS ON MIXED STANNIC ARSENATE - SILICA GEL

LAYER (1:1).

107

Name of the Compound

1. DL-Alanine

2. DL-2 Amino-n-butyric acid

3. L-Arginine HCl

4. DL-Aspartic acid

5. L-Cysteine

6. L-Cystine

7. L-Glutamic acid

8. Glycine

9. L-Histidine HCl

lO.L-Levicine

11.DL-Isoleucine

12.DL-Nor-leueine

13.L-Lysine-HCl

14.DL-Methionine

15.L-0rnithine HCl

16.DL-Phenylalanine

17.L-Proline

18.DL-Serine

19.DL-Threonine

20.DL-Tryptophan

21.L-Tyrosin

22.DL-Valine *

23.DL-Dopa 24.Hydroxy Proline

Rf Val

Si

0.56

0.53

0.60

0.60

0.56

0.56

0.53

0.56

0.36

0.23

0.46

0.20

0.36

0.20

0.26

0.66

T

T

T

T

T

T

T

T

ues in

S2

0.60

0.56

0.56

0.56

0.66

0.66

0.63

0.66

0.66

0.36

0.46

0.33

0.43

0.30

0.43

0.43

0.46

0.56

0.56

0.53

0.46

0.53

0.56

0.60

Different

S3

0.30

0.43

0'06

0.26

0.06

0.0

0.25

0.13

0.10

0.53

0.30

0.43

0.53

0.0

0.30

0.46

0.26

0.16

0.10

0.30

0.26

0.70

0.66

0.20

Solvent

S4

0.31

0.63

0.20

0.46

0.43

0.26

0.56

0.16

0.16

0.60

0.73

0.73

0.16

0.16

0.10

0.56

0.50

0.66

0.46

0.50

0.46

0.73

0.70

0.73

Systems

S5

0.28

0.30

0.10

0.06

0.03

0.06

0.30

0.08

0.03

0.63

0.53

0.83

0.03

0.26

0.0

0.93

0.46

0.23

0.16

0.56

0.63

0.60

0.33

0.50

S6

0.18

0.03

0.0

0.0

0.16

0.33

0.26

0.03

0.00

0.25

0.63

0.78

0.0

0.23

0.0

0.91

0.23

0.0

0.11

0.14

0.17

0.46

0.0

0.15

-3-(3,4-Dihydroxyphenyl)-DL-Alanine. Si-Potassium Chloride-n-BuoH(3:2),S2-Potassium Chloride-1,4,dioxane (3:2), S3 n-BuoH-1/4,dioxane— Potassium Chloride(3:1:1), S^ n-BuoH 1,4,dioxane—Potassium Chloride (2:2:1), S5 n-BuoH-1,4,dioxane Pota­ssium Chloride (5:4:1), Sg-n-BuoH 1,4,dioxane Potassium Chloride (5.5:4:0.5).

T - Tailing.

108

TABLE - 4-2

R^ VALUES OF AMINO ACIDS ON MIXED STANNIC ARSENATE - SILICA GEL

LAYER (1:1).

Name of the Compound

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

DL-Alanine

DL-2 Amino-n-butyric acid

L-Arginine HCl

DL-Aspartic Acid

L-Cysteine

L-Cystine

L-Glutamic acid

Glycine

L-Histidine HCl

L-Leucine

DL-Iso-leucine

DL-Nor-Leucine

L-Lysine HCl

DL-Methionine

L-Ornithine HCl

DL-Phenylalanine

L-Proline

DL-Serine

DL-Threonine

DL-Tryptophan

L-Tyrosin

DL-Valine

DL-Dopa

L-Hydroxy Proline

Rf va

S7

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

lues in

S8

0.15

0.15

0.0

0.08

0.20

0.23

0.11

0.0

0.0

0.73

0.33

0.86

0.0

0.63

0.0

0.50

0.15

0.23

0.21

0.33

0.20

0.60

0.20

0.93

Different

Sg

0.86

0.93

0.50

0.46

0.90

0.66

0.43

0.90

0.66

0.86

0.46

0.93

0.53

0.80

0.46

1.0

0.93

1.0

0.73

0.83

0.93

1.0

1.0

1.0

Solvent

^10

0.60

0.43

0.23

0.66

0.23

0.50

0.66

0.83

0.66

1.0

0.90

1.0

0.70

1.0

1.0

0.93

0.86

1.0

0.86

1.0

1.0

1.0

1.0

1.0

System

Sll

0.80

0.60

0.23

0.73

0.28

0.76

0.76

0.60

0.36

0.96

0.70

0.56

0.21

0.76

0.13

0.76

0.73

0.70

0.90

0.93

0.86

0.96

0.80

0.75

^12

0.60

0.50

0.13

0.41

0.40

1.0

0.41

0.21

0.10

0.75

0.86

0.56

0.13

0.73

0.10

0.50

0.40

0.33

0.56

0.93

0.56

0.60

0.53

0.50

Sy-Acetone, So - Acetone-Potassium chloride (9:1),Sg-Acetone-Potassium chloride (4:1), S;i^Q-Acetone-Potassium chloride (3:2), SjLi ~ Acetone-Potassium chloride-acetic acid ( 20 :3 :2 ) ,3-, 2-Acetone-Potassium chloride-acetic acid (45:4.0:1.0)

TABLE - 4.3 ^^^

R^ VALUES OF AMINO ACIDS ON MIXED STANNIC ARSENATE - SILICA

GEL LAYER (1:1).

Name of the Compound

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

DL-Alanine

DL-2-Amino-n-Butyric acid

L-Arginine HCl

DL-Aspartic Acid

L-Cysteine

L-Cystine

L-Glutamic acid

Glycine

L-Histidine HCl

L-Leucine

DL-Iso-Leucine

DL-Nor-leuc ine

L-Lysine HCl

DL-Methionine

L-Ornithine HCl

DL-Phenylalanine

L-Proline

DL-Serine

DL-Threonine

DL-Tryptophan

L-Tyrosin

DL-Valine

DL-Dopa

L-Hydroxy Proline

R£ vail

Sl3

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

ues in

Sl4

0.83

T

0.0

0.80

0.0

0.76

0.86

0.03

0.0

1.0

0.78

1.0

0.0

0.73

0.23

T

0.33

1.0

T

T

T

1.0

T

T

Different

Sl5

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Solvent

Sl6

0.50

0.26

0.90

0.23

0.53

0.46

0.53

0.43

0.91

0.26

0.66

0.30

0.86

0.26

1.0

0.0

0.13

0.36

0.20

0.26

0.20

0.80

0.33

0.20

Systems

Hi

0.23

0.23

0.03

0.16

T

T

0.60

0.0

0.0

0.24

0.26

0.53

0.03

0.23

0.06

T

0.26

0.20

T

0.40

0.60

0.80

0.23

0.36

ST 3-Acetone-Sodium hydroxide ( 3 : 2 ) , S-j^^-Acetone-sodiumhydroxide

( 4 : 1 ) , SHL5-Benzene-acetone(l: l) , S^^g-Benzene-acetone-methanol ( 5 : 5 : 2 ) , 3^7 -Benzene-acetone-Methanol ( 3 : 4 : 3 ) .

110

sodium hydroxide in the system (4:1) pH 9-10. As a general

rule all amino acids act as cationic species in highly

acidic medium and anionic species in alkaline medium. At

higher pH amino acids are deprotonated forming neutral or

negatively charged species (9). Neutial and acidic amino

acids in this system behave as anionic or neutral species

and hence have no electrical interaction with the exchanger

resulting high R^ values. On the other hand hasic amino

acids which exist as cationic species (10,11) are strongly

held by the exchanger and therefore remained at the point of

application. On the basis of this diffrential behaviour it

has been possible to achieve the separation of basic amino

acids from a mixture of neutral and acidic amino acids.

Besides this several binary separations have also been

demonstrated. (Table-4.5).

Citrate phosphate buffer of varying pH has also been

tried for the determination and separation of amine acids.

In citrate phosphate buffer of pH - 4 all the basic amino

acids have very low R_ values, while acidic amino acids have

high R_ value. This can also be explained on the basis of

earlier discussion. Aspartic acid and glutamic acid show

exceptional behaviour as they have maximum R- values. This

is probably due to other interactions which may be involved

besides ion-exchange which predominate the distriburion of

I l l

TABLE - 4.4

R^ VALUES OF AMINO ACIDS ON MIXED STANNIC ARSENATE - SILICA GEL

LAYER (1:1)

Name of the Compound

Rf Values in Different Solvent Systems

'18 '19 '20

I. DL-Alanine

2. DL-2-Amino-n-butyric acid

3. L.Arginine HCl

4. DL-Aspartic Acid

5. L-Cysteine

6. L-Cystine

7. L-Glutamic Acid

8. Glycine

9. L-Histidine-HCl

10. L-Leucine

II. DL-Iso-leucine

12. DL-Nor-leucine

13. L-Lysine HCl

14. DL-Methionine

15. L-Ornithine HCl

16. DL-Phenylalanine

17. L-Proline

18. DL-Serine

19. DL-Threonine

20. DL-Tryptophan

21. L-Tyrosin

22. DL-Valine

23. DL-Dopa

24. L-Hydroxy Proline

T

0 . 7 0

0 . 1 3

1 .0

T

0 . 7 3

1 .0

T

0 . 1 3

0 . 6 0

0 . 7 6

0 . 9 3

0 . 1 5

0 . 6 0

0 . 3 5

T

T

0 . 8 6

T

T

T

0 . 8 0

0 . 7 3

T

T

1 . 0

0 . 0 6

0 . 8 6

0 . 2 3

0 . 0

0 . 9 0

T

0 . 2 0

0 . 1 0

0 . 9 0

0 . 9 3

0 . 1 3

0 . 5 3

0 . 2 3

T

T

0 . 9 0

T

T

0 . 7 3

0 . 9 0

0 . 6 6

T

T

1.0

0 . 0

0 . 2 3

0 . 1 5

0 . 1 5

1.0

1.0

0 . 2 3

0 .40

1.0

T

0 .0

0 . 2 3

0 .0

T

T

T

T

T

0 . 8 5

0 .86

0 . 6 6

T

^18 ~ "ti trate phosphate buffer pH-2.6, S-Lg-Citrate phosphate buffer of pH-4.0, S20 ~ Ci t ra te phosphate buffer of pH-7.0.

112 TABLE - 4 . 5

QUALITATIVE SEPARATIONS ACHIEVED IN DIFFERENT SOLVENT SYSTEMS.

S.No. B i n a r y S e p a r a t i o n s Achieved S o l v e n t Systems

1. DL-Valine(0.70), L-Lysine (0.53) S3

2. DL-Valine(0.70), DL-Alanine (0.30) S^

3. DL-Nor-leucine(0.43),L-Hydroxy- S^ proline (0.20)

4. DL-ISO-leucin(0.73),L-Histidine(0.16) S^

5. DL-ISO-leucin(0.73), Glycine (0.16) S^

6. DL-ISO-leucin(0.73),DL-2-Amino-n-Butyric S. acid (0.63)

7. DL-Valine(0.73),DL-Phenylalanine (0.56) S.

8. DL-Valine(0.73),L-Histidine (0.16) S.

9 . L - H y d r o x y p r o l i n e ( 0 .73) /DL-Meth- ionine S. ( 0 . 1 6 )

1 0 . D L - N o r - l e u c i n e ( 0 . 8 3 ) , L - L y s i n e ( 0 . 0 3 ) S^

11. L-leucine(0.63),DL-Methionine (0.26) S_ b

12. L-Tyrosin (0.63), DL-Threonine (0.16) S^ 13. DL-ISO-leucine(0.63)/L-Glutamic acid S

(0.26)

14. DL-ISO-leucine(0.63),L-Cystine (0.33) S,

15. DL-Nor-Leucine(0.78),L-lysine (0.0)

16. DL-Nor-leucine(0.86),L-lysine (0.0) S

6

6 17. DL-Valine(0.60),L-Ornithine (0.0) Sg 18. DL-Hydroxyproline(0.93),DL-Serine(0.23) S„

o 19. DL-Nor-leucin(0.86),L-Arginine (0.0) Sg 20. DL-Methionine(0.63),L-Histidine (0.0) S^

o

21. DL-Threonine(0.73),DL-ISO-leucin(0.46) Sg

22. DL-Nor-leucine(0.93),L-Histidine(0.66) Sg

23. L-leucine(0.86),DL-ISO-leucine(0.46) Sg

24. L-leucine (0.96),L-Ornithine (0.13) S

25. L-leucine (0.96),L-Arginine (0.23) S,, 26. L-Glutamic acid (0.76),L-Histidine(0.36) S,, 27. DL-Alanine (0.80),L-lysine (0.21) S^

113

18

Table No.4.5 Contd.

28. L-leucine(0.75),L-Arginine (0.13) S,_

29. DL-Methionine(0.73), L-Arginine(0.13) S,

30. DL-Phenylalanine(0.50),L-Arginine(0.13) S,_

31. L-leucine (1.0), L-Histidine (0.0) S,

32. DL-Nor-leucine(1.0),L-Arginine (0.0) S

33. L-Leucine (1.0), L-Arginine (0.0) S,

34. DL-Nor-leucine (1.0),L-lysine (0.0) S,.

35. L-Glutamic acid (0.86),L-Histidine (0.0) S,.

36. DL-Aspartic acid (0.80),L-Lysine (0.0) S,.

37. DL-Valine (0.80),DL-Phenylalanine (0.0) S^

38. L-Cystine (0.53), DL-Nor-leucine(0.30) S,g

39. Glycine (0.53), L-leucine (0.20) S^

40. DL-2-Aniino-n-buturic acid( 0 . 70 ) ,L-Argi- S nine (0.13)

41. DL-Aspartic acid (1.0),L-Histidine S,^ (0.13)

42. L-Glutamic acid (1.0),L-Histidine(0.13) S,g

43. L-Glutamic acid (1.0),L-Lysine (0.15) S,Q

44. DL-2-Araino-n-butyric acid (1.0),DL- S, _ Aspartic acid(0.86)

45. L-Glutamic acid (1.0),L-Histidine (0.20) S,Q

46. Glycine (1.0),L-Histidine (0.23) S,_

47. D-ISO-leucine (1.0),DL-Aspartic acid S,_ (0.86)

Ternary and Mixed Separations.

1. DL-Valine(0.73),DL-Thr4onine(0.46),DL- S. Methionine (0.16)

2. DL-Phenylalanine(l.O),L-Histidine(0.66), S-DL-ISO-leucine (0.46)

3. L-leucine (0.53) from all other amino S-, acids (0.43) except DL-Valine(0.70),

DL-Dopa (0.66), L-Lysine (0.53)

4. DL-Phenylalanine(0.93) from all other S^ (0.70) except DL-Nor-leucine (0.83)

113B

Table No.4.5 Contd,

DL-Phenylalanine (0.91) from all other Sg amino acid (0.80)

S -n-Butanol-1,4,Dioxane-potassiumchloride (3:1:1)

S^-n-Butanol-1,4,Dioxane-potassiumchloride (2:2:1)

Sc -n-Butanol-1,4,Dioxane-potassiumchloride (5:4:1)

S,-n-Butanol-l,4,Dioxane-potassiumchloride (5.5:4:0.5) b

Sg-Acetone-potassiumchloride(9:1), Sg-Acetone-potassiumchloride

(4:1), S, ,-Acetone,potassiumchloride-acetic acid (20:3:2)

S,p-Acetone-potassiumchloride-acetic acid (45:4.0:1.0)

S-, .-Acetone-sodiumhydroxide( 4:1),S^g-Benzene-Acetone-Methanol

(5:5:2), S-,g-citrate phosphate buffer of pH-2.6, S^^-Citrate

phosphate buffer of pH-4.0

114

solutes between mobile and the stationary phase.

Several quantitative separations of amino acids have

also been reflected. Phenylalanine has been selectively

separated from a mixture of 20 amino acids. The results are

given in table-4.6.

It can be concluded that stannic arsenate silica gel

can successfully utilized for both qualitative and quanti­

tative separations.

115

TABLE - 4.6

QUANTITATIVE SEPARATION OF AMINO ACIDS ON STANNIC

ARSENATE-SILICA GEL LAYER (1:1)

Separtion achieved Amount taken Amount found % error Solvent ug ug System

1^ DL-Valine 50.0

L-Histidine 50.0

2. Phenylalanine from 50.0 other amino acids

3. Nor-leucine 50.0

L-lysine 50.0

4. Nor-leucine 50.0

L-lysine 50.0

5. Nor-leucine 50.0

L-lysine 50.0

4 9 . 0

4 9 . 5

5 0 . 0

5 0 . 0

4 9 . 8

4 9 . 9

5 0 . 0

4 9 . 9

4 9 . 8

2 . 0 0

1 .00

0 . 0

0 . 0

0 . 4

0 . 2

0 . 0

0 . 2

0 . 4 '14

Solvent Systems- S,-n-Butanol-l,4,Dioxane-potassiumchloride

(2:2:1)

Sc-n-Butanol-l,4,Dioxane-potassiumchloride

(5:4:1)

S^-n-Butanol-1,4,Dioxane-potassiumchloride

(5.5:4:0.5)

S,--Acetone-Sodiumhydroxide (4:1)

116

REFERENCES

1. M. Qureshi, K.G. Varshney and S.D. Shantia, Sep. Sci.

Technol., 1^ (1978) S17.

2. S.D. Sharma, T.R. Sharma and B.M. Sethi, J. Liq.

Chromatogr., ^ (1983) 1253.

3. N.S. Seth, R.P.S. Rajput, N.K. Agrawal, S.K. Agrawal

and S. Agrawal, Anal, lett., 181 (1985) 481.

4. S.A. Nabi, W.U. Farooqui, Z.M. Siddiqui and R.A.K.

Rao^J. Liq. Chromatogr., 6(1), (1983) 109.

5. S.A. Nabi, W.U. Farooqui and N. Rahman,

Chromatographia, 20(2) (1985) 109.

6. L.Lepri, P.G. Desideri and D. Heimler, J.Chromatogr. ,

268, (1983) 493.

7. K.G. Varshney, A.A. Khan, S.M. Maheshwari and U.

Gupta, Bull. Chem. Soc. Jpn. 65 (1992) 2773.

8. S. Moore and W.H. Stein, J. Biol.Chem. 176 (1948) 367

9. H.F. Walton, Techniques and Applications of

Ion-Exchange Chromatography, E. Heftmann, Ed., ed..

Van Nostrand Reinhold, New York (1975) p. 326.

10. A.S. Win,grove, and R.L. Caret Hoper and Row,. Publi­

shers, New York (1981) 1213-14.

11. H.J. Mcdonald, J. Chem. Education, Sep.(1952) 428.

CHAPTER - V

Sorption Studies of Metal ions on Modified Anion Exchange Resin, Selective Separation of

Mercury (II) from others Heavy Metals.

117

Aromatic complexing agents containing sulfonic acid

groups have already shown analy t ica l competence (1). Anion

exchange r e s i n s incorporated with these complexing agents

have been useful par t icu la r ly for the separation of metal

ion (2-11) . These compounds display a high aff ini ty for

anion exchangers. I t i s therefore worthwhile to incorporate

these ion-exchange materials with d i f fe ren t types of chela­

t ing agents in order to enhance the se l ec t iv i ty of such

materials towards metal ions and other species . The present

work was therefore undertaken as an effor t to develop

modified exchange resins and to explore t h e i r potent ia l i ty

for the s e l e c t i v e separation of metal ions . The following

work summarize my findings in t h i s d i r ec t ion .

EXPERIMENTJLL

Reagents :

Amberlite resin IRA-400 (Mesh s ize 20-50), Sodium

ace ta te , Acetic Acid, Formic acid from BDH were used.

Disodium s a l t of EDTA (S.D. fine chem), Eriochrome Black-T

and other reagents were of A.R.grade.

118

Apparatus :

Bausch and Lomb Spectronic-20 Spectrophotometer and

Elico pH meter model Ll-IOT,

Reagent Solution :

1% solution of EBT in absolute ethanol and .OlM

aqueous solution of metal ions were used.

Preparation of EBT sorbed Resin :

To obtain modified resin the Amberlite resin IRA-

400 (Cl") form was treated with 100 ppm solution of EBT for

24 hours at pH-3. The resin was then washed with several

times with demineralized water until the supernatent liquid

was free from excess reagent. The resin was finally dried in

an oven at 60 C. All the studies have been done using this

material.

Determination of Adsorption Isotherm of EBT on Amberlite

resin IRA-400 at pH-3 :

Sorption of EBT was studied under static condition.

0.3 mg of Amberlite resin IRA-400 (Cl ) form was equili­

brated for 24 hours with 20 ml of EBT solution of different

concentrations at constant pH-3 and at room temperature. The

equilibrium concentration of dye in the solution was then

determined spectrophotometrically. The results are shown in

figure-5.1

119

Effect of pH on the Sorption of EBT on

Amberlite Resin IRA-400 :

0.3 gm of Amberlite resin IRA-40 0 (Cl~ form) was

shaken with 20 ml of 100 ppm EBT solution for six hours. The

pH was adjusted either by adding 0. IM HNO^ or O.lM NaOi

solution. The equilibrium concentrtion of EBT was then

determined spectrophotometrically. The results are given in

Fig. 5.2.

Study of Metal Ion Retention as a function of pH :

0.3 gm of modified resin was loaded with 40 ml

solution of metal ion in demineralized water. The desired

pH was adjusted either by adding 0.1 M HNO^ or 0.1 M NaOH

solution. The mixture was equiliberated for 24 hours. The

metal ion concentration was then determined by titrating it

against a solution of EDTA. The results are plotted in figs.

5.3 and 5.4.

Determination of Distribution Coefficient of Metal Ions :

0.4 gm of modified Amberlite resin beads loaded with

40 ml of metal ion solution in a . 250 ml Erlenmeyer flask.

The mixture was shaken intermittantly while kept for 2 4

hours. The amount of cation before and after the equili­

brium was determined by standard method (12) using 0.01 M

EDTA disodium salt solution as titrant, Kd values were

calculated as described in our earlier publication (13).

The results of Kd values are summarized in figs. 5.5 and 5 .6

and Table 5.1.

120

Quantitative Separation of Metal Ions :

For separation elution technique was used. 2 ml

mixture of metal ion solution was added to a glass column of

diameter 0.6 cm (i.d.). It was then allowed to pass through

the column containing 1.5 g resin with a flow rate of 8-10

drops/minute . The elution of metal ions was done with

appropriate eluting reagent in the usual manner keeping the

constant flow rate 18-20 drops/minute through out the

elution process. The eluted metal ion was then determined

titrimetrically using a standard disodium EDTA solution.

Table S"2 describes results of some important binary separa­

tions. Elution behaviour for different sets of separations

are presented in figs. 5.7 and 5.8.

+2 For a selective separation of Hg from a mixture of

+ 2 other metal ions, different amount of Hg was mixed with

Fe" ^ (5.58 mg), Cr" ^ (3.06 mg), Al^^(2.83 mg) Cd^^ (1.79 mo)

+2 and Pb (17.40 mg). The mixture was transferred into the

column & allowed to pass through slowly. The mixture of

+2 metal ions except Hg was first eluted with 40 ml of DMW

+2 and pure Hg was then recovered by using 2M HCl solution.

121

RESULTS AND DISCUSSION

Amberlite Resin sorbed with eriochrome black-T have

been found useful for the separation of important and diffi­

cult pair of metal ions. Fig. 5.1 shows the adsorption

isotherm of EBT on Amberlite resin IRA-400 at a constant

pH 3. It has been found that maximum uptake of EBT is

5x10 timole/gm. Fig. 5.2 shows the retention behaviour of EBT

on the resin as a function of pH. The optimum pH for the

maximum sorption of EBT has been found to be 3. The sorption

behaviour of metal ions on EBT form of resin as a function of

+ 2 +2 pH reveals many interesting features. In case of Cu , Sr

+2 +2 . . . . . Ca and Ba , ions the reteniion increase with the increase

in pH (Fig. 5.3, 5.4). But Al"^^, Fe^^, Cr"^^, Ni"*" , Co^^,

+ 2 +2 +2 Hg , Cd and Pb , show the reverse trend. It has been

+ 2 found that among various metal ions studied Hg is most

strongly retained by the exchanger in pH-2 medium. It is

due to the fact that the ion exchange resin incorporates

chelating agent EBT by n bond, which in turn reacts with

metal ions to form metal chelates having different stability

constants. The stronger the metal chelate formed more

strongly the metal ion will be held by the resin.

The distribution coefficient of metal ions in differ­

ent concentrations of formic acid such as IM, 2M, 3M, 5M

122

IM — ^

(U iS3u^o u j 6 / 3 | o u j r r ) x a 3 >° l u n o u j v

o 2 o

Li.

to < (—

m

o O I— CL a o

fN

i n

•H

( i 3 ; 0 0 , - . , . „ u 3 , u . , , 0 3 0 t x u . 6 , a , o . . ; , e 3 , , , , . oujv

123

J3

a.

a o CO

-I L. O o

o

o

2 o

o \n u. O I a

X o. u. o z o o r u. (/I <

tn liJ

O

CD

LU

2 O I/) z o

z o

o o o

z UJ

z t >

UJ

a.

IT)

(•/.) uoiiuauw 01

-H

o 2

124

/

;

/

/

^ o "a '^

O

/

[•I.I |) j u i t i x j I';.) p»uif )>u

in

125

show some interesting results (Table 5.1 & Fig.5.5,5.6) .Distribution

+2 +2 +2 +2 coefficient of metal ions such as Cd , Ni , Mn , Co ,

Fe-y, Al and Hg decreases as the concentration of formic

acid increases, on the other hand reverse trend has been

, , „ +2 „ +2 _, +2 ^ +2 „ +2 _.+2 „ +2 .,+3 observed for Mg , Zn / Sr , Ca , Ba / Pb , Cu , Al

It is reflected from the data on Kd-values (Table 5.1,5.2 &

+ 2 figures 5.7,5.8) that Hg behaves in an exceptionally

different manner as compared to other metal ions studied.

+2 The exceptionally high Kd-value of Hg has made it possible

to separate this metal ion from, other metal ions. The

utility of this material for the selective separation of

+ 2 Hg from other metal ions has been actually demonstrated by

+ 2 by separating different amount of Hg from a synthetic

mixture of other heavy metals. (Table 5.3)

It is apparent from the results (Table 5.3) that this

method can be successfully employed for the removal and

+ 2 recovery of Hg from samples of sea water and effluent of

industrial wastes.

126

TABLE - 5.1

DISTRIBUTION COEFFICIENT OF METAL ION IN DIFFERENT CONCENTRATION

OF FORMIC ACID.

M e t a l I o n

Mg+2

Zn+2

Cd^2

Sr+2

Ca+2

Ba+2

Pb+2

Mn+2

Ni+2

Co+2

Cr+3

Fe+3

Al+3

Cu+2

H ^ 2

DMW*

8 . 6 9

1 4 . 2

6 . 5

1 0 . 0

3 . 8

1 7 . 1 4

5 1 , 7 8

3 . 7 5

1 1 . 1 1

1 0 0 . 0

2 2 . 9

3 1 . 5 7

3 4 . 6 1

4 6 . 1 5

7 7 5 . 0

I n d i f f e r e n t c o n c e n t r a t i o n o f

IM HCOOH

4 . 1 6

1 6 . 6 6

3 0 . 8 8

1 5 . 7 8

7 4 . 0 7

7 0 . 1 4

1 8 3 . 0

3 1 5 . 0

3 1 . 5

5 7 . 8

3 4 . 0 9

2 1 2 . 5

3 7 7 . 0

1 0 4 . 7 4

4 0 4 . 8

2M HCOOH

8 . 6 9

3 3 . 3 3

2 3 . 6 1

5 1 . 7 2

1 4 7 . 3 6

1 3 0 . 8

2 5 4 . 0

3 8 . 3 3

1 9 . 0

1 5 . 3 8

3 4 . 0 9

1 5 0 . 0

2 0 8 . 0

9 7 . 9

1 9 1 . 6 6

3M HCOOH

1 3 . 6

3 7 . 9 3

1 7 . 1 0

1 5 1 . 4 2

2 1 3 . 3 3

1 4 5 . 0

4 3 1 . 0

2 2 . 0 5

1 6 . 2

7 . 1 4

4 0 . 4 7

1 3 8 . 0

3 4 . 6

9 3 . 8

1 3 8 . 6

F o r m i c a c i d

5M HCOOH

2 5 . 0

4 8 . 1 4

1 0 . 1 1

2 3 0 . 8 2

4 2 2 . 0

2 0 0 . 0

6 0 8 . 0

1 6 . 3 2

1 3 . 6

1 .35

4 7 . 5

1 2 7 . 2

1 9 . 3

9 0 . 0

1 0 1 . 9 2

* DMW - Demineralized water

127

\

-J

' '

o z o

2. o

a. o

2 o

•* o

2:

(t UJ u. u.

2 O

\

2

" " I f A P I 60T ID •

in

• H

128

I ' L.

O --> — O

n u < u 7 a o u. u. o z o »— < Q:

y UJ

u 2 O o

(_) < CJ

z. K O u. U-

o z: o h -

< cr 1—

z UJ

<_) o o 1—

:z UJ

cc UJ

u. u. Q

r

in r. o _ j

< >— LU

V-I j -O

l / l

I jJ Z>

s z n i r A p)( 6on

o o

IT)

• H b4

129

TABLE - S;2

SOME BINARY SEPARATIONS ACHIEVED ON THE AMBERLITE

RESIN, IRA-400 MODIFIED WITH EBT.

s. No.

1.

2.

3.

4.

5.

Separa­tion Achieved

Cd+2

Hg-'

Pb+2

Hg+2

Fe- 3

Hg+2

Cr+3

Hg+2

Al+3

Hg^2

Amount loaded-mg

11.9706

10.5309

17.403

10.530

5.584

10.530

3.067

10.510

2.8329

10.530

Amount found mg

11.9144

10.5309

17.403

10.470

5.567

10.430

3.041

10.510

2.8329

10.530

Percent recovery

99.5%

100.0%

100.0%

99.4%

99.7%

99.0%

99.1%

99.8%

100.0%

100.0%

Volume of Effluent

40 ml

120 ml

20 ml

120. ml

20 ml

120 ml

15 ml

120 ml

25 ml

120 ml

Eluent used

DMW

2M-HCL

DMW

2M-HCL

DMW

2M-HCL

DMW

2M-HCL

DMW

2M-HCL

X

o

is u. u. UJ

u. o UJ

o >

z o

< OH

< a.

in

•H fa

X

o C or £ u.

* ^ CM

Z J3 UJ Q.

_l U. u. o

UJ

u.

o UJ 2 3

Z

o

< < Q. llJ to

ID

• H CM

(l"J ) V i a a W 10 <o iujniOA ( |u j ) v i a 3 H l O ' *o »ujn|o/\

131

. ^ I

o C a. E u.

o X

00

IT)

• H fa

X

o

7, 4,

3. 3

O

00

in

fa

( iw ) tf 1 0 3 H 10 | 0 JuiniOA (I"") V 1 0 3 W 10 | 0 >ujn|OA

132

(/< a. o

T3

O rs(

I 1 E -^ o O m

I i a. o rj

•»- (/) c c 2 6 — o

t U3

& ^ E

LO c J : E in 13

( l " ' ) V i a 9 WIO- ioaiunio/^

133

TABLE - 5.3

SEPARATION OF Hg"'' FROM SYNTHETIC MIXTURE OF Fe"*" ,

Cd"*" ON A COLUMN OF AMBERLITE RESIN IRA-400

Al+\

Cr^^ Pb ,

LOADED WITH EBT.

VOLUME OF DMW* USED FOR ELUTION OF HEAVY METALS MIXTURE IS

4 0 ml IN EACH CASE.

S. Amount No. of Hg" ^

loaded mg

Amount of Hg" ^ found mg

Percent Recovery

Volume of 2M-HCL used for complete elu-tion of Hg'''

1.

2.

3.

4.

5.

4.41

5.01

5.20

6.80

7.60

4.3

4.9

5.14

6.7

7.4

98.5

98.0

98.8

98.5

97.3

150 ml

150 ml

150 ml

150 ml

150 ml

* Dimeralized water

134

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(1980).

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(1968).

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