DETECTION, DETERMINATION AND THIN- LAYER … · fication of anions. The highlights are the systematic analysis of anions in the solid state and the development of new sorbent phases

DETECTION, DETERMINATION AND THIN-LAYER CHROMATOGRAPHIC SEPARATION

OF ANIONS

Abstract

THESIS SUBMrrTED FOR THE DEGREE OF

Bottor of $I)Uo!E(opI)p IN

APPLIED CHEMISTRY

'N^.î;-^t>

BY UNDER THE SUPERVISION OF

SHAR4D TIWARI Dr. AH Mohammad Read«r

DEPARTMENT OF APPLIED CHEMISTRY ZAKIR HUSAIN COLLEGE OF ENGINEERING AND TECHNOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

1991

The thesis comprises of six chapters in all dealing

with detection, identification, separation and quanti

fication of anions. The highlights are the systematic

analysis of anions in the solid state and the development

of new sorbent phases for thin-layer chromatographic

studies of anions.

Chapter-I presents an overview of all important

aspects of chemical analysis with detailed discussion on

spot-test analysis and thin-layer chromatography. An

extensive literature survey has been presented on the

procedures involved in the analysis of inorganic ions.

Significance, mechanism and applications of analytical

solid-state color reactions have also been discussed. The

work done on thin-layer chromatography of anions till date

has been described in tabular form, whereas the comparison

of work done on cations and anions during last decade has

been shown graphically.

Chapter-ri summarizes our efforts of utilizing

capillary solid-state spot-test technique for systematic

analysis of anions. A glass-wool plug modification has been

added to the capillary technique to make a test more

selective or even specific. Several new reagents for solid

state detection of anions have been identified. The color

reactions of powder trituration method have been compared

with those obtained in solution state. Table 1 summarizes

the solid state color reactions of anions with various

reagents. The order of selectivity and sensitivity of these

methods is given below:

Selectivity: Glass-wool plug modified capillary method >

capillary solid-state method > powder trituration method.

Sensitivity: Powder trituration method > contact capillary

solid state method > glass-wool plug modified capillary

method.

Based on the solid-state reactions two schemes for

the systematic analysis of anions in the solid state have

been proposed.

In Chapter-Ill investigations regarding thin-layer

chromatographic behavio r of 17 anions on silica gel

impregnated with inorganic salts such as copper sulfate,

zinc sulfate, cobalt chloride, hexamine cobalt III chloride

and nickel chloride using mixed aqueous-organic eluents

containing formic acid have been summarized. The effect of

mineral acids on the mobility of anions has also been

studied by substituting formic acid with HCl , H^SO, or

HCIO, in the mobile phase. In addition to microgram

separation of NO2 and IO3 from various anionic species,

(Table 2,3), some other important qualitative separations

have been realized. The effect of pH on the sample solution

and loading amount of IO3, Br03 , NO2 and I on their Rp

values have been investigated. The limits of identification

for all anions on impregnated layers have been determined.

The developed procedure has been utilized for the semiquan

titative determination of NC2 and Br03.

Chapter-IV deals with the utility of water as

eluent in thin 1ayer chromatographic separation of various

anions on plain (silica gel G, alumina and cellulose) as

well as on mixed beds containing different combinations of

silica gel, alumina or cellulose. Microgram separation of

_ _ 2-10^ from milligram quantities of IO3, Br03, MoO^ and

Fe(CN)g has been realized. Effect of pH of sample in the

separation of 10^ from accompanying ions has also been

studied. The limits of detection of anions on alumina thin-

layers have been determined, NO2 in artificial sea water

has been detected. Effect of hardness causing ions on some

separations of analytical importance has been examined and

the results are presented in Table 4.

Chapter-V deals with thin-layer chromatographic

studies of some anions on plain and impregnated silica gel

layers in aqueous-organic solvents containing acetone. The

results obtained on plain silica gel have been compared

with those obtained on copper sulfate impregnated layers.

The impregnated layers dramatically change the selectivity

and permit separations not possible on untreated silica.

Aqueous sodium chloride-acetone (9:1) and ammonium

hydroxide-acetone (9:1) were the most effective solvent

systems for differential migration of anions. Better

results in terms of clarity of detection and compactness of

spots were found with HCOOH-acetone as compared to

HCl-acetone. Finally identification limits for all anions

on impregnated layers has been determined as shown in

Table 5.

Chapter-VI summarizes results achieved on the

separation of 10^ from other anions in the presence of

common cations, using silica gel layers as stationary phase

and distilled water as mobile phase. A volumetric method

has been devised for the determination of iodate with

priliminary TLC spearation from preiodate. Some TLC

parameters such as^Rp (Rp of IO3 - Rp of 104), separation

factor ( cK ) , capacity factor (K') and resolution (R ) on

the separation of IO4 from IO3 have been computed as shown

in Table 6.

• J

Q O o

Qi

u re CTi

c 0) M TO OJ Di

OJ B 0 (/)

XI 4J •H

w C o

<

o w c o

•r l 4J O re cu

D.S

•U

w

I

o a (/)

(U 4J re 4J CO I

T ) •H

O CO

!r,

o 0 00

P re

a B

H

I f

t f

I I

4 f +

I I

J + * I I I I I + f I

I I •• I I I I + I + I

I I I ( I I t { I -h (

t- t- f f + -( I I t < f

t t I 4 y i \ (. + (. J.

+ + I I + I I + + +

+ 1 I + I + + I + •-

+ < I I + + I + +

I I I I . I I I

f I I I I I I

I I I I I I I

f I I f -) ^ +• +

I I I I I I I

t i I I I I I

1 1 1 ) 1 ) + +

1 + 1 ) 1 ) )

I I I ) ) I )

I i I I < I )

i I I f ) ) +

I I ) ) ) ) I

+ ) + + + + + + + + + + +

) ( I I ) ) + + + + + ) )

^ i + + + + + + f + + + +

I ) ) ) ) I M I ) I I I

+ + + + + + + + + + + + )

) ) ) ) I I I I ) ) i ) )

) + + ) I + + I ) ) +

) + + ) ) + + I ) + )

/ ( ( ( ( ( ( ( ( + +

+ + ) ) ) + ^ + + ( - ^ •

+ + + » - + i + + + +h +

1 + + + ) + + ) + + +

1 + + I I + + + I ) I

) ) ) ) ) + + + ) + +

I I I +'i ^ + I I + I

I I + I I + + ( I I !

I I I i I + + I I -t I

) + ) + + + + ( - ) + +

] ) I + I I ) ) I + )

I ) )

I I I

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) ) >

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) ) I ) ) ) >

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1 ) 1 ) ) ) )

I + + ) ) + )

) ) + • ) ) + I

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I + + ) ) + +

+ + + + ) + +

! I + I I I I

+ + + ) ) + H

1 + I ) ) I )

+ + + + ) ) +

) > H- ) I ) )

X a. )

i •i

Z _ n

0. Z tn

tri

+

^^ u. n r t

z (A + 0 — z

u.

n

_ j c. 0.

o'

z o '0 u. T r i

00

* X

. 0 Z

< «n r t

!2

+ •— u

^ r» ao <>v o

y " £ < < < * y H I- < < Q a a O O — n r T '

o z z

~ 01 a. — 1 " O + o < Q Q r 2 I- h Q Q t. li i. a a

^ r ^ DO ^ O

+ a a < < + o

< < < a a S 5 c — rt f^ *r *^

3 »\ r>i I - L c

( ORANGE

1 NOCOLOn

o n FEN —»-yELLOV/

CrO 2-

r

NO colon

GHEEN —»'YELLOW

f f (CN)g3"

1

NOCOLO"

1.2 r coLon NOCOLOR

. 1" PINK 5 C N -

NOCOLOR PINK j12 CO32-

BRQWN

VO3-

1 NOCOLOR

I" I

VELLOW

| -

COLOR

_J1L 1

NOCOLOR

1 NOCOLOR

I" RED

5 2 0 6 ^ -

I BROWN

BrO i "

NOCOLOR

O R A N G E - Y E L L O W NOCOLOR

Br- ] ? 6 I

R E O - - * - B L A C K

S ^ O j Z -

1 NO CO

I Y E L L O W . BROWN

I O 4 -

LOR

25

NOCOLOR Hi

I YELLOW

CH3COO-

! NOCOLOR

( BROWN

NO 2

NOCOLOR

Is

COLOR

11"

I NOCOLOR

19

I YELLOW

S 2 -

COLOR 120

1

NOCOLOR

NO COLOR | 2 3

YELLOW NO3-

( ORANGE

5 0 , ; -

23 I

BLUE—"-GREEN

IO3-

NOCOLOR

1"

1

NOCOLOR

LIGHT YELLOW

NO COLOR PO/ ,3-

h9

NOCOLOR

COLOR BROWN

1" MoO 2- i! 0

LiGH r YELLOW

5 0 2 -

YE I I OW

HCU)

NOCOLOR | , 9

I LIGHT YELLOW

NOCOI.CR ^2^^~

i' VELLOW

c r Scheme 1 Reac t ions in a l e f t - h a n d branch are performed before

those in a r i gh t -hand branch. The reagent numbers re fe r to Table 1.

SAMPLE

NO COLOR

I" RED

S206^

NOCOLOR |19

I I PALE YELLOW NO COLOR

103- 22

COLOR 1

NOCOLOR

h5

ORANGE

soa^" NOCOLOR LIGHT YELLOW

| 2 5 C 2 0 , 2 -

YELLOW • B R O W N

IO4"

Scheme 2

TABLE 2

Quantitative Separation of NO2 from I , SCN~, and Br on 0.17. CUSO,-

Impresnated Silica Gel LayersUsin« Formic Acid:Acetone(l :9) as Mobile Phase

Loading amount of individual Separations anion salt in mixture (Ri - R^)

NaN02

50 pg

0.5 mg

1.0 mg

2.0 mg

50 pg

0.5 mg

1.0 mg

2.0 mg

100 pg

4.0 mg

KI 0.125 mg

0.25 mg

0.5 mg

1.0 mg

1.0 mg

NH^SCN

0.125 mg

0.25 mg

0.5 mg

0.5 mg

KBr

0 .5 mg

1.0 mg

2.0 mg

4.0 mg

0.25 mg

0.5 mg

NO2

NO2

NO2 NO-

NO"

NO2

NO2

NO2

NQ-

NO2

NO2 NO;

N02

N02

N02

N02

NO 2

NO2

NO2

(0.63-0.43)

(0.62-0.42)

(0.75-0.55)

(0.77-0.57)

(0.65-0.39)

(0.65-0.32)

(0.67-0.11)

(0.69-0.56)

(0.65-0.43)

(0.82-0.69)

(0.70-0.50)

(0.60-0.31)

(0.63-0.26)

(0.72-0.51)

(0.82-0.64)

(0.80-0.72)

(0.79-0.62)

(0.74-0.50)

(0.87-0.56)

- I~

- l"

- l"

- l"

- r . - I~

- l'

- SCN" - SCN" - SCN" - SCN' - SCN" - SCN"

- Br"

- Br"

- Br"

- Br"

- Br"

- Br"

(1.0-0.87)

(1.0-0.91)

(1.0-0.85)

(1.0-0.83)

(1.0-0.80)

(1.0-0.78)

(1.0-0.80)

(1.0-0.88)

(1.0-0.79)

(1.0-0.89)

(1.0-0.88)

(1.0-0.79)

(1.0-0.75)

(1.0-0.9)

(1.0-0.91)

(1.0-0.95)

(0.96-0.86)

(1.0-0.85)

(1.0-0.94)

Note: Detection of Br is difficult, requiring about 20 min after

spraying the chromatogram with the reagent.

TABLE 3

Qunntltntlvo Sopnrntlon oC TO3 FronRrO", N0~, T~, Br~, mid .S(;N~ on O.JX

CuSO^-Impregnated Silica Gel Layers Using FArDMSOrAcetono (1:1:8)

Loading amount of individual

anion salt in mixture

Separations

(R L Rj)

KIO - 3 50

0.4

50

0.4

P8

mg

Mg

mg

50 pg

0.4 mg

100 pg

1.0 mg

0.4 mg

KBrO,

0.25 mg

0.5 mg

0,1 mg

NaN02

1.0 mg

2.0 mg '

0.2 mg

KI

1.0 mg

2.0 mg

4.0 mg

0.4 mg

KBr

4.0 mg

7.0 mg

4.0 mg

NH^SCN

IO3

10"

10-

IO3

10-

IO3

I03

I03

I03

I03

I03

I03

I03

(0.15-0.0)

(0.17-0.0)

(0.35-0.0)

(0.25-0.0)-

(0.27-0.0)-

(0.36-0.0)-

(0.22-0.0)

(0.21-0.0)

(0.34-0.0)

(0.35-0.0)

(0.41-0.0)

(0.4-0.0)

(0.4-0.0)

- Br03

- Br03

- Br03

- NO"

- NO2

- NO2

- I"

- I"

- I~

- I"

- Br"

- Br'

- Br"

(0.96-0.77)

(0.98-0.56)

(1.0-0.82)

(0.98-0.67)

(1.0-0.51)

(1.0-0.78)

(1.0-0.89)

(1.0-0.84)

(1... 0-0. 75)

(1.0-0.87)

(1.0-0.69)

(1.0-0.65)

(1.0-0.81)

10 pg IO3 (0.3-0.0) SCN (1.0-0.81)

Note; The synthetic mixture of IO3 with SCN'

in the formation of precipitates.

(concentration 17o) results

10

TABLE 4

Effect of CaCl2, MgCi2 and NaUCO^ on Some Selected Separations

Standard Rp value of Individual Ions are in parenthesis. I07

(0.00), IO3 (0.53), VO3 (0.00), SCN" (0.94), BrOj (0.93) and

NO2 (0.92).

Salts Separations (R, - R p)

CaCl2 IO4 (ND) - IO3 (ND) - SCN~ (1.0-0.89)

IO4 (0.02-0.0) - IO3 (0.67-0.49) - Br03 (1.0-0.82)

IO4 (0.02-0.0) - IO3 (0.73-0.54) - NO2 (1.0-0.88)

VO3 (0.02-0.0) - IO3 (0.65-0.44) - SCN" (1.0-0.88)

MgC]2 10^ (ND) - IO3 (ND) - SCN" (1.0-0.87)

I0~ (0.01-0.0) - IO3 (0.63-0.45) - BrO" (1.0-0.87)

10^ (0.03-0.0) - 10" (0,73-0.60) - NO2 (1.0-0.87)

VO3 (0.03-0.0) - IO3 (0.70-0.53) - SCN~ (1.0-0.87)

NaHC03 lO^(ND) - IO3 (ND) - SCN~ (1.0-0.87)

10^(0.02-0.0) - IO3 (0.65-0.46) - Br03 (1.0-0.84)

10^(0.03-0.0) - IO3 (0.76-0.62) - NO2 (1.0-0.90)

VO^ (0.01-0.0) - IO3 (0.70-0.50) - SCN~ (1.0-0.79)

Note: ND, not detected

11

TABLE 5

Limit of Detection and Dilution Limit of Anions and VO 2^ on

Silica Gel Impregnated with O.IZ CuSO^ with Formic Acid:

Acetone (1:9) Mobile Phase

S] .

No.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Ions

Br" 3-

4

l"

VO3

NO-

NO"

SCN"

Fe(CN)

Fe(CN)

Cro2-

Cr207"

MoO^"

woj-

4-6 3-6

Salts

KBr

Na2HP0^

Kl

V02S0^

NaV02.Il20

NaNO^

NaN02

NH^SCN

K^Fe(CN)g.

K3Fe(CN)g

K2Cr0^

^2^^207

Na2Mo0^.2H

Na2W0^.2H2

3H2O

2O

0

Limit of detection

LOO.O

10.0

10.0

10.0

1.0

1.0

1.0

1.0

1.0

0.5

0.5

0.5

0.1

0.1

Dilution

limit^

? 1:10 -

3 1:10^

3 1 :10

1. 1:10~

1:10'^

1:10'^

J rio'

4 1:10^

1: 10 "

1:2x10^

1 :2xI0^

1:2x10^

1:10^

1:10^

Dl]ution ]imit

1: Volume of test solution (ml) x 10

Limit of detection ( pg)

12

TABLE 6

TLC Parameters for the Separation of IO4 (Rp=0.01) from 10-

(Rp=0.92) in the Presence of Certain Metal Ions

MetaJ ions

Hg^^

Cd2^

Ni2^

Zn2^

Co2^

Cu2^

Fe^^

Al^^

Rp

0.90

0.87

0.85

0.86

0.87

0.86

0.87

0.87

TLC parame

• '10-

19.0

24.0

15.66

10.11

19.0

13.28

13.28

19.0

ters

380.03

244.89

159.79

194.42

220.93

210.79

210.79

220.93

R s

12.05

6.69

5.66

6.29

6.96

6.37

6.96

6.96

DETECTION, DETERMINATION AND THIN-UYER CHROMATOGRAPHIC SEPARATION

OF ANIONS

THESIS SUBMITTED FOR THE DEGREE OF

Mottox of $lîla)Biopt)p IN

APPLIED CHEMISTRY

BY UNDER THE SUPERVISION OF SHARAD TIIVARI Dr. Ali Mohammad

Readet

DEPARTMENT OF APPLIED CHEMISTRY ZAKIR HUSAIN COLLEGE OF ENGINEERING AND TECHNOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

1991

'. f

Cfl ^ ^ -2.00^

{j^-

TÎES^^ s^ct\ot^

2 5 SeP 1992

ALI MOHAMMAD Ph.D. (Aligarh)

Editor CHEMICAL AND ENVIRONMENTAL RESEARCH

Phone : Office DEPARTMENT OF APPLIED CHEMISTRY

Z.H. College of Engg, & Technology ALIGARH MUSLIM UNIVERSITY

ALIGARH-202 002 (INDIA) TLX : 564-230 AMU IN

CERTIFICATE

Certified that the work incorporated in this

thesis is the original contribution of the

candidate and is suitable for submission to the

award of Ph.D. degree.

1 . wiJ^^^^--^^ (DR. ALI MOHAMMAD)

Supervisor

ACKNOWLEDGEMENTS

At thi'i crucial juncture when a research fellow has

to acknowledge all those concerned with the thesis, I am

finding it a bit difficult to frame a sentence in honor and

praise of my supervisor, Dr. All Mohammad. His constant

encouragement, constructive criticism, a thorough back

ground of the subject and monumental patience made this

work possible. Dr. Mohammad's contribution to my career

would always be a corner stone of my life.

I place on record my profound gratitude to

Professor S.A.A. Zaidi for his unending help and support. I

express my sense of indebtedness to Professor K.T. Nasira

for his cooperation and moral support. My sincere thanks

are due to Professor Mohammad Ajraal, Chairman, Department

of Applied Chemistry, A.M.U., Aligarh, for providing all

the necessary research facilities.

I wish to express my deep sense of appreciation for

my parents and my aunty Mrs. K.R. Mahajan whose good wishes

and sacred blessings have always been a source of

inspiration in accomplishing this task.

Friends and colleagues alway play a significant

role, but I would like to single out Dr. Nairn Fatima and

Mr. Majid Khan for their invaluable help which they

rendered to me, when I needed it most.

I am thankful to Mr. H.S. Sharma for his neat and tidy

electronic typing of my thesis.

Finally, the financial assistance in the form of

Senior Research Fellowship from the Council of Scientific

and Industrial Research, New Delhi, India, is gratefully

acknowledged.

6f^dX\{Xîiw^ î

(SHARAD TIWARI)

it

LIST OF PUBLICATIONS

1. Thin-Layer Chromatographic Separation and Identifi

cation of Some Anions on Copper Sulfate Impregnated

Silica Gel Layers.

Chromatographia, 30, 405-409 (1990) (Germany).

2. Chromatography of Anionic PoHutants on Silica Gel

Layers: Selective Microgram Separation of NO^ and I0„.

Microchemical Journal, 44, 39-48 (1991) (U.S.A).

3. Analysis of Anions by Sol id-State Spot-Tests.

Talanta, 1992 (In press) (U.K.).

4. Identification and Separation of Some Anions on Plain

and Mixed Adsorbent Layers Using Water as Eluent.

Microchemical Journal, 1992 (In press) (U.S.A.).

5. Effect of Heavy Metals on Chromatographic Separation of

10^ from 10 3, BrOj , MoO and Fe(CN)g .

Journal of Planar Chromatography, 1992 (In press)

( Swi t/.cr I and ) .

Dedicated to

my Parents

"Only amateurs consider that an idea must be

brand new in order to be good; important in

reality is not he who first had the idea but

he who expressed it better"

CONTIiNTS

Page

Acknowledgement 1

List of publications ii

Chapter - I

(ICMicral J uL r o t l u c L i o n 1

C h a p t e r - I I

Analysis of Anions by Sol id-State Spot-Tests 92

Chpater - III

Microgram Separation and Semiquantitative DoL cnni na L 1 on oi' AnJinis 125

Chapter - IV

Identification and Separation of Some Anions on Plain and Mixed Adsorbent Layers 149

Chapter - V

Thin-Layer Cliromatographic Separation of Anions on Copper Sulfate Impregnated Silica Gel Layers 167

Chapter - VI

Effect of Heavy Metals on Chromatographic Separation of 10^ and Quantitative Determi-nal 1 on of \in 188

CHAPTER - 1

GENERAL INTRODUCTION

AnaJytical Chemistry, a most useful branch of

modern chemical sciences should be considered as the

'Mother of Chemistry' because the development of

traditional branches of Chemistry (organic, inorganic and

physical) depends solely on principles and new methods of

analysis which is the essence of analytical chemistry.

Analytical research, as opposed to synthetic, .Ushered in

the change from magic and alchemy to quantitative,

scientific chemistry. Analytical work led to formulate the

laws of definite and multiple proportions and made possible

Ualton's great achievement, an atomic hypothesis. The

increasing importance of environmental pollution and an

explosive technological development have combined to create

analytical problems which demand increasing instrumentation

for their solution. The trends of recent years have brought

analytical chemistry into the forefront of research in many

exciting areas. Typical examples of such problems are

purification of the environment, utilization of solar

energy, analysis of ores, recovery of precious metals from

the spent fuel or sea water, detection of unusual molecules

in polluted atmosphere of smog-bound cities, determination

of pesticide residues in food products, identifying traces

of impurities in ultra pure semi-conductor materials,

deducing the sequence of different amino acids in a giant

protein molecule and determination of complex organic

molecules in the nucleus of a single cell.

Analytical chemistry is as old, and as new, as the

science of chemistry itself. It is concerned with the

methods and techniques employed in the chemical analysis oi

substances. A chemical analysis may be either qualitative

or quantitative. Qualitative analysis deals with the

identification of substances and is concerned with what

elements or compounds are present in a sample. The

quantitative analysis is concerned with the determination

of how much of a particular substance is present in a

sample. As an example, if a pollutant in a river has been

killing fish, qualitative analysis could be used to find

out the chemical identity of the pollutant, whether the

pollutant was a heavy metal- (Pb, Cd or Hg) or any other

substance. After establishing the identity of a chemical

species, the concentration of that species in the sample

can be determined by quantitative analysis. Any physical or

chemical property characteristic of a particular element

can be made the basis of a method for its analytical

determination.

f>om a historical standpoint, the majority of early

analytical methods were either gravimetric or volumetric

which are termed as classical or non-instrumental methods

of analysis. Procedures based on the measurement of

optical, electrical, thermal and other properties were

developed later, and are called instrumental or modern

methods. In fact, fundamental differences between the two

categories (classical and instrumental methods) do not

exist, both are based upon the correlation of a physical

measurement with concentration; neither is specific and

separations often precede both types of analysis. Thus the

classification of methods as classical or instrumental is

founded largely on chronological development. The

instrumental methods are faster and generally used where

high selectivities or very low concentrations of analyte

are involved. These methods are expensive and require

special maintenance. On the other hand, non-instrumcnLa 1

methods are simple, cost effective, versatile and suited Lc

on field experimentation. These methods are of greater

importance for the developing countries like ours. It is

therefore, worthwhile to develop and strengthen non-

instrumental methods for chemical analysis.

The qualitative analysis can be conducted either in

solutions or in the solid state, employing optical,

chemical or electroanalytical techniques. Of these the

chemical methods have received wide applicability in the

detection and determination of functional groups and

inorganic ions. Qualitative analysis can be performed

easily by using wet procedures. In most cases the chemical

reagents used for the qualitative analysis either

selectively precipitate one particular substance from a

mixture or selectively react to yield a colored reaction

product.

As a result large number of color reactions have

been developed to achieve improved sensitivity,

selectivity, accuracy, reproducibility, and practical

applicability. Because of the availability of wide range ot

reagents, suitable color reactions can be deviled 't

specific cases.

SPOT-TEST ANALYSIS:

The present work is concerned with the system-itii.

analysis of anions by solid-state spot-test technique and

hence now more emphasis would be laid in the subsequent

pages on spot test methods and their significance m

chemical analysis.

The 'spot-test method' as introduced by Feigl and

Stern[l] for the detection and determination of a substmet

is a micro scale analytical procedure commonly carried out

by placing a drop of the reagent and a drop of the test

solution together on a filter paper, spot plate (a

porcelain plate with many small depressions), watch glass

or in a micro test-tube.Alternatively the test can also be

performed either by spotting the drops so that their

peripheries just meet or by bringing into contact the test

solution with a solid reagent and detecting the resulting

gaseous or colored product. Sometines the test is made bv

triturating powdered solid reactants capable of producing

colored product.

The term 'spot-test analysis' is a generic tern-

referring to sensitive and selective tests based on

chemical reactions where the use of a drop of the test or a

reagent solution is the essential step. Inorganic spot-test

analysis is the outstanding field of application of

specific, selective and sensitive reactions with the goal

of rapidly solving problems in qualitative micro analysis.

The spot reactions can also be advantageously applied to

quantitative determinations since they lead lo the

formation of colored products. The sensitivity of H test

depends upon the conditions under which it is carried out.

The first colorimetric determination using

spot-test method was reported by N.A. Tananaeff [2] in

1929. He compared the intensity of the color reactions

carried out in drops of test and standard solutions on

filter paper. In accordance with their limits of

identification and the corresponding dilution, spot

reactions can only be applied within a particui ir

concentration range of the material to be detected.

Sometimes the filter paper becomes an active participant in

the spot reactions although it does not appear in the

stoichiometric representation [3|.

The elegant variations in spot-tests have been

affected by Fujimoto [4] and Qureshi et al . [5j. These

modifications led to the birth of two new spot-test

techniques known as: (1) Resin Spot Test and (2) Capillary

Solid-state Spot-Test.

(1) Resin Spot Test:

In 1960 Fujimoto [4] used ion exchange resins as

reaction media for micro or ultramicro detection tests

which he called as 'resin spot tests'. In this method ion

exchange resin beads are added to the solution containing

ions or charged complexes to be detected. The ions or

charged complexes are taken up by the resin beads and when

these beads are brought into contact with a selective

reagent, a color reaction takes place on the surface of

beads indicating the presence of a particular species. The

important advantages of resin spot test technique are:

a) The sensitivity of the test is high because the colored

species is concentrated on resin beads.

b) It is more selective because cationic or anionic inter

ferences can be easily avoided simply by using an anion

or a cation exchanger.

c) Stabilization of the unstable colored products in the

exchanger phase [6] and the possibility of preserving

the results by drying the colored resin beads.

d) Use of a simple and inexpensive equipment.

e) No special training is required.

f) Ion exchange resin beads can be easily removed from Che

reaction mixture.

The limitation of resin spot test is that it is

applicable only to the solutions containing ions or char '.ed

species.

Resin spot-test technique was originally applied

for the detection of metal ions. Recently its utility has

been extended for the detection and determination of

organic compounds [7-10],

(2) Capillary Solid-State Spot-Test:

Chemistry of the solid state has been the most

neglected aspect of chemical sciences probably of the

feeling that 'Corpora non agunt nisi fluid' as ennncl-jted

by Aristotle. Solid-state reactions have much to offer to

the chemists concerned with synthesis, analysis, reaction

mechanism or the chemistry of natural processes.

Particularly all reactions in various geological processes

take place in the solid phase. Solid-state reactions have

some novel features which distinguish them from reactions

occuring in the fluid/solution state.

a) Solid-state reactions are free from the complicating

influence of the solvent.

b) Their rates are comparatively slower and hence are more

amenable to kinetic and mechanistic studies.

c) They are useful for the study of weak interactions and

for the discovery of new species [11-13].

d) Reactions in crystals proceed with minimum of atonic

and molecular movement and thus governed by the

structures of the reacting crystals.

e) Chemical reactivity of atoms or molecules in solids

depend both on their nature as well as on their

positions in crystals.

f) Molecules in a crystal occur in only a small number of

confrontations leading to a limited number of intcr-

molecular approaches in the crystalline state.

g) The activity of the reactants and products remain

almost the same during the reaction.

h) Solid-state reactions do not reach equilibrium except

in specific cases and are generally exothermic.

It is now fully established fl4] that reactions which

take place in the solution state also occur in the solid

state particularly if there is a close contact between the

reacting substances. Such reactions are called 'contact

reactions'. For example on contacting sodium metavanadaLe

crystals with crystalline potassium bisulfate, a reddish-

orange color develops at the contacting boundary.

The solid-state reactions may be studied ei-ther by

allowing the reactions to occur between single crystals or

by means o£ capillary technique proposed by R.P. Rastogi

[13]. In this technique powdered reactants are filled in a

capillary as shown in figure 1. The progress of a

solid-state reaction in glass capillary is followed by

monitoring the movement of the boundary of the colored

product.

The salient features of this technique are as

fol1ows:

A 10 cm graduated capillary (3 mm interna]

diameter) is partly filled with powdered reagent from one

end with the help of two iron rods and the material (well

powdered) is similarly filled from the other end. Care is

taken to maintain a reproducible packing and both the

materials should come in close contact with one another in

the middle of the capillary. The color of the product

formed at the junction, direction of movement of the

colored boundary and the length of the product boundary are

recorded at desired temperature after specific time

intervals. The start of the reaction is indicated by a

change in color. This technique is very useful to scan a

large number of reactions quickly. The reproducibility of

results mainly depend upon the uniformity of the particle

"size and the amount of pressure exerted in filling the

capil1ary.

10

Fig. 1 Capillary technique for the study of solid-

state reactions of powders.

R. and R^ are reactants and P is the product

a,b,c and d are solid boundaries

arrow indicates the direction of reaction progress

11

In 1976 Qureshi et al. [5] extended the use of this

technique for the detection of organic substances and

called it as 'capillary solid-state spot-test'. This

technique has some distinct advantages over conventional

spot test and resin spot test methods.

In the conventional spot tests, we notice the color

formed on mixing the reactants, but in this case we observe

color at the junction, the length of the product boundary

and the direction of movement of the boundary. As a re-. IL

two substances giving the same color with a reagent can be

distinguished on the basis of boundary movement, if they

move in opposite directions. Thus, the wide choice of

variables in this technique such as (i) the color (ii) the

length and (iii) the direction of movement of the colored

boundary leads to a selective or even specific detection of

compounds.

The capillary solid-state spot-test technique which

was originally applied for the detection and

semiquantitative determination of organic substances

[15-18] was later on extended for the systematic analysis

of cations [19]. We have further extended the use of this

technique in the systematic analysis of anions and a "lass

wool plug modification has also been proposed tci i^nhance

the selectivity of the test.

12

It may be appropriate at this stage to discuss the

analytical applications of solid state reactions. Only one

review by Voskresenskii [20] has appeared till now. The

various parameters affecting solid-state reactions are

summarized below:

(i) the rate of reaction generally increases with an

increase in the degree of grinding of the

reactants.

(ii) Water of crystallization or atmospheric moisture

has an unpredictable effect on the course of

reaction. Water vapor is always present ir

atmosphere and it can accelerate, retard or even

stop a reaction.

(iii) The rate of reaction generally increases with

temperature.

(iv) Effect of pH is less important in the solid-state

than that of in the solution. For example, a

reaction takes place when any aluminium salt Is

triturated with arsenazo 1 whereas a definite pH is

necessary to carry out this reaction in the

solution state.

The following characteristics of solid-state

analytical reactions have also been noted:

(i) If several ions can react with particular reagent,

13

then the ions having the highest valency react

first.

(ii) If ions of the same valency are present, all or

which react with a particular reagent, then ''^TSL

having the hi'gher atomic number or atomic weight

react first.

(iii) The ability of minerals to react depends on their

chemical structure, while the kinetic behavior

(the rate of reaction) depends on their physical

structure.

(iv) All crystal grains do not have identical therMcai

reactivity.

The use of pure metal salts is very convenient for

demonstrating solid-state qualitative analytical reactions.

Let us now illustrate a few examples of such reactions.

Beryllium may be detected by reactions with

quinalizarin or beryllon 2. A little solid sodium hydroxide

is first added to a berrylium salt followed by trituration,

then the reagent is added and the contents are triturated. A

characteristic blue coloration appears with both the

reagents. Any molybdenum compound when mixed with excess of

ammonium sulfate and heated until the evolution of SO,

ceases. A blue colored solid mass of molybdenurr blue

appears. Similarly, on mixing a tin salt and potassiurr

14

iodide, a golden yellow color rapidly appears.

Metal ions sometimes form complexes of low boiling

point on reacting with inorganic substances and thus can ho

separated easily. For example, beryllium as oxy acet-itc and

tin or nickel as dimethylglyoxime complexes have been

separated. Solid-state analytical reactions can also be

used for solid phase colorimetry both visually m-

instrumental 1y.

It is now apparent from the above discussion that

field of solid-state analytical reactions is wide open tu

accelerated research activities for qualitative testing or

minerals, alloys, mineral fertilizers and organic

functional groups. Feigl [21j has shown the existenc ot

new possibilities by using solid-state reactions iri

qualitative organic analysis. The on field application of

solid-state analytical reactions is of special importance

for testing geological samples. As yet only the reactions

which lead to the formation of colored reaction product-

have been found suitable for solid-state analytical

reactions but it may soon be possible to use reactions,

involving the formation of white products with the aid of

luminescence methods of analysis.

The following reaction steps may be assumed in

order to understand the mechanism of solid-state reactions

of inorganic substances.

15

The exchange of particles at the interface between

the two reactants and the formation of the reaction

product for ideal contact between the solids. Iherc

are three possible transport mechanisms after Lhi-

formation of a homogeneous, pore free product layer

as shown in figure 2.

Transport of the particles through the intG-''"'c

I. between the reactant and the reaction prod'u t .

Diffusion of anions and cations of the reactant MO

through the reaction product via volume diffusioii

or grain boundaries and dislocation of crystallite

of the product.

Reaction of the cations and anions of the subst m C(

MO at interface I^ with the reactant AÔ^ to for'

the reaction product. The formation of renctio-

product is progressed via heterogeneous nucleatio

followed by crystal growth.

Almost similar reaction steps are followed b\

organic reactions. The only difference is that in organic

reactions the diffusing species in most cases are molecule^

or free radicals instead of ions.

Separation is of utmost importance because in "'an\

cases detection and determination depend on the separation.

Among the most versatile analytical separation techniquos.

16

Orrt^

O

< 2

O

<

•V CO

< • • •

>>) ()

(•I



•H C ex)

o 0)

4-1

>- o ex c 1-1

•

17

ion exchange, electrophoresis, and chromatography are of

wider applicabiJity. ion-exchange processes are mostJy used

in nuclear engineerings, such as purification of fuels,

waste disposal enrichment and purification of useful

radioactive isotopes. It is because of selectivity and the

completeness of the separation.

i) Ion ICxchange:

Ion exchange is a process in which reversible

stoichLoincL ric interchange of ions of the same sign take

place between an electrolyte solution (mobile phase) or

molten salt and a solid phase (stationery phase). The

selectivity of an exchanger depends upon the nature of the

ion exchanger and the composition of the liquid phase,

which is in contact with it. it is, therefore, possible to

increase the separation potential of the exchange process

by pro[)er selection of exchanger and cluent. The exchanger

phase may be inorganic or organic in nature. The use of

solid absorbing substances to improve water quality has

been recorded since ancient times. Although most

applications involved in the removal of solid impurities

through filtration, ion exchange was also inadvertently

employed in the exchange of dissolved salts between the

water and the solid material. Earliest of the references

were found in Holy Bible which establishes Moses priority

who succeeded in preparing drinking water from brackish

18

water by ion exchange meLhod. Francis Bacon described a

method for removing salt from sea water and Hales

rccoiiiiiicMulcd LhaL sen waler \)o desalinated by filtration

through stoneware. The use of natural and artificial

aluminosL1icates to purify beet syrup was reported by harm

(i896) and Rumpler (1903). A singnifleant development took

place with the discovery of Adams and Holms [22] regarding

the use of some synthetic high molecular weight organic

polymers bearing ionic functional groups as ion exchangers.

However, organic ion exchangers have two basic drawbacks

(1) low resistance to heat and radiation and (11) their

composition cannot be varied with ease. As a result

emphasis was shifted to the synthesis of inorganic ion

exchangers [23,24] many of which exhibited expected

chemical stability and proved highly selective for certain

ions. Ion-exchange process is well suited for the

sc[)aratioii of inorganic ions (cation and anions) because

the separation is based on the exchange of Ions in

stationary phase, as shown below:

X ' Y — ^ Y I X

The barred symlx^ls denote the ion exchange phase.

An eciu i 1 ibr J uiii is eventually set up in which some of the

ions initially present in the exchanger phase have been

replaced by Y ions from the solution. Separation by ion

exchange combined with spectrophotometry can be applied for

L9

the rapid and LoLaJ analysis of numerous mixed

pharmacGUticaJ products. For example, Dowex-IXI (an anion

exchanger) can be used for the separation of morphine and

codeine [ 25 J ,

11) I'J ecLrophoresls:

I'J ectrophoresis is another separation technique

which can be defined as the migration of particles through

a solution inider the influence of an electric field. The

term 'electrophoresis' is presently applied both to the

111 i)', ra H on of i lul i v i dun 1 ions as well ns to colloidal

aggregates. This technique provides a powerful means of

fractionating the components of a mixture. Separation and

identification of micro amoiuits of high molecular weight

substances such as proteins which are often difficult to

separate by chromatography alone, are well separated and

identified by electrophoresis. Depending upon the

properLics of the medium, the separations may result

primarily from the electrophorotic effect or from a

combination of electrophoresis and adsorption, ion exchange

or other distribution ecjuilibria. Methods based upon

electrophoresis in a stabilizing medium bear a variety of

names, including el ectrochromatography, zone

electrophoresis, electromigration, and ionophoresis.

}J cctrochromatographic methods are indispensable to

the clinical chemists and the biochemists, who use them for

20

the f racLionaL iiip, oC an aiiia/.iug number oC biological

materials, e.g. cJinical diagnosis for the separation of

proteins and other large molecules present in serum, urine,

spinal fluid, gastric juices, and other body fluids. This

technique also provides a convenient means for the

separation of inorganic ions.

iii) Chromatography:

JL is said that when one of the greatest dis

coveries in the chromatographic science was, submitted for

pulil i ca L i on, Llic joiuiral of a renowned chemical society

rejected it. The importance of the work was eventually

emphasized by a Nobel Prize, in fact, such instances are

n(>( iiMcoiiiiiioM In the hl.'dory of HCICMIIM'IC soclolv

l)ub I i ca I i ons . I he paper on ' br ass Lno I Lde ' was rejected by

the journals of learned societies, the impact of which was

realized after its publication in 'Nature' and the journals

of those societies flashed news stories.

Ihe origin of the chromatography go back to Runge's

experimenL on capillary analysis. Davy observed changes in

the composition of crude petroleum when it came in contact

with rocks displaying adsorptive activity. These reports

can be considered as [>art of the development of

chromatography, it was only about 95 years ago that a

Russian botanist Michael Tswett [)ubl ished two papers

[26,27] in 1906, on the separation of plant pigments

21

achievGcJ by percolating Lhe peUroleum ether extract of

green leaves through a glass column packed with fine grains

of calcium carbonate. The separation results into a series

of g,recn and yellow bands and hence the term

'chromatography', chroma (color) + graphy (writing) was

coined by him. Despite this early description, the

importance of Tswett's work was not realized until the

beginning of the 1930s when Kuhn et al . [28,29] applied

chromatographic principles to the separation of natural

substances. Therefore, the birth of column liquid

chromal (\o raphy is ascribed to the work of Tswett.

Another milestone in the development of

chroiiiaLography was reached in 1941 when Martin and Synge

I 30, 31) it'porLcd their discovery of 1 i(juid-l iquid partition

chromatograj^hy. They used one liquid as a sorbent and

auollu'i- li(nii(l was allowed lo pc> rco I a I e L h roug.h lhe foriiior,

thus making, the tochni(|uo a chromatographic process. Their

work set a precedent for the development of other forms of

chromatography.

Mic cii rono 1 ()g, i c a 1 dcwo 1 opiuent in scjiaratlou

tcchnjciucs alter 'I'swctt's discovery of chromatography,

basetl firsL on adsorption and then partition phenomena are

presented in Table I.

Chromatography is the general name given to the

methods by which two or more compounds in a mixture

22

TABLE 1

Chroiiol ()/»] en I Dcvol ()[)nicnl in ChromnLo/;r.-iphic Separation Technic|UGS

Authors Year Separation system

1 . Ma r L i n / i iu l S y i i j ^ c

2. Consdon, Gordan and Martin

3. Craig

4. Mayer and Thompkins

5. Samuel son

6. Haugaard and Kroner

7. Izmailov and Schraiber

8. Kirchnor, MlMcr and Ket tor

9. Barrer

10 . Clacsson

11. James and Martin

1 2 . J nines <-ind M;i r l 1 n

1 3 . J a m e s and M a r t i n

1 4 . S m a l I , S t e v e n s and B.-111111 a n

1941 Partition chromatography

1944 Paper chromatography (PC)

1944 Counter current distribution (CCD)

1947 Ion-exchange chromatography (lEC), adsorption

1963 Il'C (partition)

1948 I'J ectrophoresis

1938 Thin-layer chromatography (TLC), adsorption

1951 TI.C (partition)

1945

1946

1952

19 52

19 74

1975

Gel permeation chromatography (GPC)

Gas chromatography (GO

Gas-liquid chromatography (GLC)

ll[;',h pc r foriiiance llcjuid chromatography (HPLC)

Hi gh-performance thin-1ayer chromatography (IIP ThC )

Ion chromatography

23

ptiysically separated themselves by dlstributinp, themselves

between two phases (i) a stationary phase, which can be a

solid or a liquid supported on a solid and (ii) a mobile

phase, either a gas or a liquid, which flows continuously

over the stationary phase. The separation of individual

components results primarily from differences in their

affinity for the stationary phase. There have been many

definitions of chromatography formulated according to

various classification aspects but to propose a good

definition of chromatography is difficult. It is a

collective term applied to methods which appears diverse in

some regards but share certain common features. Keulemaii's

definition serves as well as any: 'Chromatography is a

physical method of separation, in which the components to

be separated are distributed between two phases, one of

these phases constituting a stationary bed of large surface

area, the other being a fluid that percolates through or

along the stationary bed'. The stationary phase may be

cither a solid or a licjuid, and the moving phase (i.e.

mobile i)hase) may be either a 1 iciuid or a gas.

CLASSIFICATION OF CHROMATOGRAPHIC SYSTEMS:

According Lo the [)hysical arrangement chromato

graphic systems can be divided into planar or column

depending on the geometry of the column support, the

24

columnar systems can be divided as shown in Table 2. The

planar arrangement are represented by paper and thin-layer

chromatography. According to development procedures the

planar systems can be further classified" as ascendent,

horizontal, descendent and occasionally, centrifugal.

Furthermore the development is termed as isocratic if the

composition of the mobile phase remains unchanged during

the development. On the other hand, when the composition of

the mobile phase varies, it is called gradient development.

The chromatographic systems can be classified according to:

1. The state of aggregation of the phases.

2. The physical arrangement of the phases and

3. The mechanism underlying the distribution

equilibrium.

The four possible chromatographic systems derived

from solid, liquid and gaseous phases are: (a) liquid-

liquid, (b) 1 icjuid-sol id, (c) gas-liquid, and (d)

gas-solid. Of these, liquid-liquid and liquid-solid systems

constitute 'liquid chromatography'. A summary of typical

chromatographic systems according to this classification is

given in Tab!e 3.

The overall process of chromatography is a

differential migration phenomenon. The separation of the

components of mixture depends upon their differential

25

TABU' 2

Chromatographic Systems Based on the Geometry of the

Chromatographic Column

Columna r PIanar

Packed coJuiniis

Capi]J ary co]umns

Liquid-] i<iuicl columns

Paper chromatography (PC)

Thin-]ayer chromatography (TLC)

TABLE 3

Chromatographic Systems Classified According to the State of

Aggregation of the Phases

Stationary Phase Mobile Phases

Liquid Gas

Solid

Sol id + Liquid

Liquid

LSC

LSLC

LLC

CSC

GSLC

GLC

L: Liquid S: Solid G: Gas and C: Chromatography

26

pencLration into t;he porous sorbenL. This migration is

produced by a non-selective driving force, the flow of the

wash licjuid. The differential migration results from a

selective resistive action, namely, the selective sorption

of the components of the mixture [32].

Analogous differential migration from a narrow

initial zone of mixture forms the basis of several related

separatory methods. With suitable combinations of driving

force and resistive action, either one or both of which

must be selective, effective separations have been made

under a variety of conditions [32-39].

In column chromatography the separation may proceed

isocratical1y or with a programmed gradient of composition

of the mobile phase, isothermally or with programmed

changes of column temperature, and isobarically or with

programmed changes of mobile phase pressure at the column

inlet. in planar chromatographic systems the solute

compounds are usually not eluted from the chromatographic

bed but rather detected directly in it.

LIQUID CIIROMATOGKAPHIC TECHNIQUES:

It is clear from the above discussion that several

types of licjuid chromatographic techniques have been

developed. Ihe more important liquid chromatographic

techniciues have been illustrated in Fig. 3 . The common

27

1/1 LLI

3

o z I u U l 1—

>-X a < a. o o 1— < 2 O a. X o Q

3 a

cc < 2

Q: UJ >-

—• < — _ j

- J Q. ~

1

2 O

cc < 2

• < —

a

$

2 2 3

1

O I w )

— Q

(_) cr o u.

u u cc o >•

— a. —

_)

a cc <

 — cc <

_J

a < o

Q

U.

U

(—

u _J

1—Q-o u .—1

IT)

X

u • CL

CC

U — _ l

t—

— u CL

o 4J

0 0

-0 m

• H t/) 4 3 0) O 13 E cr

• H 14-1

c o X. O 4-1 (U l-i

•u o

a o "" •H S

1? ^

o ° f^ 0) ^ ^

4-1

•H -O 13 C cr n)

^ 4J C

o C W

o •r-t 14-1

4-» O CO

O OJ •r^ (X

M-l CO

•H X. en w en ca Qj

O 4J

CO

•

ClH

^ >. x; a CO

O 4-J CO

E

o X: CJ

X ) •r-l

cr • H ^ j

OJ V-i

;3 (/) en 0) V-j

0-,

00

X

II

o i-J

*> x: ex CO

00 o

4-1

CO

B O M

J3 O

c E

P r—i

o o

II

o

QJ

4J 0 z

QJ cn l-l

QJ

> QJ

Pi

II O 0-, Pi

> x; ex CO

00 o 4J CD E 0 -

x: o V-i

QJ

CO

1 1

C •r-l

x:

II

o K J H

»> x: ex CO

00

o 4J CO

e o

o V-i

QJ CL CO

D-

II

a.

OJ 1-1

D (/}

en QJ

0-

QJ

> O

II

Cu O O ^

U QJ

- 4J O (1)

H Q H

C T) O QJ • ' ^ QJ ^

DO _ N

x: o 0 0 M

X QJ E CO

r\ ^

^ II ^ Q X |_-I

Cu -^ ca CX u ^ 00 * o °o 4J O

E ^

2 O

^ o QJ U if) QJ CO >-,

x: CO a . , - j

28

liquid coluiiiii chroma togrnphic techniques arc being

described in the following paragraphs.

Adsorption:

In adsorption chromatography the retention of the

solute is a consequence of the interaction with the surface

of the solid adsorbent. The adsorbent surface has a rigid

structure making this type of chromatography useful for

separations of geometric and structural isomers with

molecular weights up to about 1000.

Partition:

Licjuid-l iquid partition chromatography was first

described by Martin and Synge [30,31] in 1941. The

distribution of solutes takes place between two immiscible

solvents. In normal phase (straight phase) chromatography

the more polar liquid often water rich is the stationary

phase, whereas the opposite is true in reversed phase

partition. The stationary phase may be situated on a

variety o^ supports depending on the polarity of the

stationary phase. Partition chromatography is used for

separation of solutes with molecular weights up to a few

thousands, and is a powerful tool in the separation of

scries of homologs.

29

Bonded Phases:

Most applications of liquid column chromatography

are now made on silica which has been chemically modified

(bonded phase chromatography), the modification is made by

chemical reaction between the silanol groups and a

chlorosilane compound. The carbon radicals of the

chlorosilane compound determines the nature of the final

column material. Using silanes containing alkyl carbon

chains with 8-22 carbon atoms gives the particle

hydrophobic surfaces, but more polar surfaces may be

obtained by incorporation of alcohol, amino, cyano and

other groups in the alkyl chain.

The column materials bearing bonded alkyl cliains

are used for reversed phase chromatography, while some of

the more polar, chemically bonded phases may be used in the

straight phase mode as well as in the reversed phase mode,

giving more possibilities for selection of the appropriate

chromatographic system.

Ion Exchange:

'Hie stationary pliase in ion exchange chromatography

is made of a porous polymer to which anionic or cat ionic

exchange groups have been attached. The retention and

separation of solutes are performed according to the degree

of ionization of the solute and its affinity to the ionic

30

sites on the stationary phase. The eluent is usually an

aqueous buffer and the retention may be controlled by

changes in ionic-strength.

Size Exclusion:

In size exclusion chromatography the solid support

is a porous polymer with a controlled pore size, and the

solute molecules are separated according to their size in

solution. The large molecules are excluded and they have

the shortest retention time. The size exclusion may be

performed in aqueous systems (gel filtration), where water

soluble macromolecules can be separated or in non-aqueous

sysLoms (',e 1 permeallon). liy calibration the method can

also be used for determination of molecular weight or

molecular weight distribution.

Affinity:

In order to achieve a high degree of selectivity

special groups with a high affinity to solute or a group of

solutes may be attached to a solid matrix. The ionic

exchange groups in ion exchange chromatography are the most

well known example of this, but many column materials even

more selective have been developed (e.g. immobilized

enzymes). The field of bio-affinity chromatography is

expanding rapidly.

31

Most of the initial developments in chromatography

were for the separation of organic compounds. The beginning

of inorganic chromatography can be attributed to the work

of Runge (1850) on PC; Beyerinck (1889) on thin layer of

gelatin and Schwab (1937) on alumina column.

Since most of the work carried out by us is based

on thin-layer chromatography (TLC), it is justified to

discuss the salient features of TLC in detail. The

following paragraphs will present an overview of all

important aspects of this widely popular and versatile

separation technique.

THIN-LAYER CHROMATOGRAPHY:

Thin-layer chromatography (TLC) together with paper

chromatography comprises 'Planar' or 'flat bed' chromato

graphy. IL is a rapid, inexpensive and highly effective

analytical technique applicable for the analysis of a great

variety of multicomponent mixtures. The stationary phase in

TLC is an active solid termed as the sorbent whereas a

liquid containing a single solvent or a mixture of solvents

is used as mobile phase. A suitable closed vessel

contaLiiiiig iiiobUe jihase and a [ilaLe (gjass or plasLic)

coated with a suitable sorbent (silica, cellulose, alumina,

polyamidc or ion exchanger) are all that required to carry

out qualitative and semiquantitative separations. Ihe

32

mobile phase (solvent;) is usually allowed to migrate up the

plate 10 to 8 cm from the starting line on a TLC plate. The

conventional one-dimensional ascending technique is usually

used for the development of chromatographs. Multiple, two

dimensional, centrifugal and gradient devel oi)ment

techniques have also been used.

Basic TLC is carried out as follows. A drop volume

(0.5-10 pi ) of a sample mixture is spotted on a TLC plate

at about 2 cm from the lower edge of the layer. The spot is

completely dried at room temperature or at an elevated

temperature and the plate is developed with a suitable

mobile phase inside a closed chamber. The components of the

mixture migrate at different rates during the migration of

mobile pliaso Lhrough the stationary phase. After the

devel o[)ment is over, the stationary phase is removed, the

plates are dried and the zones arc detected using suitable

detection reagent. Differential migration results because

of varying degrees of affinity of the mixture components

for the stationary and mobile phase.

Compound identification in TLC is based on Rp, value

which is a measure of the ratio of the distance traveled by

the solvent from the starting line to the distance traveled

by the solute i.e.

distance moved by the solute

^ distance moved by mobile phase front

33

The R,, values are generally noL exactly

reproducible. The contributing factors for this are:

Chamber dimensions, purity and flow direction of mobile

phase, size and nature of layer, humidity, equilibration

conditions, temperature and sample preparation methods.

Further characterization of separated substances can be

achieved by scraping the layer and elution of the analyte

follov 7cd by spectroscopic methods.

History of TLC:

The History of TLC is marked by three dates.

In 1938, Izmailov and Schraiber [40] separated

certain medicinal compounds on binder free

horizontal thin layer of alumina spread on glass

plates. Since the solvent was applied as drops on

the glass plates, containing sample and sorbent,

their method was called 'drop chromatography'.

In 1949 Meinhard and Hall [41] demonstrated that

powdered adsorbent, fixed to a microscope slide by

moans of a suitable binder, [provides a system for

microchromatography called 'surface chromatography'

I'hey separated Fe from Zn on microscope slides

coated with a mixture of alumina and starch

(binder).

34

3. About 1958, Stahl [42,43] introduced the term

'thin-layer chromatography' and standardized

procedures, materials and nomenclature.

Kirchner [44], Heftmann [45], Stahl [46] and Pelick

[47] have nicely reviewed the history of TLC. After the

pioneering work of J. Kirchner [48] and E. Stahl , TLC

became important for the separation of samples not

amenable to analysis by GC. The rapid growth of TLC was

slowed down during the 1970s with the corresponding rise in

popularity of HPLC. The capacity factors in HPLC are more

reproducible than R , values in TLC. However, recent

improvements in TLC have removed many of its limitations.

As a result of the recent improvements in TLC

several new techniques such as high-[)erf ormance (HP) TLC,

over pressurized (OP) TLC, centrifugal layer chromatography

(CLC) and reversed phase (RP) TLC came in light. The

quantification by densitometric scanning have also improved

the efficiency of classical TLC.

lil'TLC layers are smaller, thinner, contain sorbent

of more uniform particle size and are developed for a

shorter distance. All these factors lead to faster

separations, reduced zone diffusion, lower detection

limits, less solvent consumption and better separation

efficiency. Figure 4 shows the practical advantages of

35

Separa t i on d i s t a n c e ( so lvent m ig ra t i on )

100 mm

5 0 mm

30 m i n ^ Runn ing time

F i g . 4 C a i ) i J ] a r y Clow d iag ram

36

HPTLC over conventional TLC.

OPTLC was introduced by Hungarian scientists ,in

1970s. In OPTLC,the vapor phase has been eliminated and the

sorbent layer is completely covered with an elastic

membrane under external pressure. The mobile phase migrates

through thin layer due to the 'cushion system' at over

pressure. Thus, OPTLC combines advantages of the continuous

development technique and elimination of free space in the

chromatographic chamber.

In CLC, the eluent flow is induced by centrifugal

force. The sam])le is applied near the center oC a rotating

disk covered with adsorption material. Concentric zones of

substance migrate towards the outside of the plate during

elution. The circles elute sequentially from the disk and

can be recovered separately. Figure 5 illustrates a

comparison between efficiences of fine and coarse particle

layers as a function of migration distance and development

technique. The detailed comparisons of TLC, HPTLC, and CLC

have been well documented in recent literature [49-52].

Advantages of TLC:

TLC is the most versatile and flexible chromato

graphic method. IL is rapid because precoatcd layers are

available to be used as received, without preparation. It

has highest sample throughput because upto 30 individual

37

E

70

60

50H

01

•^ 40

-.i 3 0

en f<

^ 20 > <

10

-L 2

±

H P T L C Plate

f o r c e d 1^°^

HPTLC Plate

Forced f low development

.L

>• Convent ional TLC Pla te Norma l development

4 6 8 10 12

M i y r a t i o n d i s t ance ( c m )

U 16

Fig. 5 Comparison between efficiences of HPTLC and 'I LC 1 ayers

38

samples and standards can bo apjilied to a single plaLc and

separated at the same time. The automated sample

applicators and developers allow high accuracy and

precision in quantification. There is a wide choice of

layers, developers and detection methods. The wide choice

of detection reagents leads to unsurpassed specificity.

Less pure samples can be applied as the layers are normally

not reused. Being an 'off line' method, different steps of

the procedure are carried out independently.

Chromatographic Systems:

The optimum conditions for separation in TLC are

yielded through mutual harmonization of the stationary and

mobile phases. The following separation mechanisms are

expected to operate, depending upon the nature of analyte,

sorbent layer and the type of mobile phase.

(1) Adsorption (physical sorption of solutes from solution

onto the active surface groups of the sorbent), (2)

Partition (dissolution of solutes into a stationary liquid

held on the layer), (3) Ion exchange (attraction of ions

to sites of opposite charge on the layer) and (4) Size-

exclusion or gel permeation (retention or rejection of

solutes on the basis of their size and/or shape).

Adsorption TLC is wel1 suited to the separation of

structural isomers whereas partion TLC is useful in the

39

separaLion of homologs.

As we have used TLC for the analysis of inorganic

ions, LL is therefore fruitful to say a few words about the

chromatographic systems used for TLC of inorganics in

recent past. The following paragraphs are devoted to bring

out the developments which took place in the last ten years

in this regard.

Stationary Phase (Layer Sorbents):

A large number of sorbents are available whicli can

be used in '1LC, but the need for a perfect sorbent has been

always felt. In the absence of an ideal sorbent the searcli

for a stable, inexpensive, reproducible and readily

avail al)lc sorbent [phases continue. Among the sorbent phases

used, silica gel has been the most favored layer material.

Silica gel, a sorbent of the widest range for TLC

applications, is an amorphous and porous adsorbent referred

to as silica, silicic acid, or porous glass. At the surface

of silica gel the free valencies of the oxygen are

connected either with hydrogen (Si-OH, silanol groups) or

with another silicon atom (Si-O-Si, siloxanc groups) as

shown i n 1igurc 6 .

The silanol groups represent adsorption active

surface centers that are able to interact with sample

40

Fig. 6 Chemical structure of silica gel

41

molecules. The ability of the silanol groups to react

chemically with appropriate reagents is used for controlled

surface modifications. Therefore, silica gels are most

suitable as stationary phases in chromatography.

The various types of sorbent layers used may be

classified as follows:

(I) Unmodified or Untreated Sorbents:

Silica gels, aluminas, inert silicon dioxides

(silica 50,000 and kieselguhrs), cellulose, polyamides

(polymide 6 and iôlyamide II) and scphadex (cross linked

polymeric dcxtran gels ). Kicsclguhr matrix consists of

Si02, AI2O., MgO, Na20, K2O, CaO, Ti02 etc.

(II) Bonded by Chemically Modified Sorbents:

In recent years the importance of using surface-

modified sorbents in ILC has increased. Both hydropliobic

and hydrophilic modified sorbent phases have been used.

(a) Hydrophobic Modified Phases (RP phases):

Ihe non-modified sorbents show polar surface

characteristics, therefore they are not of much practical

utility for ch rom/i Log raph i c separations of solutes having

identical polar characteristics. This problem has been

42

solved using hydrophobic interactions of the stationary

phase with compounds o£ appropriate molecular weight. The

most popular such organo lunctional groups are methyl

(RP-2), Octy] (RP-8), dodecyl (RP-12), Octadecyl (RP-18)

and IMuMiyl residues.

(b) Hydrophilic Modified Sorbents:

Hydrophilic modified sorbents possess amino-,

cyano- and diol residues as a functional group. The polar

functional groups, in each case, are bonded via short-chain

non-[:)olar spacers to the silica matrix.

(Ill) Impregnated Sorbents:

Besides the possibility of changing the

selectivity of sorbent by chemical modification,

iiiiproveniont in selectivity can also be achieved by

impregnating the matrix with suitable organic or inorganic

substances (physisorption).

(a) Organic Imprcgnants:

Silica gel lias been impregnated with diethyl ene-

trianiine, sul f aguanidine , 8-hydroxyquinol ine and

t-buty]amine, 2 , 2-dipyridal , iminodiacetic acid, tributyl-

aminc, ED I'A, pyridinium tung stoarsenate, p-toluidine etc.

MicroLrysta 1]ine cellulose impregnated with chitosan

formate, bis-(2-ethy1hexy1 ) orthophosphoric acid, trioctyl

phosphine oxido-bis-(2-ethy1hoxyl) orthophosphoric acid and

43

dibuty] butyl phosphonate.

(b) Inorganic Impregnants:

Ceric molybdate, sodium molybdate, NH, CI , Ba(NO-,)^,

NaNOr,, NaNO^, potassium ortho dihydrogen phosphate and

CuSO, have been used as the impregnants for silica gel.

(IV) Miscellaneous Sorbents:

In this category one can report numerous types of

sorbent phases, that have been used in TLC. Some of such

sc)rbci\lj; are suiiima r I/.ecl below:

Silica gel II slurried in 47„ ammonium nitrate solution

containing VL sodium carboxy methyl cellulose (CMC), silica

gel G-starch, Silica gel G-Ceric molybdate, silica gel R-

vionite CS ion exchanger, silica gel G- Na carboxy methyl

cellulose, kaolin, chitosan, kieselgel G -chitosan,

microcrystal 1 ine cellulose mixed with powdered chitosan,

diethyl aiiiinoethyl (DEAE) - cellulose layers in H form,

cellulose phosphate in thiocyanate form, p-cellulose +

inicroc ry sL a 11 inc cellulose (2:1) in free hydrogen form,

cellulose phosphate • microcrystal1ine cellulose (3:1),

cellulose (chemapol) with azopyrocatechol group, alumina

with plaster of paris binder, staimic arsenate, stannic

antimonaLc, hydrated sLainiic oxide, /Irconluiii tnngsLaLe In

44

H foi"m, P-stannic arsenate in H form, Ti(IV) antimonate

in H form, styragel R 60 A polystyrene dVB copolymer,

Dowcx 50 WX8 + silica gel (1:1) and Amberlite IRA-400 +

slllea gel (1:1).

Both commercially available (precoated) layers as

well as home made sorbent layers have been used.

Mobile Phases:

Separation of ions by TLC is governed by the

physical interactions of the adsorbent and the coordinative

properties of the mobile phase. The composition of a mobile

phase is usually altered in order to achieve a desired

separation with improved selectivity, resolution -and

clarity.

Mobile phases are most often selected by consulting

the pertinent literature to find suitable solvent for the

separation of interest. Ihis is followed by a trial-and-

crror approach to modify the mobile phase for the

particular layer and other local conditions being used, if

necessary. Based on solvent strength and selectivity

parameters systematic approaches to mobile phase selection

and optimization have been developed. A praiseworthy work

in this direction has been done by 3nyder [53] and Kirklaiid

[54]. In general, solvent systems used for TfC of

45

inorgiinics arc (a) mixture of organic solvcnL with some

acids or a buffer (b) acjueous solution of mono or polybasic

acids or their alkali metal salts and (c) various organic

solvents ranging from low to high boiling points. An

important advantages of TLC with volatile mobile phases

such as acetone, methanol or benzene lies in the fact that

they quickly evaporate from the sorbent layer, after

development, but reproducibility in such cases suffers due

to the presence of mobile phase components in the vapor

phase over the surface. Conversely, TLC with mobile phases

of lower volaLiliLy g,ives better reproducibility, but the

continuous slow advancing of the mobile phase after the

withdrawal of the plate from the chamber may lead to an

additional zone broadening. With different sorbent phases

the organic solvents such as acetyl acetone,

trif1uoroacetyl acetone, monotetradecyl phosphate, tributyl

phospliatc, tctrahydrof uran (TllF), dimethyl sul f oxide ( DMSO ) ,

dimethylformamide (DMF) , and 2-ethyl hexyl phosphate in

combination with other solvents have proved very useful for

the separation of inorg,anic ions. '1 he mobile phases used

durin*', last decade may be categorized into fol it)wing

groups:

1. Inorganic Solvents: (a) acids, (b) bases, (c) salt solu

tions, (d) mixture of acids and bases and their salts.

46

2. Organic Solvents: (a) acids, (b) bases, (c) alcohols,

(d) aldehydes and ketones, (e) esters, (f) mixture of the

above .

3. Mixed Solvents: (a) organic solvents mixed with mineral

acids (b) organic solvents mixed with inorganic bases (c)

organic solvent mixed with water (d) organic solvents mixed

with salt solutions.

4. Complex-forming Organic Solvents

Sample Preparation:

Cations are generally dissolved in water

maintaining a concentration of nearly O.IM. In some cases,

the corresponding acid is added to prevent hydrolysis. Rare

earth solutions are prepared by dissolving their nitrates

in O.IM HNO^ or by fusion followed by dissolution in dilute

HCl or HNOo. Anions are usually taken as their water

soluble sodium, potassium or ammonium salts. Methanol and

ethanol are used as solvents for preparing the sample

solutions of organometal1ics .

Detection and identification:

The detection methods used for inorganic ions fall

into three riajcr categories: (a) Chemical, (b) Physical,

(c) Kni'ymatic or biol ogical.The chromatopl ates are air. dried

prior CO tne deteccioii of ions.

47

Chemical tiicChods of detection involve the spraying

of chromaLoplates with a suitable reagent, which forms

colored compounds with the separated species. Reagents

giving clear and sufficiently sensitive color reactions

with several ions are preferred. Both selective and non

selective reagents may be chosen for detection purposes.

Among the physical methods, visualization in ultraviolet

(UV) light is most common. This method is highly sensitive,

non destructive, and a\ .lable to the visualization of sptits

before undertaking quantitative studies. Enzymatic

detection iiietliods are also quite simple and selective.

For the detection of inorganic ions standard sjjot

test reagents (general as well as specific) have been used

by most of the workers. The reagents used most extensively

are; alizarin Red S, benzidine, dithizone, diphenyl-

carbazide, 8-hydroxyquinoline, Rhodamine-B, potassium

1 or roc vanide, y(Ml(.)w ammonium sulfide, chromotropic acid,

alkaline ;.Uyoxal di thiosemica rbazone , and rubeanic acid for

cations- arsenazo III, chl orophosphonazo-m-NOg,, alizarin,

and t ribroinochl oroph(> -ihonazo for rare earth elements; and

ninhydrin, diphenylamine, alizarin, silver nitrate,

F8CI3 , pyrogallol and 2,6-dichlorophenolindophenol for

anions. Y -specLroscopy or autoradiography [55j for the

detection of rare earths appearing as fission products in a

freshly irradiated fuel. X-ray fluorescence microanalysis

48

with a scannidj:.; collimated primary X-ray beam [56] for

metal ion detection, and Rhodamine-B £or the detection of

silicon in edible oils [57] have also been used. Crystal

violet solutions of standard acidity have been employed for

the detection of phosphoric acid derivatives [58]. Nanda

and Devi have reported an enzymatic method [59] for the

detection of heavy metal compounds in fresh water.

In some cases the detection of certain inorganic

ions has been carried out according to the color reaction

appearing in the sorption zone [60-62] of the element on

the plate during the chromatographic run. Selenium in food

samples has been identified as 2,3-diaminonaphthalene Se

complex, v/hich upon exposure to U.V. light (360 nm) gives

pink fluorescence on a TLC plate [63]. An indirect

fluorimetric niethod for the detection of non - fluorescing

anions has recently been reported [64]. Fluorescent morin-

aluminium complex as a detection reagent for various anions

has been reported [65] by T. Okumura. The Rp values of the

detected zones are used to identify the various ions

present in the sample.

Quantitation:

In recent years quantitative TLC has taken the

world by storm due to the development of ne\^'

instrumental techniques as a result of which a TLC for

49

quantitative analysis has surged to dramatic heights, and

has gathered momentum. The three main approaches associated

with such analysis are: Visual estimation, zone elution

and scanniiig densitometry.

Visual Estimation:

The simplest for semiquantitative analysis by TLC

is to develop a definite sample aliquot alongside standards

containing known weights of analyte. After detection, the

weight of analyte in the sample is estimated by visual

comparison of the size and intensity of the standards and

sample zones. This method has accuracy and reproducibility

in the range of 10-307o which is adequate for the purpose

intended. Visual comparison works well if amounts close to

the detection limit are applied on the chromatoplates and

the sample is accurately bracketed with standards.

In an attempt to standardize the quantification in

TLC, Moha mmad and Fatima [66,67], Mohammad and Tiwari [68],

Nanda and Devi [69] and Mlodzikowski [70] h;r'e established

a linear relationship between the spot size and the amount

of solute.

Zone Elution:

The zone elution method involves:

a) Drying the layer

50

b) Locating the separated analyte zones

c) Scraping the portion of layer containing the

anaJyte from the sorbent and

d) Measurement against standards by an independent

microanalyticaJ method such as solution

spectrometry, gas chromatography or voltametry.

This quantification method is tedious and tine

consuming and seems inaccurate because of difficulties in

locating the exact z . boundaries loss of sorbent during

scrapping and collection, non-reproducible elution from the

sorbent and background interferences due to oluted

impurities from the sorbent. These errors can be minimized

if standards and samples are chromatographed, scrapped and

eluted with full consistency, and an equal size blank area

of layer is scrapped and eluted in an identical fashion.

Scanning Densitometry:

In situ measurements of zones wil , a scanning

densitometer is the preferred technique for quantitative

TLC. Substances separated by I'LC or HPTLC are qualified bv

in situ measurement of absorbed visible or U.V. light or

emitted fluorescence upon excitation with U.V. light.

Absorption of U.V. light is measured either on regular

layers or on layers with incorporated phosphor.

51

A double bed densitometer equipped with a TLC

scanner, an integrator, and a microcomputer have been used

for the simultaneous determination of light rare earth in

monazite sand, CAMAG turner fluorometric scanner for the

estimation of cadmium ion [71], Aminco SPF 125 for spectro-

fluoronietric determination of selemium in food [63], KM-3

densitometer [72] (Zeiss) for HPTLC determination of Se in

biological matrices, ERI-10 densitometer (Carl Zeiss, Jena,

4-GDR) for the determination of NOo and Fe(CN)^ in molasses,

and other anions that produce a blue color with

dipheny]amine have been used [73].

It is clear from what has been discussed above that

there is a necessity of developing non-instrumental methods

of analysis such as solid-state spot-tests and TLC. A look

at the literature presented above shows that very little

work has been done on the TLC of anions and almost no work

on capillary solid-state spot-tests of anions has appeared

til] date. In fact a systematic study on anions regarding

their detection, determination and separation is lacking.

It, therefore, appears quite interesting to couple

capillary solid-state spot-test technique with thin-layer

chromatography to cover a wide spectrum of analysis of

anions present in solid or fluid samples. These techniques

provide a rapid method for the screening of a large number

of anions for their quick detection, identification,

quantification und separation.

52

The present work was taken up with the following

aims:

1. To develop new mixed and impregnated sorbent layers for

some useful separations of anions.

2. To make a comparative study on color reactions of

anions in the solid-state and in solutions.

3. To develop new selective and specific capillary solid-

tests for anions.

4. To devise a scheme for the systematic analysis of

anions in the solid state.

Some exciting results achieved by the present

studies aie being presented in the following chapters.

Literature:

The voluminous work done on TLC of inorganic and

organic substances has been well documented in the form of

several reviews, monographs, books and articles. The work

done on TLC of inorganics till 1972 has been admirably

reviewed by Brinkman et al . [74] whereas the work appeared

during 1972-80 has been summarized by Volynets and Kuroda

[75]. The latest work on thin-layer chromatographic studies

of inorganics and organometal1ics has been presented in a

chapter, in Handbook of Thin-Layer Chromatography published

by Marcel Dekker in 1990 [76].

53

The daca available on the published work on TLC of

cations and anions during the last 10 years has been

compared and graphically shown in Figure 7. Surprisingly

very little work has been carried out on anions as compared

to the cations. Table 4 based on the available data from

reviews, chemical abstracts, monographs and books,

summarizes the work done on TLC of anions till date.

54

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CHAPTER - II

ANALYSIS OF ANIONS BY SOLID-STATE SPOT-TESTS

92

The capillary solid-state spot-test technique

reported by this laboratory [l] offers a simple and

inexpensive tool for identification of many organic and

inorganic solids |2-4], and in some cases for their

determination. I he screening procedure can easily be

adapted for use by mobile laboratories in the field. Ihc

choice of indicative parameters in this technique, such a^

the color, length and direction of movement of the colored

boundary leads to selective or even specific detection,

even of chei'iically closely related compounds. The tests can

11 so be ipi^lied bv trituration of the test compound and the

reageni on i spot-plate.

Another novel feature of capillary solid-state

spot-tests is that the boundary between the product and

reacL.mts can be clearly distinguished even when their

r.l- Z color, rire similar. Colored anions such as CrGf, CroO-,' 4 ' z /

Fe(CN)^~ etc. can be clearly detected in a glass capillary

even il the product is yellow or orange. Likewise, colored

reagents can be easily utilized for detection purposes in a

glass capillary. The enhanced selectivity due to the

surface contact of the reactants is an added advantage.

Reactivity in the solid-state depends not only on the

che'iicil nature of the crystal, but also on the positions

occupied b\ ions, atoms or molecules in it [5],

93

Earlier studies [2] showed that the color produced

by a reaction in solution is not always the same as that

Cor the sati.e reaction in the solid state, since the latter

occur with a minimum of atomic and molecular movement.

Considerable work has been done on the detection and

determination of anions in solution [6,7], but none on

systematic detection of anions in the solid state.

Ihe present work summarizes our efforts to utilize

the capillary solid-state spot-test technique for selective

cietectLon and systematic analysis of anions. It has already

proved Uboful for common cations [2]. The technique has

i)een Modified by use of a glass-wool plug to make the test

inore -êlective or even specific.

EXPERIMENTAL

Reagents

All reagents used were of BDH analytical or

laboratt)ry reagent grade, and ground to 50-100 mesh size.

Ihe rcrigenis used were p-dimethyl aminobenzal dehyde (p~DAB).

p diiiicthvl aminocinnamaldehyde (p-DAC), diphenyl amine ( DPA ) ,

p-toluidme (p-IU), benzidine (BD), benzidine hydrochloride

iBDHCi, diethyl amine hydrochloride (DEAHC), barbituric acid

(BTA), chromotropic acid (CA), sodium nitroprusside (SNP),

Phenolphthalein (PPL), potassium hydrogen sulphate (PHS ) ,

94

ferric chjnrjdo (KeClo), silver nitrate (AgNO^), and 507

w/u int i"iat ' mixtures of p-DAB, DPA, p-DAC, p-TD, BTA, CA,

Kl, N,I^MM()^^, l''f'( 1,, FoSO,(NH/^ ^ Z^'^^ ^^'^ AgNO^ with F HS , and

ot SNf' and PPL with NH^Cl .

For detection in solution, ethanol or

deinineral i zed water, or a mixture of the two when

required, was used as the solvent, and PHS and ammoniun

chloride were replaced by dilute hydrochloric acid and

ammonia solution respectively.

Procedures

Sulid-stale detection. About 5-10 mg of the

powdered test macerial was triturated with several mg of

reageiiL lu a depression on a white spot-plate with a glass

rod. I he colors developed at 30 or 40° and on heating to a

higher temperature (60-100°) were recorded.

Detection in solution. One or two drops of an

aqueous solution of the test material were mixed with a few

drops of a concentrated solution of the reagent in a

depression on the spot-plate. The colors developed at 40'

and at higher temperatures were recorded.

Solid-state detection in capillaries. A 10-cm

length of graduated capillary (3 mm internal diameter) was

partly filled with powdered reagent from one end with the

95

help of two iron rods, and the powdered sample was

similarly packed from the other end. Care was taken to make

the packing reproducible and that the two materials were in

close contact in the middle of the capillary. The color of

the product formed at the junction, and the length and

direction of movement of the colored boundary at the

desired temperature after specified time intervals were

recorded,

Solid-state detection in capillaries modified with

,1 glass -wool plug. An air gap between the reagent and the

test material was created by placing a glass-wool plug (4-8

mm) in the middle of the capillary before filling it with

the reactants. I'he test material and the reagent were then

added from opposite ends of the capillary as usual. The

capillary was kept at the desired temperature in an

electrically heated oven. The color formed either at the

glass~wool/reagent junction or the glass-wool/test material

junction was observed after a fixed time interval and the

thickness ol' the colored boundary was also recorded.

RESULTS AND DISCUSSION

The results obtained for the trituration reactions

are summarized in Table 1. They were compared with those

obtained in solution, and in many instances these colors

96

<

E O O a:

ro to •u C OJ 00

0)

e o w

c o

•r-(

<

o in c o

•H •U o re cu

oi

•U

a; c— " l •u o a

00

0) j j ra u GO I

T5 •r-l r-H

o

o CC

TO I-0) Q-e

f < I

I + I

I ^ I

( i •

f f f

+ + +

I -( 4 I I + +- I I I +

I -t ( I I f + I I f I

I I I I I I I I I + +

t ( I I I 4 4 f 4 I f

+ + + • - + 1 + + + +* - +

+ + + I + + I + + +

+ + I I + + + I I I

i I I I + + + I f +

I I 4 I -t + I I t I

I • ( - I I + + I I I I

I I I I + + I I -4 I

t I f ( + •- h I t t

I I f I f I I I f I

I I I I I I I I t I

I I I I I I I I I I

- ( I I I I I I I I I

I I I I • ( + I I • ( I

I I I I I f I I + I

I I I I f +- I I + I

I I I I -t- f I I + +

t • + + ^ + + f - ^ l ^ l • ^ + t - + l + +

I I I I I I I I I I I I I -I I I I I I

+ + + + -t + f + + (- + + + ) I I f - 4 + + | 1 +

I I I I I I I I I I I I I I I I I I -I I I I I

I I I

I I I

I I I

i < I

I- -t ^

I + I

+ I + + I +

I + t ( +

I I I I I I

I I I I I I

I I I I I I

I t I < t *

I I I I I I

I I I I I I

I I I I f -1-

{ I I I I I

I I I I I I

I I I I I I

I ( I I f I

I I I I I I

t

d

h iii

3 ' ? -Z u- U. X <

-c

0 .

+

O

z z u - !_

I I I I I I

Z Z

0 = ^ 5 - - -•: t/i Z a. i , u; o r » = o

— 1/1

y " r < < < + iJ H I- < < Q CJ a Cj U — r l f ^ T v^

— 1/1 a. — ) ^

O + u < • G = 3 H H Q Q o r- 3o c o

</i

t

< < < ^ ~ a a x i G (L C L G G i . — r i '--1 'T - ^

< < <

97

we re entirely different. For example, VO-. reacts with

p-DAB+PHS Lo give a brown product in the solid state but a

3-red precipitate in solution. Similarly, Fe(CN), gives an

orange-yellow product with p-DAB + PHS at 60° in the solid

state, and a brown product in solution. With the same

reagent in the solid-state Fe(CN). yields a green product

which changes to blue-green, while in solution a yellow

2-color is observed. At 40°, Cr20^ reacts with p-DAB+PHS to

give a red product in the solid-state, which is immediately

converted into a dark brown product, but at 60° only a dark

brown product appears. A red color does not occur in

solution at any temperature. It appears that the red

product is unstable and its formation is only possible in

the solid state at lower temperature.

V r(>:

UFA can be used tor the specific detection oi'

in the solid state because this is the only one of

the anions studied that gives any color (green-yellow at

40 ). In .-.olution DPA forms a bright yellow product with

CrO ", . orange with Cr20-, and green-yellow with Fe(CN), 3-

Fhe colors produced by DPA * PHS with NO" Br0~ 10"

SO, cind Cr20^' are different in the solid state from those

in solution. When BrO^ reacts with DPA + PHS the initial

blue color changes to green within few minutes. In solution

only a yellow-brown precipitate is obtained. lOZ behaves

similarly in the solid state but in solution gives a green

98

product.. 10 " /Jives a green color which changes into

yellow in the soJid state, and in solution a stable green

product is fonned. DPA+PHS reacts with MoO^ only in

2- d solution, to give a green product. With Cr20y an

Fe(CN)^ it gives a yellow color in the solid and green in

solut i on.

p-I)AC produces a green-yellow product with most

anions in solution. The brown species formed with SCN in

the solid state differs from that formed in solution

ives an orange color in the solid

2-

( grcen-yel 1 ov;) . SOo

state but docs nut react in solution. SOt and N0„ produce 4 i

a light orange color in the solid at 40° but in solution

both anions giv - a greenish yellow color. For the detection

r)f most anions on a spot-plate p-DAC + PHS was not suitable

because ef Its 0'jji\ color. However, the colors formed with

S- , MoO^ , Fe(CN)^ , VO and Fe(CN)^ in the solid state

differ fro'i those formed in solution. p-TD+PHS gives

colored species with NO2 (light yellow), NO3 (red brown),

l" I red-black), J O3" (blue-black), 10^ (red-violet), MoO' "

( blue-viol et j , VO 3 (violet) and Fe(CN)^'" (pink) in solution

l)ut does not react with these anions in the solid state. In 2- 2 -

the s o l i d s t a t e , CrO^ and CrÔy g i v e p a l e ye l l ow and

o r a n g e - y e l l o w c o l o r s r e s p e c t i v e l y wi th p-TD+PHS whereas in

s o l u t i o n they p roduce v i o l e t and brown c o l o r s r e s p e c t i v e l y .

99

Fe(CN)^ gives a brown-yellow product in the solid state,

but a red-violet product in solution.

With BD only two anions, CxÔhj' and FelCN)^" react

in the solid state at 40°, giving brown-yellow and green-

yellow colors respectively, allowing their selective

2- 3-detection. In solution CrÔy and FelCN)^^ give violet and

4-green-yellow products respectively, and FeCCN)^^ produces a

2~ brown precipitate. CrO^ forms a green-yellow precipitate

2~ in Jiolution. which interferes in the detection of (^.rJ^n i

whereas in the solid state it does not interfere, because

of its conj^lete inertness. At 80° BD reacts with SÔ,

ivellow solution), 10 „ (yellow precipitate) and 10, (brown

precipitate) only in solution. N0„ produces a beautiful red

precipitate with BDHC at 80°, which can be utilized for its

selective detection. I0„ and 10, give yellow and green-

yellow precipitates respectively at 40°, whereas at 80"

they produce brown and blue precipitates respectively,

indicating formation of different species at the two

temperatures. CrO, gives a blue-violet precipitate at 40'

but at 80 it produces a dark brown precipitate along with

a green solution. However, in the solid state only a green

producl is obtained at both temperatures. CrÔ-, gives a

brown product in the solid state but in solution gives

brown-violet (40") and brown (80°) precipitates. Fe(CN)^~

100

does not react in the solid but gives light blue and yellow

precipitates at 40 and 80" respectively. BDHC reacts to

2-îve colored species with only three anions, CrO/^ (green),

9- 3-

CrÔy (brownj and Fe(CN), (orange-yellow) in the solid

state at 40° and can be used for their selective detection.

DEAHC was found to be much less reactive towards

anions both in the solid state and in solution. However, it

can be used for the selective detection of CH^COO and

9 -

S.,OT which produce yellow products in the solid state at

40". At 80 \ NO^ gives a yellow product,and I~ and SCN

[live light pink products.

BTA can be used for the selective detection of

SCN and V0„ at 40". SCN does not give any color in

solution but gives a pink color in the solid state. VO-

gives a brown color in the solid state and a yellow color

in scjlution. None of the other anions studied gives any

folor in the ,s(jlLci state with IJ'l'A at this tenipc ra L ure.

Ilowevc!-, ii! sol til ion tlie selectivity of Bi'A is reduced and

seven cUiions ]:)roduce colored products at 40"; MO2 ,

producing a violet solution can be distinguished from the

4-rest ot the anions. At 80", I''e(CN)g gives a green-yellow

solut-ion but does not react in the solid state.

Solid-state color reactions of BTA+PHS can be used

for the selective detection of Br (orange-yellow). I

101

(yellow), SCN tvioJet), Cr207 (brown), Fe(CN)^ (red) and

Fe(CN)t^ (blue). Fe (CN), can be distinguished from 6 6 / — 7 — 9 — —

Fe(CN)^ , and (C.r r^-j from CrO^ in the solid state. CI

gives no color with BTA + PHS and hence does not interfere

in the detection of I and Br in the solid state. SCN

gives a red-vilet color with aqueous BTA + PHS, but no

colored species was observed in solution when PHS was 2-

repl aced by dilute hydrochloric acid. Cr jO-, produces a

blue-green color with aqueous BTA+PHS, which changes to

blue. Similarly, the yellow color initially formed with

Fe(CN), changes to green, but Fe(CN)p gives only a

green-blue color. With an aqueous solution of Bl A

containing hydrochloric acid, NO2 produces a violet color

which is quickly converted into yellow. A red-brown

2- 4-precipitate is obtained with I . CrÔ-, and Fe(CN)^ gi ve

very stable blue species whereas the green-yellow color

formed with Fe(CN), changes to green-blue. BrO^, VO ,,,

2- 2-SO^ and S,-,0o also give colored species with BTA in the

presence of hydrochloric acid, but these anions do not

react with aqueous BTA+PHS. CA is a selective reagent for

3-

the detection of Fe(CN)^ . The colors obtained in the solid

state with NO^ (brown), CO^" (brown), and Fe(CN)^" (red-

yellow) are different from those obtained in solution. At ?- 4-

8U°, SÔ^ and Fe(CN), give light blue and yellow colors 2-

respectivel y, but only in solution. Cr„0-, does not react

102

in the solid state but in solution it gives a dark brown

color at 80°. CA-* PHS gives color reactions with many

anions, and hence it can be used as a general reagent for

detection of anions. It gives a brown color with VC' ,

3- 4-

yellow with Fe(CN)g and light yellow with Fe(CN)(^ in the

solid state, but in solution gives a yellow color with VO^,

green-yellow with Fe(CN)g and blue with Fe(CN) . Thus Fe(CN)^ can be distinguished from Fe(CN), in solution.

6 6

SNF can be used as a selective reagent for the

detect Loii of C'rO - , Cr20^ , 1 and Fe (CN) in the sol id

state, and in solution SO can be detected selectively by

means ot the red color formed. NH,C1 + SNP can be used for

the selective detection of I , S20^ , CrO^ and ^r^^^^ in

the solid state at 40°. Fe(CN)^~, Fe(CN)';~ and S2O?" can

also be selectively detected at 80°. In solution this

reagent produces colored products with most of the anions

and can be used as a general reagent.

It is also clear from Table 1 that PPL which

produces a pink color with most anions in solution, can be

2- 2-used tor the selective detection of CO 3 (pink), CrO^

2- 3-(green-yellow), Cr207 (orange) and Fe(CN)^ (yellow) in

the solid state. Amongst all the anions tested, only CO3

and HCO3 give colored products, violet and pink

respectively, in the solid state at 100°, leading to their

specific detection.

103

.2-in particular SCN , VO^ , CrO^ , 612©^ , i^w.^/.

In solid-state spot-tests PHS has been used as a

source of hydrogen ions [8]. We have observed that it is a

good reagent for the selective detection of a few anions,

Fe(CN)?" and

Fe(CN)^ . it produces an unstable violet product with SCN

at 30". With VO3 an orange color in the solid state and a ) _

dark brown precipitate in solution are observed. CrOt

shows an interesting pattern of color changes with PHS at

30". U gives orange product which changes through yellow

to brown in the solid state, whereas in solution a stable

orange-yellow color is obtained. However, at 80°, a green-3-

yellow color appears in solution. FeiCN). forms a yelJou

product in the solid state and a green one in solution.

4-Fe(CN)j. gives a blue product in both the solid state and

in solution.

KIMMHS does not react with NO3 in Che solid statu

but gives red color with it in solution. CI and I react

only in solution, to give a yellow color and a dark brown

precipitate respectively. BrOo gives blue precipitate in

solution at 30" and in the solid state reacts to give an

orange ' red product. Other anions which react only In

2- 2- 2 -solution arc SO, (red-yellow), COo (yellow), Ô'Â

(yellow), PO^" (yellow) and Fe(CN)^~ (red-yellow). The

colors tormed with IO3, VOo,S , SO^ S„0^ , CrÔ-, and

Fe(CN)^ are different in the solid state from those in

104

solution. AL 30', ^nO. gives a brown precipitate along

with a violet gas but at 80° a red solution. Similarly,

MoO, gives blue precipitate along with a brown gas at 30",

and a green solution at 80°

Na„MoO/+PHS produces yellow-brown and violet

products with I and SCN respectively in the solid state,

whereas in solution it gives a green-blue color with 1 and

veilow with SCN . In the solid state HCOo produces a

.2-colorless gas and no color, whereas CO- gives a light

vellow product along with evolution of a colorless gas. Br

does not give any colo; in solution, but in the solid state

Mives a yellow-brown (30°) or blue-yellow (80°) product.

VO3 produces a blue-red product at 30° and a red-brown one,

2-dl 80 bLiL only a yellow color in solution. Cr.-Oy gives

vellow and blue products at 30" and 80° respectively in the

solid state, and an orange-yellow color in solution. At

elevated temperature Na^MoO,+PHS cannot be used

satisfactorily as a solid-state reagent, because of

possible interferences by its own color.

Feci 3 and FeCK+PHS can be used as general

reagents because they produce colored species with most of

the anions, but they require special care to ensure non-

hygroscopic conditions. Furthermore, their utility in

analytical solid-state spot-tests is restricted because of

105

their own color. The color reactions of FeCl-, with Br , I ,

SCN", VO3, SO3", Fe(CN)^" and Fe(CN)^~ in the solid state

3-differ Lroni those obtained in solution. Fe(CN)(^ and

Fe(CN ) 4- react with FeClT+PHS to give brown and green

products respectively in the solid state, and in solution

both give a blue precipitate. I and SCN give a red

precipitate in solution, whereas in the solid state I

produces dark brown (30°) and green-yellow (80°) products.

2-SCN gives a violet product in the solid state. 32©^ gives

a blue color in solution at 30" and a yellow product in the

sol id state at 80°.

FeSO , ( Nil, ) ,,S0, f PHS can be used for the selective

3- 4 detection o\' VO o (buff), Fe(CN);: (blue) and Fe(CN):

(blue) in the solid state at 30". At this temperature, T

the solid state but produce colors in solution.

2- 2-Cr20-7 and MoO, do not react in

AgNOo reacts with I to give a light yellow color

in the solid state, which changes to black, but in solution

a grey precipitate is obtained. SÔ^ shows similar / D

behavior in the solid state but produces an orange brown 2-

precipitate in solution. With 820^ the red product

initially formed becomes dark-brown and then black.

AgNO^+PHS gives violet and yellow-brown products with SCN

at 30" and 80" respectively, but only a white precipitate

106

is formed in solution. I gives light yellow (30°) and grey

(80') products in the solid state whereas in solution only

4-a grey precipitate (30°) is produced. Fe(CN)g gives light

yellow (30°) and green(80°) products in the solid state and

a grey precipitate in solution.

Capillary solid-state tests

In the solid-state spot-tests, the test material is

triturated with the reagent to form an intimate mixture but

in the capillary solid-state spot-tests the reactants are

only in contact at the interface. Thus, many color

reactions which occur on trituration are usually not

noticed in the glass capillary tests. Therefore, the anions

that give color reactions by the trituration method were

selected for further study in glass capillaries. Tables 2

and 5 summarize some of the results obtained for the glass

capillarv reactions. It is observed that many anions giving

a cul(;r reaction on the spot-plate do not react in the

glash capillary, showing the greater selectivity c:if t. he-

latter lechnique. For example, CrO^~, CrÔy , Fe(CN)^"' and

4 FP((;N)^J do Dot react with p-DAB in the glass capillary but

form colored products on the spot-plate. Thus, p-DAB can be

used for the specific detection of SCN by the capillary

test (lable 3). It gives a yellow product at the junction,

which moves towards reagent, giving a 6 mm length (of color

after 1 hr at 60 ).

c o C < o B o i/1

t -

o 4-1 u: •U)

tu

I

4J O

a 0)

•u •u

I TJ •H

CO

> i

TO

D. m

(N

IT)

rsi

O CM

vO

CM

1-

0 pa O Q 00 O

o CQ

o c 00 U

c •H .-^ E

x:

o o 00 O

c •r-l E

o

o c

1-1

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'• I .

o 00

,—, »

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• - - ,

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PQ O Q

r o (T:;

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pQ Q O

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107

PC

PQ I

I -CQ I

1 ^ PQ

PQ

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> 1

o

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l-j

PQ

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PQ Q PQ 2^ PQ Z

1- 2 : CQ Zi

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>

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PQ Q

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a: o 2;

0 z z o

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• 0

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PQ 1

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CN » •

0< H

> o •

• H

• Pi H

)-< PC 1

p ;

o

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o PQ

o

O z

ro O

P3 U

ro 0 !-

CQ

1 CO 0 1—1

1 <r 0 *—\

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I-5

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CN

PQ S 2 O

,—, s

^ CQ

l-j CQ 1

1 ^ pq

S 2

t ^ l S CQIZ

t ^ P3 e

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r i G ;!

r—1

»- -r-l 1 CO Q . O TO Z U

4-) O P

X I 0

ex.

X3 0)

0 1—1

o o

cu x: 4-1

o 4-)

to V-I

0) U-l 0) V-I

C/5 4J 0)

^ u TO (-1

X)

C •H

•r4

3

C O

• H 4-1 TO E 1-1

0 i-w

T-H

QJ

TO 0

c c •H 3

" x: 4-1

(/I

0 ^ •H 4.) " ^ TO QJ

•H E

> ^ OJ O VH 4 ^

TO ' ~ ' 1—1

TO U -1-1 0) 4J

x; -H 4-1 C O -H

110

I l l

p-DAC can be used for the specific detection of

S I vol low product) at 80° and for the selective detection

2-ol NOo (pink-brown product) or SC3 (yellow product) at

120 by the capillary method. Furthermore, NO3 can be 2-

cl early distinguished from SO 3 on the basis of the length

of the colored boundary: NO3 gives a 4.0 mm boundary length

and SO3 only 1.0 mm after 1 hr at 120°. In both cases the

colored boundaries move towards the test material, showing

that p DAC is the only species diffusing. SCN , SO,, ,

SO, and Cr, 0-, do not react in the glass capillary.

p-TD can be used for the selective detection of

CrO, and Fe(CN)^ by means of the products, green and buff

1espectively, obtained at the junction in capillaries. 1 he

boundary zone moves towards the test material in both

, as for Fe(CN,. . 4 6 2- 4

cases, but is twice as long for CrO, as for Fe(CN, 2- 3-

Interestingly, CrÔ-, and Fe(CN). do not react in 2-

capillaries and hence CrO. can be detected in the presence

4-Similarly, Fe(CN), can be detected in the

3-

of CrÔ 2 2^7

presence of Fe{CN)^

BTA is found to be the most suitable reagent for

f ht' specif [i- deteclion of N0'_; by the capillary method. None

"t Iht anions which give colored products in the

trituration spot-test produces any color at the junction in

the capillary, except N02~. It forms a pink boundary which

112

moves towards the test material, to give a length of 4.0 mni

after 3 hr a I 120'.

Fe(CN) can be specifically detected by observing

the orange product formed at the junction by reaction with

BDHC. NO^ and SCN" can be selectively detected at 80' with

DEAHC as reagent. l", Fe(CN)^~, Cro|~ and Cr20^" do not

react in the capillary at this temperature but at 120° CrO,

3-gives a 1.0 mm red-brown boundary and Fe(CNi, a 2.0 mm

dark green boundary after 1 hr. In all cases the reagent is

the diffusing species, since the colored boundary always

extends towards the test material.

Of all the anions which produce colored products

with CA on the spot-plate, only NOo gives a red-orange ring

at the junction in the capillary within 10 min at 30",

leading to its specific detection. This color reaction is

also specific at 80°. At this temperature NO 2 gives a

violet boundary which develops away from the reagent.-

Hov.ever. at 120 VC3 , MoO'4~ and FelCNjg" also react with CA

to give thin black, red-brown and brown-black rings,

respectively, at the junction, within 3 hr. Under these

conditions NO2 gives a 6.0 mm red-violet boundary which is

specific .

NO2 and N0„ react with p-DAB + PHS to give yellow-

orange products at the junction. In the case of NO^ the

113

product changes to green-brown on keeping for half an hour

at 60", giving a boundary length of 2.0 mm on the reagent

side, and NO2 can be distinguished from NO3 on the basis of

either boundary length or color. Simil arl y , Br~ aixl I~ can be

distinguished from each other by the color of the junction;

I and Br give orange and yellow products respectively

(CI does not produce a color). 10/ gives an unstable

yellow product which at 60° changes to brown within half an

hour, whereas 10 3 forms a quite stable orange product,

which distinguishes it from 10^. The red-brown boundary

(4.0 mm] formed by BrO^ at 60° within half an hour with

p-l)AB^PHS can be utilized to distinguish Br03 from VO3

.. _ 2-K hiii ring), 10, or 10,, (0.5 mm). Similarly, CrO, (thin

ves a

J I \j I JL v_y Q \ \j * .J III III / * t j j _ HI jL J a i -i y 5 \^ 1. \^ ,

orang.e ring) can be distinguished from CrÔ-, which gi

red boundary 4 mm in length. Fe(CN), reacts with p-DABM^HS

to iive a transient orange color which quickly changes to

greenihh-bl ue, v^horeas Fe(CN), gives only a blue product.

The boundary length after half an hour at 60° is 3.0 mm for

the former and 4.0 mm for the latter. In all cases the

colored boundaries move towards the reagent, showing that

the test materials are the only diffusing species.

[3-DAC + PHS gives colored products at the junction

onJv with SCN" (violet), CrO^~ (dark brown) and Fe(CN)^"

(dark brown) within 10 min at 30°. Amongst these only SCN

shows a boundary length of 10.0 mm. Therefore. SCN can be

114

seJecLiveiy delected. CI reacts at 80° to give a dark

brown product (2,00 mm) towards the reagent and a pink

product (6.00 mm) towards the test material after Ihr ; I

and Br do not form colored species, however. Thus, CI can

be detected in the presence of I or Br . NO 2 and NO3 both

react with p-DAC + PHS, NO2 to give a brown boundary (2.00

mm) towards the reagent and NO 3 a violet boundary (2.0 v'^)

towards the test material. Thus, NO 2 can be distinguished

froii NO3 by means of the position of the boundary.

DPA+PHS gives a violet boundary with SCN , yellow

with iip\' and blue with FelCN)"^", NO3, Br03, IO3 . CrO^'

and I r C "" all produce green products. However, the green

boundary moves only in the case of N0„ (1.0 mm) towards

the reagent. Ihus, NO^ can be selectively detected on the

basis of boundary length. The colored boundary formed v ith

SCN gives the greatest length, leading to the selective

detection of SCN .

Capillary solid-state spot-tests can be utilized

for the specific detection of SCN and Fe(CN)^~ with

BTA+PHS and SNP+NH,C1 respectively as reagents. Similarly,

2 -PPI tNH,Cl can be used for the specific detection of CO3 or

HCO3, which form pink products at 120".

PHS can be utilized for the specific detection of

VO at 40° and the selective detection of Fe(CN)^~ or

115

Fe((,N)^ ,\i 80 . I he colored boundaries formed with VO3

(brown) ruid Fr'(rN), or Fe(CN), (blue) do not show anv

niovemenL durinj', 1 hr at 80". It is interesting to note

that o\ 11 anions which produce colored products with

2- 2-Agi\(j , on tiituration, only S^0„ and SÔ , form bl ack-

broun pioducl U I he junction within 10 min at 40 . 1 he

{'o 1 01 e d t:i . r.2-

wh

oundary formed with Sr,0, shows no movement,

ereas ^nOo gives a 2.0 mm boundary length, which

distinguishes between them. At 80°, AgNO^ gives colored

l~ (red), PO " products with Fe(CN)-: (red), PO- , (black-brown), I

(black-brown), CrO" ^ (black) and CrÔ^-^ (black) at the

junction. The colored boundary moves only in the case of

PO^" (2.0 mm, 1 hr ).

Ferrous ammonium sulfate can be utilized for the

2 y a..^ iiww / . Lw ^ g i v e s a r e d r i n g - • e l e c t i v e d e t e c t i o n of NO^ and MoC"/ . NO

(80 , 1 h r ) and a r e d - b r o w n b o u n d a r y 1 .0 m.m. i n l e n g t h M

h.r <it 100 l .A[ i i ongs t a l l t h e a n i o n s w h i c h p r o d u c e c o l o r e d

2-

products (lable 2) at 80°, only MoO/^ gives a boundary

length of 2.0 mm, which can be used for its selective

detec tion.

I can be selectively detected with Na^MoO,+PHS on

the basis of the length or direction (which is towards the

test material) of the yellow-brown boundary formed at the

2-junction move towards the test material . S„0^ forms a

116

brown-black boundary (4.0 mm) at 100° which moves towards

2-the reagent. Thus, S O ^ can be distinguished from I on

this basis, but i ot from the color. VOo gives a red ring at

40 but a brown ring at 80 or 100° . Of all the anions

tested (jiiJy SOo 1 orms a blue ring at 40 or 80". Therefore,

it can be selectively detected. However at 100°, PO/ also

forms a blue ring at the junction.

At 40", SCN reacts with KI + PHS to give a violet

boundary (8.0 mm) towards the test material within 10 min.

Ihe other anions which give colored products at the

junction such as N0„ (red), BrO^ (yellow), 10^ or 10,

(orange), S2O3" (red), Fe(CN)^" (blue) and CrO^~ (brown)

show no boundary movement. Thus, SCN can be selectively

detected on the basis of the boundary length. At 80° the

formation of a 10.0 mm thick blue boundary by Fe (CN)

allows its selective detection.

It is apparent from Table 2 that the color

reactions of some anions are temperature- dependent. For

example S^0„ gives only a red product with KI+PHS at 40",

v;hereas at 80°, it gives two colored species. The red

product formed at 40° shows no movement. However, at 80° in

addition to the red ring, a yellow product (2,0 mm) is also

formed which moves towards the test material, and gives

2-selective detection of SÔo .

117

FeCl ., can be used for the specific detection of

SCN" which gives a red violet ring at 40°. CI , Br and I

can be distinguished from each other at 80° on the basis of

the boundary length measured after half an hour (Table 2).

NOT can be detected in the presence of NO2 because at 80' a

2-red ring appears only with NO^. At 80°, CrO, gives a

2-]ight brown boundary (3.0 mm) after 1 hr , but CrJD-j does

2-not react. Thus, CrO, can be detected in the presence of

2- 2 - 2 - 2-Cr.,Ot and SO,, . S gives a red ring and SO,. forms a

yellow ring with FeClo.

it is clear from the discussion above that the

capillary solid-state spot-tests are more selective than

the conventional solid-state spot-tests and many anions can

be selectively or specifically detected by performing the

tests in a capillary. Table 3 summarizes the results

obtained i or the sel ective/specif ic detection of some

anions by tlie capillary method. The selectivity of the

capillary technicjue can be further enhanced by introducing

a glass-wool plug into the middle of the capillary, between

tli£ reactants. In this way the reactants do not come in

direct contact and the reaction, if any, proceeds by vapor

phase diffusion of the reagent or test material through the

glass-wool plug. Such reactions may be termed solid-vapor

phase reactions rather than solid-solid reactions. The

glass-wool technique was applied to the anions which gave

118

TABLE 3

Specific/Selective Capillary Solid-State Spot-Test for some Anions

Anion

.se>r

s^-

SX)f

NO 3

CrO^'

Fe(CN)

FL'(CN)

XO^

S( N

NO 9

SC N

NO

Fo(CNj

I

VO^

SON"

CO'J"

H(0^ J

4-6 3-6

4-6

Tern

Reagent

1

5

5

5

7

iU

1 ]

11

I i

14

17

17

19

22

25

27

perature,

°C

60

80

LOO

120

40

100

80

120

30

120

80

80

120

40

40

40

120

Time

1 hr

1 hr

1 hr

1 hr

12 hr

1 hr

1 hr

3 hr

10 min

1 hr

1 hr

1 hr

1 hr

10 min

10 min

10 min

1 hr

Color of boundary

Y

Y

LY

RBr

G

Buff

0

Y

V

PK

PK

PK

V

Bl-BK

0

Br

R-V

BK Br

BK-Br

LFK

LPK

Direction of movement

TR

NM

NM

TM

TM

TM

TM

NM

TM

TM

NM

TR and

TM

TM

NM

NM

NM

NM

TM

NM

TM"

TM"

Length,

mm

6.0

-

-

4.0

4.0

2.0

4.0

-

2.0

4.0

-

1 6 . (J

10.0

4.0

-

-

~

2 0

-

"The portion of the tube filled with the test material becomes

oo-[)leLcly i ink. All abbreviations as in Table 2.

119

TABLE 4

Glass-Woo] Plug Modified Capillary Solid-State Spot-Test for some

Anions

.-\n i on

NO 2

NO"

Br"

Hie J

\\('0 ,

HrO

'^'\

Si K

V(i

co:

MoO;

•' ' 6

•f ( ( N )

Rc aoeiiL Teinper ature, Time, Color of °C hr boundary

2

4

2

2 7

.2 0

2

2

2

20

21

27

2

65

40

65

120

80

80

65

65

65

65

80

80

100

120

65

65

25 80

65

65

65

8

8

8

]

0.5

1

8

2

G at GW/R

G-)B1 at GW/R

Y at GW/M

LPK" at GW/M

Y at GW/M

0 at GW/M

G at GW/R

DBr at GW/M

8 DBr at GW/M

0.5 0 at GW/R

0.5 0 at GW/M

1 Y--)Br at GW/M

1 Y- Br at GW/M

1 LPK" at GW/R

0.5 0 at GW/R

2 Y at GW/M

1 BK at GW/R

2 G at GW/M

8 G at GW/M

8 Bl at GW/M

Length, niiii

1.0

2.0

5.0

2.0

6.0

2.0

4.C)

2.0

6.0

GW/R ' glass wool/reagent junction CW/M glass wool/test material junction ^_ " Whole tube containing test material (CO^ or HCO- ) beco-nies pink — > indicates a change in color Other abbreviations as in Table 2

Reaênt 2 / NH, C] +PP1 4

120

colored products in glass capillaries, and the color

reactions observed are shown in Table 4 and show that the

glass-wool technique is superior to the ordinary capillary

technique in terms of selectivity and specificity.

Applications

For obvious reasons (the main one being the

complexity of the ork thatwouldbe involved) the investigation

was restricted to the sodium and potassium salts of the

anions involved. It is a fairly simple matter to devise a

systematic scheme for identification of one of these salts

in the absence of the others, as shown in Scheme 1. It is

also possible to device a systematic scheme for analysis of

a mixture containing almost all the anions examined in this

work. The major problem in devising such schemes is that in

contrast to schemes based on physical separation by

precipitation or extraction, each test is applied to a

j:)ortion of the original sample, so all the components are

present in every test performed. Hence the classification

i .- based on elimination of groups of anions from

consideration, by their failure to give a color with a

jjarticular group reagent. For instance, if no color is

obtained with DPA+PHS in the trituration test, acetate,

molybdate and phosphate are the only anions (of the 25 in

the scheme) that may be present. However, if a color is

S A M P L E

ORANGE

2 -C r ^ O /

1

r

1 NOCOLOR 121

IL G R F E N i ' Y E L L O W

C r O 2-

NO COLOR

O R E E N — K ' Y E L L O W NOCOLOR

3 - | l 2 ftiCU)^-'

COLOR

PINK

5 C N -

NOCOLOR PINK

NOCOLOR

NOCOLOR

12 CO 2 -

BROWN

VO3-

i l COLOR

In

1 NOCOLOR

YELLOW NOCOLOR

I " f?EO

( 3 R 0 W N

B r O - r r

N O C O L O R

1" O R A N G E - Y E L L O W N O C O L O R

B r ' 26

r R E O — « - B L A C K

S 2 O 3 2 -

n NO CO LOR

25

Y E L L O W - B R O W N NO COLOR

1 ) 1

( Y E L L O W

C H 3 C 0 0 "

( BROWN

NOf

NO COLOR

111 NO COLOR

Is r '

COLOR NOCOLOR

C 0 L 0 W

.All-N O C O L O R

NO C O L O R

V t L L O W NO3-

2 3 ( '

B L U E — a - G R E E N

I O 3 -

r

N O C O L O R

L IGHT Y E L L O W

NO COLOR P O / , 3 -

h9

NO COLOR

ORANGE

^ 0 , 2-

I

N O C O L O R

L IGHT Y E L L O W

B R O W N COLOR

20

MoO 2-

SO 2 -

n Y F 1 l o w

H C O i

„^J NOCOLOR

LIGHT YELLOW 2-

NOCOIbR C20^

I' YELLOW

cr Scheme 1 Reactions in a left-hand branch are performed before

those in a right-hand branch. The reagent numbers refer to Table 1.

I — REU

SpOe^

SAMPLE

NO COLOR

NOCOLOR

PALE YELLOW NO COLOR

i2i COLOR

1

NOCOLOR

15

122

ORANGE

5 0 3 ^ '

NOCOLOR LIGHT YELLOW

25 CpO^^-

Y ELLOW •'BROWN

Scheme 2

123

obtained, these three anions may also be present in

subsequent tests, so their reactions with all subsequent

reagents have to be taken into account in working out the

scheme.

On the other hand, it is comparatively easy to use

I able 1 to find small groups of anions that do not give a

color with a given reagent, and then to devise

Identification schemes such as that in Scheme 2.

[he capillary tests can similarly (and more easily)

be organized into a detection scheme, since additional

information is provided by boundary movement of the

products. Devising such schemes is an excellent exercise in

logical thinking, as a part of student training.

As far as we are aware, this is the first attempt

at systematic solid-state analysis for anions. Further

publications will be concerned with the chemistry of the

new reactions described.

124

REFERENCES

1. M. Qureshi, H.S. Rathore and A. Mohammad, Talanta, 23,

874 ( 1976) .

2. M. Qureshi, A. Mohammad and G. Ganga Raju, Talanta,

28, 817 (1981).

3. M.N. Akhtar, H.S. Rathore and M. Qureshi, Talanta, 25,

235 (1978).

4. A. Mohammad and N. Fatima, Microchem. J., 37, 161

(1988) .

5. M.D. Cohen and B.S. Green, Chem. Brit., 9, 490 (1973).

6. F. Feigl , Spot Tests in Inorganic Analysis, 6th Ed.,

r. Isevier, Amsterdam, 19 72.

7. W.J. Williams, Handbook of Anion Determination,

Butlerworths, London, 1979.

8. P.I. Voskresenskii, Talanta, 12, 11 (1965).

CHAPTER - III

MICROGRAM SEPARATION AND SEMIQUANTITATIVE DETERMINATION OF ANIONS

125

In I'ecent years thin-layer chromatography (TLC) has

grown much in status and has experienced a dramatic surge

due to its simplicity, versatility, and low cost. TLC

provides good resolution and is comparatively fast. TLC,

with optimization of techniques and materials, can be

applied for the quantitation of various compounds present in

environmental, geological, and biological samples. Some

recent applications of TLC such as the identification of

perchlorates in explosive residues [l] and the determination

ol' selenium in foodstuffs [2], total heavy metals in

industrial and waste waters [3], ortho- and polyphosphates

in soft drinks [4], and Hg{II) in river and industrial waste

waters [5] have shown its utility as an effective, rapid,

and simple separation technique. Some important separations

involving microgram to milligram quantities of elements have

also been reported recently [6-8].

As evident from the literature survey, few workers

have attempted the TLC of anions [9-12], in contrast to that

of cations [13-20]. Thin layers of stannic chloride,

cellulc)se, silica gel, silufol , and alumina have been used

for the separation and identification of anionic species. In

most of the cases ammonia [21,22] in combination with

alcohols and ketones has been selected as the mobile phase.

Aqueous salt solutions and aqueous-organic systems

containing mineral or carboxylic acids have also been tried.

126

Recent investigations by Mohammad et al . [23] regarding the

effect of solvent composition on the mobility of anions show

that two component systems containing formic acid mixed with

acetone, ethyl methyl ketone, butanol, or isopropanol were

most useful in producing differential migration of anions.

Our work on TLC of cations [17,24,25] in mixed

organic solvents containing formic acid has clearly

established the practical applicability of these systems as

eluents for several analytically difficult separations on

plain silica gel as well as on slice gel layers impregnated

with aqueous salt solutions. The improved separation

possibilities of cations on impregnated silica gel layers

suggested us to study the applicability of impregnated

layers in the analysis of anions. The present paper

describes a thin-layer chromatographic study of anions on

silici gel impregnated with some aqueous salt solutions.

Mixed iqueous organic solvents containing formic acid have

been used as eluents.

It has been possible to clearly separate the anions

at microgram to milligram levels over a wide pH range oi

sample solutions,

EXPERIMENTAL

Apparatus. A thin-layer chromatographic apparatus (Toshniwal,

India), ^0 x 3.5 cm glass plates, and 24 x 6 cm glass jars

127

were used. An El ico Model LI-IOT pH meter was used for pH

measurements.

Test solutions. The test solutions (17o) were either sodium

or potassium salts of ferrocyanide, ferricyanide, chromate,

dichromate, tungstate, iodide, bromide, phosphate,

molybdate, vanadate, nitrate, nitrite, bromate, iodate,

permanganate, oxalate, except SCN (ammonium thiocyanate).

Double-distilled water having a specific conductivity

K - 1.5 X 10 ohm cm at 25°C was used for solutions.

Reagents. All the reagents used were of analytical grade.

Detectors. For the detection purpose the following reagents

were used:

Sc '0, aturated AgNO., solution in methanol for Br , I , PC, ,

CrO, and CroO^ .

2. DiphenyJamine (0.2-0.5%) in 4M H2SO, for lO" BrO" NO^,

NO^, VO" W0,^~, and MnO" J J 4 4

3. Ferric chloride (10%) in 2M HCl for SCN~, Fe(CN)^~, and

Fe(CNl-^~. b

4. Alcoholic pyrogallol (O,57o) solution for MoO , .

2-5. Aqueous potassium ferrocyanide (1%) for CoO , .

Mobile phase. The following solvent systems were used as

mobile phases:

128

S - Formic acid:acetone (1:9)

S2 DMSO:acetone (1:8, 3:6, 6:3)

S^ - Formic acid:DMSO:acetone (1:1:8, 3:1:6, 1:3:6,

5:1:4)

S^ - H2S0^:DMS0:acetone (1:1:8)

S^ - HclO^:DMSO:acetone (1:1:8)

S^ - HCl:DMSO:acetone (1:1:8)

In all the solvent systems, HCl, HCIO,, H^SO,, and

formic acid (FA) were O.IM aqueous solutions while the

acetone and DMSO (dimethylsulfoxide) were used as received.

Stationary phase. The stationary phases were:

(a) Plain silica gel,

(b) Silica gel impregnated with 0.17o aqueous

solutions of CuSO/ , ZnSo., NiCl^ , CoCl^ , a ncl

CO(NHT).C1T. 3 6 3

Preparation of TLC plates.

(a) Plain silica gel plates: Silica gel was mixed with

conductivity water in the ratio 1:3 with constant

shaking for 5 to 10 min. The resultant slurry wab

coated on well-cleaned glass plates to give a layer

approximately 0.25 mm thick. The plates were dried at

room temperature (30°C) and then heated at 100jt 5°C for

1 hour. After activation the plates were stored in an

air-tight chamber.

129

(b) The impregnated plates were prepared by mixing

an aqueous solution of 0.17„ copper sulfate,

zinc sulfate, nickel chloride, cobalt chloride,

and hexamine cobalt III chloride with silica

gel in 3:1 ratio. Thin layers were then

prepared in a similar fashion as described lor

plain silica gel plates.

Procedure. One or two drops of anion solutions were spotted

on the plates with fine glass capillaries. The spots were

dried and the chromatoplates were developed, allowing the

s(5lvenl to ascend to 10 cm from the starting line in all

cases. After drying the spots were visualized using the

appropridte reagent.

For the study of the loading effect on the K,-,

values, the known volumes of standard solutions of anion

were spotted on the chromatoplates with a micropipette. The

plates were developed with 5^(1:1:8). The spots were

detected and their R. (R„ of leading front) and Rrp(R„ of L F ° I F

trailing front) values were determined. The areas of the

spots produced at different concentrations of anion were

also calculated. Standard solutions of 10., and BrO^

(2.5-107.], NO " (2.5-407o), and T (2.5-80%) were used.

For semiquantitative determination of BrOZ and N0.~,

0.01 ml of various standard solutions of KBrO^(1-10%) and

130

NaNO. (2.5-407„) were spotted on silica gel impregnated with

0. 1';' CuSO, layers. The chromatograms were developed with

S^fl:l:H). After detecting the spot, it was copied onto

tracing paper from the chromatopl ates and then the spot area

was ca1cu1 a ted.

In order to achieve the separation of anions at

different pH values, the pH of the test samples were brought

to the required value by the addition of either glacial

acetic acid or dilute sodium hydroxide solution.

Ihe liiiiits of detection of various anions were

(ietermined by spotting different amounts of anionic

solutions on the chromatoplates. The plates were developed

and detected. The method was repeated with successive

lowering of the amount of anion until no detection of the

spot was achieved. The minimum amount of anion just

detectable was taken as the limit of detection.


Ihe silanol group of hydrated silica gel is weakly

acidic and imr'iersion in an aqueous salt solution causes some

catlDH exchange through a reversible reaction. This cation

M"' + m(- SiOH)^=iM{OSi-)";;;'" + mH".

exchange in the normal pH range of 4-7 is very smail ; n is

the charge of the unhydrolyzed ion and is equal to m for

131

trionovalenL ions. Thus the formation of a metal-surface

complex on the surface of silica gel brings about a change

in the retention behavior of the silica gel surface toward

inorganic species.

The results of this study have been shown in Figs.

1-4 and Fables 1-4 . In many cases it was found possible to

separate one anion from several anions. The R^ values for

all anions on copper sulfate-impregnated layer showed

excellent reproducibility (variation does not exceed 51 of

the average value), except NO^ which gave a variation of

8-1 (); from the average K„ value. Among the solvent system

used. FA:DMSO:acetone (1:1:8), i.e. S2(l:l:8) was the best

and the silica gel layers impregnated with 0.1?o copper

sulfate gave better results compared to other impregnants.

Therefore, the chromatographic system consisting of 0.1%

copper sul fate-impregnated silica gel layers as the

stationary phase and S. (1:1:8) as the mobile phase was

selected for detailed study. Figure 1 illustrates the

dependency of R„ values of anions on the nature of

impregnants. Aqueous solutions [O.Vi) of the chlorides of

24 3+ 2+ 2+ 2+

Co , Co , or Ni and the sulfates of Cu or Zn were

used as impregnants and anions were chromatographed on the

impregnated silica gel layers with the S^ (1:1:8) solvent

system. There is a group of anions that are strongly

absorbed or have a little mobility, and a group of anions

132

100

o o

X

a:

L L

rr sz

o o

CE

Z inc s u l p h a t e

N icke l c h l o r i d e

o o

ex

r.

100 r

100

60

20

r C o b a l t ch lor idt H e x a m m ? c c b a H III c h l o r i d e

_ l . J.

o 7

: 4 i—

o O O ° c a > u 2

Anions

F i g . 1 Coni [ )a r i son of R v a l u e s of a n i o n s on d i f f e r e n t

i i i p r e g n a n t s w i t h FA : DMSO : A c e t o n e ( 1 : 1 : 8 ) s y s t e m .

& 2 Compact s p o t w i t h R, -R^ < 0 . 3

k A B d d l y t a i l e d s p o t s v ; i t h R -R > 0 . 4

133

(NO" SCN", r , and BrO~) migrating with the solvent front

giving high K,. values. Br~ could not be detected on the

impregnated layers, whereas it was easily detectable on

plain silica gel layers. In most of the cases CrO^,Cr2(J7,

and Fe(CN).^~ produced tailed spots. The tailed spots in the

case of Cr0| and Cr20^~ are possibly caused due to the

coexistence of the following species in equilibriun as

expected in acidic media.

Cr20^" + UÔ :^-^. 2HCrO^

HCrO: r± H' ^ CroJ"~ 4 4

3-However, the tailing of Fe (CN) spot seems due to

3-the incomplete precipitation of Fe(CN)(^ by the zinc present

as inpurity in silica gel.

Figure 2 summarizes the adsorption behavior of

anions in DMSO:acetone systems containing varying concen

trations of DMSO and acetone in their mixture. DMSO being an

aproLic dipolar solvent with hard oxygen and soft sulfur is

a good solvating agent for anions, while acetone does not

solvate the ions. There is a gradual and slow increase in

the Kp values of anions with increasing DMSO concentration.

NO2 and BrO^ showed a sharp increase in their R^ values uith

increase in DMSO concentration. DMSO, being a stronger

solvent than acetone, interacts strongly with the-solute,

decreasing its adsorption and causing faster migration.

134

rn U3

CD

CO

_i__ o T " "

..J. _ __ J. ao

O

.i_._^ I.,

o o o o 6

c o

- • ->

o <

o to

o

c o

u <

o

Q

1

-^

o

1

CI

o

L rs |

O

•r-<

c o

-v (U

ex

oc o •U 03 E O 1-1

o

(J) c o

•r-l

c CD

o

1—I

03

> Di a

0 1

CO

o

c o

4-1

c 0) u c o u

o

o OJ

CN

00

ex o o

o

CU

cx

OJ

CO

OJ

00

CU

o 0)

4-J

CU ~0

u o

o

o o

u

135

However, the slow evaporation of DMSO from chromatoplates

• iftc-r (.heir cievc 1 opincnt . longer dcvel opiiicnt Lime, .md i oor

detection of anions hamper its applicability at higher

concentration.

There is a little effect on the R^ values obtained r

for KI, KBrO,^, NaN02, and KIO^ between the pH limits of 2.5

to 12 (Fig. 3). I he formation of highly compact spots at d1 1

pH values permits a reliable and reproducible separation of

IO3- 1

i.iethocl can be appl fed for the separation and removal of 10^

from acidic, neutral, and saline waters.

3 iron' NO^ , Br03and I over a wide range of sample pH. I his

An attempt has been made for semiquantitative

deterniination of anions by the measurement of the spot area,

l.ie spots were directly drawn on a piece of transparent

ivtper t'roi;! Lhin layer chroma tograms and the area of each

spot vvjs calculated. A linear relationship was only obtained

for N(K and Br0 7 when the amount of the sample spotted was

plotted against the area of the spot (Fig. 4 ) . The precision

and accuracy is always below + 15%. A similar relationship

has been reported for cations [26] and°^-tocophenols [27].

The results of chromatographic behavior of anions in

robilc phases containing variable proportions of DMSO,

acetone, and formic acid are shown in Table 1. The clarity

of detection and compactness of spots increase with the

136

u

0 8 10 12

PH

Fig. 3 rffect of pH of sample on Rp values of anions

J L OD

o o o

o o 00

137

o o CO

O o <r

en =L_

^ •*->

c D O

E <

o o CNl

-v CO

O

O O

o o

o

on

o o CNl

c D O

E <

o c CO

o 4-) c o E CO

05

> CO CU •H

CO

4-) O a •J)

o 4-1

O

cn O

pa

o

•r-l fa

f UJO) D 2 ) J V

138

I

o CO

O

c o

u <

t3

c o

o <

c TO

:- s

c •H c TO

0 o

c > o

o •H c <

in

;2

o

<r o in

I/)

0)

; 03

-J •o 0)

TO

c 00 CD u

a E

u

O 00

<

c o CU 0 0 u •• ro ,-1

o ^ en — Q

0) u. o •IJ .— dJ ex; o ••

i n o

o o

LO

U-1

ON

TO 00 CNi

TO

Q

LTl

i n o o a> z

X ) u c o o

LO

o c o u — (1) OC

o •• TO - J

O (73

i n

o o o

i n

00 OO

TO r--rs)

TO i n

m rs)

Q 2

i n

a> o o

r^ r--

m

i n CO

i n i n TO

rsi CNI

O o

l / ^

a

TO

r^ ro

TO

m CO Q Z

. ^ a

o o

. r^ 0 0

Q Z

^

T H

r o

00

T , (D

vn

m c^

•> o

i n

• rsj i n CTN

CO O

i n

<r en

r-

c

00 Q

m

i n

c r

O m a

i n a^

o o

i n

• m ON

i n

• r~-c^

i n

in m

rsi

o o

m T-H

CM

O

i n CTN

i n

1-1

a>

C7> 00

TO O en

TO i n

vC

TO i n

o ro

TO O CO

TO i n

^ CO

TO

o CO

Q Z

m <r o

Q z

Q Z

v£>

i n

CX> 00

o o

o o

o o

Q z

i n

T—1

a

ro ON

G Z

o z

i n

• < ) •

00

1 0 0 ^

Z o ^' o

U H

1

z o • — •

QJ LM

1 Z o 00

1 csi<r

o ;-. o

1

O r\(

)-. o

; v-

DQ

1 CO < t

1 C t—I CL.

1 O l

o z

CO

z

i n C

o 0^

c o

o o

o o

o o

m o

oc c o

o c

o o

c c

c o

lO

o c

;^

cr X

c o

^ ^

L ' -

. r^

— m ' J cr-

X O O I I

I I

I "^ C O "•J-J" I ' ^ O C C >- C C C OJ C

X) 0) 4J

o CU

u 0)

X )

4-1

c c *-

Q z;

•' •U

c a w 0)

,—1

- D 3 O -o

• 00 Q

• • Q) U 0 2

. <t

• o A

1 J

i J •H

' 4-1 O

a CD

•o OJ

, — 1

•H 03 i J

> , — 1

-o 03 pq

CO

• CO

o

'X \

oT x: XJ •r-l

3

4-1

o a en

-o OJ

1—1

•H n3

H X i

139

140

TABLE 2a

Quantitative Separation ofIO3 fromBrO , NO , I , Br , and SCN on 0.11

CuSO,-Impregnated Silica (Jel Layers Using FA:DMSO: Acetone (1:1:8)

Loading aiount of individual

anion salt in mixture

Separations

(R L R,

^'3

30 ug

0.4 m g

")0 ug

' ) . - . .10

KBrC)^

0.2 5 mg

0.5 mg

0. 1 mg

NaN02

1.0 mg

2.0 mg

0.2 mg

1O3 ( 0 . 1 5 - 0 . 0 :

1O3 ( 0 . 1 7 - 0 . 0 :

1O3 ( 0 . 3 5 - 0 . 0 :

- BrO" (0.96-0.77

- BrO^ (0.98-0.56

- BrO^ (1.0-0.82)

NO; IO3 (0.25-0.0)

IO3 (0.27-0.0)- NO2

IO3 (0.36-0.0)- NO2

(0.98-0.6 7

(1.0-0.51)

(1.0-0.78)

KI

K) pg

0.4 mg

lUO pg

1.0 •• g

1 .0

2.0

4.0

0.4

KBr

4.0

7.0

mg

mg

mg

mg

mg

mg

4.0 mi

I03

I 0 3

I 0 3

I03

I 0 3

I03

I 0 3

(0

(0

(0

(0,

(0,

(0.

(0.

.22-0.0)

.21-0.0)

.34-0.0)

.35-0.0)

,41-0.0)

,4-0.0)

4-0.0)

- I

- r - i~

- i~

- Br~

- Br"

- Br~

(1

(1

(1,

(1

(1,

(1.

(1.

.0-0

.0-0

.0-0

.0-0

.0-0,

,0-0,

,0-0.

.89)

.84]

.75)

.87)

,69)

,65)

81 )

0.4 ro

NH^^SCN

10 yg IO3 (0.3-0.0) SCN (1,0-0.81)

Note: The synthetic mixture of IO3 with SCN (concentration 17o) results

in the formation of precipitates.

141

TABLE 2b

Quant i t ati vo Separation of NOT, from I , SCN , and Br on 0.17o CUSO,-

Inipro.' nat-cd Silica GeJ ] >ersUsi)ii/ Formic Acid : AcetoneC 1: 9 ) as Mobile Phase

Loading amount of individual Separations

anion salt in mixture (R D L "T'

N iNO, K[

^0 jj^,' 0.125 mg NO" (0.6 3-0.43) - I~ (1.0-().87)

0.25 mg NO^ (0.62-0.42) - l" (1.0-0.91)

0.5 mg NO" (0.75-0.55) - T (1.0-0.85)

1.0 mg NO2 (0.77-0.57) - l" (1.0-0.83)

0.3 -.g 1.0 mg NO" (0.65-0.39) - l" (1.0-0.80)

1.0 mg NO" (0.65-0.32) - I~ (1.0-0.78)

2.0 mg NO2 (0.67-0.11) - I~ (1.0-0.80)

NH^SCN

•;0 Mg 0.125 mg NO" (0.69-0.56) - S C N " (1.0-0.88)

0.25 mg NO2 (0.65-0.43) - S C N " (1.0-0.79)

0.5 mg NO2 (0.82-0.69) - SCN~ (1.0-0.89)

0.5 mg 0.5 mg NO2 (0.70-0.50) - S C N " (1.0-0.88)

l.O mg NO2 (0.60-0.31) - SCN" (1.0-0.79)

2.0 mg NO2 (0.63-0.26) - SCN" (1.0-0.75)

KBr

100 Mg 0.5 mg NO2 (0.72-0.51) - Br" (1.0-0.9)

1.0 mg NO2 (0.82-0.64) - Br" (1.0-0.91)

2.0 mg NO2 (0.80-0.72) - Br" (1.0-0.95)

4.0 mg NO2 (0.79-0.62) - Br" (0.96-0.86)

^•0 ,iig 0,25 mg NO2 (0.74-0.50) - Br" (1.0-0.85)

0.5 mg NO2 (0.87-0.56) - Br" (1.0-0.94)

Note: Detection of Br is difficult, requiring about 20 min after

spraying the chroriatogram with the reagent.

142

TABLE 3

Separations Achieved Experimentally at Different pH Values of

Sample Mixture on Silica Gel Layers Impregnated with 0.17o Copper

Sulfate Using FA:DMSO:Acetone (1:1:8) as Mobile Phase

Sar-ple pH value Separations achieved R ^ - R ^ :

2.6 IO3 (0.12-0.0) - BrO^ (0.95-0.85)

5.4 I0~ (0.12-0.0) - BrO^ (1.0-0.801

11.7 IO3 (0.18-0.0) - BrO^ (1.0-0.85)

2.5 IO3 (0.06-0.0) - I~ (1.0-0.95)

6.2 IO3 (0.05-0.0) - I" (1.0-0.93)

11.5 I0~ (0.22-0.0) - r (1.0-0.96)

2.4 10^ (0.07-0.0) - NO^ (1.0-0.90)

6.4 10^ (0.09-0.0) - NO2 (1.0-0.831

11.0 IO3 (0.18-0.0) - NO2 (1.0-0.85

Note: Ihc amounts of I and NO^ are taken in twofold excess in

the r;iixture to ensure sharp detection.

143

increase in the concentration of acetone in the mobile phase

and hence FA:DMSO:acetone (1:1:8) was the best solvent

system in this regard. CrO?" , Cr20^~ , and FelCN)^" gave

double spots when eluated with FA:DMSO:acetone (5:1:4). Ihe

Kp value of anions obtained in O.IM mineral acids (H^SO, or

HCIO, or HCl ):DMSO:acetone (1:1:8) are also recorded in

lable I. These acids can be put in the following preferred

order if used as eluent in combination with DMSO and

acetone:FA>HC10,>HoSO, cr^ HCl . Results of quantitative 4 Z 4 '— ^

separation of lOZ and NO^ from large excess of I , SCN ,

Br , and BrO-T and vice versa are given in Tables 2a and

2b. The proposed method is very convenient for separating

milligram quantities of an anion from microgram to milligram

amounts of other anions.

Ln order to widen the applicability of the important

separation of 10^ from I , N0~ and BrO^ , its separation

from synthetic mixtures of different pH values has been

investigated. Table 3 reveals that 10^ can be easily

separated from I, NOj and BrOo" in the pH range 2,4-11.0 of

sample soUition. Thus, the method can be utilized Lo

separate iO ' from acidic, neutral, and alkaline (natural or

sviithetici samples containing I , NO2 , and BrO"; without

adhering to close control of sample pH.

144

TABLE 4

Limits of Detection and Dilution Limits of Anions as Their Salts

on Silica Ge] Layers Impregnated with 0.17o CuSO, Solution, Using

HCIO, .-DMSOrAcecone (1:1:8) as Mobile Phase 4

Sample Ions Salts

10

1 I

l.

1 3

14

1 T

V4

MoOr NaoMoO, .ZHÔ 4 z 4 z

WO^" Na2W0^.2HÔ

Côf Na2C20^

1 KJ

VO^ NaV0..H20

l'o(tN)/" KjFetCN)^ o J b

h ''( h /

10^ KI0

BrO^ KBrO.^

S( N~ NH-SCN

NO; NaNO^ z

MnO/!" KMnO,

CrO;" K,,CrO, 4 / 4

LrÔ^" K2Cr20^

Limit of detection

( Mg)

100.0

100.0

100.0

10.0

10.0

10.0

10.0

10.0

^.0

1.0

1.0

1.0

0.5

0.5

0.5

Dilution 1imit^

1 ilO"

1 :10^

1 ilO'

1 : ] 0

1:10-

1 :10-^

1:10^

1:10^

1 :2xl() '

1 .-10^

1 ilO'^

] :10^

1 :2xl(/*

1 :2xl0"^

1 :2xl0'^

"Dilution limit - l:(Volume of test solution x 10 )/

Limit of detection ( pg)]

145

lable L\ summarizes the limits of detection of soiiie

anions as their metal salts along with their dilution

limits. I(. is evident from this table that the proposed

method is highly sensitive for the detection of several

anions,

146

REFERENCES

1. J,E. Chorstowski, W.O. Thurman and J.J. JaworskL,

Arson Anal. Newsl., 5, 14 (1981).

2. leresd Moreno - Deminguez, Concepcion Garcia - Moreno

and Abe] Marine - Font, Analyst, 108, 505 (1983).

i. M.P. Volynets, L.P. Kitaeva and A.P. Timerbaev, /h.

Anal . Khiin. , 41, 1989 (1986) .

4. Y. lonogai and M. Iwaida, J. Food Prot., 44, 835

(1981) .

3. M. Ajmal , A. Mohammad, N. Fatima and A.H. Khan,

Microchem. J., 39, 361 (1989).

b. •1. A|nial , A. Mohanimad and N. Fatima, Microchcm. 1.,

37. 314 (1988).

/. A. Mohaiiiinad and N. Fatima, J. Liquid Chromatogr. , 10,

1 34 i (1987) .

8. M. AJmal, A. Mohammad, N. Fatima and J. Ahmad, I.

liquid Chromatogr., 12, 3163 (1989).

9. H.M. Chawla, N.N. Ralhan, N.K. Garg and S.S. Chibber,

J. High Resolut. Chromatogr. Chromatogr. Commun., 3,

651 11980J.

10. K. Kawanbc and K. Maruyama, Yakugaku Zasshi, 101, 912

(1981 ) .

147

11. r,. Buchbauer and R.E. Markis, Sci. Pharm., 51, 48

(1983) .

12. K. Ravindra Nath and P.B. Janardhan, Chem. Abstr., 97,

65573ni (1982); Ind. J. Chem., 21A(2), 150 (1982).

13. K.r. DenBleyker and T.R. Sweet, Chromatographia, 13,

Ll^ (1980).

14. M. Ajmal , A. Mohammad, N. Fatima, and J. Ahmad, J.


15. 1. Shimizu, H. Tanaka and T. Ohsawa, Chromatographia,

15. 177 (1982).

lb. 1. Shimi/u. E. Uehara and M. Ohtani, Chromatographia,

2K 1 75 (1986) .

1/. A. Mohammad and N. Fatima, Chromatographia, 25, 536

{ 1988) .

18. K. Ishida, S. Ninomiya, Y. Takeda and K. Watanabe, J.

Chromatogr., 35, 489 (1986).

19. N. Fatima and A. Mohammad, Sep. Sci. Technol., 19, 429

( 1984).

20. D.S. Gaibakian, J.K. Rozylo and M. Janicka, J. Liquid

Chromatogr., 3, 2969 (1985).

21. r. Okumura, 'lalanta, 26, 171 (1979).

2 . ^ . J. Franc and E, Kosikova, J. Chromatogr., 187, 462

( 1980) .

148

Z ^ . M. Aj'nal N A, Mohammad, N. Fattma and J. Ahmad, J,


24. A. Mohnnimaci and N. Fatima, Chromatographia, 23, fi3J

(1987).

25. A. Mohammad and N. Fatima, J. Liquid Chromatogr., 9,

1903 (1986).

26. A. Mohammad and N. Fatima, Chromatographia, 22, 109

(1986).

/ . -A Scher. Mikrochim Acta, 308 (1961)

CHAPTER - IV

IDENTIFICATION AND SEPARATION OF SOME ANIONS ON PLAIN AND MIKED ADSORBENT

LAYERS

149

Thin - Layer Chromatography (TLC) today is a

dyna:iiica 1 1 y developing modern analytical technique because

of its simplicity, rapidity, wide spectrum of selectivity

and iniproved efficiency. The separation of ions in TLC is

generally governed by the physical interactions of the

adsorbent and the coordinative properties of the mobile

phase. As a general practice, the composition of mobile

phases is usually altered to achieve a desired separation

on a particular adsorbent. From the literature [1-4] the

following four main approaches concerning the mobile phases

currently in use are identified.

(ij Inorganic solvents (acids, bases, salt solutions,

mixtures of acids and bases or their salts).

(ii) Organic solvents (acids, bases a 1cohols

ilclehvdes, ketones, esters and their mixtures).

', i i i i l-!ixed aqueous organic solvents (organic solvents

'Lxed with mineral acids, inorganic bases, salt

solutions or water).

(iv) rt)mplex forming organic solvents.

I he modern chromatographers are of the opinion that

the I'lObilc phases consisting of more than four components

should be avoided because of problems associated with

150

ruprocliic i bl e preparation. Our recent studies [5] on TLC of

anions vviith single and mixed organic solvent systems also

point out that three component systems are not of much

practical utility for chromatographic separation of anions

owing to the formation of diffused spots.

It is, therefore, amenable to select mobile phase

as simple as possible and prepared from pure grades of

solvent. Keeping this in view, the present work is

undertaken to explore the possible application of distilled

water in chromatographic separation and identification of

some anions on thin layers of pure as well as mixeci

adsorbents. Several favorable features such as easy

availabilitv at low cost, non-toxic nature, high purity,

low viscosity and volatility of water make it fit for

chromatographic studies. Interestingly, a single phase

scjlvent can be repeatedly used.

This paper describes a simpler method for

i (lent 1 r i c IL i on and separation of anions under variable

"\peri cut a! conditions to make it applicable for

L'nvi roiinuMit al samples. NO 2 in artificial sea water has been

identified on chromatoplates.

EXPERIMENTAL

Test solutions. 11 test solutions were either sodium or

151

[)otassiuin salts of al] anions studied, except SCN which

was Laken as animonium thiocyanate. Double distilled water

was used tor the preparation of solutions.

Reagents. Alumina and cellulose microcrystal1ine from CDH

laboratories (India), silica gel 'G' and methanol froti

(>'J axo laboratories (India), and all other reagents were oi

analar grade obtained from BDH, E. Merck or s.d. fine

chemicals (India).

Detectors. The detection reagents for various anions were

used as reported in chapter III. Diphenylamine (0.2-0.5" )

prepared in 4M H^SO, and saturated solution of AgNO^ in

methanol were used for the detection of 10/ and CI

r ( s p e c t i \' e 1 V .

M o b i l e p h a s e . D i s t i l l e d w a t e r w a s u s e d as m o b i l e p h a s e for

the entire study.

Stationary phase. The stationary phases were:

S,,

So

Silica gel 'C

Alumina

Cellulose microcrystal1ine

Alumina + Silica Gel (1:1, 1:2, 2:1

Alumina + Cellulose (1:1, 1:2, 2:1)

Preparation of TLC plates. The plates were prepared by

152

"lixing silica gel or alumina with conductivity water in 1:3

ratio bv weight. The resultant slurry was mechanically

shaken for 10 minutes after which it was applied on well

cleaned glass plates to give a layer of 0.25 mm thickness.

Ihe [11 Uc^s were air dried at room temperature and then

heated at lOO^^'X for Ihr.After activation the plates were

kept in an air tight chamber. Cellulose coated plates were

similarly prepared using a slurry made in 1:4 ratio o!

cellulose to water by weight. No additional binder was

added to the mixture used for the preparation of plates.

Procedure. Ihe procedures for the development of plates,

detection of anions, qualitative as well as quantitative

separation of anions and pH studies were followed as

described in chapter III.

In order to widen the applicability of the

propo.- eci ii'ethod, it was tested for the detection of NO2 in

sea water in the presence of other anions. For this purpose

1 sodiuiii nitrite was prepared directly in artificial sea

water. Ihe sea water was prepared by mixing together 100 iii 1

each c>l the following solutions:

( a 1 0.6 M NaCl

3 (b) 10 M KBr

(c) 3x10^^ M NaHCO 3

153

I'he pH vaJue (8.3) of the synchetic sea water was verv

close to the pH (8.1) of sea water collected from Arabian

sea. An aliquot (0.01 ml) of the synthetic mixture

containing NO^ <ind 10/ , VO- or MnO^ in 1:1 ratio w is

spotted on the chromatoplate. However, in the case of PO^

3-the mixture consists of NO 2 and PO^ ions in 1:2 ratio. The

plate.s were developed after complete drying of the spots.

NO2 (Kj.. 0,95) was detected as highly compact and we^

separated spot. 10^, VO3, MnO^ or PO remained at the

point of application.

To examine the feasibility of the proposed method

for tine separation of certain anions from hard water

•o.imples some important separations ( IO4 or VC^ - lO-

SCN , BrO o or NO2 ) were carried out in the presence of

hardness causing salt solutions (CaCK, MgCl2 and NaHCO^) .

The sample spotting procedure involves loading of 0.02 ml

of anionic mixture containing 10/^ or VO3 - 10^ " SCN ,

HrOi or NOM (1:1:1) on the chromatoplates followed by the

spotlii,;' ot 0.02 "il of CaCAy, MgCK or NaHCO.. The spots

.vere cc [decelv dried before the development of the plates.

Che anions were detected and their R„ values were compared

with their standard K„ values as determined in the absence

of hardness producing salt solutions.

154


The results of this study are presentee! in

figures 1-2 anci tables 1-3. All the anions except CI and

NOo were v;ell detected on all sorbent phases. Generally,

highly compact sjjots for anions on all sorbent phases were

observed. However, occasional tailed spots (K, -K,p > 0.3]

ior Fe(C;N)f^", FelCN)^", CrO^~ and Cr207'' also appeared on

some sorbent layers. The results showed excellent

reproducibility (variation is less than 107„ of the average

K, value) for all anions. The development time for 10 c,

run on silica gel or alumina containing layers was 30-40

niinutcs whereas it was 10-12 minutes on cellulose

VHMU a 1 n i ng, lied .

Fig. 1 illustrates the dependency of R , values on

f he n.iture and composition of sorbent phases. The anions

producing only single spot on all adsorbents have been

taken in figure 1 . Br , I , SCN , Br03 and NO2 move with the

solvent front (R = 0.92-0.96) regardless the composition

of the adsorbent. On silica gel Fe(CN)^ goes with the

solvent front yielding single spot (Rp = 0.95) whereas it

produces double spots corresponding to R^ values about 0.95

and 0.1 on alumina and mixed beds (alumina + silica gel).

Ihe double spots persist also on alumina - cellulose (2:1)

bed showing the dominating effect of alumina on the

155

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CD

f ^

o

in

n

i^ I- _1 L

1

o -

1

CD

O

_ 1 L ^ J

O

-- L . J

: o

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O

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o o

c £1

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<

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4-J

C

c o en c o C CT3

M - l

O



DS

o > o c a) c 0) G. 01

Q

00 • f - l

C

<

<

a

<

re

c

3 1 — I

<

O CO

0) 00

03

O

CO

o +

f

o <

OJ

o

«

t

CJO

+

<

vD

CO +

z

CO

J-J

D

(X

u

156

3-niobiliLy of Fe(CN) . However, the higher proportion ot

cellulose in alumina causes the conversion of well resolved

double spots into a badly tailing spot (R^ = 0.0-1.0).

Fe(CK). is strongly adsorbed (Rp

Tailed spot is also produced on pure cellulose layers.

0.1) on al umina and

aluriina - cellulose (1:1, 1:2 2:1) layers. However, it goes

with the solvent (R„ = 0.93) on silica gel layers. It tails

on cellulose and yields double spots (Rp = 0.15 and 0.95

on mixed beds containing alumina and silica gel in 1:1 and

2:1 ratio. A tailed spot on alumina-silica gel 1:2 was

[)roduced by FelCN)^^ . Chromate or dichromate produces

singU- compact spot on alumina (Rp = 0.1) as well as on

silicii ',el and cellulose layers (Rp =- 0.95) whereas taiK-d

spots result on mixed beds. MnO, is strongly adsorbed (R,,

0.081 on silica gel, cellulose and alumina-cellulose

layers bul tails on silica gel - alumina layers. Molybdale

moves with the solvent on silica gel or cellulose layers.

-strongly retained by alumina and alumina-silica gel (2:1 )

and produces tailed spots on alumina-silica gel layers.

10, exhibits strong sorption (Rp = 0.0) on all sorbent

phases except cellulose and cellulose-alumina bed.

Conversely, it goes with the solvent front on cellulose. It

produces double spots (Rp - 0.0 and 0.90) on mixed bed

containing alumina and cellulose (1:1, 1:2, 2:1). The

formation of double and tailed spots may be attributed to

157

the adsurption/precipitation phenomenon taking place

betv-jeen the surface active centers of adsorbent and the

test subs L ance.

Ihe interesting behavior of I0„ facilitates its

scpar.JtLon from several anions. It migrates to the top

fRp 0.93} with the solvent on silica gel and cellulose

layers, just leaves the point of application on alumina

layers (R,, -- 0.23) and exhibits variable adsorption

tendencies on mixed bed containing alumina and silica gel

or cellulose in different ratios. The resulting R^ values

fall in the range of 0.34 to 0.70 and permits some

inportant ternary separations.

Based on the Rr. values of the individual ions on i

different adsorbent layers, several separations are

possible. A few of the separations realized experimentally

are >',iven in Table 1. In addition to qualitative

separations (50 pg) of 10^ was quantitatively separated

Iron 0.) ng of \i)~ or BrO", 3.0 mg of Fe(CN)f^"" and 2.0 iiy

'>' '''<-''l ''1 silica gel layers. The separation of 10, from

r.>K \

t lies I ions

l'( ((-N'f , 10^, BrO.^, I and SCN is important a^

usually interfere in the sepectrophotometric

procedures !6,7j applied for the determination of I0~. The

reproducibility of I0~ - 10^, BrO~ or Fe(CN)^~ separations

was also checked on commercially available precoated plates

158

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160

fAnchroni F.nte rp r ibes , Lndia). The results (Fig. 2) are

LclenLLcal to those achieved on hand coated plates.

Ihe Ionic species in solution, in most of the cases

varies with the pH of sample solution. For excjmple.

chroniuhi above pH 8 exists as CrO, ions whereas in acidic

2- - 2-medium (pH 2-6) CrÔ-, , HCrO, and CrO, co-exist in varying

2-amounts. '"''loO/, species predominate in strong alkaline

medium whereas MoÔ-^ and Mo-,0, / exist in solutions of

interi ediate pH value. 10^ is reduced to iodine in

soluLions of moderate acidity (0.1-2.0 M HCl ) whereas in

ilka line [uediuni, iodine reacts with OH ions to give iodide

and io".

10^ t 51 6H -) 31^ t 3H„0

I2 - 20H

310

} I + 10 + H2O

(Unstable)

-} 2I~ + 10"

Because ol this fact we carried out the separation

of 10] from 10" BrO^, l". Mooi "' and Fe(CN)^~ over a wide

pH range of sa,iiple solution. The results are presented in

2-l.il lc J. I he separ.ition of 10, from MoO, at pH 3 is not

10

chromatographed as mixture with MoOf . It seems that

periodaLe it this pH value is masked with molybate to forr

possil)le. 10, could not be detected at pH 3 when

.2-

161

Fig. 2 Separation of 10^ Crom 10^, BrO^ and FeCCN)^'

on precodted plates.

(1)

(2)

(3)

IO4 - 103

ro~ - BrO;

10 7 Fe(CN)

162

TABLE 2

S>eparations at Different pH values of Sample Mixture on Silica

Ge\ Layers Using Distilled Water as Mobile Phase

'J

it)

S i i i p J e pH v a l u e s S e p a r a t i o n s (R. - Rr,. pH Vd

]

1 /

9

i'l

1

0

< • )

] 1

3

6.

8.

1 ue

.0

.0

.0

. 0

.1

0

9

1

9

9

s

IO4

1«4

I«4

104

^04

to.

IO4

^04

IO4

IO4

I«4

(0.14-0.0)

(0.1-0.0)

(0.0-0.0)

(0.0-0.0)

(0.0-0.0)

'0.0-0.0)

(0.0-0.0)

(0.0-0.0)

(ND)

(0.07-0.0)

(0.1-0.0)

- IO3

- IO3

- 10^

- I0~

- BrO^

- BrO~

- BrO-

- BrO^

- MooJ~

- MooJ"

- MoO^~

(1

(1

(1

(1

(1

(1

( 1

(1

(1

(1.

(1.

.0-0

.0-0

.0-0

.0-0

.0-0

.0 0

0-0

0-0.

0-0.

0-0.

0-0.

.85 1

.85 )

.90^

.90)

9 2

90 •

90 '

91

7 V<

89)

90)

n . ' i 10^ ( 0 . 0 6 - 0 . 0 ) - MoO^ ( 1 . 0 - 0 . 8 9 )

i . l I 0 7 ( 0 . 1 7 - 0 . 0 ) - F e ( C N ) f " ( 1 . 0 - 0 . 8 8 '4 6

' . 0 10'^ ( 0 . 0 - 0 . 0 ) - F e ( C N ) ^ " ( 1 . 0 - 0 . 8 8 :

iU_^ ( 0 . 0 - 0 . 0 ) - F e ( C N ) ^ ' ( 1 . 0 - 0 . 9 0 :

1 1 . ^ l i \ ( 0 . 0 - 0 . 0 ) - F P ( C N ) ^ ' ' ~ ( 1 . 0 - 0 . 8 6 ;

163

6-Mol ybdoperiodate [8j which migrates with the solvent

front. I'he separation of 10, from I (sample pH 3-12) could

not be achieved. 10. at all pH values and I at pH 3 could

not be detected on the chromatopl ates. IC, was detected

on 1V when its loading amount was kept three fold in che

sample mixture with I . However, the increasing of 10,

amount offers deleterious effect causing very poor

detection of I .

The lower limit of detection of some anions were

determined on alumina layers. The lowest possible

detectable amoLtnt of anions (given in parenthesis) is as

f O I 1 OV.'S .

MnO, (0.7

SCN^ (7.6

4-5 ^ g ) , VO^ (0.70 pg), Fe(CN)^ (0.54 pg

3 pg), 10^ (8.71 jjg), and 10^ (8.30 pg)

An interesting aspect of this study is to

investigate the effect of most frequently encountered ions

e.g. Oa , Mg , Na , CI and HCO„ in the aquecRis

environment on I07 or V0~ - I0~ - S C N " , BrOo or NO"

4 3 3 ' 3 2

separations. In the case of 10, - 10,. - NO „ separation the

spot of 10 J was perfectly round and compact, but the spot

"! '•'';,: bui-uiie (.dongated. A pronounced effect v;as observed

Jii iO^ iO ^ • SCN separation. In this case only SCN is

derected whereas 10 and 10,. could not be detected. As

164

TABLE 3

Kffect of GaCl^, MgCl2 and NaHCO^ on Some Selected Separations

Standard R„ value of Individual Ions are in Paranthesis. 10/

(0.00), IO3 (0.53), VO3 (0.00), SCN~ (0.94), Br03 (0.93) and

NO2 (0.92).

S <i 1 t s Separations (R, - R,,,

CaCl IO4 (ND)

IOA (0.02-0.0)

IO4 (0.02-0.0)

VO3 (0.02-0.0)

IO3 (ND)

IO3 (0.67-0.49

10- 0.73-0.54)

lOo (0.65-0.44:

SCN (1.0-0.89

Br03 (i.0-0.82

NO2 (1.0-0,88

SCN~ (1.0-0.88:

M"uJ 10^ (ND)

10^ (0.01-0.0)

10; (0.03-0.0)

VO^ (0.03-0.0)

10^ (ND) - SCN

IO3 (0.63-0.45)

10^ (0.73-0.60)

IO3 (0.70-0.53) - SCN

(1.0-0.87)

- BrO^ ( 1.0-0.87)

- NO2 ( 1.0-(. .87)

(1.0-0.87)

N iHCO I04(ND)

10^(0.02-0.0)

10^(0.03-0.0)

VO^ (0.01-0.0)

IO3 (ND) - SCN

IO3 (0.65-0.46

IO3 (0.76-0.62

IO3 (0.70-0.50) - SCN

(1.0-0.87)

Br03 (1.0-0.84

NO" (1.0-0.90

(1.0-0.79)

Note: ND, not detected

165

o idem Iron uit le ^ i ] 1 other separations are not effected

h\ the pre'-ence ot Cd" , Mg and Na etc. The K., of 10,

llucLuite between 0.30 to 0.70 in the mixture.

166

REFERENCES

1. I'.A. rh. Brinkinan, G. De Vries and R. Kuroda, J.

Chromatogr. , 85, 187 (1973) .

2. R. Kuroda and M.P. volynets, in CRC Handbook of

Chroniatography: Inorganics, Vol. I (M. Qureshi, ed. ) ,

CRC Press, Boca Raton, Fla., 1987, p. 89.

i. A. Mohammad and K.G. Varshney, in Handbook of Thin

layer Chromatography (J. Sherma and B. Fried eds.),


4. N. Fatima and A. Mohammad, Sep, Sci. Techno!., 19, 429

(1984).

5. M. Ajmal , A. Mohammad, N. Fatima and J. Ahmad, J.


6. C.h. Hendrick and B.A. Berger, Anal. Chem., 38, 791

(1966) .

7. A.M. Escarrilla, P.F. Maloney and P.M. Maloney, Anal.

Chim. Acta, 45, 199 (1969).

S. R. Belcher and A. Townshend, Anal. Chim. Acta, 41, 395

{ l'-)6^ I .

CHAPTER - V

THIN-LAYER CHROMATOGRAPHIC SEPARATION OF SOME ANIONS ON COPPER SULFATE

IMPREGNATED SILICA GEL LAYERS

167

Krot'i the literature the TLC of anions has been much

less extensive co'ipared to that of cations fl-3l. Silica

^]e\ [4 8i, cellulose 19-Ll!, alumina [12], silufol [13] and

hydrated stannic oxide layers [14] have been used for the

separation and identification of anions although no work

has been reported on the use of silica gel impregnated with

inorganic salts.

In the present work, the thin-layer chromato

graphic behavior of some common anions on plain and copper

sulfate - impregnated silica has been investigated in mixed

aqueous-organic solvent systems containing acetone.

EXPERIMENTAL

Test solutions. IX test solutions were either sodium or

potassium salts of all anions studies, except SCN which

was taken as ammonium thiocyanate. Double distilled water

was used for the preparation of solutions.

Reagents. Silica gel (E. Merck, India), formic acid,

hydrochloric acid, hydrobromic acid, sodium chloride.

aiimoniuiii hydroxide and acetone (B.D.H., India) were used.

These and all other reagents were analar grade.

168


used as reported in chapter III.

Mobile phases. The following solvent systems were used as

I!:obi 1 e phases :

M HCl: Acetone (1:9)

HCl: Acetone (9:1)

NaCl: Acetone (1:9)

NaCl: Acetone (9:1)

HBr: Acetone (1:9)

HBr: Acetone (9:1)

NH,OH: Acetone (1:9)

NH,OH: Acetone (9:1) 4

Formic Acid: Acetone (1:9)

Formic Acid: Acetone (9:1)

"2

M,

M-

Mr

>\r

M 10

In all the solvent systems, HCl, NaCl, HBr, NHÔH and

formic acid were O.IM aqueous solutions while the acetone

\.'as pure .

Stationary phases,

S, Plain silica gel G

S. Silica gt'l impregnanted with O.l-'j.O/

aqueous solution of copper sulfate.

Preparation of TLC piates-

(i) Plain silica gel plates: Silica gel was mixed with

169

conductivity water in the ratio 1:3 with constant shaking

for 5 to 10 inin. The resultant slurry was coated on well-

cleaned glass plates to give a layer approximately 0.25nim

thick. The plates were dried at room temperature (30°C)

and then heated at 100+ 5°C for 1 hour. After activation

Che plates were stored in an air-tight chamber.

(it) Copper sul fate-impregnated silica gel plates: To

prepare impregnated silica gel layers, a slurry was made

by mixing an aqueous solution of O.l-5.07o, copper sulfate

with silica gel in the ratio 3:1. Thin layers were then

prepared as described above for plain silica gel plates.

Procedure. For qvialitative analysis, one or two drops of

the anion sulutions were spotted on the plates with glass

capillaries. The spots were dried and the plates developed

by the ascending technique, the ascent of the solvent was

fixed ac 10 cm in all cases. After development, the plates

were dried and the anion spots visualized with the

appro[)rlate spray reagent. Rp values of the leading front

(R, ) and of the trailing front (R,p) were measured and

reported as (R,-R^),

Rr. values were calculated from R, \ ' T

The limits of detection of the various anions were

determined as reported in chapter III.

summ

170


The main points which emerge from this study are

arized below:

1. A small change in Rp values was sometimes observed

when mixtures of anions were developed as compared

with single substances.

2. The development time for a 10 cm run ranged between

45-70 min depending upon the mobile phase.

3. Silica gel impregnated with CuSO, gave excellent

results. Thin layers were of good quality. Generally,

the spots were compact and well formed in all solvent

systems at 0.1-17o impregnation. Plates impregnated

with 2-57o copper sulfate solution deformed during

development.

4. R,-, values reported in this paper represent the

averages of triplicate tests and were measured to the

center of the spots on the plates.

5. Sodium chloride-acetone (1:9 and 9:1), NH,OH-acetone

(1:9 and 9:1) and HCl-acetone (1:9) systems were found

most suitable for separations. A few anions showed

occasional tailing. HCl-acetone (1:9) was found to be

the best solvent system for multicomponent separation

with 1% copper sulfate impregnation. Formic acid-

171

acoLcjiu (1 :y) produced highJy compact spots of anions

on (). 1 "', copper sulfate layers,

b. Ihe copper sulfate travels with the solvent front upto

the iiicidle ot the plate in solvents M., , M, and M-IM-

Ihcse systems are therefore unsuitable for iinpregn.i t eci

1 ayers.

7. Solvent.^ containing 907o acetone gave better results

than those containing lOX acetone.

2- 2- 3-o. In all solvent systems, CrO^ , Cr20y , Fe(CN)^ and

l- eiLN'i, produced colored spots on impregnated layers

and thus were self detecting.

9. NO. could not be detected on 17o impregnated layers

while it was clearly detected on plain silica gel as

well as on O.IX impregnated layers.

10. In addition to the other ions, VO2 was also chromato-

graphed to assess the possibility of separating VO 2

fro!!, VOT-

Ihe results have been summarized in Figures 1-3 and Tables

1 and Z. Figure 1 summarizes the results of AR^ values

' A Kp Rp )>T plain silica - R,, on impregnated silica)

with various mobile phases. It is evident from Figure 1

that i.iipregnated layers are more selective (strongly

sorbing) than plain silica for most of the anions, as

.0 r

0.8

0.6

-0.2 -

-O.A -

H C i : A c e t o n e ( 1 : 9 )

( a )

1

1 r^t

O Z

1 1 2 U t / l

1

1 i_

00

1

rsi rx/

O >

1

1

O a

1

1 r-1

r> >

I

172

J Nl ^« O U

O

fvl ^

o o 2

• ^ - ^ O

l i . o

Q: <3

O.A

0?

0.0

-0 .2

-O.A

<3

O.A

0.2

0.0

- 0 2

-O.A

HBr ; A c e t o n e ( 1 : 9 )

( b )

_L _L _l

1 rsi U 2

1

Z U LO

PNI C%*

o >

1 n -t

O a

1 n O >

rsi ^

o L ,

f-J

r-i Z ^ -J ;?: ^ "X^^ y U O

u

F o r m i c A c i d ; A c e t o n e ( 1 : 9 )

(c )

A n i o n s F i g . l contfl

173

N a C i : A c e t o n e ( 1:9

 o

>

o

1 r~j v j

O O i

' • ' 7 U t>> u_

•-T ^

z U c u.

o

u o

A n i o n s

Fig . 1 contd.

U-cr < j

174

NH4OH : Ace tone ( 9 ; i )

--1 l- ( -. 1 _ _J I :z u in CD

(-Nj r s i

0 >

m vj 0 Q.

1 r~>

0 >

O U

• z u q

u

An ions

Fig. 1 Plot of ARp (Rp on plain silica gel - Rp on

CuSO, impregnanted silica gel layers) vs.

anions

o 1 Copper sulfate impregnation

A 0 .17„ Copper sulfate impregnation

175

indicate d by positive ARp values. There was no

significant ditt'ercnce in the mobility of anions when

chromatographed cm silica impregnated with \% or 0.1°

copper sulfate using HCl , HBr, NaCl or formic acid-acetone

(1:9) solvent systems. With formic acid-acetone, NO2 showed

increased mobility on VL impregnated layers ( Rp = -0.3)

compared to its mobility on plain silica or silica

impregnated with 0.17o CuSO^ ( A R p = +0.1). The reverse

trend was observed for I which moves faster on plain

silica ( ARp = 0.24) compared to W layers. However, 0.1%

CuSO, impregnation was found to be ineffective in changing

the mobility of I which behaved similarly in NH,OH-acetone

(1:9) where it moves faster on plain silica as compared to

1; CuSO/ layers. Sodium chloride-acetone (9:1) and

NH,OH-acetone (9:1) systems were found the most effective

in changing the mobility of most anions on impregnated

layers and thus open numerous possibilities for

separations. However, the tailed spots produced by some

anions limit their separation from other anions. With

NHÔH-ace tone (9:1), most of the anions were strongly

ndsorbecl on V, layers compared to plain or 0.1% layers.

Figure 2 summarizes the hR„ values of anions

chromatographed on silica gel impregnated with II CuSO,

and developed with solvents M. M„,M,,M^,M^,Mg and MQ.

176

o o r—

X

h. a: • — '

U-

rr X

100

80

60

AO

20

H C I : A c e I o n e ( 1:9 )

0

o o » — •

X Li.

cr —

[i-

ir JZ

100

8 0

60

AO

z'O

1 r-J O z.

t\^

-

-

-

7:

u ID

O L

T O O r

CO

4

r-i fsi O >

1

m <t O a

^ 1 m (J >

o

u ^

o O

u u o u

U

H Br : A c e t o n e ( i : 9 )

F o r t i l ic Ac i d ' A c e t o n e ( 1*.9 )

A ri I o n s

F i g . 2 contci .

100

o o < — X

u_ (T —^ LL

u.

80

60

AO

20

0 L_

I - N a C I : Ace tone ( i: 9 )

: i - N a C i : A c e tone ( 9:1 )

177

100

o 80 o

>< 6 0 tier - AO a: X 20

0

N H / ^ O H ; A c e t o n e ( 1:9 )

N H / O H: A c e ( o n e ( 9:1 )

A n i o n s

Fig. 2 Plot of hRp versus anions

htationarv phase: l7o copper sulfate imprrjniat cd

silica ii e 1

O Compact spots with R. -R. <0.3

L Tailed spots with K, "H^ >().3

¥: Badly tailed spots \M.th R,-R^ >0.4

178

IL is evident that the different mobile phases are able

to bring about different retention sequences of anions

leading to several binary, ternary and quaternary

separations. The mid R^ (Rp - 0.4-0.6) values of NO 2 in

M. and Br in M , My and M^ can be used for their

separations form all other anions with higher or lower Kp

Vi 'alues. Vol^, PO4", Mool", Cr207 , Fe (CN) 5 , Fe ( CN) 5 and

WO^ are strongly adsorbed on the impregnated layers (hRp

(J-IO) in all solvent systems containing 907o acetone

(Figure 2). The mobility of a few anions is increased when

the concentration of sodium chloride or NH/OH is increa' ed

2-ile phase (M, and Mo). However, CrO^ and in the mob

Cr ,0^ showed tailing. Likewise, M. and M^ can be utilized

for the ser)aration of MoO, (hR^ = 90-95) from the anions ' 4 F

with low R„ values. SCN , I , Br and NO,-, gave higher Rp

values in most of the .mobile phases and thus can be

separated from anions with lower Rp values.

In Figure 3, the results obtained on O.IZ copper

sulfate layers in various solvents have been summarized. A

major advantage of this adsorbent is that NO3, which could

not be detected on VL copper sulfate layers, was very

clearly detected on 0.17„ layers. Comparison of Figure 2

and 3 slio\.' almost identical chromatographic behaviour of

tht anions on both the adsorbents in HBr-acetone and

loTi'iic acid-acetone. Conversely, most of the anions do \'0\i

100

c o ; > - ' Lt

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179

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' 0 0 ( 1

80 [ I

' 0 [

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2i)

tU 1 : Ac e t o n e 1:9

A n i o n s

Fig . 3 contd

180

^-~•

o o X

u cr

u. Q:

r

100

fi 0

6 0

A 0

2 0

0

C)--

in O

NaC

NaC

Ac e t o n e 1:9

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o I

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<f?'^^^'" r j -J-

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c> <)

1 '••'

C)

^

1

z u 1/1

1 r-1 O 7

4-o >

Fig. 3 Plot of hRp versus anions

Srationary phase: 0 .1/„ copper sulfate i npre,"ii i ti H

silica gel

O Compact spots with R,-R^ <0.3

A lailed spots with R,-R^ >0.3

* Badly tailed spots with Rj-R^ ^0.4

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184

TABLE 2

Limit of Detection and Dilution Limit of Anions and VO „ on

Silica Go! Impregnated with 0.1X CuSO, with Formic Acid:

Acetone (1:9) Mobile Phase

S] .

No.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

a Dilution

, . Voli

Ions

Br~

3-PO

l'

2

V0~

NO-

NO'

SCN"

Fe(CN]

Fe(CN)

Cro2-

Cr202-

MoO?~ 4

wo^-

] imit

ame of

't 3-6

test

Salts

KBr

Na2HP0^

KI

V02S0^

NaV02.H20

NaNO^

NaN02

NH^SCN

K^Fe(CM)g.

K3Fe(CN)^

K2CrO^

K2Cr207

Na2Mo0^.2F

Na2W0^.2H2

solution (m

3H2O

I2O

0

1 ) X

Limit of detection

100 .0

10 .0

10 .0

10 .0

1.0

1.0

1.0

1.0

1.0

0 .5

0 .5

0 .5

0 .1

0 . 1

10^

Di1utton

11 n 11

2 1:10^

1:10^

1:10^

1:10^

iiio"^

1:10^

1:10^

IrlO'"''

i.-io''

1 :2x10'^

1 :2xl0'^

I -.ZxHy'

1 : 1 0 '

1:10^

Limit of detection { pg

185

behave similarly on both sorbeni:s in NaCl-acetone and

NH, OH-acctone. Thus, in addition to the separation of \'0 - ,,

from numerous anions on 0.1% layers many other m'-ti

separati(^ns oi' anions can be realized in NaCl -•icotf)n(. r

NH,OH-acetone.

The chromatographic systems developed pro\'ide

numerous possibilities for rapid, reproducible and cloTn

separations of anions from mul ticomponent mixtures. Sn- c

separations experimentally achieved have been .sumnari/ed

in Fabl e 1 .

Table 2 summarizes the limit of detection md

dilution limit of various ions. The proposed mcLhod is

highly sensitive for most ions except Br .

186

REFERENCES

1. U.A. Th. Brinkman, G. De Vries and R. Kuroda. f.

Chromatogr., 85, 187 (1973).

2. R. Kuroda and M.P. volynets, in CRC Handbi ol-- o:

(Chromatography: Inorganics, Vol. 1 (M. Qurcshi cd.),

CRC Press, Boca Raton, Fla., 1987, p. 89.

3. A. Mohammad and K.G. Varshney, in Handbook of Fhii

Layer Chromatography (J. Sherma and B. Fried eds.).


4. J. Benes, Collect. Czech. Chem. Commun., 44, 103^

(1979) .

5. J. BeuGs, Collect. Czech. Chem. Commun., 44, 'iQh

(1979) .

6. H.M. Chawla, N.N. Ralhan, N.K. Garg and S.S. Chibber,

J. High Resolut. Chromatogr. Chromatogr. Commun., 3.

651 (1980).

7'. K. Kawanbe and K. Maruyama, Yakugaku Zasshi, 101, 912

(1981);Chem. Abstr., 96, 45420K (1982).

8. J. Zuanon Netto, A.F.C. Graner and M. lonashiro.

Ecletica Quim , 9, 45 (1984).

9. G. Buchbauer and R.E. Markis, Sci. Pharm., 51. 48

(1983) .

10. R. Gallego, J.L. Bernal and A. Martinez, Quim Anal..

31, 3 (1977) .

187

11. R. Gal lego, J.L. Bernal and A. Martinez, Quir,. Anal.

31, 69 a977)

12. K. Ravindra Nath and P.B. Janardhan, Indian J. Cher'.

21A{2), 150 (1982).

13. J. Franc and E. Kosikova, J. Chromatogr., 187, 46;

(1980).

14. A.K. Sen and U.Gh. Ghosh, J. Liquid Chromator., 3. 7

(1980).

CHAPTER -VI

EFFECT OF HEAVY METALS ON CHROMATOGRAPHIC SEPARATION OF 10^ AND

QUANTITATIVE DETERMINATION OF lOf

188

Since its inception in 1950 by Stahl , thin-layer

chromatography (TLC) is on a regular upswing as a result of

which many reviews, monographs, books and scientific papers

dealing with separation, identification and quantification

of micro amounts of different classes of compounds have

appeared in literature fl-8]. TLC in combination with

sensitive quantitative techniques has widen its scope in

the analysis of various natural and industrial microscale

samples. According to recent literature the TLC of anionic

mixtures is much less extensive than for cations or

organometal1ics. As regards the separation conditions, the

influence of counter ions and varying amounts of ions Lo be

separated on the efficiency of the separation have largel\

been neglected.

In continuation of our earlier studies on TIC of

anions the present work is aimed to the development of a

simple and inexpensive method for the selective separation

of 10^ from IO3, Br03, MoO^~ and Fe(CN)^~ on silica eeJ

layers using distilled water as mobile phase. The effect of

more commonly encountered cations such as Pb , Hg , Cd

Ni^^, Co^^, Zn^^, Cu^^, Al-^^, Fe^^, Bi"^^, Tl and Ag" on

the separation efficacy of 10/ from other anions has been

investigated. Volumetric determination of iodate with

preliminary thin-layer chromatographic separation from

periodate has also been examined.

189

EXPERIMENTAL

Test solutions. The test solution 1% were potassium salts

of periodate, iodate ,'iodide , bromate ferrocyanide except

molybdate which was taken as sodium molybdate. 17o solution

of nitrates of lead, bismuth, aluminium, thallous and

silver, chlorides of cadmium, nickel, cobalt, mercury and

iron, sulfates of copper and zinc were prepared in double

distilled water containing small quantities of correspon

ding acids to prevent hydrolysis.

Reagents. Silica gel 'G' and methanol were from Glaxo

Laboratories (India). All other reagents were of also

analar grade-


used as report in chapter III.

Mobile phase. Double distilled water was used as a mobile

phase.

Stationary phase. Plain silica gel was used as a

stationary phase.

Preparation of TLC plates. Plain silica gel plates were

prepared as reported in chapter III.

Procedure. To study the metal ion effect on 10^-10^, 10/ -— _ 9 _ ^ / _„

BrOn, lO^-MoO^ and IO/-Fe(CN)g separations, the samplr

190

containing the mixture of 10, , separating anion and neta]

ion in 1:1:5 ratio were used. The synthetic mixture was

prepared by adding five fold excess of metal solution Into

a mixture containing 10/ and the separating anion (10^,

Br03, MoO^ and Fe(CN)g in 1:1 ratio followed by thorough

mixing to get homogeneous solution. About 10 yl of the

synthetic mixture so obtained was spotted on the

chromatoplates. The spots were completly dried at rooT

temperature and then the plates were developed by the

ascending technique, keeping ascent to 10 cm from the

starting line in all cases. After the development the

plates were air dried and the spots were visualized using

the appropriate spray reagent. The R, and R^ values for

both the anions present in the mixture were determined. For

microgram separation of periodate from milligram quantities

of iodate, 1.0 ml solution of periodate was mixed with 1.0

ml of l-107o solutions of KIO^ in a test-tube. 0.01 ml

volume of the resultant mixture was spotted on the

chromatopl ate with the aid of a micropipettc.

Alternatively, 0.01 ml of KIO^ was first spotted on the

chromatoplate followed by the spotting of 0.01 ml of

standard solutions of KIO^. The spots were dried, plates

were developed and detected. The R, and R^ values for both

the anions were determined.

A volumetric procedure was adopted for the

quantitative determination of iodate after its separation

191

from periodate on thin layer chrona'coplates. A standard

volumetric method [9] was set up using O.OIM sodium

thiosulfate solution as intermediate solution. For the

determination of IOT in the presence of IO4, various

samples containing the mixture of KIO^ and KIO, in variable

amounts were prepared. With the help of a lambda pipette 0.1

ml solution of the resultant mixture was loaded on the

chromatoplates. The plates were developed with distilled

water. A pilot plate was employed simultaneously in order

to locate the exact position of the spot on the working

plate. The area covering IO3 was scratched and lOo was

eluted with l.OM HCl , The adsorbent was separated froc

the solution and washed with l.OM HCl to ensure complete

elution of IO3. The filtrate was added to the blank

prepared by mixing 2 ml KI (1%), 2 ml cone. HCl and 0.2 ml

KIOo (1%). The contents were titrated with O.OIM sodium

thiosulfate solution. The blank was also simultaneously

titrated with O.OIM sodium thiosulfate and the difference^

between the volumes of sodium thiosulfate solution consumed

in both the cases was taken for the determination of 10^ in

the sample.


Data collected in Table 1 demonstrate that all

anions produce well formed and compact spots over a

reasonable ranee of loading amount of anion salts. All

192

TABLE 1

Rp Values of Anions on Silica Gel Layers Using Distilled Water as

Mobile Phase

Anions Range of loading Range of R„ Lower detection

amount (pg) obtained limit of anion(jLig)

10^ 50-250 0.00-0.05 4.15

IO3 25-500 0.93-0.95 A.08

BrO" 20-500 0.90-0.95 0.76

MoO?" 100-2000 0.70-0.95 6.61 'k

50-3000 0.94-0.96 7.65

Fe(CN)^ 50-3000 0.72-0.92 5.24 D

The effect of loading amount on Rp values was examined usins:

standard solutions of KBrO^ or KIO3 (1-107J, KIO^ (1%), Na2Mo()^

(l-407c) , K^ Fe(CN)^.2H20 (l-307o) and KI (l-607o).

The limit of detection was determined by spotting different

volumes of anionic solutions (0.01-]%).

193

anions except IOA show greatest affinity towards the mobile

phase and move with the mobile front. However, 10/ is

retained at the point of application and thus ic is well

separated from accompanying anions. The lowest possible

detectable amount of anions on the chromatoplates as shown

in Table 1 indicates the working limit of the procedure,

Br03 less then 1 pg in a sample can be easily detected

using the proposed method. The small difference in the R .

values over a reasonable range of loading amount of anions

given in Table 1 shows the formation of highly compact

spots.

The most interesting aspect of this study is the

separation of 10/^ from other anions in the presence of

counter ions/metal ions. A pronounced effect of metal ions

on most of the separating anion pairs was observed. IO4-

Fe(CN)g separation was badly effected as all added cations

cause precipitation. Similarly, Ag, Pb, Al and Tl hamper

- 2-the separation of 10, from Br03 or MoO^ by producing

precipitation with anionic mixture. The separation of IO4

from IO3 is not achieved in the presence of Pb, Ag, Bi and

Tl due to the formation of precipitates. However, the

presence of other metal ions do not effect the separation

of anions.

In order to present a clearer picture regarding the

separation of 10^ from IO3 , some TLC parameters such as

194

TABLE 2

TLC Parameters for the Separation of IO4 (Rp=0.01) from 10^

(Rp=0.92) in the Presence of Certain Metal Ions

Metal ions

Hg^^

Cd2^

Ni2^

Zn2^

Co2^

Cu2^

Fe3^

Al3^

Rp

0.90

0.87

0.85

0.86

0.87

0.86

0.87

0.87

TLC parame

""'104

19.0

24.0

15.66

10.11

19.0

13.28

13.28

19.0

ters

380.03

244.89

159.79

194.42

220.93

210.79

210.79

220.93

R s

12.05

6.6 ^

5.66

6.29

6.96

6.37

6.96

6.96

195

ARp (Rp of IO3 - Rp of 107), separation factor ( < ) .

capacity factor (K') and resolution (R ) have been

calculated. The numerical values of these parameters have

been encapsulated in Table 2. The following relationships

were used to calculate the values for c< and R . ^ s

4' 3 R.

where, K' =

(ii Ai

where, /jX is the distance between the center of

spots of the separating ions and d. and d^ are their

respective diameters. Two ions are just separated if R =1.

A perusal of data given in Table 2 shows that IC3 is

well separated from IO4 in the presence of Hg, Cd, Ni, Zn,

Co, Fe, Cu and Al as indicated by high values of A R ^ , and

R . The best separation is achieved in the presence of

mercury. The high value of separation factor and fairly

large value of R (R J 6) are indicative of well resolved ^ s s

spots of IO4 and IO3. The large positive value of capacity

factor for IO4 (K' >10) confirms its strong attraction to

the stationary phase compared to the solvent.

20-50 pg of IO4 can be easily separated from ten

fold amount of IO3. The proposed method clearly demonstrate

1%

TABLE 3

Determination of lodate with Preliminary TLC Separation from

Periodate

Amount of 10^ Amount of 10^ Amount of 10^ '/, Error

taken (mg) loaded (mg) found (mg)

0.415 0 .405 0 .412 -1 .7

0 .365 0 .45 0 .441 +2.0

0 .307 0 .50 0 .489 +2.2

0 .232 0 .59 0 .601 - 1 . 9

197

the influence of sample composicion on the separation of

periodate from other anions. The extension of this work to

other species seems worthwhile to pursue.

Finally the results of quantitative determination of

iodate in the presence of periodate have been presented in

Table 3. The results show that the method used is accurate

and also reproducible. It is applicable for microgram

determination of 10- if present in the presence of 10^.

198

REFERENCES

1. J. Sherma, Anal. Chem., 58, 69 (1986).

2. R.G. Ackman, C.A. McLeod and A.K. Banerjee, J. Planar

Chromatogr., 3, 450 (1990).

3. B. Kratochvil and J. Peak, in Analytical Methods for

Pesticides and Plant Growth Regulators, Vo!. XVII

(J. Sherma, ed. ) , Academic Press, New York, 1988, Chap.

1.

4. F.A.A. Dallas, H. Read, R.J. Ruane, and I.B. Wilson,

eds. , Recent Advances in Thin-Layer Chromatography.

Plenum, New York, 1988.

5. L.R. Treiber, ed. , Quantitative Thin-Layer Chromato

graphy and Its Industrial Applications, Marcel Dckker,

New York, 1987.

6. M. Ajmal, A. Mohammad, and S. Anwar, J. Planar

Chromatogr., 3, 511 (1990).

7. T. Shimizu, Y. Suzuki and C. Inose, Chromatographia,

23, 648 (1987).

8. M. Brown, M. Sutherland and S. Leharne, J. Chem. b:duc . ,

64, 448 (1987).

9. W.W. Scott, in Standard Methods of Chemical Analysis,

5th ed. , Vol. 1 (N.H. Furman, ed. ) , D. Van Nostrand

Company, Inc., New York, 1939.

MKROCHIMKAl JOI RNM 44, <9^!< ( I99I I

Chromatography of Anionic Pollutants on Silica Gel Layers; Selective Microgram Separation of N02^ and IO3

Ai I MOHAMMAD* AND SHARAD TIWARI

Anatxlual l.ahimiu>i\ Dcpiulmenl i>l Applied C'hemisli\. /.akir Hiissain C'dllcvc <>t l.niiinecrim; ami Ici hnoliivx, Alii;tifh Miisliin Universilw Aliiiarli-2()20l)l. India

Received November 15. I'WO; accepted February 10, 1991

investigations regarding lhin-la\ei chromatographic behavior of seventeen anions have been conducted on sihca gel impregnated with inorganic sails such as copper sulfate, zinc sulfate, cobalt chloride, hexamine cobalt ill chloride, and nickel chloride using mixed aqueous-organic eiuents Lonlaimng formic acid. The effect of mineral acids on the mobility of anions has .ilso been studied by substituting formic acid vnth HCl. H,S().,, or H("l()4 in the mobile ph.isc In addition to mrcrogram separation of NO, and lO, from various anionic species some other important quahtative separations have also been realized. The effect of pH ot the sample solution and loading amount of 1(), . BrO, , NO, , and 1 on their /?, values has been investigated. I he limits of identification for all anions on impregnated layers have been determined. Semiquantitative determination of NO-, and BrO, have been attempted usiiii; the spoi .uea mcisuiement method. ' iwi VL.Kk-mu Press, im

INTRODUCTION

In recent yeais thin-la\er chromatography (TLC) has grown much in status and has experienced a dramatic surge due to its simplicity, versatility, and low cost. TL.C provides good resolution and is comparatively fast. TLC, with optimization of techniques and materials, can be applied for the quantitation of various compounds present m environmental, geological, and biological samples. Some recent applications of I l.C such as the identification of perchlorates in explosive residues (/) and the determination of selenium in foodstuffs (2). total heavy metals in industrial and vîste waters (J), ortho- and polyphosphates in soft drinks (4). and Hg (111 in river and industrial waste waters (5) have shown its utility as an effective, rapid, and smiple separation technique. Some important separations involving microgram lo tnilligram quantities of elements have also been reported recently (6-A).

As evident from the literature survey, few workers have attempted the TLC of anions (9-/2), m contrast to that of cations {13-20). Thin layers of stannic chloride, cellulose, silica gel. sijufol, and alumina have been used for the separation and identification of anionic species. In most of the cases ammonia (2/, 22) in combination with alct)hols and ketones has been selected as the mobile phase. Aqueous salt sokitions and aqueous—organic systems containing mineral or car-boxylic acids have also been tried. Recent investigations by Mohammad ct al. {23) regarding the eftect of solvent composition on the mobility of anions show that two component sv stems containing formic acid mixed with acetone, ethyl methyl ketone, butanol, or isopropanol were most useful in producing differential migration of anions.

^9

()()26-265X'91 $1.50 ( opMi.Kht < IWI b\ Acidemia Press. Iru \li !ij:lils of reprodiiLlion in .in\ l()rm reserved

40 MOHAMMAD AND IIWARI

Our work on TLC of cations (24-26) in mixed organic solvents containing formic acid has clearly established the practical applicability of these systems as eluents for several analytically difficult separations on plain silica gel as well as on silica gel layers impregnated with aqueous salt solutions. The improved separation possibilities of cations on impregnated silica gel layers suggested we study the applicability of impregnated layers in the analysis of anions. The present paper describes a thin-layer chromatographic study of anions on silica gel impregnated with some aqueous salt solutions. Mixed aqueous organic solvents containing formic acid have been used as eluents.

It has been possible to clearly separate the anions at microgram to milligram levels over a wide pH range of sample solutions.

EXPERIMENTAL

Apparatus. A thin-layer chromatographic apparatus (Toshniwal, India), 20 x 3.5-cm glass plates, and 24 x 6-cm glass jars were used. An Elico Model LI-IOT pH meter was used for pH measurements.

Test .solutions. The test solutions (1%) were either sodium or potassium salts of ferrocyanide. ferricyanide, chromate, dichromate, tungstate, iodide, bromide, phosphate, molybdatc, vanadate, nitrate, nitrite, bromate, iodate, permanganate, oxalate, except SCN (ammonium thiocyanate). Double-distilled water having a specific conductivity Af = 1.5 x 10 ''ohm ' cm ' at 25°C was used for solutions.

Rcuiênts. All the reagents used were of analytical grade. Detectors. For the detection purpose the following reagents were used: 1. Saturated AgNO, solution in methanol for Br , I , PO] , CrOj and

Cr,0^ . 2. Diphenylamine {0.2-0.5Vc) in 4 M H,S04 for 10, , BrO, . NO^" . NO," , VO,",

WO5 , and Mn04 . 3. Ferric chloride (10%) in 2 M HCI for SCN , Fe(CN)^ . and Fe(CN)(^ . 4. Alcoholic pyrogallol (0.59f) solution for MoO^ . 5. Aqueous potassium ferrocyanide (1%) for CÔj . Mobile phase. The following solvent systems were used as mobile phases:

S| Formic acid:acetone (1:9) S, DMSO:acetone (1:8, 3:6, 6:3) S^ Formic acid:DMSO:acetone (1:1:8, 3:1:6, 5:1:4) S4 H.S04:DMSO:acetone (1:1:8) Ss HCI04:DMSO:acetone (1:1:8) S„ HCl:DMSO:acetone (1:1:8).

In all the solvent systems, HCI, HCIO4, H2SO4, and formic acid (FA) were 0.1 M aqueous solutions while the acetone and DMSO were used as received.

Stationary pha.se. The stationary phases were: (a) Plain silica gel. (b) Silica gel impregnated with 0.1% aqueous solutions of CUSO4, ZnS04,

NiCl., CoCU. and Co(NH,)f,CI,. Preparation of TLC plates. (a) Plain silica gel plates were prepared using the method reported earlier (27).

http://pha.se

C HROMAIOCRAPHY OI ANIONIC POl.l.U I AN I S 41

(b) The impregnated plates were prepared by mixing an aqueous solution of ().\9c copper sulfate, /inc sulfate, nickel chloride, cobalt chloride, and hexamine cobalt III chloride with silica gel in 3:1 ratio. Thin layers were then prepared in a similar fashion as described for plain silica gel plates {27).

Procedure. One or two drops of anion solutions were spotted on the plates with tine glass capillaries 1 he spots were dried and the chromatoplates were developed, allowing the solvent to ascend to 10 cm from the starting line in all cases. After drying the spots were visualized using the appropriate reagent.

For the stud\ of the loadmg effect on the /?, values, the known volumes of standard solutions of anion weie spotted on the chromatoplates with a micropi-pette. The plates were developed with .S, (1:1:8). The spots were detected and their/?[ (/?, of leading front) and/î {Ry of trailing front) values were determined. The areas of the spots produced at different concentrations of anion were also calculated. Standard solutions of lO, and BrO, (2.5-10^), NCK (2.5-4()9f), and I (2.5-8()9(^) were used

For semiquantitative determination of BrO, and NO; . O.OI ml of various standard solutions of KBrO, (l-l()'/r)and NaNO^ (2.5-409f) were spotted on silica gel impregnated with O.l'f C'uS04 layers. The chromatograms were developed with S, (1:1:8). After detecting the spot, it was copied onto tracing paper from the chromatoplates and then the spot area was calculated.

In order to achieve the separation of anions at different pH values, the pH of the test samples were brought to the required value by the addition of either glacial acetic acid or dilute sodium hydroxide solution.

The limits of detection of various anions were determined by spotting different amounts of anionic solutions on the chromatoplates. The plates were developed and detected. The method was repeated with successive lowering of the amount of anion until no detection of the spot was achieved. The minimum amount of anion just detectable was taken as the limit of detection.


The silanol gioup of hydrated silica gel is weakly acidic and immersion in an aqueous salt solution caLises some cation exchange through a reversible reaction.

\r • w( SiOH) ^ MtOSi-)"„/" + w H ' .

This cation exchange in the normal pH range of 4-7 is very small; n is the charge of the unhydroly/ed ion and is equal to m for monovalent ions. Thus the formation of a metal-surface complex on the surface of silica gel brings about a change in the retention behavior of the silica gel surface toward inorganic species.

The results ot this studv have been shown in Figs. 1-4 and Tables 1 ^ . In many cases it was found possible to separate one anion from several anions. The /?, values for all anions on copper sulfate-impregnated layer showed excellent reproducibility (variation does not exceed 59^ of the average value), except NO; which gave a variation of 8-10''^ from the average /^, value. Among the solvent system used, FA:DMS():acetone (1:1:8), i.e., S, (1:1:8) was the best and the silica gel layers impregnated with ().\'4 copper sulfate gave better results compared

42 MOHAMMAD AND I IWARl

to Other impregnants Therefore, the chromatographic system consisting of 0.15f copper sulfate-impregnated sihca gel layers as the stationary phase and S, (1:1:8) as the mobile phase was selected for detailed study. Figure 1 illustrates the dependency of Rf values of anions on the nature of impregnants. Aqueous solutions (0.19?) of the chlorides of Co"^, Co^ * , or N r " and the sulfates of Cu"* or Zn" were used as impregnants and anions were chromatographed on the impregnated silica gel layers with the S, (1:1:8) solvent system There is a

o o X u.

ct

LL Ct

100 r

60 -

20

8>-4

\

' ' c

f 1 1 /

A ' /

A/ ) 6 —1—X—

^ \

\

\ 1 1 ».y

Zinc sulphate

Nickel chloride

vx eg >f

O

100 r o o

X

U-tr

u. en

o o

u. tr

u. a. SI

Plain silica gel

- Copper sulphate

Cobalt chloride Hexomme cobalt I I I chloride

Anions PIG 1 Compaiison ot K, values ot anions on ditterenl impregnants with the FA DMSO Acetone

(I 1 8) svsteml ACompaLl spot with « , - / ? , < 0 1 (A) Badly tailed spots with « , - « , 0 4

( HROM \ lOCiRM'HY Ol ANIONIC P O I I U I A N I S 43

0 i -

0 8

0 2 ^

0 L

~ x »

i h

MS A eto

Fe(C N ) ,

Fe(CN)g

" ' 3 ) MoO/ WO,

0 i

0 2

0 0

B rO ,

(1 8) (3 6) (6 3)

DMSO Acetone

1 ic 2 ^ttti.1 ot DMSO Lonti-nli ition i)n the R, \ IIULS ot anions thi()mcitot,idphccl on sihea j,cl l.iycrs impiegn.iltd wilh 0 I Loppti Mill ilt Nl) not dtttclccl

group ot anions ihdt die slionglv adsorbed or have a little mobility, and a group ot anions (N()^ SC N I and BiO, ) migiating with the solvent tront giving high /Ji values Bi could not be detected on the impregnated layers whereas it was easily detectable on picun silica gel lavers In most ot the cases Ci()4 CrÔy , and Pe(CN),' pioduccd tailed spots I he tailed spots in the case of Cr04 and CrÔy aie possibly caused due to the coexistence ot the following species in equilibiium as expected in acidic media

Ci ,() ' + H ,0 ;=± 2HCr04 HCi()4 — H + Cr()4

However the tailrng ol h e(C N),' spot seems due to the incomplete precipitation ot Pe(CN)^ b\ the /inc present as impurity in silica gel

higure 2 summaii/es ihe adsorptron behavior ot anrons m DMSO acetone systems containing varving concentrations of DMSO and acetone in their mixture

LJ. CE

1

0

0

0

u n

0

9

8

2

-

1 ^ 0 1 1 1 1 1 1 1 1 1

— • KI

• NaN02

_ - ^ K I 0 3

1 1 1

PH

I K ' 1 ticel ol pll ot sample on R, values ot anions

44 MOHAMMAD AND TlWARl

DMSO being an aprotic dipolar solvent with hard oxygen and soft sulfur is a good solvating agent for anions, while acetone does not solvate the ions. There is a gradual and slow increase in the Rp values of anions with inci easing DMSO concentration NO^ and BrO, showed a sharp increase in their /?[. values with inciease in DMSO concentration. DMSO, being a stronger solvent than acetone, interacts strongly with the solute, decreasing its adsorption and causing faster migration However, the slow evaporation of DMSO from chromatoplates after their development, longer development time, and poor detection of anions hamper Its applicability at higher concentration.

There is a little effect on the /?[ values obtained for KI, KBrO,, NaNOj. and KIO, between the pH limits of 2.5 to 12 (Fig. 3). The formation of highly compact spots at all pH values permits a reliable and reproducible separation of 10, from NO, , BrO, and I over a wide range of sample pH. This method can be applied for the separation and removal of 10, from acidic, neutral, and saline waters

An attempt has been made for semiquantitative determination of anions by the measurement of the spot area The spots were directly drawn on a piece of

TABLE I IJR) Values ot Anions in Solvents Containing DMSO and Acetone Mixed with 0 1 M Acids on

0 r-f CuSOj-lmpregnated Layers

hR,

0 I M H,SO. 0 1 M HCIO, 0 I M HCI

' \n ions

Fe(CN)^

Fe(CN)^

SCN

C i o ;

Cr,()^

Br

1

POj N O ,

N O ,

ID,

BrO

M o O j

W O j

vo. CO]

MnO^

0 1

1 1 S

12

02

89

^0 V

^ 0 '

N D

8X S

0(1

9^

84 ^

07

8^

00

00

00

— —

M hormic

^ 1 6

^S

n 91 S

% v

16 5"

94 "i

96

00 91 S

N D

21 9^ "i

00

00

00

— —

acid D M S O

1 1 6

<; <i

00 9S

M)"

Mr

N D

N D

00

N D

N D 5 5

89

00

N D

^ s — —

acetone

•i 1 4

DS 0 9S •;

2 5

9^

DS

0 94 S

DS

0 9"; s

9"

9S

(K) 9S 5

97 "i

80 5''

97

00

(K)

N D

— —

D M S O

ace tone

( 1 1 8 )

22"

00

9*; V"

^'^"

N D

96 "i

00

87 •!

N D ^ "i

80

00

00

00

00

10 ^

D M S O

ace tone

(1 1 8)

10 "i

00

88 "i

27"

21 S"

N D

9'i

00

77

8li DS double spot Nt) not detected ' Badly tailed spot wilh « , - /?, ^ 0 4 ' I ailed spot with /?, - /?, > 0 1

l A R I t 2a

Quanti tdi ivc Stp.u. i l ion i>t l O , from B r O , N O , I Br and SC N on 0 1*^

C u S d j Impicuna teJ Silica Ciel 1 avers Using hA D M S O Acetone (1 I Hi

I Odding uiioiinl ol nidividual

anion salt in ni ixtui t

K B t O

0 2^ nig

(1 s nig

0 I nig

N a N O

1 0 nig

2 0 nm

0 2 nig K l

1 0 niK

2 0 nm

4 0 nig

0 4 nm

KBi 4 0 mg " (» nig 4 0 mt;

N H , S( N

IO | ig

l O , S ( ) |

0 4

•>()

0 4

sO|

0 4

IDC

1 0

0 4

u-g

mg

M-g

mg

u-g

mg

t |Xg

mg

mg

Sepai . i l ions

( « , - R,)

l O , (0 1 M ) 0 ) BrO , (0 96-0 77)

l O , (0 17-0 0) - B r O , (0 9 8 - 0 ^ 6 )

l O , (0 ^s -0 0) - B rO , (1 0-0 82)

l O , (0 2s-()0) N O , (0 9X-0 67)

l O , (0 27-0 0) N O , (1 0-0 M) l O , (0 M-^)0) N O , (1 0-0 78)

l O , (0 22-0 0) I

l O , (0 21-0 0) 1

l O , (0 ^4-0 0) I

l O , (0 ^s-c) 0) 1

l O , (0 41-0 0) Br

(1 0-0 89)

(1 (M) 84)

(1 0-0 7" )

(1 0-0 87)

(1 0-0 69)

l O , (0 4-0 0) Br (1 0-0 6S)

l O , (0 4-0 0) Br (1 0-0 81)

l O , (0 M ) 0) SC N (1 0-0 81)

\Mth S( N ( toncen t ra l ion I 'c) results m the tormat ion ot \(>l( 1 he s\nthLliL niivluK ot lO

precipi tates

1 \ B 1 F 2b

Quanti tat ive Sepai ition ot NO t iom I SC N and Br on 0 \'i C u S O j Impregnated Silica Ciel

1 a \ e i s I sing Hoimic Acid Acetone (1 9) as Mobile Phase

1 oadinc imoiinl ot individual

anion salt in nnxtu ie

N a N O

S()|Xg

0 s mg

1 0 mg

2 0 mg

s O n g

0 s mg

1 0 mg 2 0 mg

100 |ig

4 0 mg

Kl I) 12s nm

0 2s mg

0 ^ mg

1 (I nm

I 0 mg

NIl^SC N

0 12s mg

0 2 s mg

0 s nm

0 s mg

IsBi 0 s mg

1 0 mg

2 0 nm

4 0 mu

0 2^ mg

0 s nm

N O ,

N O ,

N O ,

N O

N O ,

N O ,

N O

N O

N O ,

N O ,

N O ,

N O , N O ,

N O ,

N O ,

N O

N O ,

N O ,

N O ,

Sepal

(«,

(0 6 M ) 4 1 )

(0 62-0 42)

(0 7 M ) SS) -

(0 77-0 ^7) -

(0 6S-0 W) (0 6S-0 ^2)

(0 67-0 11)

(0 69-0 %)

(0 6 M ) 4^)

(0 82-0 69)

(0 70-0 M))

(0 60-0 M) -

(0 6 M ) 26) -

(0 72-0 M)

(0 82-0 64) -

(0 8-0 72) -

(0 79-t) 62)

(0 74-0 ^) -

(0 87-A) ';6)

al iens

«,)

1

1

1

1

1

1

1

SCN

SCN

S t N

SCN

SCN

SCN

Br

Bi

Bi

Br

Hi

Bi

11 0-0 87)

(1 0 -0 91)

(1 0-0 S i)

(1 (M) 81)

(1 0-0 80)

(1 0-0 78)

(1 (M) 80)

(1 0-0 88)

(1 0-0 79)

(1 0-0 89)

(1 0-<)88)

(1 0-0 79)

(1 (M)7<i)

(1 0-<) 9)

(1 0-0 91)

(1 0-0 9S)

(0 96-0 86)

(1 0-0 8'i)

11 (M) 94)

\(>!t Detection ot Bi istlilliLull i cqui img about 20 mm after spiaying the ch romatogram with the

leagent

4s

46 MOHAMMAD AND IIWARl

JABLE 1 Separations Achieved txperimentallv at Different pH Values of Sample Mixture on Silica del

Layers Impregnated with 0 I'/r Copper Sulfate Usmg FA DMSO Acetone (I I 8) as Mobile Phase

Sample pH value

t

^ 4 s (l

7 8 9

\(>ic Ihe

detection amounts of 1 and

"i 4 II 7

2 *>

6 2

II " 2 4 6 4

11 0

NO,

lO, K), lO, K), lO, lO, 10, K) K),

Separations achieved

(«, (0 12-0 0) (0 12-0 0) (0 IS-0 0) -(0 06-0 0) -(0 OS-O 0) -(0 22-0 0) -(0 07-0 0) -(0 09-0 0) -(0 18^) 0)

- « , )

BrO, BrO, BrO, 1 1 1 NO, NO, NO,

0 9M) 8*; 1 0^) 80) 1 (M) 8*)) 1 (M) 9<i) ) (M) 9"*) 1 (M) 96) 1 0-0 90) 1 (M) 8 ) 1 0-0 8^)

are taken m twofold excess in the mixture to ensure sharp

transparent paper from thin layer chromatograms and the area of each spot was calculated. A linear relationship was only obtained for NO, and BrO, when the amount of the sample spotted was plotted against the area of the spot (Fig. 4) The precision and accuracy is always below ±l59f. A similar relationship has been reported for cations {28} and a-tocophenols (29).

The results of chromatographic behavior of anions in mobile phases containing variable propoitions ot DMSO, acetone, and formic acid are shown in Table 1. The clarity of detection and compactness of spots increase with the increase in the concentration of acetone in the mobile phase and hence FAiDMSOacetone (11:8) was the best solvent system in this regard. CrO^ , Cr O^ . and Fe(CN)^ gave double spots when eluated with FA.DMSOracetoe (5:1.4). The Ry. value of anions obtained in 0 1 M mineral acids (H2SO4 or HCIO4 or HCI):DMSOacetone (I 1:8) are also recoidcd in Table 1. These acids can be put in the following

100 200 300 i.00 500

Amount ( | j g )

18

U

10

6

2

0

- ® -

• ^^ •-"""^

^ ^ ^ ^ 1

^

• ^ • - ^

1 1 1 1

200 400 600 800 1000

Amount C ug )

FIG 4 Plot of spot area vs amount of anion salt loaded (a) KBrO, (b) NaNO,

( HROM \IOCiRAPHY Ol ANIONIC P O I I U T A N I S 47

I \m h 4 Limits ot DeteLlion ind Dilution I iniits of Anions as Ihcir Salts on Silica (jel Ld\trs Impregnated

with 0 I ( nSO, Solution I sing HC l()4 DMSO Acetone (118) as Mobile Phase

Sample

1 • >

^ 4 s

6 7 8 9

10

II

i :

n 14 | s

Dilution limit

MoO

W O ,

( (), 1

\() I'O

1 e(( N)

l e ( ( \ )

l o

HiO

S( \

NO

MnO

( lO

( I O

1 (Vo

Ions Salts

Ni MoO, 2H O Na WO, 2H O Na C O, Kl N iVO H O N I H P O , K,he(( N), k4Fe(C N), 2H O KIO KBrO, NHjSC N NaNO KMnO, k CiO, K Ci O

I (Volume ot lest si)lution ^ l()'')/|l imit ot detection (n,g)|

Limit ot detection

(|JLg)

100 0 100 0 100 0

10 0 10 0 10 0 10 0 10 0

S 0

1 0

1 0

1 0

0 ^

0 s

0 ^

Dilution

limit

1 10

1 10

1 10

1 10

1 10

1 10'

1 10'

1 10

1 2 >

1 10^

1 lO-"

1 K)-*

1 2 A

1 2 X

1 2 >

10

K)-'

K)-*

K)-*

pretericd order it used .is elucnt in combiridtion with DMSO and dtelone FA > HCIO4 HoSO^ HC 1 Results ot quantitative separation ot 10, and NO^ from large excess ot 1 SC N Bi and BiO, and vice veisa are given in Tables 2a and 2h I he proposed method is veiy convenient tor separating milligram quan titles ot an anion trom microgiam to milligram amounts of other anions

In oidei to widen the applieability ot the important separation ot 10, trom 1 NO^ and BiO, its separation trom synthetic mixtures ot ditterent pH values has been invest mated I able ^ leveals that 10, can be easily separated trom I NOT and BiO in the pH langc 2 4-110 ot sample solution Thus the method can be utilized to separate lO, trom acidic, neutral and alkaline (natural or synthetic) samples containing I NO^ and BrO, without adheiing to close control ot sample pH

Table 4 summaiizes the limits ot detection ot some anions as their metal salts along with then dilution limits It is evident trom this table that the proposed method is highlv sensitive toi the detection ot several anions

ACKNOWLEDGMENTS

Piotcssoi K 1 \ isim i hiiiman Depaitmenl ot Applied Chemistry is thanked toi his generous cooper ilion in pun iding the necessiiv lesearch I icilities 1 hanks ire also due to Anand Rawat toi his help

REFERENCES / Choisli)v.ski I 1 Ihiiiinin W O Jawoiski J J Anon Anal V M H / 1981 5 14 2 leres I Moicno DommgiK/ C oncepeion Ci iieia Moieno Abel Marine Lonl AniUwt 198'? 108

SOS

48 MOHAMMAD AND 1 IWARl

i Voivnels M P Kitaeva I P I imefbaev A P Zh Anal Khim 1986 41, 1989 4 ronogcu Y Iwaida M J hood Piot 1981 44, 8 -i •i Aimal M Mohammad A Falima N Khan, A H Muroclum J 1989 39,^61 6 Aimal M Mohammad A Falima N Munxhem J 1988 37,^14 7 Mohammad A Fatima N J Liquid Chromatof>i 1987 1 0 , 1 ^ 9 fi Ajmal M Mohammad A Fatima N Ahmad J J Liquid Chromulof;i 1989 12,^16^ 9 Chawla H M Ralhan N N Garg N K Chibber S S y Hi>>h Riwhit Choimilom (hio

inaloiii C oniiniin 198(1 3, 6'il 10 Kawanbe K Mamyama K Yakugaku 7 1981 101, 192 Chun Ahsu 1982 96 4M2(1K // Buchbauer O Markis R E S< / Phcinn 1983 51,41 12 Ravindia Nath K Janaidhan P B Chcm Ahsti 1982 97, 6'i<i73m /? DenBlevkei K 1 Sweet T R Chiomatof;raplua 1980 13 114 14 •\]mal M Mohammad A Fatima N Ahmad J J Planui Chromaroni 1988 1,239 h Shimi/ii T lanaka H Ohsawa I Chiomalographm 1982 15, 177 /6 Shimi/ii T Vchara F Ohtani M Chiomcitoi>niphia 1986 21, 17"! 17 Mohammad A Fatima N Chiomiitotiit'pltui 1988 25, S36 /.S Ishid.i K Ninomiya S laked.i Y Walanabe K / Chromalogi 1986 35,489 (9 Fa\mv\ N MohammAj A Stp Sii Tttfinoi WU W, 429 20 Gaihakian 1) S Ro/ylo J K JanicRa M J Liquid C hioimiloKi 198S 8 2969 21 Okiimuia T laUiiUa 1979 26, 171 22 Franc I Kosikova F J Chiomalo^l 1980 187, 462 2< Aimal M Mohammad A Fatima N Ahmad J J Plaiiai C hioiniilovi 1990 in press 24 Mohammad A Fatima N Chionuilo^Kiphui 1988 25, 'i36 2^ Mohammad A Fatima N ChionuiloVi'iphia 1987 23, 6* 3 26 Mohammad A Fatima N J I iquid C hioinaloi;i 1986 9, 1903 27 \)md\ M Mohammad A Fatima N Ahmad J J Phinui C hiomaloin 1990 3 '!96 2<S Mohammad A Fatima N Chroinalovniphia 1986 22, 109 29 Sehei A Mikunhim Ami 1961 308

CHROMATOGRAPHIA

An International Journal for Rapid Communication in Chromatography and Associated Techniques

REPRINT

Editors

E R Adiard Burton United Kingdom

H Engelhardt Saarbrucken Federal Republic of Germany

L S Ettre Norwalk Conn USA

G Schomburg Mulheim Federal Republic of Germany

R Stock Keyworth United Kingdom

Publisher

F Lube Wiesbaden Federal Republic of Germany

Friedr. Vieweg & Sohn Pergamon Press

Vieweg

r UD AMI iiTtf% ADA Dill A . £. « . . » I M - W / V M . ^

CHROMATOGRAPHIA An International Journal

for Rapid Communication in Chromatography and Associated Techniques

Distributed internationally, Chromatographia provides a medium for the rapid and widespread communication of current information in the field of chromatography. This includes associated techniques and combined methods, as well as the application of computers and modern data systems.

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The editorial board of Chromatographia consists of internationally renowned scientists from Austria, P. R. China, Czechoslovakia, France, both the German Democratic and Federal Republics, Hungary, Israel, Italy Japan, The Netherlands, Sweden, Switzerland, the United Kingdom, the United States of America and the U.S.S.R.

Chromatographia is published monthly by Friedr. Vieweg & Sohn Verlagsgesellschaft mbH, R O. Box 58 29, D-6200 Wiesbaden 1, F R. G. and is distributed in the United States by German Language Publications, Inc., 560 Sylvan Avenue, Englewood Cliffs, NJ 07632, USA.

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K. Jinno, Toyohashi, Japan B. Karger, Boston, MA, USA M. L. Lee, Provo, UT, USA W. Lindner, Graz, Austria K. Markides, Provo, UT, USA M. Martin, Paris, France H. Poppe, Amsterdam, NL R A. Sewell, Liverpool, UK A. M. Siouffi, Marseille, France C. F Simpson, London, UK P J. Schoenmakers, Eindhoven, NL K. Unger, Mainz, FR Germany D. Westerlund, Uppsala, Sweden

Honorary Editorial Board

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FR Germany G. Schay, Budapest, Hungary W. Simon, Zurich, Switzerland E. Smolkova-Keulemansova,

Prague, CSFR K. Tesarik, Brno, CSFR G. Widmark, Stockholm, Sweden A. A. Zhukhovitskii, Moscow, USSR A. Zlatkis, Houston, TX, USA


Thin-Layer Chromatographic Separation and Identification of Some Anions on Copper Sulphate Impregnated Silica Gel Layers

A. Mohammad*/S Tiwaii

Analytical Laboratory, Department of Applied Chemistry, Zakir Hussain College of Engineering and Technology, Aligarh Muslim University, Ahgarh-202002, India

Key Words Thin layer chromatography Copper sulphate impregnated silica gel Anion separation Acetone elucnts

In the present work, the thin-layer chromatographic behaviour of some common anions on plain and copper sulphate-impregnated silica has been investi gated in mixed aqueous-organic solvent systems con taming acetone

Summary Thin layer chromatographic behaviour of thirteen anions on plain silica gel and sihca gel impregnated with copper sulphate solution has been investigated in aqueous organic solvents containing dcetonc some of which have achieved reliabk and reproducible sepa rations The effect ot copper sulphate concentration on the mobility of anions has been examined I he results obtained on plain silica gel have been com pared with those obtained on copper sulphate impregnated layers 1 he impregnated layers drama tically change the selectivity and permit separations not possible on untreated silica Aqueous sodium chloride acetone (9 I) and ammonium hydroxide ace tone (9 1) were the most effective solvent s>stems for differential migration ol anions Better results in terms of clarity of detection and ccmipactness of spots were found with HCOOII acetone as compared to HCl acetone

The etiect oi anion loading on Rj values has been investigated and identification limits on impregnated layers determined

Experimental

Apparatus A TLC apparatus (Toshniwal, India) was used for the preparation of 20 x 3 5 cm glass plates The chroma tography was performed in 24 x 6 cm glass jars

Reagents

Silica gel (E Merck, India), formic acid, hydrochloric acid, hydrobromic acid, sodium chloride, ammonium hydroxide and acetone (B D H , India) were used These and all other reagents were Analar grade

Test Solutions

The test solutions (1 %) were either sodium oi potassium salts of nitrate, nitrite, vanadate, molybdate phosphate, bromide, iodide, tungstate, ferrocyanide ferncyanide, chromate and dichromate except SCN (ammonium thiocyanate) Water, double distilled from all-glass apparatus and having a specific conductivity K = 2 x \(y^ mhos cm ' at 25 °C was used for solutions

Introduction 1 rom the literature the TLC of anions has been much less extensive compared to th it ot cations |l-3[ Silica gel [4-S] cellulose [9 11], alumina [12] silufol [13] and hydrated stannic oxide layers [14] have been used for the separation and identification ot anions although no work has been reported on the use of silica gel impregnated with inorganic salts

Detect ion

The following reagents were used

1 Saturated solution of AgNO^ in methanol for Br

I , CrO^ , Cr20-, and PO^ The brown spot due to

Br appeared 30 mm after spraying

2 02 % Diphenylamine m 4M H2SO4 for NO, and

( hronidtographia Vol ' O No " s Otlohcr 1990 Oriiînals

0009 189V90/10 ()40s Os $()^,()()() ^ 1990 Priedr Viewe" <t Sohn VerlnPsoesolKt h^fl nihH

40-

3. 10% Ferric chloride in 2M HCl for SCN",

Fe(CN)ândFe(CN)^".

4. 0.5 % Alcoholic pyrogallol solution for MoO?"

and WO4".

5. 1 % Aqueous potassium ferrocynide for VO^;

VOj^ was also detected with this reagent.

Mobile Phases

The following solvent systems were used as mobile phases:

Ml = HCl : Acetone (1:9) M2 = HCl : Acetone (9:1) Ml, =NaCl : Acetone (1:9) M4 =NaCl : Acetone (9:1) Ms = HBr Acetone (1:9) M6 = HBr • Acetone (9.1) M7 = N H 4 0 H : Acetone (1:9) M8 = N H 4 0 H : Acetone (9:1) M9 = Formic Acid : Acetone (1-9) Mio = Formic Acid : Acetone (9:1)

In all the solvent systems, HCl, NaCl, HBr, NH4OH and formic acid were O.IM aqueous solutions while the acetone was pure.

Stationary Phases

Si ~ Plam sihca gel G S2 = Silica gel impregnated with 0.1-5.0 % aqueous

solution of copper sulphate.

Preparation of Thin-Layer Plates

(i) Plain silica gel plates Silica gel was mixed with conductivity water m the ratio 1:3 with constant shaking for 5 to 10 min. The resultant slurry was coated on well-cleaned glass plates to give a layer approximately 0.25 mm thick. The plates were dried at room temperature (30 °C) and then heated at 100 ± 5 °C for 2 hours. After activation the plates were stored in an air-tight chamber.

(/;) Copper sulphate-impregnated silica gel plates To prepare impregnated silica gel layers, a slurry was made by mixing an aqueous solution of 0.1-5.0%, copper sulphate with silica gel in the ratio 3:1. Thin layers were then prepared as described above for plain silica gel plates.

Procedure For qualitative analysis, one or two drops of the anion solutions were spotted on the plates with glass capillaries. The spots were dried and the plates developed by the ascending technique. The ascent of the solvent was fixed at 10 cm in all cases. After development, the plates were dried and the anion spots visualized with the appropriate spray reagent. Rp values of the leading front ( R L ) and of the trailing front ( R T ) were measured and reported as ( R L - R T ) -

Rp values were calculated from Rp = RL + R ,

The limits of detection of the various anions were determined by spotting different amounts using standard solutions, developing the plates, and estimating the spots. This was repeated with successively smaller

amounts of the test substance until spots could no longer be detected. The smallest amount of anion just detectable was taken as the limit of detection.

Results and Discussion The main points which emerge from this study are summarized below:

1. A small change in Rp values was sometimes observed when mixtures of anions were developed as compared with single substances.

2. The development time for a 10 cm run ranged between 45-70 min depending upon the mobile phase.

3. Silica gel impregnated with CUSO4 gave excellent results. Thin layers were of good quality. Generally, the spots were compact and well formed in all solvent systems at 0.1-1 % impregnation. Plates impregnated with 2-5 % copper sulphate solution deformed during development.

4. Rp values reported in this paper represent the averages of triplicate tests and were measured to the centre of the spots on the plates.

5. Sodium chloride-acetone (1:9 and 9:1), NH4OH-acetone (1:9 and 9:1) and HCl-acetone (1:9) systems were found most suitable for separations. A few anions showed occasional tailing. HCl-acetone (1:9) was found to be the best solvent system for multicomponent separation with 1 % copper sulphate impregnation. Formic acid-acetone (1:9) produced highly compact spots of anions on 0.1 % copper sulphate layers.

6. The copper sulphate travels with the solvent front upto the middle of the plate in solvents M2, M^ and MiQ. These systems are therefore unsuitable for impregnated layers.

7. Solvents containing 90 % acetone gave better results than those containing 10 % acetone.

8. In all solvent systems, CrO^", CrjO^", Fe(CN)^

and Fe(CN)g produced coloured spots on im

pregnated layers and thus were self detecting.

9 NOj could not be detected on 1 % impregnated

layers while it was clearly detected on plain silica gel as well as on 0.1 % impregnated layers

10. In addition to the other ions, VOj^ was also

chromatographed to assess the possibility of

separating V02^ from VO3.

The results have been summarized in Figures 1-3 and Tables I and II. Figure 1 summarizes the results of ARp values (ARp = Rp on plain silica - Rp on impregnated silica) with various mobile phases It is evident from Figure 1 that impregnated layers are more selective (strongly sorbing) than plain silica for most of the anions, as indicated by positive ARp values. There was no significant difference m the mobility of anions when chromatographed on silica impregnated with 1 % or 0.1 % copper sulphate using HCl, HBr, NaCl or formic acid-acetone (1:9) solvent systems (e.g. la) . With formic acid-acetone, NO^ showed increased mobihty on 1 % impregnated layers

406 Chronidtogrdphid Vol 30, No 7/cS October 1990 Originals

Table I. Some experimental separations

Stationary phase Mobile phase Separation achieved ( R L - R I )

Silica Gel impregnated with 1 % CUSO4 solution

HCl Acetone 19

Silica Gel impregnated Formic Acetone with 0 1 % CUSO4 solution dcid

19

Silica Gel impregnated with 1 % CUSO4 solution

HCl Acetone 19

Fe(CN)6 (0 0-0 0)-NO2 (0 38-0 21) - SCN" (0 97-0 78)

Cr04 {00-00)-NO2 (0 43-0 23) - Br" (0 70-0 54)

PO4 (00-00) NO2 (0 50-0 23) r (10-0 85)

M0O4 (00-00) VO, (0 31-O26)-SCN-(10-0 9)

Fe(CN)6 (0 0-0 0)-NO2 (0 62-0 56) - NO3 (0 92-0 83)

CrjOT (0 24-00)-NO2 (0 80-0 60) - NO3 (0 97-088)

POf (00-0 0)-NO2 (0 68-0 4 9 ) - r (10-0 95)

WOi (00-00)-NO2 (0 67-058)-Br-(0 9-075)

Fe(CN)i (0 0-0 0) - NO2 (0 53-0 39) - Br~ (0 76-0 64) - SCN" (1 0-0 87)

Fe(CN)6 (0 0-0 0)-NO2 (0 56-0 4) Br" (0 74-0 63) - SCN (10-0 88)

Cr04 (OO-OO) NO2 (0 50-0 35) - Br-(072-0 59)-SCN'(10-0 85)

CrjO? (0 0-00)~NO2 (0 49-0 35) - Br" (0 75-0 59) - SCN (10-0 87)

PO4 (00-O0)-NO2 (0 56-0 41)-Br (0 72-0 65) SCN-(10-0 9)

v o l (00-0 0)-N02 (0 49-0 35)-Br (0 70-0 65)-SCN-(1 0-086)

M0O4 (00-00)-NO2 (0 6-0 5) - Br" (0 75-0 62) - SCN (10-0 9)

Amount of I and Br in mixture three to Inc lold m excess for sharp detection

Table II. Limit of detection and dilution limit ol anions and VO2 on silica gel impregnated with 0 1 % CUSO4 with formic acid acetone (1 9) mobile phase

2+

SI No

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Ions

Br-

PO4

r vof VO3

NO3

NO2

SCN-

Fe(CN),'

Fc(CN)6^

Cr04

CrjO?

M0O4

wo|

* Dilution limit

= 1

Salts

KBr

Na2HP04

Kl

VO2SO4

NaVO-i Fl20

NdNO^

NaN02

NH4SCN

K4Fe(CN)6 ^H20

K3Fe(CN)(,

K2Cr04

K2Cr207

Nd2Mo04 2H2O

Nd2Vv'04 2HiO

Volume ol test solution (ml) x lo''

Limit ol detection (|ig)

I unit ol detection

(Hg) KXK)

10 0

10 0

10 0

10

10

10

10

10

0 5

0 5

OS

01

01

Dilution limit*

1 1()2

1 103

1 lO-''

1 10-

1 104

1 11)4

1 10*

1 10^

1 Uf

1 2x10"*

1 2x10-*

1 2xU)4

1 103

1 10-''

(ARp = -0.3) compared to its mobility on plain silica or silica impregnated with 0 1 % CUSO4 (ARp = +0.1). The reverse trend was observed for I which moves faster on plain sihca (ARp = 0 24) compared to ) % layers However, 0 1 % CUSO4 impregnation was

found to be ineffective in changing the mobility of 1" which behaved similarly in NH40H-acetone (1 9) where it moves faster on plain silica (Figure Ic) compared to 1 % CUSO4 layers. Sodium chloride acetone (9.1) and NH40H-acetone (9:1) systems were found the most effective in changing the mobility ol most anions on impregnated layers and thus open numerous possibilities for separations (Figures lb and d). However, the tailed spots produced by some anions limit their separation from other anions With NH40H-acetone (9:1), most of the anions were strongly adsorbed on 1 % layers compared to plain or 0.1 % layers.

Figure 2 summarizes the hRp values of anions chromatographed on sihca gel impregnated with 1 % CUSO4 and developed with solvents Mi, M-;, M4, Ms, M7, Mg and M9. It is evident that the different mobile phases are able to bring about different retention sequences of anions leading to several binary, ternarv and quaternary separations. The mid Rp (Rp = 0 4 -0 6) values of NOj in Ml and Br" in Mi, M7 and M<s can be used for their separations form all other anions with higher or lower RF values. VO^" , PO] Mo04^, CrjO^^, Fe(CN)^, Fe(CN)^^ and W o j a r e strongly adsorbed on the impregnated layers (hRp -0-10) in all solvent systems containing 90% acetone (Figure 2). The mobility of a few anions is increased when the concentration of sodium chloride or NH4OH IS increased in the mobile phase (M4 and Ms). However, C r O ^ ' a n d Cr20.y~ showed tailing Likewise, M4 and Mg can be utilized for the se paration ofMo04~(hRp = 90-95) from the anions with low Rp values. SCN", I", Br^ and N 0 2 g a \ e higher RF values m most of the mobile phases and thus can be separated from anions with lower R[ values.

Chronidtographid Vol 30, No 7/(S Oclohcr 1990 Originals 40'

1 0

0.8

0 5

a:

<i 0 2

0 0

- 0 2

- 0 4

H C i : A c e t o n e (1 9 )

a)

r - N a C I . Ace tone ( 1 9 )

I I - N Q C I A c e t o n e ( 9 1

NaCI A c e t o n e ( 1 9 )

100

o 80

I - N H ^ O H A c e t o n e ( 1 9 )

I I - N H 4 O H A c e t o n e O 1 )

Anions

HCI A c e t o n e d 9 )

0

0 4

0 2

0 0

- 0 2

- 0 4

en <

N H ^ O H - A c e t o n e d 9)

- 0 4 I-

o O 2 •^ U S

A n i o n s

NH4OH A c e t o n e O 1)

A n i o n s

Figure 1 ARp (Rp on plain silica gel - Rp on CUSO4 impregnated silica gel layers) vs anions ° 1 % copper sulphate impregnation A 0 1 % copper sulphate impregnation

Figure 2

hRp vs. anions. Stationary phase: 1 % copper sulphate impregnated silica gel. o Compact spots with R L - R T < 0.3 A Tailed spots with RL - RT > 0.3 * Badly tailed spots with RL - RT > 0.4

408 Chromatographia Vol 30, No 7/8. October 1990 Oneinals

1 - N a C l ; A c e t o n e i1 , 9 )

1 1 - N a C I : A c e t o n e ( 9 . 1 )

HCI . A c e l o n e i I - 9

A n i o n s

Figure 3 hRp vs. anions. Stationary phase 0 1 '\> copper sulphate impregnated silica gel. Other details as m Figure 2

In Figure 3, the results obtained on 0.1 % coppci sulphate layers in various solvents have been summarized. A major advantage of this adsorbent is that NOj, which could not be detected on 1 "/o coppci sulphate layers, was very clearly detected on 0.1 ",. layers. Comparison of Figures 2 and 3 show almost identical chromatographic behaviour of the anions on both the adsorbents in HBr-acetone and formic acid acetone. Conversely, most of the anions do not beha\c similarly on both sorbents in NaCl-acetone and NFÔH-acetone. Thus, in addition to the separation of NO^, from numerous anions on 0.1 % layers main other mutual separations of anions can be realized m NaCl-acetone or NH40H-acetone.

The chromatographic systems developed provide numerous possibilities for rapid, reproducible and clean separations of anions from multicotnponent mixtures. Some separations experimentally achieved have been summarized in Table L Table II summarizes the limit of detection and dilution limit of various ions. The proposed method is highly sensitive for most ions except Br^.

Acknowledegement We wish to express our sincere thanks to Prof. K. I. Nasim, Chairman, Department of Applied Chemistry, for providing the necessary research facilities. We also thank Dr. Anand Rawat for his valuable help.

References | 1 | U.A. 'I'h. Brinkman. G. De Vries, R. Kuroda, J. Chromatiitr ,

85,187(1 y?.'?). |2] R. Kuroda, M.P. Volynels, CRC Handbook ol Chroma

tography: Inorganics {M. Qureshi, ed.) Vol I, CRC press. B o o Raton, Fla, 1987.

|3J A. Mohammad, K.G. Varshney, Handbook ol I'hin-l a\er Chromatography («. Fried and J. Sherma, etis), Mare.-I Dekker, Inc., USA (m press).

|4] ./. Benes. Collect. Czech. Chem. Commun.. 44. 1(B4 (1979) l-SJ ./. Benes, Collect. Czech. Chem. Commun., 44. 14(X) (1979; |6| II.M. Chawla, N.N. Ralhan, N.K. Carg, S.S. Chibbcr, J lli <h

Res. Chromatogr./Chromatogr. Commun., 3, 6,S1 (1980). |7| K. Kawanhe, K. Maruyama, Yakugaku Zusshi 1981, 101. 192,

Chem. Abstr., 96,4.5420K, 1982. [S] / Zuanon Nelto, A.I-'.C. Graner, M. lontvihiro, Fcletic Ouini,

9, 1984,4,5; Chem. Abstr., KM, 141201 u, 1986. |9] G. Buchhauer, R.E. MarkLs, Sci. Pharrn., 51, 41 (198.?).

|10] R. Gallego, J.L. Bernal, A. Martinez, Ouim Anal., 31, ,3. !97 \ Chem. Abstr., 1978,88, 1.302267.

|11] R. Gallego, J.L. Bernal, A. Martinez, Ouim Anal., 31, 69, ' ,9/ ; . Chem. Abstr., 18181 la, 1978,

[12| K. Ravindra Nath, P.B. .lanardhan, Chem. Abstr., 97, 6.S,- ' MII, 1982.

|13| J. Franc, F. Koslkova, J. Chromatogr., 187, 462 (1980). [14] K. Sen. il.Ch. Ghosh. J. Liqd. Chromatogr., 3, 71 (1980).

Received: May 22, . f'lO Revised manuscripi received: July 7, I'^'O Accepted: July 18, i9H0 C

Chromatographia Vol 30. No. 7/8, O u o h c r 1990 Originals 40^)

Preparation of Anhydrotrypsin-Immobilized Diol Silica as a Selective Adsorbent for High-Performance Affinity Chromatography of Peptides Containing Arginine or Lysine at Their C-Termini

T. Ohta*/T. Inoue/Y. Fukumoto/S. Takitani Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12 Ichigaya-Funagawara Machi, Shmjuku-Ku, Tokyo 162, Japan

Key Words Column liquid chromatography Affinity chromatography Anhydrotrypsm C-termmal Arg- or Lys-containing peptides Diol silica

Summary Anhydrotrypsm (AHT), a catalytically inert derivative of trypsin in which the active site serine residue was converted to dehydroalanine residue by chemical modification, was immobilized onto diol silica through the activation with tnfluoroethanesulfonyl chloride, and an AHT-diol-silica column was used for high performance affinity chromatography separation of peptides containing arginine or lysine at their C-termini from the others Improved separation in terms of speed was accomplished.

Introduction Anhydrotrypsm (AHT), a catalytically inert derivative of trypsin in which the active site serine residue is converted to dehydroalanine residue by chemical modification, exhibits affinity toward peptides containing arginine or lysine at their C termini [1] Kumazaki et al. [2] reported that immobilized AHT prepared through the activation of agarose with cyanogen bromide was useful for the selective isolation of C terminal peptide fragments from tryptic or chymotryptic digests of proteins. However, several hours are usually required for the separation In addition, immobilization with cyanogen bromide, though employed widely for affinity chromatography, results m formation of charged isourea groups, and coupled hgands tend to leak from the support [3].

High performance affinity chromatography (HPAC) introduced by Ohlson et al [4] has been used for the fast and efficient purification of a number of bio logical molecules In this study, we immobilized AHT onto diol silica, a widely used support in HPAC [5], through the activation with 2,2,2-trifluoroethanesulf onyl chloride (tresyl chloride) that was developed tc overcome the disadvantage arising from the use ol cyanogen bromide [6], and used an AHT-diol-silica column for the HPAC separation of peptides containing arginine or lysine at their C-termini from the others. Improved separation in terms of speed wa' accomplished without reduction in resolution.

Experimental

Materials Tresyl chloride was obtained from Nacalai Tesque (Kyoto, Japan). Bovine pancreas trypsin (type III) and phenylmethanesulfonyl fluoride were from Sigma (St Louis, MO, USA). Peptides were from Peptide Institute (Osaka, Japan), Sigma and Aldnch (Milwaukee, WI, USA). Peptides solution were prepared in water and stored at -20 °C until used.

AHT was prepared by the method of Ishii et al [7] The remaining trypsin activity in the purified AHT was 0.2-0.7 % of the original activity when benzoyl DL-arginine p-nitroanilide was used as a substrate. Ir certain experiments, crude AHT prepared without purification by affinity chromatography [7] was used.

Diol silica was prepared by silanization ol LiChrospher Si 300 (10 (im, E Merck, Darmstadt FRG) with 3-glycidoxypropyltrimethoxysilane undei anhydrous conditions followed by hydrolysis of tht epoxy groups with 0.01 M HCl [8]. In certain experi ments, SEP-PAK diol (Waters Associates. Milford MA, USA) was used as did silica

Deionized water (Millipore RO-Q system) was usee throughout this study

410 Chromatogrdphid Vol 30, No 7/8. October 1990 Oriaiiidls

{)()()9 5893/90/10 0410 04 $03 00/0 © 1990rncdr Vieweg & Sohn VcrldgsgescllsthdU mbli

CHR0MA10GRAPHIA An International Journal

or Rapid Communication in Chromatography and Associated Techniques

Some important papers published during 1989 Characterization of Animal Fats via the GC Pattern of Fame Mixtures Obtained by Transesterification of the Triglycerides L Matter/D Schenker/H Husmann/G Schomburg

Evaluation of Non-Polar Bonded-Phases for the Clean-up of Maize Extracts prior to Aflatoxin Assay by HPTLC K I Tomiins/K Jewers/R 0 Coker

New Approach to the GC-Separation of Hydrocarbons by Using LC-like Microcolumns T Takeuchi/K Ohta/D Ishii

Size Exclusion Chromatography on Porous Fractals F Brochard/A Ghazi/M LeMaire/M Martin

Plananty Recognition of Large Polycyclic Aromatic Hydrocarbons by Various Octadecylsilica Stationary Phases in Non-Aqueous Reversed-Phase Liquid Chromatography K Jinno/S Shimura/N Tanaka/K Kimata/ J C Fetzer/W R Biggs

Preparative HPLC of Carotenoids M Isaksen/G W Francis

Applications of HPLC with Evaporative Light Scattering Detection in Fat and Carbohydrate Chemistry A Bruns/H Waldhoff/W Winkle

Synthesis and Characterization of Novel Bonded Phases for Reversed-Phase Liquid Chromatography A M Stalcup/D E Martire/L C Sander/S A Wise

Determination of Alternaria Mycotoxins in Foodstuffs by Gradient Elution Liquid Chromatography with Electrochemical Detection F Palmisano / P G Zambonin/A Visconti/ A Bottalico

Polymer Encapsulated Stationary Phases Advantages, Properties and Selectivities H Engelhardt/H Low/W Eberhardt/M MauB

A Chemically Bonded Liquid Crystal as a Stationary Phase for High Performance Liquid Chromatography Synthesis on Silica via an Organochlorosilane Pathway J Pesek/Teresa Cash

Hydrodynamic Chromatography of Macromolecules on 2 urn Non-Porous Spherical Silica Gel Packings J C Kraak/R Oostervink/H Poppe/U Esser/ K K Unger

Applications of Electrosorptive Detection in Ion Chromatography T Ramstad

The Strength of Interaction of Highly Retentive Silanols with Hydrocarbons on Porasil C J Nawrocki

Uncertainty Resulting from Inconstancy in the Slope of the Plot of Homologous Series S J Hawkes

Derivatization of Carboxylic Acids with 9-Bromo-methylacridme Using Micellar Phase-Transfer Catalysis F A L van der Horst/M H Post/J J M Holthuis/ U A Th Brinkman

Application of Diode Array Detectors for Solute Identification in Toxicological Analysis H Engelhardt / Th Konig

Physico-Chemical Modelling of Solute Retention in Reversed-Phase HPLC with Ternary Mobile Phases Teresa Kowalska

lon-Pair Chromatogrphy with Divalent Counter Cations in Reversed-Phase Systems C Pettersson/G Schill

Isolation and Quantitative Analysis of Hydroxylysine Glycosides R M Napoli/B S Middleditch / N M Cintron/ Y-M Chen

Optical Resolution HPLC Column Packings Carrying Penicillin Sulfoxide Nucleus Y Saotome/T Miyazawa/T Endo

Simultaneous Determination of Pseudoundme and Creatinine in Urine by HPLC with Polarographic Detection F Palmisano/A Guerrien/T Rotunno/ P G Zambonin


CHROMATOGRAPHIA An International Journal

for Rapid Communication in Chromatography and Associated Techniques

Editors E. R. Adiard, Delryn, Vicarage Lane, Burton, South Wirral L645TJ, UK

H. Engelhardt, Angewandte Physikalische Chemie, Universitat des Saarlandes, D-6600 SaarbriJcken, FR Germany

L. S. Ettre, Belden Avenue Station, RO. Box 21 75, Norwalk, CT 06852-21 75, USA

G. Schomburg, Max-Planck-lnstitut fiJr Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-4330 MLilheim/Ruhr, FR Germany

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Documents

DETECTION, DETERMINATION AND THIN- LAYER … · fication of anions. The highlights are the systematic analysis of anions in the solid state and the development of new sorbent phases