SEPARATION STUDIES OF ORGANIC AND INORGANIC …ir.amu.ac.in/3652/1/T 5538.pdf · 2015. 7. 21. · separation of zinc (11), cadmium (II), and mercury (11) ... solvent extraction and

SEPARATION STUDIES OF ORGANIC AND INORGANIC COMPOUNDS

T H E S I S SUBMITTED FOR THE AWARD OP THE DEGREE OF

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VINEETA AGRAWAb

DEPARTMENT OF APPLIED CHEMISTRY F A C U L T Y OF ENGINEERING & TECHNOLOGY

A L I G A R H M U S L I M UNIVERSITY AL IGARH ( INDIA)

2001

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DEPARTMENT OF APPLIED CHEMISTRY

Dr. Ali Mohammad Ph.D., D.Sc. (F.N.A.Sc.)

EDITOR : Chemical & Environmental Research

Faculty of Engineering & Technology, Aligarh Muslim University, Aligarh-202 002, India 564-230 AMU IN (Telex) . 91-571-700920, 700921 (Tel) . 456 (Ext.) . [email protected] (E-mail)

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Residence : "AL-RAIYAN" Iqra Colony, New Sir Syed Nagar, Aligarh-202002 (INDIA), 8 : 0571-408196

mailto:[email protected]

CONTENTS

Acknowledgements List of Publication CHAPTER!

Page No.

CHAPTER-11

CHAPTER-III

General Introduction

Micellar Thin-Layer Chromatographic Separation and Identification of Amino Acids • Introduction • Experimental • Results and Discussion • Tables • Figures • References

Use of Water-in-Oil Microemulsion in Thin-Layer Chromatographic Detection and Separation of Amino Acids • Introduction • Experimental • Results and Discussion • Tables • Figures • References

1-64

65-89

90-103

CHAPTER-IV

CHAPTER-V

Thin-Layer Chromatography of Aromatic 104-119 Amines with Hybrid CTAB-Alcohol-Water Mobile Phase • Introduction • Experimental • Results and Discussion • Tables • Figures • References

Surfactant-Modified Mobile Phase in 120-139 Thin-Layer Chromatography of Transition Metal Cations • Introduction • Experimental • Results and Discussion • Tables • Figures • References

CHAPTER-M Enhanced Separation Selectivity of Transition 140-154 Metal Ions with Cationic Micellar Eluents in Normal-Phase Thin-Layer Chromatography • Introduction • Experimental • Results and Discussion • Tables • Figures • References

CHAPTER-VII Micelles Activated Planar Chromatographic 155-168 Separation of Coexisting Iodide, lodate and Periodate Ions • Introduction • Experimental • Results and Discussion • Tables • Figures • References

CHAPTER-Vm Separation of Coexisting Hexacyanoferrate 169-184 (II), Hexacyanoferrate (III) and Thiocyanate with New Mobile Phase • Introduction • Experimental • Results and Discussion • Tables • Figures • References

ACKNOWLEDGEMENTS

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(VlNEETA A G R A W A L ) I ^/^t

LIST OF PUBLICATIONS

1. Applicability of surfactant-modified mobile phases in thin-layer chromatography of transition metal cations : identification and separation of zinc (11), cadmium (II), and mercury (11) in their mixtures, A.Mohammad and Vineeta Agrawai, J.Planar Chromatogr. - Mod. H X (Hungary), 13 (2000), 210-216.

2. Micellar thin layer chromatographic separation and identification of amino acids: separation of L-proline from some aliphatic and aromatic amino acids, A.Mohammad and Vineeta Agrawai, J. Planar Chromatogr. - Mod. TLC (Hungary), 13 (2000), 365-374.

3. Thin-layer chromatography of aromatic amines with hybrid CTAB-alcohol-water mobile phase: separation of indole from diphenylamine and p-dimethylaminobenzaldehyde, A. Mohammad and Vineeta Agrawai, Separation Science & Technology (U.S.A) In press

4. Thin-layer chromatography of anions: separation of coexisting hexacyanoferrate (II), hexacyanoferrate (III) and thiocyanate with new mobile phase, A. Mohammad and Vineeta agrawai, J. Planar Chromatogr.-Mod. TLC, (Hungary) communicated.

5. Use of water-in-oil microemulsion in thin layer chromatographic detection and separation of amino acids, A. Mohammad, S. Kumar and Vineeta Agrawai, Separation Science <6 Technology (U.S.A) communicated.

6. Micelles activated planar chromatographic separation of coexisting iodide, iodate and periodate ions, A. Mohammad and Vineeta Agrawai, / . Quimica Analitica (Spain) communicated.

7. Enhanced separation selectivity of transition metal ions with cationic micellar eluents in normal-phase TLC: simultaneous separation of associated zinc, nickel, mercury and cadmium or manganese cations, A. Mohammad and Vineeta Agrawai, / . Chinese Chemical Society (China) communicated.

-I

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1.1 ANALYTICAL CHEMISTRY

Analytical chemistry is one of the most important branches of

chemistry which is mainly concerned about detection, determination

and separation of traces of organic and inorganic substances in air,

water and soil environments. Owing to the greater importance of

pollution in modern life, the separation chemistry is becoming more

and more relevant to us. To meet the challenges of the modern world,

the classical methods have to be modified according to the need of the

situation and new methods have to be developed for solving the

problems created by explosive technological developments and

increasing environmental pollution. Consequently, the need for

purification of the environment, recovery of precious metals from the

spent fuel or sea water, analysis of ores and the determination of

pesticides and toxic metals in food products is intensifying day by day.

Thus, analytical chemistry has much to offer to chemical technology

which is growing with unhindered pace. In fact, the use of a particular

substance in various fields of science (chemical, biochemical,

physiological and engineering etc.) depends solely on its chemical

analysis which may be qualitative or quantitative. A qualitative

analysis deals with methods used for the determination of nature of the

constituents of a substrate whereas the quantitative analysis is

concerned with the methods dealing with the determination of actual

amount of a given species present in sample. Both instrumental and

non-instrumental (classical) methods are used in analytical chemistry.

Instrumental methods are usually faster and more sensitive, whereas

non-instrumental methods, which form the basis of standardization of

the instruments, are considered more accurate. However, it is difficult

to draw a clear border line between instrumental and non-instrumental

analytical methods.

Despite distinct advantages of instrumental methods in many

directions, their wide spread adoption has not rendered the classical

methods obsolete. The non-instrumental methods should be

strengthened because these are simple, inexpensive and versatile. In

fact, a close critical comparison of all available analytical methods

demonstrate the truth of well known rule of life that " nobody (here:

nothing) is perfect". The team is always stronger than the individual

and nothing can be replaced completely without loss. Co-operation is

best. It is, therefore, concluded that all the analytical techniques should

be developed continuously without discrimination.

There are several methods for the analysis of elements but the

simplest of them, characterized by high selectivity, rapidity and

reliability and requiring no expensive reagent and instrumentation is

usually preferred. This is especially important in the determination of

very small quantities of elements. One has to look into the possibilities

of using various separation techniques such as precipitation,

distillation, dialysis, ring-oven technique, ion-exchange,

electrophoresis, solvent extraction and chromatography while handling

a new system.

1.2 CHROMATOGRAPHY

It is a physical method of separation in which the components to be

separated are distributed between two phases, namely (i) stationary

phase, which can be a solid or a liquid support on a solid and (ii)

mobile phase (a gas or a liquid) which flows continuously over the

stationary phase. The separation of individual components results

primarily due to differences in their affinity for the stationary and

mobile phases. The word "chromatography" was first used by Russian

botanist, Michael Tswett (1906) to describe separation of plant

pigments, which was effected by passing an extract of green leaves

through a column packed with fine grains of calcium carbonate. Since

the separation results into formation of a series of coloured zones, the

name "chromatography", derived from the Greek words chromatus and

graphein, meaning "colour" and "to write" was used by him. After the

initial work of Tswett, a wide variety of independent techniques that

have little or nothing to do with colour have come to be called

chromatography. From its infancy, chromatography has grown in

significance and popularity to become a leading technique of analysis.

The salient features of common chromatographic techniques are

summarized in Table 1.1.

Since the work summarized in this thesis is based mainly on the

use of thin - layer chromatography as a separation technique, it is

therefore worthwhile to provide the important aspects of this

technique. The following pages are devoted to outline the development

and current state - of - art of thin layer chromatography (TLC) as used

to the analysis of inorganics, amino acids and amines.

1.3 THIN LAYER CHROMATOGRAPHY

TLC is a subdivision of liquid chromatography in which the mobile

phase (a liquid) migrates through the stationary phase (a thin layer of

porous sorbent on a planar inert surface) by capillary action. It is a

rapid, simple, versatile, reasonably sensitive and inexpensive analytical

tool which is applicable for both qualitative and quantitative analyses

of several compounds.

1.4 HISTORY OF TLC

The history of TLC has been reviewed by Stahl (1) Kirchner (2,3) and

Pelick et al. (4). In fact, the beginning of TLC may be attributed to

Beyerinck who reported the separation of sulphuric and hydrochloric

acids in the form of rings on thin layer of gelatin (5). Following the

same technique, Wijsman separated enzymes from malt diastase (6). In

1938, Izmailov and Shraiber separated certain medicinal compounds on

binder free horizontal thin layer of alumina spread over a glass plate

(7). As the development was carried out by placing solvent drops on

the glass plate- containing sample and adsorbent, their method was

called ""drop chromatography. However, this method could not catch

the eyes of scientists until two American chemists, Minhard and Hall

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used a mixture of aluminium oxide (adsorbent) and celite (binder) as a

layer on a microscopic slide to separate inorganic ions (8). They called

this technique as "surface chromatography" and this was the first

application of layer chromatography in the separation of inorganics.

Since 1958, when Stahl introduced the term "thin layer

chromatography" and standardized procedures, materials and

nomenclature (9,10), the effectiveness of this technique for separation

was realized.

A major breakthrough in the field of TLC came in the early

1960's with the availability of precoated plates (11). It had recently

been realized that modern high performance thin layer chromatography

(HPTLC) initiated in 1975, rivals high pressure liquid chromatography

(HPLC) and gas chromatography (GC) in its ability to resolve complex

mixtures and to provide analyte quantification.

1.5 COMPARISON OF TLC, HPLC AND HPTLC

According to recent literature (12), TLC has distinct advantages over

HPLC, e.g. greater detection possibilities, more rapid through put, use

of disposable plates, easier sample preparation, low solvent

consumption and lower operating cost. The poorer separation

efficiency and the influence of environmental conditions on the

reproducibility of Rp values have, however, been major disadvantages

of TLC compared with HPLC and GC.

The above-mentioned advantages of HPTLC/TLC have made it

the premier method for assessing atmosphere, aquatic and residual

pollution. It has been successfully applied for the analysis of

wastewater for total heavy metal content (13), characterization of

hazardous wastes (14), identification of metals in sludge sample (15),

and quantification of toxic metals in industrial sewage (16). Compared

to conventional TLC, HPTLC provides faster separations, reduced zone

diffusion, better separation efficiency and higher sensitivity. However,

HPTLC is not in common use because of high capital investment.

procedural difficulties and instrumental complications. A comparison

of TLC and HPTLC characteristics is summarized in Table 1.2 and the

distribution of TLC/HPTLC publications among the most important

fields of application is shown in Figure 1.1.

1.6 TLC METHODOLOGY

The complete process of TLC is summarized in Figure 1.2.

Solute identification in TLC is based on Rp, known as

retardation factor which is calculated as

Distance travelled by the compound from the origin

R F = -Distance travelled by the mobile phase from the origin

The Rp values in TLC are dependent upon many variables (nature of

the sorbent, layer thickness, layer-activation temperature, chamber

saturation, nature of mobile phase, pH of the medium, development

technique used, room temperature, sample size and relative humidity)

which must be regulated carefully during the preparation and

evaluation of the chromatogram to obtain reproducible results. Rp

values vary between 0.0 (solute remaining at the point of application)

and 0.999 (solute migrates up to the solvent front) and have no unit.

The differential migration results because of varying degrees of

affinity of components in a mixture, for the stationary and mobile

phases.

1.7 PRINCIPLE AND TECHNIQUES

In TLC separation of components in a mixture is achieved by

optimizing the experimental conditions. The desired separation can be

achieved by proper selection of adsorbent (stationary phase) and

solvent (mobile phase).

1.8 NATURE OF PHASE INTERACTIONS

Some of the important physical and chemical characteristics that

determine the degree of interactions of mobile phase - solute, sorbent -

Table 1.2 Comparison of TLC and HPTLC

Parameter TLC HPTLC

Plate size

Average particle size

Adsorbent layer thickness

Plate height

Sample volume

Solvent migration distance

Separation time

Samples per plate

Detection limits

Absorption 1 - 5 ng 0.1 - 0.5 ng

Fluorescence 0.05 - 0.1 ng 0.005 - 0.01 ng

Diameter of separated spots 6-15 mm 2- 6 mm

HPTLC provides faster separation, reduced zone diffusion, better separation efficiency and higher sensitivity.

20 X 20 Cm

20 |im

100 - 250 nm

30 \im

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10 - 15 cm

30 - 200 min

10

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5 ^mi

200 ^mi

12 nm

0.1-0.2 iL

3 - 6 c m

3 - 2 0 min

18 or 36

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1 1 1 1 1 1 Inorganics Foodanalysis liRvinmnKnial liiochcniistn I'hanrucy Misccll.-UK<>us

and Medicine

Figure 1.1 Distribution of TLC/HPTLC publications in important field of application

Preparation of Sample

Applied on Chromatoplate By Spotting or Streaking

Development

Drying of Chromatogram

Detection Visual, UV-scanning, reagent spray

Component Removal (optional)

Documentation

Figure 1.2 The process of thin-layer chromatography

10

solute and mobile phase - sorbent are given in the following

paragraphs.

(a) Intramolecular Forces: which hold neutral molecules together

in the liquid or solid state. These forces are physical,

characterized by low equilibrium and results in good

chromatographic separation.

(b) Inductive Forces: exist when a chemical bond has a permanent

electrical field associated with it (e.g. C-Cl, C-NO2 groups).

Under influence of this field, the electrons of an adjacent atom,

group or molecule are polarized so as to give an induced dipole

moment. This is a major contributing factor in the total

adsorptive energy on alumina.

(c) Hydrogen Bonding: makes a strong contribution in adsorption

energies between solute or solvents having a proton donor group

and a nucleophilic polar surface such as that of alumina or silica

gel

(d) Charge Transfers: between components of the mobile phase and

the sorbent can also take place to form a complex of the type S"

A' (where S = solvent or solute, and A = surface site of sorbent).

This is prominent in ion - exchange chromatography.

(e) Covalent Bonds: can be formed between solute and / or the

mobile phase and the sorbent. These are strong forces and result

in poor chromatographic separation.

1.9 CHROMATOGRAPHIC SYSTEMS

The optimum conditions for separation in TLC are yielded through

mutual harmonization of stationary and mobile phases

I. STATIONARY PHASE (ADSORBENT)

A large number of sorbents are available which can be used in TLC .

However, more commonly used sorbents are silica gel, alumina,

cellulose and kieselguhr (diatomaceous earth)

11

Silica gel is the most frequently used layer material. It is slightly

acidic in nature. 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, Siloxane groups) Figure 1.3. The

silanols groups represent adsorption active surface centers that are able

to interact with solute molecules. The ability of the silanol groups to

react chemically with appropriate reagents is used for controlled

surface modifications. Hence, silica gel is considered as the most

favoured layer material in chromatography.

Alumina (aluminium oxide, AI2O3) is also widely used as a sorbent.

It is more reactive than silica gel, but for a given layer thickness it will

not separate quantities of material as large as can be separated on silica

layer. Adsorption is the separation mechanism in both silica gel and

alumina. The surface - active centers of alumina are hydroxyl groups

and oxide ions. In aqueous suspension, alumina surface is capable to

bind with metal ions, anions or metal complexes. Chromatographic

properties of alumina are influenced by the adjustment of pH value.

Three ranges of pH values have proved suitable for aluminas i.e. pH

values of 9-10 (basic aluminas), pH value 7-8 (neutral aluminas) and

pH values 4 - 4.5 (acidic aluminas).

Cellulose can be used as a sorbent in TLC when it is covenient to

perform a given paper chromatographic separation by TLC in order to

decrease the time required for the separation and increase the

sensitivity of detection. In general, two types of cellulose such as (i)

native cellulose and (ii) microcrystalline cellulose are in use. Native

cellulose is fibrous and has a degree of polymerization of 400 - 500

glucose units whereas microcrystalline cellulose consists of

approximately 40 - 200 of glucose units. Cellulose are mainly used in

partition TLC for the separation of relatively polar compounds.

Kieselguhr is a chemically neutral sorbent consisting of about 90%

SiOj alongwith AI2O.,, Fe.O,, MgO, Na20, K2O, CaOi and TiOj in

12

OH OH

Si

0 S i ^ " ^0^ 0

1

/

Figure 1.3 Structure of silica gel

13

different proportions representing, the remaining 10% of the kieselguhr

material. Kieselguhr has very low surface activity and hence used

mostly to separate herbicides and aflatoxins in a partition

chromatographic mode.

The various types of sorbent layers presently in use may broadly

be clubbed together as follows:

(a) Unmodified or Untreated Sorbents: In addition to silica gel,

alumina, cellulose and kieselguhr as discussed above, sorbents

like polyamides (polyamids 6 and polyamide 11) and sephadex

(cross linked polymeric dextran gels), may be put under this

category.

(b) Bonded or Chemically Modified Sorbents: In recent years the

importance of using surface - modified sorbents in TLC has

increased. Both hydrophobic and hydrophilic modified sorbent

phases have been used.

(i) Hydrophobic modified phases (RP phases): The non-modified

sorbents show polar surface characteristics and are of little

practical utility for separations of those solutes having identical

polar characteristics. This problem has been solved using

hydrophobic interactions of the stationary phase with compounds

of appropriate molecular weight. The most popular such organo-

functional groups are methyl (RP-2), octyl (RP-8), dodecyl (RP-

12), octadecyl (RP-18) and phenyl residues.

(ii) Hydrophilic modified sorbents: Hydrophilic modified sorbents

posses amino-, cyano- and diol - residues as a functional group.

The polar functional groups, in each case are bonded via short-

chain non-polar spacers to the silica matrix.

(c) Impregnated Sorbents: Besides the possibility of changing the

selectivity of sorbent by chemical modification, improvement in

14

selectivity can also be achieved by impregnating the matrix with

suitable organic or inorganic substances.

(i) Organic impregnants: Silica gel has been impregnated with

diethylene-triamine, sulphaguanidine, 8-hydroxyquinoline and t-

butylamine, tributylamine, EDTA, pyridinium tungstoarsenate,

p-toluine etc. Microcrystalline cellulose impregnated with bis-(2-

ethylhexyl) orthophosphoric acid or trioctyl phosphine oxide-bis-

(2-ethylhexyl) orthophosphoric acid has also been used as layer

material.

(ii) Inorganic impregnants: Ceric molybdate, sodium molybdate,

ammonium chloride, nitrites of sodium, barium and thorium,

sodium nitrates, potassium ortho dihydrogen phosphate and

copper sulphate have been used as the impregnants for silica gel.

(d) Miscellaneous Sorbents: In this category one can report

numerous types of sorbent phases, that have been used in TLC.

Some of such sorbents are summarized below:

Silica gel H slurried in 4% ammonium nitrate solution containing 1%

sodium carboxy methyl cellulose (CMC), silica gel G + starch, silica

gel G + ceric molybdate, silica gel R + vionite CS ion exchanger, silica

gel G + sodium carboxy methyl cellulose, kaolin, chitosan, kieselgel G,

chitosan, microcrystalline cellulose mixed with powdered chitosan,

diethylaminoethyl (DEAE) cellulose layers in H" form, cellulose

phosphate in thiocyanate form, p-cellulose + microcrystalline cellulose

(2:1) in free hydrogen form, cellulose phosphate + microcrystalline

cellulose (3:1), cellulose (chemapol) azopyrocatechol group, alumina

with plaster of paris binder, stannic arsenate, stannic antimonate,

hydrated stannic oxide, zirconium tungstate in H" form, P-stannic

arsenate in H" form, Ti (iv) antimonate in H* form, polystyrene +

divinylbenzene copolymer, Dowex 50W-X8 + silica gel (1:1) and

amberlite IRA -400 plus silica gel (1:1)

15

Both commercially available (precoated) layers as well as

laboratory made sorbent layers have been used by chromatographers.

However, more emphasis has been on precoated HPTLC plates.

II. MOBILE PHASE (SOLVENT SYSTEM)

In liquid chromatography including TLC, the mobile phase exerts a

decisive influence on the separation. Various optimization schemes

(Windows diagram. Overlapping resolution maps, Simplex method and

PRISMA model) are proposed for normal phase and reversed-phase

TLC. A large number of mobile phases have been reported, of them

some are enlisted below.

(a) Organic Solvents : The single component mobile phase

including acetone, acetonitrile, benzene, carbon tetrachloride,

chloroform, dioxane, ethanol, ethylacetate, methanol, o-xylene,

petroleum ether, toluene, n-octanol, n-nonane, cyclohexane and

binary / ternary mixtures of alcohols, amines, ketones, phenols,

haloalkanes have been used.

(b) Inorganic Solvents: Being non-toxic and non-volatile, solvent

systems of this group have been widely used in TLC of

inorganics and organometallics. This group includes the solution

of mineral acids, alkalies and inorganic salts prepared in double

distilled water or water - methanol mixture.

(c) Mixed Solvents; Mixtures of two or more different solvents,

most of which have either a base (NaOH, NH4OH and amine) or

an acid (mineral or carboxylic) as a component, are used to

develop the TLC plate.

(d) Surfactant - Mediated Solvents: Solutions of surfactants (SDS,

CTAB, or Triton X-100) have also been used to lesser extent as

mobile phase in TLC.

16

SURFACTANT-MEDIATED SYSTEMS

These systems contain surfactant as one of the components of the

mobile phase. Surfactants in the aqueous mobile phase can be used in

the following ways:

(a) as monomer surfactants where the concentration of surfactant in

aqueous mobile phase is restricted to well below the critical micelle

concentration (CMC) of the surfactant. These mobile phases are

most suited to separate ionic species by ion-pair chromatography

(IPC). In this technique, a small concentration of ion-pairing

reagent, which has an opposite charge to the ionic solutes (i.e.

cationic surfactant for anionic solutes and anionic surfactant for

cationic solutes) is added to the aqueous mobile phase and its

concentration is kept low to avoid the formation of micelles.

(b)as surfactant micelles where the surfactant concentration is kept

well above its CMC value. In such cases, the mobile phase is

composed of surfactant molecules in the form of monomers and

aggregates (or micelles). These mobile phases are very useful for

simultaneous separation of ionic and non-ionic compounds by

micellar liquid chromatography (MLC).

(c) as microemulsion where surfactant in the presence of water, an oil

(hydrocarbon) and cosurfactant (i.e. medium chain length amine or

alcohol) is used as transparent solution.

Since the major portion of the work presented in this thesis is

related to the use of surfactant containing mobile phases, it is

worthwhile to mention the salient features regarding the behaviour of

surfactants in aqueous media as well as their utility in chromatography.

Surfactants are long chain amphiphilic organic or organometallic

molecules containing a highly polar (hydrophilic or lipophobic) or

ionic "head group" attached to a non-polar (hydrophobic or lipophilic)

hydrocarbon tail of varying chain length. The "head group" is either

cationic (e.g. ammonium or pyridinium ion), anionic (e.g. hydroxy-

17

compounds) or zwitterionic (e.g. amine oxide, carboxylate or

sulphonate betain) and the hydrocarbon tail which may contain at least

8 carbon atoms. Since surfactant molecules are surface - active i.e.

capable to lower the surface tension of water when dissolved in it,

these are sometimes called surface-active agents or detergents. Owing

to the presence of both non-polar (hydrophobic) and polar

(hydrophilic) groups, these substances have also been referred to as

amphipathic, tensioactive tenside, heteropolar or polar-nonpolar

substances.The dual character (polar-nonpolar nature) of surfactant

molecules is responsible for their unique properties in solution which

render possible applications in dispersion, emulsification, wetting,

catalysis, organic synthesis, reaction kinetics and separation

technology. The factor responsible for desired surface activity is the

balance between hydrophobic and hydrophilic characteristics of these

molecules. Depending upon the nature of the hydrophilic group,

surfactant can be classified as anionic [R - X'M" ]; cationic [R - N*

(CH3)3 X"]; zwitterionic [ R - (CH3)2 N" CH2X"] and nonionic

[R(OCH2CH2)]mOH where R is a long aliphatic hydrocarbon chain, M"

is a metal ion, X' is a halogen, CO2 or S04^' and m is an integer. A list

of some common surfactants is provided in Table 1.3.

MICELLES

Surfactant (or amphiphilic) molecules comprising of hydrophobic and

hydrophilic moities tend to exhibit a considerable degree of self

organization when dissolved in aqueous solutions. Above a certain

concentration level, termed as critical micelle concentration (CMC) ,

the surfactant molecules in solutions (water or organic solvents)

aggregate to form micelles. The process of micelle formation is called

"micellization". Micelle do not exist at all concentrations and

temperatures. There is a very small concentration range below which

aggregation to micelles is absent and above which association leads to

micelle formation. This narrow concentration range during which

micelle formation occurs is called the CMC. At low concentration i.e.

18

CMC (M)

8.1 X 10"

3.0 X 10-

2.0 X 10"

Aggregation Number

62

c

81

Kraft Point

9

25.6

<0

Table 1.3 Typical Surfactants and their CMCs, Aggregation Numbers, Kraft Point Values*

Surfactant

Aqueous (normal) Anionic Sodium dodecyl sulfate (SDS), CH3(CH2)i,OS03Na* Potassium perfluoroheptanoate, C T F I S C O O K *

Sodium polyoxyethylene(12)dodecyl ether, CH3(CH2),i(OCH2CH,2),20S03Na"(SDS 12E0)

Cationic Cetylpyridinium chloride, ' ' 1 . 2 x 1 0 " 95 c C,6H33N'C5H5Cr Cetyl trimethyl ammonium bromide (CTAB), 9.0 x lO"* 78 23 CH'(CH2),5N"(CH3)3Br

Nonionic Poiyoxyethylene(6)dodecanol, 9.0 x 10"' 400 c CH3(CH2)n(OCH2CH2)60H Polyoxyethylene(23)dodecanol(Brij-35), 1.0x10"* 40 c CH3(CH2),,(OCH2CH2)230H

Zwitterionic N-Dodecyl-N,N-dimethylammonium-3- 3.0 x 10'^ 55 <0 propane-1-sulfonic acid (SB-12), CH3(CH2)nN*(CH3)2(CH2)3S03-N,N-Dimethyl-N- 25 x 10"^ 24 <0 (carboxymethyl)octylammonium salt, C8H,7N^(CH3)2CH2COO(octylbetaine)

Nonaqueous (reversed) Bis(2-ethylhexyl) sodium sulfosuccinate 6.0 x 10" c c (AOT),' Na03SCH(CH2COOC8H,7)COOCgH,7

"values for aqueous solution at 25"C ''Temperature at which the solubility of an ionic surfactant is equal to the CMC. 'Not available or not defined 'În 0.0175 MNaCi "In hexane.

19

below CMC and at temperatures above the criticle micelle (e.g. Kraft

temperatures), the surfactant is dispersed in the aqueous media at the

i»olecular level as a monomer. The average number of monomers per

micelle is called the aggregation number (N). At 25* C and 1 atm., the

CMC is typically less then 20 mM, with each micelle - consisting of 40

- 140 monomers.

Regardless of the structure of the hydrophilic moiety (cationic,

anionic, nonionic or zwitterionic), it is generally accepted that

hydrophobic interactions are the main driving force for micelle

formation in aqueous media. A conventional model of micelle is that

proposed by Hartley (Figure 1.4) which is very useful for visualization

of a micelle. The various structures formed in aqueous solution on

increasing the concentration of surfactant is illustrated in Figure 1.5.

Micellar structure is also affected by experimental parameters such as

temperature, pressure, pH, ionic strength and the presence of additives

in the surfactant solution.

There are mainly two types of micelles

(a) Normal Micelles: The molecular organization of surfactant

molecules in aqueous solutions resulted in the formation of

normal micelles. Above CMC, the surfactant molecules are self

aggregated in such a manner that the hydrophobic moities (i.e.

hydrocarbon tails) are oriented inward forming a non-polar core

and hydrophilic (polar) head groups are oriented outward

keeping themselves in contact with the bulk aqueous phase.

Normal aqueous micelles are generally formed from single-chain

surfactants and chain-branching inhibits micellization.

Micelles are considered to be dynamic in nature, with continuous

exchange of surfactant molecules in and out of the aggregates

occurring in the milliseconds to microseconds range. Thus individual

surfactant molecules (called monomers) are thought to be distributed

throughout the aqueous phase surrounding the micelles or monomers

20

— • ^

>;?> / v'\-/- \ • _ _

— Hydrocorbcn -7' interior

:_ Aqueous exterior

Gouy - Chapman layer

Stern layer

Figure 1.4 Hartley model of a spherical micelle

21

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22

and micelles are visualized as being uniformly distributed throughout

the aqueous phase. When more than one surfactant are present in

aqueous solution, mixed micellar system is produced. The surface

activity of the mixed micelles system has been reported to be superior

than the single surfactant micelle system (17).

(b) Reverse Micelles: In contrast to the normal micelle which are

formed in polar (i.e. aqueous media) solvents, reverse micelles

are formed in non-polar solvents like hexane or chloroform and a

trace of water where the polar head groups of the surfactant are

directed towards the interior of the aggregate and the

hydrocarbon chains are in contact with the non-polar solvent.

Compared to normal micelles, reverse micelles are more complex

and less understood. Reverse micelles offer the same potential

advantages for analysis as do normal micelles i.e. the ability to

solubilize polar species that would be excluded from normal

micelles. An interesting aspect of reverse micelles is their

capability to solubilize water in the interior of micelle structure.

In many instances water actually promotes the formation of

larger and more stable micelles.

From macroscopic perspective, micellar solutions are homogeneous

and can not be filtered. However, the unique characteristics of micellar

aggregates stem from their microscopically non-homogeneous nature

i.e. they provide a microenvironment which is distinctly different from

the bulk solvent. The most important property of micelles is their

ability to solubilize substances that are otherwise insoluble (or

sparingly soluble) in water.

MICROEMULSION

Microemulsions are transparent thermodynamically stable systems

containing oil (nonpolar solvent), water, surfactant and a cosurfactant

which is generally a medium chain length alcohol, amine or similar

polar organic molecule (18). The microemulsions are also called

23

"swollen micelle" (oil-in-water microemulsion) or reversed micelle

(water-in-oil microemulsion). The term "microemulsion" was first

proposed by J. H. Schulmen and co-workers in 1959 (19). A

microemulsion is formed when a co-surfactant is added into coarse-

emulsion (water-surfactant-oil) upto clarity (20). Microemulsions

permit the separation of aqueous and non-aqueous phase divided by a

surfactant membrane in a homogeneous medium. Mainly two types of

microemulsion such as water-in-oil microemulsion (w/o ME) and oil-

in-water microemulsion (o / w ME) have been recognized. The droplet

diameter in microemulsions range from iOO to 1000 A°. The droplets

are stablized by a mixed interfacial film of surfactant and cosurfactant.

Schematic representation of micelles and microemulsion is given

in Figure 1.6 and their characteristics features are summarized in

Table 1.4.

The use of micellar solutions as mobile phases in TLC was first

suggested by Armstrong et al. (21,22) who described the possible

advantages of micellar mobile phase (MMP) over traditional pure and

mixed solvent systems. Using aqueous SDS solutions in combination

with polyamide and alumina thin layers they successfully separated

pesticides and the pollutant decachlorobiphenyl by normal-phase TLC.

However, nucleosides could be separated by reversed-phase TLC using

a reversed micelle solution as mobile phase. It was demonstrated that

the partitioning of substances to the reversed micelles involved both

electrostatic interactions with the polar head groups of the surfactants

and solubilization by the "water pool" in the hydrophobic core. Despite

several advantageous features of MMP its use, in TLC has been less

intense compared to HPLC (23). Micellar TLC has been used to

separate substituted benzoic acids, polynuclear aromatic hydrocarbons

and vitamins (24); phenols (25,26); amino acids (27-29); alkaloids

(30); dyes (25, 30-32); aromatic amines (33); drugs (34) and inorganics

(35,36).

24

Koler(---)

Co-8u^clonl L-3 ( - - )

Micelle 0/w Microemulsion

Jurfoctanl aonomers

oilC—)

Co-surfodant

Reversed micelle xA> Microemuliion

Figure 1.6 Schematic representation of organised assemblies

25

Table 1.4 Comparison of Micelles, Microemulsion and Vesicle

Characteristic

Weight average molecular weight

Diameter (Ao)

No. of solubilizates per segregate

Kinetic stability (leaving rate of a monomer)

Solubilizate residence time

Dilution by water

Micelle

2.6x10'

30-60

Few

10

10-' - 10-'

Destroyed

Microemulsion

lO'-lO*

50-1000

Large

10'

10-' - 10-

Altered

Vesicle

10

300 - 5000

Large

Sec

Sec

Stable

26

The separation of TLC with MMP is based on selective

dissolution of polar and nonpolar substances as a result of combined

effect of electrostatic, hydrophobic and donor - acceptor interactions.

Secondary interactions resulting from partition inside the mobile phase

(i.e. between aqueous and micellar phases) play the predominant role

in the separation selectivity of MLC. However, inorganic MLC differs

from organic MLC in the sence that in MLC of inorganic ions, the

electrostatic interaction is the primary and in most cases the only

separation mode. The retention behaviour can be described with an ion-

exchange model, the retention is principally determined by the

concentration and a counterion. On the other hand in MLC of organic

compounds, hydrophobic or lipophilic interactions determine the

retention pattern of solutes.

From the above discussion it looks that micellar mobile phases

have much to offer to physical, biochemical and analytical chemists.

Even a cursory look at the recent literature points out that MMP have

been the focus of numerous studies related to chemical separations and

all signals indicate this heightened interest will continue. It is hoped

that these unique mobile phases will provide a powerful alternative to

conventional hydro-organic eluents to solve difficult analytical

problems. It is also perceived that TLC has more to offer to the study

of micelles than MMP have to offer to chemical analysis performed by

TLC. This aspect of micellar TLC will be a rewarding field for future

research.

2.0 SAMPLE APPLICATION

Sample application is one of the most important steps in the technology

of TLC. Improperly applied samples result in poor chromatograms.

Samples are applied as spots or stripes on the sorbent layer, about 2-3

cm above from the lower edge of the TLC plate so that only sorbent

layer makes contact with the mobile phase and the sample does not

dissolve in the mobile phase. The sample should be completely dried

27

before placing the plate in the development chamber. Dilute solutions

can be applied to the layer either with sorbent drying between

successive applications or after bringing the sample solution to proper

concentration.

Micropipette, microsyringe, melting point capillaries etc. have

been used to apply the sample on the plates. A number of automatic

spotters of varying design are also available in the market.

2.1 DEVELOPMENT

Development in TLC is the process in which the mobile phase moves

across the sorbent layer to effect separation of the sample substances.

Ascending development or linear development is the commonly used

mode of development in TLC in which the mobile phase moves up

(ascends) the plate. Any close container that will hold the plate upright

is usable. While performing the development one should take care of

the angle of the development and saturation of chamber apart from

other factors. It has been observed that the angle of development, that

is the angle at which the plate is supported, effects the rate of

development as well as the shapes of the spots (37). An angle of 75* is

optimum for development. If a desired separation is not achieved by

simple development, some development options such as (i) multiple

development, (ii) stepwise development, (iii) continuous development,

(iv) two-dimensional development (v) circular development and (vi)

reversed-phase partition development, are available to achieve the

desired separation.

2.2 VISUALIZATION

The methods of visualization (detection) used in TLC are of three

major types, (i) physical (ii) chemical and (iii) enzymatic or biological.

Among the physical methods, visualization in UV-light is most

common. This method is highly sensitive, non-destructive, and

amenable to the visualization of spots before undertaking quantitative

studies. Chemical methods of detection involve the spraying of

28

chromatoplates with a suitable reagent, which forms coloured

compounds with the separated species. Reagents giving clear and

sufficiently sensitive colour reactions with several species are

preferred. Both selective and non-selective reagents may be chosen for

the location of the separated zones. Nanda and Devi have reported an

enzymatic method (38) for the detection of heavy metals in fresh water.

Nicolaus and Coronelli (39) have reported a microbiological method,

called bioautography for the detection of antibiotics on TLC plates

using triphenyltetrazolium chloride (TTC) and a micro-organism that is

sensitive to the antibiotic in question.

2.3 QUANTITATION

The three main approaches associated with quantitation in TLC are (i)

visual estimation (ii) zone-elution (iii) in-situ densitometry. Amongst

these, in-situ densitometry is the preferred technique for quantitative

TLC. Substances separated by TLC or HPTLC are quantified by in-situ

measurement of absorbed visible or UV-light, or emitted fluorescence

upon excitation with UV-light. Absorption of UV-light is measured

either on regular layers or on layer with incorporated phosphor.

2.4 LATEST DEVELOPMENT IN TLC

As a result of the recent innovations, several new techniques such as

high-performance thin layer chromatography (HPTLC),

overpressurized thin layer chromatography (OPTLC), centrifugal layer

chromatography (CLC), reversed-phase thin layer chromatography

(RPTLC), radial chromatography, hot plate chromatography,

bioautoradiography, immunostaning and enzyme inhibition techniques

came into light. HPTLC layers being thinner and made of sorbent with

more uniform particle size 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.

29

2.5 COMBINATION OF TLC WITH OTHER ANALYTICAL

TECHNIQUES

The careful combination of TLC with other analytical techniques is

more useful to collect information regarding the analysis of a complex

sample. Spectrophotometry, high-performance liquid chromatography

and gas chromatography, in conjugation with TLC are the three most

widely used techniques. However, mass / GC, infrared and thermal

analytical techniques in combination with TLC have also been used.

One of the newest techniques used in combination with TLC is

photoaccoustic spectrometry, which is capable to locate compounds in-

sitii on the plate. Issaq and Barr (40) combined TLC with flameless

atomic absorption spectrometry (FAAS) to identify an inorganic

compound in an impure organometallic complex and to determine the

recovery and purity of organometallic samples.

The examples cited above reveal, how the separation method of TLC

complement the analytical methods necessary for the absolute

identification of a substance. TLC provides an excellent purification

method for separating a substance of interest from other contaminants

in the sample. Analytical techniques can then be applied to identify the

separated substances.

30

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64

-II

2.1 INTRODUCTION

Micellar liquid chromatography involving the use of surfactant ions

above their critical micelle concentration (CMC) as mobile phase for

controlling the retention of a solute has been the focus of numerous

studies (1-5) since it was first proposed by Armstrong and coworkers,

in 1977 (6). The multiplicity of interactions (hydrophobic, electrostatic

and hydrogen-bonding) associated with micellar systems provide

unique separation possibilities of structurally similar solutes. Micelles

are capable of differentially solubilizing and binding a variety of

solutes leading to their potential usefulness for achieving several new

separations, including the resolution of optical isomers. The

preferential binding (or partitioning) of compounds to micelles has

been investigated by several chromatographers for understanding

solute - micelle interactions by using micellar solutions as mobile

phase (7-11). The solubilization properties of a micellar system for a

specific solute can further be modified by addition of appropriate

additives such as organic solvents, ionic salts, cyclodextrins, and ion-

pairing and complexing agents. Organic additives (alcohols, ketones,

acetonitrile) are considered to control solvent strength thus,

influencing the retention behaviour of solutes.

Thin layer chromatography (TLC), being an efficient and

inexpensive technique, has been widely used for the separation of

organic and inorganic substances using various combinations of non-

micellar mobile phases and stationary phases of different polarities

(5,12-15). Because of their biological, medicinal and pharmaceutical

value, analysis of amino acids has been a subject of importance for

analytical scientists. Amino acids such as lycine, threonine, phenyl

alanine, tryptophan etc. have extensive use in medical practice and

these also have application as food additives in poultry farming as well

as cattle breedings. Several workers have resolved complex mixtures of

amino acids on silica gel (16-18), alumina (19-21), cellulose and

65

cellulose derivatives (22,23). polyamide (24), chitin and chitosan

(25,26) and Cig-bonded (27) layers using mixed aqueous-organic

mobile phases containing, generally.alcohol, ketone, amine, acetic acid

or acetonitrile as one of the components. However, less work has been

done on separation studies of amino acids using micellar systems. It is,

therefore, worthwhile to explore the possibility of utilization of

micellar mobile phases in TLC analysis of amino acids. This report is

an attempt in this direction. We have developed a simple and rapid

TLC method for some important separations, including that of proline

from hydroxy-proline on alumina layers developed with a hybrid eluant

of micelle -water- butanol. Proline and hydroxy proline have been

earlier separated by TLC (28) on silica-coated glass fibre sheets using

2-propanol-water (7:3) as mobile phase. The separated spots were

detected by spraying ethanolic ninhydrin reagent and autoradiography.

As far as we are aware, no TLC work on alumina using micellar mobile

phases for the separation of amino acids has been done and hence the

present study bears practical as well theoretical importance.

2.2 EXPERIMENTAL

Apparatus'.A. thin layer chromatographic applicator (Toshniwal, India),

20X 3 cm glass plates, and 24x 6 cm. glass jars were used for the

development of chromatographic plates. A glass sprayer was used to

spray reagent on the plate to detect the spot.

Chemicals and Reagents: Aluminium oxide 'G', kieselguhr 'G',

propanol, amino acids, ninhydrin, N-cetyl-N, N, N-trimethyl

ammonium bromide, CTAB (CDH, India); sodium dodecyl sulfate

(SDS), methanol , butanol ,silica gel 'G'(Qualigens, India); and

pentanol (Fluka, AG, Buchs SG, Switzerland ) were used. All reagents

were of Analar Reagent grade.

Amino-Acid Studied: glycine (Al), L-hydroxy proline (A2), DL-

alanine (A3), DL-serine (A4), L-proline (A5), DL-valine (A6), DL-

threonine (A7), L-cysteine hydrochloride (A8), L-leucine (A9), DL-iso

66

leucine (AlO), DL-nor leucine (All), DL-methionine (A12). L-tyrosine

(A13), DL-tryptophan (A 14), L-cystine(A15), DL-phenyl alanine

(A 16), 3-(3,4,dihydroxy phenyl)DL-alanine, (A 17), L-ornithine

monohydrochloride (A 18), L-lysine monohydrochloride (A 19),

Lhistadine monohydroch!oride(A20), L-arginine monohydrochloride

(A21), DL-aspartic acid (A22), L-glutamic acid (A23) and DL-2-

amino-N-butyric acid (A24).

Test Solution: Test solutions (1%) of amino acids were prepared in

double distilled water.

Detector: A 0.4% ninhydrin solution in acetone was used to detect all

the amino acids. The appearance of a dark violet spot shows the

presence of amino acids. DL-phenyl alanine-ninhydrin complex

appeared as blue fluorescence spot on TLC plate on inspection under

UV light.

Stationary Phases: The various sorbents used were

Si -Aluminium oxide 'G'

52- Alumina 'G' impregnated with different concentrations (1, 3, 5 and

10%) of aqueous LiCl.

53- Silica gel 'G'

54- Cellulose

55- Kieselguhr 'G'

Mobile Phases: The following surfactant-containing solvent systems

were used as mobile phases

M,- 1% aqueous CTAB + methanol [20+80, 50+50, 80+20, 95+5]

M2- 1% aqueous CTAB + propanol [50+50, 70+30, 80+20, 95+5]

M3- 1% aqueous CTAB + butanol [95+5, 98+2]

M4- 1% aqueous SDS + methanol [20+80, 50+50, 80+20]

M5- 1% aqueous SDS + propanol [50+50, 70+30, 80+20]

67

Mf,- 1% aqueous SDS + butanol [95+5, 98+2]

M7- 1% aqueous SDS + pentanol [95+5, 98+2]

Mg- 1% CTAB in buffer solution* of pH 2.3 + butanol [95+5]

M9- 1% CTAB in buffer solution of pH 5.7 + butanol [95+5]

M,o- 1% CTAB in buffer solution of pH 9.9 + butanol [95+5]

M,i-1% CTAB in buffer solution of pH 11.8 + butanol [95+5]

* Phosphate - boric acid buffer.

Preparation of TLC Plates:

(a) Plain Thin Layer Plates: The plates were prepared by mixing

the desired adsorbent with water in 1:3 ratio with constant shaking

until homogeneous slurry was obtained. The resultant slurry was

applied on the glass plates with the help of an applicator to give a 0.25

mm-thick layer. The plates were first dried at room temperature and

then activated at 100 ± 5°C by heating in an electrically controlled

oven for 1 h. The activated plates were stored in a close chamber at

room temperature until used.

(b) Impregnated Alumina Thin Layer Plates: Impregnated plates

were prepared by mixing alumina with 1-10% of aqueous LiCl

solution in 1:3 ratio and shaking for 5-10 minute. Thin layer of the

resultant slurry was prepared by following the procedure as described

above in (a).

Procedure: About 10 iL of test solution was spotted on thin layer

plate with the help of a micropipette. The plates were developed in the

chosen solvent system by the ascending technique. The solvent ascent

was fixed to 10cm in all cases. After development was complete, the

plate was withdrawn from glass jar and dried at room temperature

followed by spraying with freshly prepared ninhydrin solution. All

amino acids except L-proline appeared as violet spots on heating TLC

plates for 15-20 minutes at 100 + 5°C. L -proline gives yellow spot.

68

The R| (RF of leading front) and Ry (Ri of trailing front) values for

each spot were determined and the Rr value was calculated as

Rp = [RL+ RT]/ 2

The limit of detection of amino acids were determined by spotting

different amounts of amino acids on the TLC plates, developing the

plates and detecting the spot. The method was repeated with successive

lowering of the amount of amino acids until no spot was detected. The

minimum amount of detectable amino acids on the TLC plates was

taken as the limit of detection.

For the separation, equal amounts of L-proline and L-hydroxy

proline and impurities were mixed and 20 iL of the resultant mixture

was loaded on the TLC plates. The plates were developed, the spot were

detected and the RF values of the separated amino acids were

determined.

To separate and identify amino acids in biological samples (human

blood, liver and stomach tissue), the samples were spiked with mixtures

of L-proline and L-hydroxy proline, DL-alanine or DL-phenyl alanine

separately. TLC was performed with lOjiL of sample and the separated

amino acids were detected on TLC plates. The samples were prepared by

the following method.

Liver and Stomach Sample: 10 g liver tissues were dipped in sufficient

volume of cone. H2SO4 and kept overnight. It was digested in a flask

after adding cone. HNO3 till the solution becomes clear. The contents

were washed thrice with distilled water and then 25 mL of saturated

ammonium oxalate was added. It was diluted with water to the required

volume.

Blood Sample: The sample was treated with a mixture of 10% solution

of NaOH and sodium tungstate and a little quantity of H2SO4 was added

to destroy the precipitate. The contents were filtered and the filtrate was

used for detection.

69

Semiquantitative Estimation of DL-aspartic acid: For semiquantitative

determination of DL-aspartic acid, 0.01 mL of various standard solutions

of DL-aspartic acid (0.5-2%) were spotted on alumina layers. The plates

were developed with 1% CTAB in water+butanol (95+5). After

detection, the spots were copied onto tracing paper from the

chromatoplates and the area of each spot was calculated.

2.3 RESULTS AND DISCUSSION

Alumina was selected for TLC separation of aliphatic or aromatic

amino acids because of the following reasons:

(a) The pH of alumina slurry was around 7.0 and at this pH most of

the aliphatic amino acids exist as zwitterions.

(b) The hydroxyl groups (OH) and oxide (O^) ions both constitute

active sites on the surface, which are responsible for selective

chromatographic separations.

(c) Alumina undergoes following cation-exchange reaction when it

is treated with aqueous solution of lithium chloride.

Al2-0-H^ + Li" ^?=^ Al2-0-Li' + H'

(d) Because of a rigid structure, alumina undergoes little swelling or

shrinking in water or solutions containing electrolyte and organic

modifiers.

In order to select a most appropriate surfactant, we used 0.1-2%

aqueous solutions of SDS (anionic) and CTAB (cationic) surfactants.

This concentration range was selected to keep the concentration of

each surfactant below, near and above critical micellar concentration

(CMC) values. The reported CMC value of SDS and CTAB are 0.24%

and 0.46% respectively (29). The results summarizing the effect of

concentration of surfactant on the mobility of amino acids using

alumina layer are shown in (Figure 2.1a & 2.1b). It is apparent that

SDS and CTAB behave differently in controlling the mobility of amino

acids. In all cases the mobility of amino acids was higher with SDS-

70

alumina system compared to CTAB-alumina system. This is possibly

due to the micellar structural difference that exists as a consequence of

charge, head-group size and steric requirements. The positive charge

residing on the quaternary nitrogen atom of CTAB offers strong

interaction to carboxylate ion of amino acids and hence amino acids

are more strongly retained by CTAB adsorbed on alumina surface.

From separation point of view, CTAB was found better than SDS as the

development time (for 10 cm ascent) with 1% CTAB (5 min) was less

than 1% SDS (16 min). Therefore, CTAB (1%) was preferred for

detailed study. It is assumed that at this concentration level, CTAB is

mostly present as micelles. To examine the role of pH of mobile phase,

amino acids were chromatographed using 1% CTAB solution prepared

in buffered solutions of different pH (2.3, 5.7, 9.9 and 11.8) values.

This wider pH range was selected keeping in mind that cationic

surfactants are generally stable in both acidic and alkaline pH changes

(30). The results obtained with 1% CTAB pH 5.7 were better in terms

of detection clarity and spot compactness than those obtained at other

pH values. However, these results were comparable to those obtained

with 1% non-buffered aqueous CTAB (pH % 6.9). Therefore, 1%

aqueous CTAB (non-buffered) was used as mobile phase for examining

the suitability of alumina as layer material for TLC separation of amino

acids.

TLC studies of amino acids on alumina with water and aqueous

surfactant (1% SDS or CTAB) solution show dramatic changes in

retention sequence of amino acids providing new opportunities of their

separations in micellar mobile phases. The results obtained with water

i.e. non-micellar and micellar (1% SDS or CTAB) mobile phases have

been compared (Figure 2.2). In Figure 2.2 ARp values (ARp = Rp with

1% SDS or CTAB -Rp with water) have been plotted. The positive and

negative ARp values are indicative of significant influence of micellar

systems on the mobility of almost all amino acids investigated. The

positive ARp values (faster mobility) Al, A3, A9, A16, and A18 and

71

negative ARp values (slower mobility) for A4, A5, A19 and A23 amino

acids with micellar mobile phase show that alumina is more selective

(strongly sorbing) for A4, A5. A19 and A23 amino acids in micellar

systems irrespective of the nature of the surfactant used. However, the

mobility of certain neutral amino acids (A6, A7, AlO, Al l , A12 and

A14) is strongly influenced by the nature of the surfactant used. These

amino acids show positive ARp in SDS and negative ARp in CTAB.

A20 basic amino acid shows identical behaviour and acidic amino

acids (A22, A23 and A24) with close ARp values in both SDS and

CTAB systems are least effected by the nature of surfactants. The

differential migration of amino acids depends upon their relative

magnitude of partitioning to the micellar or non-micellar mobile phase

compared to the stationary phase. Amongst basic amino acids, A18 and

A20 experienced moderate effect of the nature of surfactants whereas

A19 and A21 show almost identical mobility in both SDS and CTAB

systems.

To modify the retention of amino acids on alumina layer, hybrid

mobile phases comprising of micelle (1% CTAB)-water-alcohol

(methanol, propanol or butanol) were also used and better

chromatographic performance of these mobile phases over aqueous

micellar systems was observed. The Rp values of amino acids were

determined with micelle-water-alcohol (by volume) mobile phases

consisting of methanol (5, 20, 50 or 80%), propanol (5, 20, 30 or 50%)

or butanol (2 or 5%) and 1% aqueous CTAB. In all cases a general

trend of decreasing Rp (or increasing k, capacity factor) value with the

increase in alcohol concentration was noticed. The representative's

plots are given (Figure 2.3). In general, the better separation

possibilities were noticed with mobile phases consisting of 1% CTAB

prepared in 95:5 water-alcohol. It appears that alcohols modify the

retention behaviour of amino acids on alumina layer by reducing

adsorption capability of alumina towards surfactants. The micelle-

water-alcohol mobile phase contain 5% butanol proved to be the best.

72

Thus, moderately polar alcohols (e.g. butanol) provide better

separations of amino acids in micellar systems by virtue of their

association with the core of micelles compared to more polar (e.g.

MeOH) alcohol which is supposed to be present in the outer regions of

the core. In (Figure 2.4a), ARp values (ARp = RF with 1% CTAB in

95:5 water-butanol - RF with water or 95:5 water-butanol) have been

plotted. Amino acids such as A9, Al l , A19 and A24 are more strongly

retained (positive ARF values) by alumina layer when water is used as

developer instead of surfactant added hydro-organic system whereas all

other amino acids except A17 show reverse retention behaviour

(negative ARp value) indicating higher mobility with water mobile

phase. A17 did not migrate and remained at the point of application

(RF=0.0) regardless of the nature of mobile phase (micellar, non-

micellar, hydro-organic or surfactant added hydro-organic). Thus A17

can be selectively separated from several amino acids. It is the evident

from (Figure 2.4b) plot of ARp value (ARp = Rp with 95:5 water-

butanol -Rp with water) that the presence of butanol in water hydro

organic system decreases the mobility of most of amino acids (negative

ARp values) on alumina layers.

TLC results of amino acids with the mobile phase 1% CTAB in

95:5 water-butanol at fixed pH values of 5.7 were comparable with

those obtained with unbuffered mobile phase (Figure 2.5) which has

been utilized for achieving important separations of L-proline from

aliphatic (glycine, L-hydroxy proline and DL-alanine) as well as

aromatic (DL-tryptophan and DL-phenyl alanine) amino acids. It is

evident from (Table 2.1) that separation of L- proline from other

amino acids with the selective micellar mobile phase can only be

achieved on alumina layers compared to silica, cellulose and kieselguhr

layers. The values of TLC parameter listed in (Table 2.2) for these

separations show that all such separations could be achieved in the

presence of acidic (carboxylic acids) and basic (aromatic amines)

impurities. A small change in Rp values was observed when mixtures

73

of amino acids with or without impurities were chromatographed and

compared with individual Ri. of amino acids. In all cases, L-proline

moves faster (k = 0.36-0.42) compared to the mobility of glycine,

hydroxy proline, alanine, phenyl alanine or tr>'ptoj)han (k = 1.0-1.5).

Similarly, DL-nor leucine, a neutral amino acid is successfully

separated from acidic (DL-aspartic acid) and basic (L-arginine

monohydrochloride) amino acids in the presence of heavy metal ions

(Table 2.3). Cn^* is the only metal ion which exerts adverse effect on

the separation. A linear relationship between Rp value and number of

carbons of glycine (C2H5NO2), DL-alanine (C3H7NO2), DL-valine

(C5H11NO2) and L-leucine (C6H13NO2) show that the mobility increases

as the number of carbons or the size of the molecule increases

(Figure 2.6).

In order to demonstrate the utility of Li^-impregnated alumina,

amino acids were also chromatographed on LiCl impregnated alumina

layers using 1% CTAB dissolved in 95:5 water-butanol. Out of entire

concentration range (0.01- 10% LiCl), the optimum concentration

providing compact spots of amino acids was 3% LiCl. The results of

ARp values (ARp = RF on plain alumina - Rp on alumina impregnated

with 3% LiCl) presented in (Figure 2.7) demonstrate how the

selectivity of alumina is altered on impregnation with LiCl. The

positive ARp values (Figure 2.7) for most of amino acids indicate that

Li-impregnated alumina layer is more selective (strongly sorbing) for

amino acids than plain alumina layer. Thus, impregnated alumina can

be used to get certain additional separations (Table 2.4) of amino

acids. However we focussed our attention on the use of unimpregnated

alumina layers for achieving quicker separation, as the impregnated

layers require more time for the same distance of development.

The data listed in Table 2.5 show that the present method can be

successfully applied to identify L-proline, L- hydroxy proline, DL-

phenyl alanine and DL-alanine in biological sample with preliminary

separation on alumina layer.

74

The lower limits of detection of some amino acids were

determined on alumina layers using ninhydrin as chromogenic reagent.

The lowest possible detectable amount (ng) of amino acids (given in

parenthesis) is as follows: DL-aspartic acid (16.0), L-arginine

monohydrochloride (32.0), DL-iso leucine (24.0), L-cysteine

hydrochloride (32.0), DL-threonine (24.0), L-histadine

monohydrochloride (48.0), DL-serine (32.0), glycine (48.0), L-

glutamic acid (32.0) and DL-phenyl alanine (16.0). On inspection of

ninhydrin- amino acid complexes under ultra-violet radiation, phenyl

alanine-ninhydrin complex was found to give a light blue fluorescence

and this phenomenon was utilized to detect phenyl alanine (14^g), The

limit of detection under UV light in the presence was about 4-fold

excess (amount given in parenthesis) of DL-aspartic acid (48^g), L-

arginine monohydrochloride or L-cysteine hydrochloride or DL-

methionine (56^g) and DL-threonine (55^g). In addition to qualitative

analysis, quantitative evaluation of amino acids is often required. We

have attempted semiquantitative determination of amino acids by the

measurement of the spot area. A linear relationship (following equation

y=mx+c) was obtained for DL-aspartic acid when the spotted amount

of the sample (50-200^g) was plotted against the area of the spot

(Figure 2.8). The accuracy and precision was around ± 15%. Following

similar relationship, a-tocopherol (31), cations (32) and anions (33)

have been semiquantitatively determined.

Though more efficient and highly instrumentalized GC and HPLC

techniques are available for the analysis of amino acids but TLC is

especially suited for economical routine analysis as it permits the

monitoring of several compounds simultaneously on a single plate and

provide side-by-side comparisons of substances of identical nature.

75

Table 2.1 Mobility (or Rj value) of Amino Acids on Various Stationary Phases Using 1% CTAB in Water + Butanol (95 + 5) as Mobile Phase

Amino acid

DL - aspartic acid

L - glutamic acid

DL - 2 - amino - n-butyric acid

L - histadine monohydrochloride

L - lycine monohydrochloride

L - ornithine monohydrochloride

L-cystine

L - proline

DL - alanine

DL - phenyl alanine

DL - valine

L - cysteine monohydrochloride

DL - threonine

DL - iso leucine

DL-serine

Glycine

Silica

0.97

0.95

0.97

0.77

0.48

0.53

ND*

0.75

0.90

0.82

0.92

0.90

0.97

0.87

0.97

0.92

Mobility (Rr) value

Cellulose

0.97

0.95

0.95

0.97

0.97

0.95

ND

0.92

0.95

0.95

0.92

0.95

0.95

0.90

0.92

0.92

Kieselguhr

0,95

0.92

0.95

0.95

0.95

0.95

ND

0.97

0.92

0.90

0.95

0.92

0.92

0.95

0.95

0.92

Ahimina

0.08

0.14

0.64

0.30

0.35

0.27

ND

0.86

0.58

0.54

0.62

0.17

0.22

0.70

0.31

0.34

• ND: Not detected

76

Table 2.2 The Value of « (Separation Factor), Rs (Resolution) and ARF (Difference in Rp Values) for the Separation of L-proline from DL-tryptophan, DL-phenyl alanine, glycine, L-hydroxy proline and DL-alanine on Alumina Layer Using 1% CTAB in Water + Butanol (95+5) Mobile Phase in the Presence of Acidic and Basic Organic Impurities.

Stparatlon

L-proline - DL-tryptophan

L-proline - DL-phenylalanine

L-ptoline - glycine

L-proline - L-hydroxy proline

L-proline - DL-alanine

ARF

ac

R.

ARF

oc

Rs

ARF

cc

Rs

ARF

«

Rs

ARF

cc

Rs

Acetic Acid

0.27

3.09

II

0.2!

2.5

Zl

0.35

4.8

2.8

021

3.7

1.7

020

2.42

1.4

Formic Add

0.34

4.2

3.0

0.20

2.2

1.6

0.34

3.2 .

3.0

0.19

2.7

1.9

0.20

2.3

2.0

Added Impurity

Aniline

0.32

3.8

3.1

0.20

2.2

2.0

0.33

3.0

3.3

0.21

3.6

1.8

0.20

2.3

2.0

3-cliloro aniline

0.35

4.2

3.0

0.22

2.5

1.6

0.29

3.4

2.0

0.22

3.5

1.7

0.21

2.3

1.7

c-mpiitliyl •Mine

0.35

3.03

3.3

023

2.6

1.6

0.33

4.1

2.7

0.20

2.7

1.8

0.20

2.4

1.4

Withaui iapurtty

037

4.9

2.9

0.27

3.3

2.4

0.34

4.2

3.0

024

3.6

2.3

022

2.6

2.4

ARf = Difference in RF values (ARfr= Rf of L-proline - Rp of DL-tryptophan. DL-phenyl alanine, glycine, L-hydroxyproline or DL-alanine) ac = k2/ki, kj is the capacity factor of proline (kj = I-Rf/Rp) and kj is ccpacity factor

of amino acid separated (k2 = 1-RF/RF)

[Rs = AX/0.5 (dj+d^), AX is center - to ~ center distance of separated spots, di is diameter of proline spot anddi is diameter of separated amino acid spot.

cc •',v. T '5^38'

77

Table 2.3 Separation of DL-nor leucine from DL-aspartic acid and L-arginine monohydrochloride from their Mixture in the Presence of Heavy Metal Cations on Alumina Layer Developed with 1% CTAB in Water + Butanol (95 + 5)

Amino Acid

DL-aipartic acid

&DinR, Rf >ilu( (*-5)

Without At* Cu" Uo," VO" Nl" C»" Cd" Pb" Hf" Zn" F*" Cd^ Zn" lapurity

0.08 0.06 O06 O07 006 006 0.07 0.07 0.06 007 0.05 0.05 00)0 0.014

L-irginine O40 037 0.30 0.31 0.31 038 0.36 0.34 0.36 0.35 0.32 038 OOIO 0.002 monohydrochloride

DL-nor leucine 0.64 0.58 0.39 0.60 0.51 0.61 0.59 0.62 0.61 0.59 0.51 0.58 0.014 0.075

78

Table 2.4 Experimentally Achieved Separations of Amino Acids on Plain Alumina and Impregnated Alumina Layers Developed with Mobile Phase 1%CTAB in Water + Butanol (95+5)

Stationary Phase Separation (Rp)

Plain alumina plates L-hydroxy proline (0.66) — DL-serine (0.23) / glycine

(0.36) / monohydrochloride of L-arginine (0.31) / L-

histadine (0.30) /L-lycine (0.31)

L-arginine monohydrochloride (0.31) — DL-2-amino-n-

butyric acid (0.68) / DL-valine (0.65)

Impregnated alumina plates*

L-proline (0.80) — glycine (0.34) / DL-aspartic acid

(0.10) / glutamic acid (0.13) / L-ornithine

monohydrochloride (0,28) / DL-methionine (0.48) / DL-

serine (0.31)

L-leucine (0.77) — glycine (0.34) / L-histadine

monohydrochloride (0.30)

DL-2-amino-n-butyric acid (0.64) — DL-aspartic acid (0.10) / L-ornithine monohydrochloride (0.28)

These separations are not possible on plain alumina layer

79

Table 2.S Identification and Separation of L-proline - DL-alanine, L-proline -DL-phenylalanine and L-proline - L-hydroxyproline from Spiked Biological Samples

Biological Samples

Liver

Stomach

Blood

1

L- proline -DL> alanine

(0.80)-(0.59)

(0.83)-(0.62)

(0.80)-(0.59)

Separation of Amino Acid Pair (Rp)

L- proline - DL-phenyl alanine

(0.80)-(0.56)

(0.81)-(0.59)

(0.82)-(0.59)

L-proline —L-hydroxy proline

(0.84)-(0.63)

(0.86)-(0.66)

(0.85)-(0.65)

80

L-hydfoxyprolino • OL-thr«onin«

08 J ' L- lutwriic aad

• L-ornilhine mono-hydrochloride • DLlryptophan

Q6j

L-«»Udir» mono--hydrochtoido

• t<y»t«ln« hydrocMorid* L-phenyl tlanuM

3-(3.4-dihydroxyph«ny)).DL-•alanine

1-

06^

L-fMiidin* mono-• hydroehlorid*

• Lcysieino hydrochlorid*

• L-argnin* moitohydra chloridn • Ol-2-amino-W.buiyric

tod

Wi

L-proline DL-methioTMN

1.

-•-OL-vB!«t-»-L-ieacrfi*-*-L-lyiina mooohydf o-0B< cNoridt

CTAB concfnuauon I

• OL-phtnyi aianui*

• 3»<-<»'hydro«ypr>«nyi).OL..aianin«

Figure 2.1a Effect of the concentration of CTAB on the mobility of acids on almina ammo

81

• OL-alanine l-ptolint - OL-isoieucint ^1 -•-DU-aspanic - • • L-giuiamtc - • -01 •2-amino-N-botyric

acid scK) ac d OB-

:i y- OL-maihionine - I - OLphanytaianin* t- Lornilhina mono-

QB; hydrochloride

1>

OBJ OL-me- - f • OL-phanyl thionrne alanina

L-srnitMne monohydro-chlorlda

01 05

1

M

04

02

04-

Lhyoioxy proline

01

OL-sonne -*- OU-lhiaonina

05

• DL-vaiT.o ' L-hiaiidine iiionohyCrochlor

L-cy»lair J h) frocWorido -•-3-{3.4-dihydr(wyph«nyl)-Dt

05 1 SDS corwanualion (%;

-••• Glycine - • - L-leuclne - * - DL-noriaueino

06 1 808 conctrMretton (%)

Figure 2.1b Effect of the concentration of SDS on the mobihty of amino acids on alumina

82

9i 4 - ) CD ^

J3 4-»

•»M

^ ( I .

Oi 1

< H U ^ o^ ^^ j a i->

> b.

C II

u. oc: <

ha

o 't-t ed ^

^

^ u.

1

CT) Q CO

-^ ^ +-> • « - ^

^ u.

cd 11

u. oi <l

H 4

<

•

• 1

•

. 5

• ^

s?'



<

O SI

s Ml

CS C5 r i "^ vo 00

<T <T <T5 ^

•^v

83

• 5" 0 Ntalvuwl • - * • - 20% Melhajiol • 50" oMdlvuiol - -o - 80% McGiaiiol

0 ^ If ^<f - ^ ^ - f S f * i ' ^ < r i \ c r ^ o c o ^ o - - r s m * ^ » n \ o r ^ o o a v o ^ ^ < N r ^ < < < < < < < < < < < < < < < < < < < < ! 5 3 ^ ^

o'oPrapanol • ••& • -20°'oPropanol • 30°oPropanol - -O — 50°oPropanol

< < < < < < < < < - - - - ^ - 5 - - - 5 ^ ^ 5 3

-2°baitanol •5%Butanol

Amino adds

Figure 2.3. Mobility of amino acids in 1% non-buffered CTAB containing different concentrations of alcohol

84

• • " ARy=Rp with 1% CTAB in 95:5 water/butanol-R^ with 95:5 water/butonal •k- AR^=Rp with 1% CTAB in 95:5 water/butanol-R^ in water

Amino acids

ARp=Rp with 95:5 water/butanol-R^ with water

Amino acids

Figure 2.4 Plot of amino acids Vs AR,

85

1% CTAB in 95:5 water/butanol

<

• -• 1% CTAB in 95:5 buffer/butanol

-I \ 1 1 1 1 1 \ 1 1 1 1 \ 1 1—

A 1 / 2 A 3 M A 6 / 1 6 A 7 A 6 AG A10 A11 A12 A13 AW A15 A16 A17 A18 A19/CD A21/i22 A 2 3 / »

Amino acids

Figure 2.5 Mobility of amino acids in bumered and non-buffered micelle-ivater-butanol mobile phase

a O C5

No. of carbon atoms

a

Figure 2.6 Effect of no of carbon atoms in the molecule of amino acids on their mobility Amino acids : C2H5NO2

C3H7NO2 C5H11NO2 CfiHjaNOz

86

Figure 2.7 Plot of A RF = on plain alumina - Rp on alumina impregnated with 3% LiCl, for the amino acids.

Aspartic acid concentration (%)

Figure 2.8 Plot of spot area v/s amount of DL-aspartic acid loaded

87

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(1996) 37.

18. I. I. Malaknova, B. V. Tyaglov, E. V. Degterev, V. D. Krasikov

and W. G. Degtiar, J.Planar Chromatogr. Mod. TLC, 9 (1996)

378.

19. R. Bhushan and G. P. Reddy, J.Liq.Chromatogr., 10 (1987) 3497.

20 A. Niederwieser. Methods EmymoL, 25B (1972) 60.

21. J. Rosmus and Z. Deyl, J.Chromaiogr.,70 (1972) 221.

22. E. Norfolk, S. H. Khan, B. Fried and J. Sherma, J. Liq.

Chromatogr., 17(1994) 1317.

23. B. P. Sleckman and J. Sherma, J. Liq. Chromatogr., 5 (1982)

1051.

24. R. M. Metrione. J.Chromatogr., 154 (1978) 247.

25. J. K. Rozylo and I. Malinowska, J.Planar Chromatogr.-Modi.

TLC, 6(1993)34.

26. I. Malinowska and J. K. Rozylo, Biomed. Chromatogr., 11

(1997)272.

27. I. Baranowska and M. Kozlowska, Talanta, 42 (1995) 1533.

28. D. C. Demeglio and G. K. Svanberg, J.Liq. Chromatogr., 19

(1996)969.

29. M. R. Porter, Handbook of Surfactants, Blackie Academic and

Professional, an Imprint of Chapman & Hall Wester Cleddens

Road, Bishopbriggs, Glasgow G64 2NZ, 2"** ed., pi 19 & 37.

30. M. R. Porter, Handbook of Surfactants, Blackie Academic and

Professional, an Imprint of Chapman & Hall Wester Cleddens

Road, Bishopbriggs, Glasgow G64 2NZ, 2"** ed., p251.

31 A. Seher, Mikrochem Acta, (1961) 308.

32 A. Mohammad and N. Fatima, Chromatographia, 22 (1986) 109.

33 A. Mohammad and S. Tiwari, Microchem. J., 44 (1991) 39

89

-Ill

3.1 INTRODUCTION

The role of 2,2-dihydroxy-lH-indane-l,3(2H)-dione (ninhydrin) has

been well recognized since its discovery for the detection and

quantitative estimation of a-amino acids (1). The ninhydrin- amino

acid reaction gives a purple coloured product,

diketohydrindylidenediketohydrindamine (DYDA), known as

Ruhemann's purple (2). It was found that the colour intensity of

DYDA diminishes on keeping at room temperature and hence attempts

to stabilize the coloured product were made. The effect of metal ions

on ninhydrin-amino acids interaction was examined with this view by

Moore and Stein (3). Kawerau and Wieland (4) investigated the effect

of metal ions on the DYDA formation on a filter paper chromatogram.

Micellar solutions were first used as mobile phase in thin-layer

chromatography (TLC) by Armstrong and Terrill in the late seventies

(5). The low efficiency of the method is mainly due to a slow mass

transfer between the surfactant adsorbed layer and the micellar mobile

phase (6). The addition of small amount of 1-propanol or 1-pentanol to

the mobile phase greatly improves efficiencies by reducing the

adsorbed amount of surfactant (7,8). Addition of alcohol to micellar

solutions is the first step toward microemulsion formation (9).

Microemulsions are clear water-in-oil dispersions stabilized by mixed

film of surfactant with or without cosurfactant (medium chain

alcohols). These systems have some inhomogeneous character at the

microscopic down to molecular level. It has recently been shown that

water-in-oil (w/o) could be used as mobile phase (10,11) in liquid

chromatography.

In spite of the development of highly sophisticated and

expensive analytical equipments, thin layer chromatography (TLC) is

currently enjoying extensive popularity as a simple, rapid, inexpensive

and highly effective separation technique for analysing complex

mixtures into individual components (12,13). The separation

90

possibilities in TLC are largely depend upon the proper selection of

mobile and stationary phases. As a result numerous brands of

adsorbents and even greater number of mobile phases are being

developed to achieve improved chromatographic performance in terms

of selectivity and resolution. TLC of amino acids using various solvent

systems as mobile phase is well documented (14,15). Most of these

studies involve the use of alcohol as one of the components of mixed

mobile phase. Rozylo et al. have demonstrated excellent separation

possibilities of optical isomers of amino acids on chitin-Cu layers

using methanol-water-acetonitrile as mobile phase (16). However, none

of these studies reports the use of microemulsion system as mobile

phase in TLC of amino acids. It was therefore, considered worthwhile

to use water-in-oil microemulsion as mobile phase for achieving

selective separation of amino acids on silica gel layers modified by Cu

(II) ions. To the best of our knowledge this is the first attempt of using

water-in-oil microemulsion as mobile phase for amino acids

separations.

3.2 EXPERIMENTAL

Chemicals and Reagents: Amino acids, heptane, propanol, inorganic

salts (CDH, India); silica gel 'G', sodium dodecyl sulfate (SDS),

methanol, butanol (Qualigens,India); pentanol (Fluka

AG,Buchs,SG,made in Switzerland), and Brij-35 [Polyoxyethylene (23)

dodecyl ether] (Loba-chemie, India) were used. All reagents were also

of Analar Reagent grade.

Amino Acid Studied: As described in chapter 2

Test Solution: As described in chapter 2

Detection: As described in chapter 2

Chromatographic Sationary Phase or TLC Plates:

S,: Silica gel 'G'

S2: Silica gel impregnated with 2% aqueous CUSO4 solution

91

S3. Silica gel impregnated with a mixture of 2% aqueous CUSO4 and

3%Brij-35 (1:1. v/v)

S4: Silica gel impregnated with a mixture of 2% aqueous CUSO4 and

3%Brij-35(l:2. v/v)


3%Brij-35 (2:1, v/v)

Mobile Phase: The water-in-oil microemulsion, used as mobile phase

was prepared by titrating a coarseêmulsion of n-heptane (160 mL),

deionized water (8 mL) and SDS (8 g) with n-pentanol (24 mL) as

reported earlier (11). Water content in this microemulsion was varied

using 6 or 10 mL instead of 8 mL to see the effect of microemulsion

droplet size. The microemulsion was produced at 30°C.


(a) Plain Thin Layer Plates: As described in chapter 2

(b) Impregnated Silica Gel Thin Layer Plates: Impregnated plates

were prepared by mixing silica gel with a mixture comprising of 2%

aqueous CUSO4 solution and 3% Brij-35 (1:1, 1:2, 2:1), in 1:3 ratio by

shaking for 5-10 min. Thin layer of the resultant slurry was prepared by

following the procedure as described above in (a).

Procedure: For the determination of Rp value and limit of detection of

amino acids as described in chapter 2.

For the separation, equal amount of DL-phenyl alanine and DL-

alanine and impurities were mixed and 20 |iL of the resultant mixture

was loaded on the TLC plates. The plates were developed, the spots

were detected and the Rp values of the separated amino acids were

determined.


This section is divided in to four parts in order to understand amino

acid separations as well as effect of each factor in improving the

92

S?: Silica gel impregnated with a mixture of 2% aqueous CUSO4 and

3%Brij-35 (1:1, v/v)

S4: Silica gel impregnated with a mixture of 2% aqueous CuSOa and

3%Brij-35 (1:2, v/v)


3%Brij-35(2:l, v/v)

Mobile Phase: The water-in-oil microemulsion, used as mobile phase

was prepared by titrating a coarse-emulsion of n-heptane (160 mL),

deionized water (8 mL) and SDS (8 g) with n-pentanol (24 mL) as

reported earlier (11). Water content in this microemulsion was varied

using 6 or 10 mL instead of 8 mL to see the effect of microemulsion

droplet size. The microemulsion was produced at 30^C.


(a) Plain Thin Layer Plates: As described in chapter 2

(b) Impregnated Silica Gel Thin Layer Plates: Impregnated plates

were prepared by mixing silica gel with a mixture comprising of 2%

aqueous CUSO4 solution and 3% Brij-35 (1:1, 1:2, 2:1), in 1:3 ratio by

shaking for 5-10 min. Thin layer of the resultant slurry was prepared by

following the procedure as described above in (a).


amino acids as described in chapter 2.

For the separation, equal amount of DL-phenyl alanine and DL-

alanine and impurities were mixed and 20 (xL of the resultant mixture

was loaded on the TLC plates. The plates were developed, the spots

were detected and the Rp values of the separated amino acids were

determined.


This section is divided in to four parts in order to understand amino

acid separations as well as effect of each factor in improving the

92

process: (a) separation on plain and CUSO4 impregnated silica gel

layers in the absence of Brij-35; (b) separation on silica gel

impregnated with different aqueous salt solutions with added Brij-35;

(c) comparison of water-in-oil microemulsion and other mobile phases;

(d) specific detailed studies.

(a) Separation on Plain and CUSO4 Impregnated Silica Gel Layers

in the Absence of Brij-35: On plain silica gel layer all amino acids

show little mobility without providing any possibility of their

separation. Few amino acids (DL-alanine, DL-serine, L-leucine, DL-iso

leucine, DL-tryptophan, DL-phenyl alanine, 3-(3,4 dihydro-

xyphenyl)DL-alanine and DL-2-amino-n-butyric acid) produce tailed

spots. These observations suggest that plain silica gel is not suitable

for separation of amino acids. The Rp data presented in Table 3.1 show

that CUSO4 is the most suitable in terms of separation possibilities of

amino acids. This is probably due to the fact that Cu " can form

molecular complex with amino acid (17). The retention behaviour of

amino acids was also examined on silica gel layers impregnated with

different concentrations of CUSO4 (0.5-3%). The increase in CUSO4

concentration leads to the increase in Rp value (i.e. increased

mobility). At 3% CUSO4 inpregnation, Cu^ comigrates with mobile

phase (water-in-oil microemulsion) and hence 2% CUSO4 was

considered optimum for impregnation.

(b) Separation on Silica Gel Impregnated with Different Aqueous

Salt Solutions with Added Brij-35: In order to find out a better

impregnant, silica gel layers were impregnated with 2% aqueous salt

solution with added 3% Brij-35 in 1:1 ratio and the Rp values of amino

acids were recorded using water-in-oil microemulsion as mobile phase

(Table 3.2). It should be mentioned that the separation potential was

improved when the silica gel was impregnated with a mixture of CUSO4

(2%) and Brij-35 instead of aqueous copper sulfate solution (Tables 3.1

and 3.2). This observation was the result of a detailed study of the

effect of the concentration of Brij-35 in the solution which is used for

93

impregnation. With different concentrations of Brij-35 in stationary

phase, the mobility of amino acids decreases with the increase in

concentration of Brij-35 from 3 to 10% (Figure 3.1) However at/or

lower than 1% concentration of Brij-35, a reverse trend is observed.

Improvement in separation possibility in the presence of Brij-35

may be due to two factors, first Brij-35 can check the possible

adsorption of SDS of water-in-oil microemulsion with the concomitant

effect on microemulsion stability. Secondly, Brij-35 can form mixed

micellar droplets. These interrelated factors undoubtedly affect the

separation efficiency and the spot appearance. However, excess of

Brij-35 could affect the microemulsion composition or retention

behaviour of silica gel with a simultaneous effect on the separation

process. The data summarized in Figure 3.2 indicate that silica gel

impregnated with a mixture of 2% CUSO4 and 3% Brij-35 (1:1) is the

best stationary phase for selective separation of amino acids. This

stationary phase referred as S3, has been used for detailed study.

(c) Comparison of Water-in-Oil Microemulsion and Other Mobile

Phases: water-in-oil microemulsion was used as mobile phase and its

behaviour was compared with few pure components as summarized in

Table 3.3. Water-in-oil microemulsion has spherical droplets

surrounded by SDS monomeric layer and oil (n-heptane) continuous

mantle. This internal environment of water-in-oil provides a small

aqueous volume (18) (water molecule trapped into reverse micelles)

where molecular complex of amino acid (or pure amino acids whatever

the case may be) can react with ninhydrin to give a coloured spot. This

may well be attributed to the fact that in water-in-oil microemulsion,

molecular complexes of Cu-amino acids are localized in the

hydrophilic aqueous core, which is responsible for some sort of

preconcentration of amino acids. Thus, amino acids are easily available

to interact with ninhydrin for detection. The droplet size was varied by

changing the water content in the system (19-21). However, it has

insignificant effect on the mobility (Rp values) of amino acids. When

94

water-in-oil microemulsion mobile phase was replaced by water,

heptane, alcohols (methanol, 1-propanol or butanol) or pure Brij-35

(35% solution, w/v) as mobile phase and amino acids were

chromatographed on S3, all the amino acids remained at the point of

application (RF=0) in the case of heptane, propanol and butanol.

Conversely, all amino acids except DL-iso leucine show reasonable

mobility with water and Brij-35 providing an opportunity for selective

separation of DL-iso leucine from other amino acids. A differential

migration of amino acids with methanol as mobile phase opens certain

possibilities for the separation of amino acids. But water-in-oil

microemultion was the best mobile phase among others used in the

study and could be exploited in a better way for the future work on

amino acids separations.

(d) Specific Detailed Studies: In order to provide more clear view,

some TLC parameters for the separation of DL-alanine froni DL-phenyl

alanine have been calculated and presented in Table 3.4. From these

data it is clear that the separation of DL-phenyl alanine from DL-

alanine is always possible in the presence of all the impurities. The

separation is improved by the presence of p-chloroaniline as evident by

higher value of ARp (0.51) and a (15.3). However, poorer separation

was realized in the presence of m-chloroaniline and I" as indicated by

lower ARp value. In all cases the Rs values are higher than 1.0

indicating well-resolved spots from their mixture. Two spots are

considered non-separable if the value of Rs is below 1.0. Thus higher

value of Rs is indicative of better separation efficiency. The separation

is not hampered by the presence of impurities and a reliable separation

of DL-alanine from DL-phenyl alanine is always possible.

The lowest possible microgram amounts (given in parenthesis)

detectable on different stationary phases (i.e. limit of detection) of

alanine and phenyl alanine were found as plain silica gel (0.06, 0.09)

and metal salts impregnated silica gel, HgCl2 (0.28, 0.28), Zn(N03)2

(0.66, 0.28), CdClj (0.5, 0.5), NiClj (2.0, 1.0) and CUSO4 (2.0, 2.0)

95

respectively. It is clear that the highest sensitivity (lowest detectable

amount) is possible on plain (or unimpregnated) silica gel layer.

Conversely, the lowest sensitivity is associated with copper sulfate

impregnated silica gel layers which provide the best separation

possibilities. The investigated stationary phases may be arranged in the

following preferred order for separation and detection purposes of

amino acids:

Separation: Cu-silica gel > Cd-silica gel > Zn-silica gel > Ni-silica gel

> Hg-silica gel > plain silica gel

Sensitivity: plain silica gel > Hg-silica gel > Cd-silica gel > Zn-silica

gel > Ni-silica gel > Cu-silica gel

The lower sensitivity of amino acids on metal ion impregnated

silica gel layer is due to the fading of colour of ninhydrin - amino acid

complexes in the presence of metal ion (22).

96

Table 3.1 RK Values of Amino Acids on Plain (S]) and Copper Sulfate Impregnated (S2) Silica Gel Layers Developed with Water-in-Oil Microemulsion

Amino Acid S, S2

Al

A2

A3

A4

A5

A6

A7

A8

A9

AlO

All

A12

A13

A14

A15

A16

A17

A18

A19 A20 A21

A22

A23 A24

* : Tailed spot (RL-ND: Not detected

- RT ^0.3)

0.13

0.07

0.23*

0.16*

0.11

0.11

0.12

0.16

0.16*

0.16*

0.13

0.14

0.08

0.21*

ND

0.24 *

0.17*

0.31

0.33 0.18 0.32

0.12

0.11 0.15

0.30

0.16

0.21

0.18

0.14

0.32

0.18

0.00

0.19

0.32

0.00

0.00

ND

0.40

ND

0.49

0.00

0.17

0.17 0.23 0.27

0.18

0.18 0.00

97

Table 3.2 R|. Value of Amino Acids on Silica Gel Layers Impregnated with a Mixture of 2% of Aqueous Salt Solutions and 3% Brij-35 in 1:1 Using Water-in-Oil Microemulsion as Mobile Phase

Amino Acid

Al

A2

A3

A4

A5

A6

A7

A8

A9

AlO

All

A12

A13

A14

A15

A16

A17

A18

A19

A20

A21

A22

A23

A24

Li*

0,19

0.22

0.20

0.20

0.08

0.14

0.24

0.30

0.31

0.28

0.32

0.20

ND

0.32

ND

0,31

0.00

0.16

0.12

0.09

0.15

0.10

0.11

0.13

Cu'-

0.45

0.31

0.44

0.39

0.38

0.67

0.37

ND

0.70

0.62

.0.23

0.40

ND

0.62

ND

0.87

0.00

0.49

0.47

0.42

0.51

0.36

0.35

0.03

Zn -

0.14

0.11

0.15'

0.15-

0.12

0.11

0.24

0.20

0.19

0.15

0.28

0.14

ND

0,15'

ND

0.26

0.00

0.16*

0.15"

0.10

0.14

0.15-

0.14

0.15

Cd'*

0.29

0.24

0.14

0.14

0.12

0.22

0.13

0.11

0.35

0.30

0.46

0.29

ND

0.21

ND

0,53

0.00

0.15

0.30

0.16

0,35

0.30

0.25

0.16"

Hg'*

0.26

0.23

0.22

0.20

0.19

0.27

0.27

ND

0.28

0.31

0.30

0.23

ND

0.22

ND

0.22

0.00

0.28

0,33

0,32

0,29

0.28

0.22

0.17

Th'*

0.15"

0,22

0,20

0,21"

0.09

0,18

0,25

0,17'

0,32

0,18"

0,31

0,25

ND

0,17'

0,04

0,18

0,00

0.25

0.20

0.25'

0.29

0.15'

0.15'

0.17'

Br

0.42

0.17

0.29"

0.35*

0.15"

0.25

0.28'

0,46

0,52

0,38'

0.43

0.36'

ND

0,45'

ND

0.45

0,00

0,34

0.23'

0.37

0.23

0.25'

0.27

0.29"

I

0,18

0,14

0,20

0,28

0,11

0,28

0,37

0,21

0,22

0,26

0,28

0,42

ND

0,47

ND

0,21

0.00

0.24

0.17"

0.17"

0.10

0,19"

0,12

0,15"

* ; Tailed spot (RL-RT ^0.3)

ND: Not detected

98

Table 3.3 Ry Value * of Amino Acids on Plain Silica Gel Layers Developed with Different Mobile Phases

Amino Acid

Al

A2

A3

A4

A5

A6

A7

A8

A9

AlO

Al l

A12

A13

A14

A15

A16

A17

A18

A19

A20

A21

A22

A23

A24

Water

0.98

0,96

0.91

0.92

0.91

0.89

0.67

ND

0.72

0.00

0.85

0.82

0.88

0.88

0.77

0.38

0.61

0.93

0.71

0.80

0.93

0.94

0.86

0.79

Methanol

0.25

0.35

0.85

0.04

0.15

0.15

0.25

ND

0.75

0.00

0.86

0.83

0.67

0.25

0.49

0.84

0.84

0.39

0.48

0.74

0.30

ND

0.29

0.35

Brij-35

0,91

0.91

0.91

0.94

0.92

0.95

0.90

0.92

0.89

0.00

0.88

0.71

0.87

0.87

0.85

0.90

0.94

0.92

0.94

0.94

ND

0.84

0.72

0.89

Microemulsion

0.45

0.31

0.44

0.39

0.38

0.67

0.37

ND

0.70

0.62

0.73

0.40

ND

0.62

ND

0.87

0.00

0.49

0.47

0.42

0.51

0.36

0.35

0.63

* All amino acids were simply retained on silica gel layer (Rf = 0.0) with propanol, butanol or heptane

99

Table 3.4 TLC Parameters for the Separation of DL-Phenyl alanine from DL-Alanine in the Presence of Impurities (Mobile Phase: Water-in-Oil Microemulsion and Stationary Phase: Se)

Added Impurit}'

Co^^

Ni'^

Hg^^

ZT?'

Cd'*

Fe'^

SO4'

NOj' -

Br

r 0-nitrophenol

Picric acid

Pytogallol

ift-chloroaniline

a-naphthyl amine

p-chloroaniline

Without impurity*

DL-Alanine

Kl'

2.2

1.6

2.1

2.8

2.7

4.0

3.1

4.0

3.7

4.8

2.7

2.1 •

2.5

2.7

1.7

10.1

1.5

d2

1.0

02

0.7

0.6

0.8

0.4

0.9

0.9

0.3

0.7

0.3

02

0.8

0.3

0.5

0.8

0.7

DL-Phenyl

K2'

0.44

0.58

0.61

0.61

0.61

0.75

0.56

0.85

0.92

1.60

0.72

0.69

0.72

0.85

0.72

0.66

0.25

alanine

dl

0.1

0.1

0.2

0.2

0.2

0.7

0.2

0.4

0.2

0.2

0.2

0.1

0.2

0.4

0.2

1.0

0.3

Separation Factor

(a=KI'/K2')

5.0

2.7

3.4

4.5

4.4

5.3

5.5

4.7

4.0

3.0

3.7

3.0

3.4

3.1

2.3

15.3

6.0

Resolution

lRs=D/0^dl+d2)

4.6

8.3

3.0

4.5

3.6

3.3

3.3

2.4

1.0

2.4

6.2

%2

3.0

4.1

3.1

2.1

6.6

Difference in Rf Values

(ARK)

0.38

0.25

0.30

0.36

0.35

0.37

0.40

0.34

0.31

0.21

0.31

0.27

0.30

0.23

• 0..26

0.51

0.40

*Data achieved for the separation of DL-phenyl alanine form DL-alanine in the absence of impurities ARF = Rf of DL-phenyl alanine minus RF on DL-alanine dl = diameter of DL-phenyl alanine spot d2 = diameter of DL-alanine spot KI' = capacity factor of DL-alanine spot K2' = capacity factor of DL-phenyl alanine spot D = distance between the centers of spots of DL-alanine and DL-

phenyl alanine Capacity factor was calculated using the equation

K= 1-RF

RF

for respective amino acids.

100

< 5 5 5 5 < 5 5 | ? 10/ n '• f Amino acids

1% Brij-35

3%Brij-35

5% Brij-35

10% Brij-35

Figure 3.1 Mobility of amino acids on silica gel layer impregnated with a mixture of 2% aqueous CUSO4 with added Brij-35 (1, 3, 5 and 10%) in water in 1:1 ratio

Amino acids

2% aqueous CuSO^ + 3% Brij-35 (1:1)

2% aqueous CUSO4 + 3% Brij-35 (1:2)

2% aqueous CUSO4 + 3% Brij-35 (2:1)

Figure 3.2 Combined effect of concentration of aqueous CUSO4 solution and Brij-35 on the Rp value of amino acids

101

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1402.

11. A. Mohammad, S. Tiwari, J. P. S. Chahar and S. Kumar, J. Am.

Oil. Chem. Soc, 72 (1995) 1533.

12. J. Sherma and B.Fried (eds). Handbook of Thin-Layer

Chromatography, 2"** ed.; Maral Dekker, Inc: New York (1996).

13. J. Sherma,. Anal. Chem., 70 (1998) 7 R.

14. J. K. Rozylo, I. Malinowska and A. V. Musheghyan, J. Planar.

Chromatogr. -Mod TLC, 2 (1989) 374.

15. I. I. Malakhova, B. V. Tyaglov, E. G. Degterev, V. D. Krasikov

and W. G. Degtiar, J.Planar Chromatogr.-Mod. TLC, 9 (1996)

375.

102

16. 1. Malinowska and J. K. Rozylo, Biomcd. Chromalogr., 11

(1997)272.

17. C. B. Mank, 7>a/;.v. Faraday. Soc, 47 (1951) 285.

18. A. Berthed and M. De. Carvalho, Anal. Chem., 64 (1992) 2267.

19. K. Kojikano, T. Yamagudi and T. Ogawa, J. Phys. Chem., 88

(1984) 793.

20. W. D. Weatherford, J. Dispersion Sci. Technol., 6 (1985) 467.

21. J. L. Cavallo and H. L. Rosano, J. Phys. Chem. 90 (1986) 6817.

22. A. D. Aniello, G. D. Dnofrio, M. Pischetola and L. Strazzullo,

Anal. Biochem., 144 (1985) 610.

103

-IV

4.1 INTRODUCTION

The analysis of nitrogen - containing compounds has assumed

importance because of their carcinogenic and pesticidal properties. As

a result, several chromatographic techniques (1-6) have been used for

their separation and identification at microgram level. Though gas

chromatography with packed columns (specific for nitrogen

compounds) has been extensively used for the more accurate

quantitative analysis of complex mixtures of amines but thin layer

chromatography (TLC) being inexpensive is more suitable for routine

qualitative analysis of organic as well as inorganic substances. TLC

separation of amines via charge transfer complexation with polynitro

compounds has been achieved (7-9) with mixed organic mobile phases.

Amines being n-donor, contribute electrons to the partially filled

orbital of nitro groups resulting in the formation of charge-transfer

complexes. The major drawback of this method is that the weak

physical bond formed between amines and aromatic nitro compounds

during charge transfer complexation is readily ruptured by polar

solvents and adsorption forces. Alternatively, silica gel layers

impregnated with various metallic salts have been tested for realizing

mutual separations of aromatic amine isomers (10-12) using mixed

organic eluants. The practical utility of metallic salt impregnated

layers as stationary phase is restricted due to (i) formation of

occasional tailed spots, (ii) lesser stability in acidic solvent systems

and (iii) tendency to absorb moisture.

On the other hand, TLC methods involving the use of mixed

organic solvent systems containing benzene, carbon tetrachloride,

dioxane or hexane (13-14) are not useful for routine analysis because

of their toxic effect. It is therefore, important to develop an

inexpensive and reliable TLC system for rapid analysis of amines using

relatively non-toxic mobile phases. Considering the advantageous

features of surfactant-mediated mobile phases such as non-toxicity,

unique separation selectivity, non-flammability and enhanced detection

104

capability, an extensive investigation has been initiated in our

laboratory to utilize the analytical potential of surfactant containing

mobile phases with added small quantities of organic additives for

rapid TLC separation of complex mixtures.

Despite evident advantages, surfactant mediated systems have

not been fully utilized in TLC separation of organic compounds. The

present communication describes a systematic approach for achieving

improved separations of aromatic amines using a cationic cetyl

trimethyl ammonium bromide (CTAB) surfactant containing mobile

phases.

4.2 EXPERIMENTAL

Chemical & Reagents: Silica gel 'G', n-butanol [Qualigens, India] N-

cetyl-N,N,N-trimethyl ammonium bromide (CTAB), amines, inorganic

salts, methanol, ethanol and 2-propanol [CDH, India], Brij -35 and

TritonX-100 [Loba chemie, India] were used. All other reagents were

Analytical Reagent grade.

Amines Studied: Diphenylamine (DPA), o-chloroaniline (o-CAL), m-

chloroaniline (m-CAL), p-chloroaniline (p-CAL), a-naphthylamine (a-

NPA), p-bromoaniline (p-BAL), p-anisidine (p-ASD), p-

phenylenediamine (p-PND), aniline (AL), N-N-dimethylaniline

(DMAL), o-toluidine (o-TLD), m-toluidine (m-TLD), p-toluidine (p-

TLD), o-nitroaniline (o-NAL), m-nitroaniline (m-NAL), p-nitroaniline

(p-NAL), indole (ID), p-dimethylaminobenzaldehyde (p-DAB) and DL-

tryptophan (DL-TTP).

Test Solutions: The test solutions (1% w/v) of all amines were

prepared in methanol.

Detection: All amines were detected by exposing the TLC plates to

iodine vapours. DL-tryptophan was detected with 0.5% (w/v) ninhydrin

solution prepared in acetone.

Stationary Phase: Silica Gel 'G'

105

Mobile Phases: The following solvent systems were used as mobile

phase

Composition

H2O 0.16% (w/v)CTAB in water 0.32% methanol in water 0.08 mL (CTAB + methanol) * +99.92 mL water 0.5 mL (CTAB + methanol) + 99.5 mL water 1.0 mL (CTAB + methanol) + 99.0 mL water 1.5 mL (CTAB + methanol) + 98.5 mL water 2.0 mL (CTAB + methanol) + 98.0 mL water 3.0 mL (CTAB + methanol) + 97.0 mL water 0.5 mL (CTAB + ethanol) + 99.5 mL water 1.0 mL (CTAB + ethanol) + 99.0 mL water 1.5 mL (CTAB + ethanol) + 98.5 mL water 2.0 mL (CTAB + ethanol) + 98.0 mL water 0.5 mL (CTAB + propanol) + 99.5 mL water 1.0 mL (CTAB + propanol) + 99.0 mL water 1.5 mL (CTAB + propanol) + 98.5 mL water 2.0 mL (CTAB + propanol) + 98.0 mL water 0.5 mL (CTAB + butanol) + 99.5 mL water 1.0 mL (CTAB + butanol) + 99.0 mL water 1.5 mL (CTAB + butanol) + 98.5 mL water 2.0 mL (CTAB + butanol) + 98.0mL water 0.5 mL (TritonX-100 + methanol) + 99.5 ml water 0.5 mL (Brij-35 + methanol) + 99.5 mL water 0.5 mL (CTAB + methanol) + 99.5 mL aq. NaBr (0.01-0.2 0.5 mL (CTAB + methanol) + 99.5 mL aq. NaCl (0.01-0.2 0.5 mL (CTAB + methanol) + 99.5 mL aq. Urea (0.01-0.2M

* CTAB + organic solvent taken as 1:2, w/v in all cases and required volume of

water was added into the mixture of (CTAB + organic solvent)

Preparation of Plain TLC Plates: TLC plates were prepared as

described in chapter 2.

Procedure: About 10 ^L of test solution was spotted on a thin layer

plate with the help of micropipet. The plates were developed in the

chosen solvent system by the ascending technique. The solvent ascent

was fixed to 10cm in all cases. After development, the plates were

withdrawn from glass jars and dried at room temperature. TLC plates

106

were then exposed to iodine vapors for about 10 min. The amines were

detected as yellowish brown spot. The Rj (Rj of leading front) and Rj

(RF of trailing front) values for each spot were determined and The Rj.

value was calculated

RL "*" RT

RF =

2

Procedure for the limit of detection of amines as described in

chapter 2 for amino acids.

To study the effect of the presence of heavy metal cations and

phenols on the separation of indole from p-DAB, a synthetic mixture

was prepared by adding 0.5 mL of heavy metal or phenol solution

(1.0% w/v) into 0.05 mL mixture of p-DAB and indole (l:lv/v), about

0.01 mL of the resultant mixture was spotted on TLC plate and the spot

was dried at 30° C. The plates were developed, the spots were detected

and the Rp values of the separated amines were determined from their

RL and Rj values.

For semiquantitative determination by spot-area measurement

method, 0.01 mL from a series of standard solutions of p-anisidine

(0.5-2%) were spotted on silica layers. The plates were developed with

M5. After detection the spots were copied onto tracing paper from the

chromatoplates and then the area of each spot was calculated.


The mobile phases were prepared by the addition of water (different

volumes) into 3 mL (approximately) of alcoholic CTAB obtained by

dissolving 1 g CTAB in 2 mL alcohol. Compared to pure water (Mi),

higher mobility of most of the amines was observed with aqueous

CTAB (M2), water containing 0.32% MeOH (M3) and methanolic CTAB

+ water mixture (M5) mobile phases (Table 4.1). The trends of mobility

of 0-, m- and p- chloroanilines in Mi and M3 are in the order of o-CAL

> m-CAL > p-CAL and m-CAL > p-CAL > o-CAL respectively. This

107

behaviour is possibly due to the coordination between methylene

oxygen and amino hydrogen causing the association between the two

and increasing the bulk around the substituents. Though such

association is also possible in case of p- and m-CAL but no

corresponding steric phenomenon arise in their cases. Thus, the steric

factor in the case of o-CAL is responsible for reversing the order of its

mobility when Mi is substituted by M3. The higher mobility of m-CAL,

p-CAL and ID in M3 compared with their corresponding mobilities in

Ml may be due to increased solubilizing tendency of methanol

compared to water. The Rp values (i.e mobility) of amines differ

marginally when M3 (alcoholic water without surfactant) is replaced by

M2 (aqueous CTAB) or M5 (CTAB in alcoholic water) and the

development time increases for 13 min (Mi) to 17 min (M5). The higher

mobility of ID (Rp = 0.9) compared to its mobility in Mi - M3

facilitates a good separation of ID from p-DAB and DP A (Table 4.1).

This separation is important from analytical point of view, as p-DAB

forms coloured products with both ID and DPA in solution (15). On the

other hand these amines react with 2,4-dinitrotoluene (DNT) in the

solid state to produce yellow products (16) and hence the presence of p-

DAB or DPA hampers the detection of ID with DNT.

The data presented in Table 4.2 demonstrate how the mobility of

amines is controlled by the composition of a three-component (i.e.

water - MeOH - CTAB) mobile phase. The concentration of each

component i.e. MeOH, CTAB or water was simultaneously varied in

mobile phases (M4 - M9) keeping total volume constant (i.e. 100 mL).

Depending upon the composition of the mobile phase, the development

time for 10 cm ascent was 15 - 35 min. It is clear from Table 4.2 that a

good separation of indole from p-DAB and DPA can be achieved with

M5 containing 0.16% CTAB. At other CTAB concentration levels i.e.

very low (M4) or higher (Mg - M9) relatively poor separation of indole

from p-DAB and DPA is possible. As the reported CMC value of

CTAB (17) in water is 0.46%, it is therefore concluded that hybrid

108

mobile phases composed of a cationic surfactant (CTAB), methanol

and water are most suitable for separation of aromatic amines, if the

CTAB concentration in the mobile phase is kept below or just above to

its CMC value. The separation possibility of ID from p-DAB and DPA

is increased as a result of some specific interactions of cationic CTAB

monomers with % electrons of aromatic rings and the non-bonding (i.e.

lone pair) electrons residing on nitrogen atom of indole, p-DAB and

DPA. As evident from Table 4.2 the separation of indole from p-DAB

or DPA is also possible at CTAB concentration higher than its CMC

value (M9). In such case, the separation is again controlled by the

interactions of n or TC electrons with ionic head group of CTAB

micelles.

Taking into consideration of better separation possibilities, spot

compactness and detection clarity of amines on silica gel layer, M5

containing 0.16% CTAB was selected as mobile phase for detailed

investigations. At this concentration level CTAB is present as cationic

monomer.

In order to demonstrate the effect of type of alcohol on the

mobility (Rp values) of amines results obtained with M5, Mio, M H and

M18 mobile phases which are composed of CTAB- H2O- alcohol

(methanol, ethanol, propanol or butanol) were plotted in Figure 4.1.

From this figure following trends are noticeable.

(a) Mobility of amines is influenced by the nature of the alcohol

used. Generally most of amines move faster in MeOH containing

mobile phases. Conversely, mobility of amines was lower in

ethanol containing mobile phases.

(b) Compared to the mobility in MeOH and ethanol containing

mobile phases, amines show intermediate mobility in mobile

phases containing propanol or butanol.

(c) N-N-dimethyl aniline is more strongly retained by stationary

phase (Rp « 0.08) irrespective of the type of alcohol used in

109

mobile phase and hence it can be selectively separated from all

other amines studied. Conversely, tryptophan shows almost no

interaction with stationary phase and migrates with mobile phase

giving Rp of about 0.90. Thus, tryptophan can easily be

separated from amines having lower Rp values.

(d) Indole (Rp = 0.90) can be very well separated from most of

amines in MeOH containing mobile phase.

To understand the migration trend, amines were

chromatographed on silica layer using mobile phases containing

different concentrations of ethanol (Mn - Mn), propanol (M15 - M17)

and butanol (M19 - M21). The magnitude of Rp value of individual

amine shows a variation of ± 0.16 on simultaneous increase in the

concentration of alcohol and CTAB in the mobile phases (Mn - M13,

Mi5 - Mi7 and M19 - M21) irrespective of the type of alcohol (ethanol,

propanol or butanol) used. For example, the difference of 0.24 in Rp

values was noticed in the case of p-DAB on using M12 or M13 mobile

phase instead of Mio. Similarly, p-bromoaniline shows a difference of

0.35 if TLC plate is developed with M12 instead of Mjo.

In order to examine the effect of nonionic surfactant on the

mobility of amines, CTAB in M5 was substituted with Triton X-100 or

Brij -35. The mobility of almost all amines lowered marginally in

mobile phases containing Triton X-100 or Brij -35 compared to their

mobility in CTAB. The lowering in mobility of amines with non-ionic

surfactants exert an adverse effect on the separation of amines. Our

observation is in consonance of earlier findings where non-ionic

surfactants have been found unsuitable for TLC separation of

fluorescein (18), pesticides (19) and metal diketonates (20) because of

their poor eluant strength.

In consonance with earlier studies on TLC of amines with non-

surfactant mediated mobile phases (21), the Rp values of certain

amines in the present case were also found to decrease with the

110

increase in their basicity. From the data summarized in Table 4.3 the

increasing order in R|. values of amines e.g.

AL<o-CAL <o-NAL;

AL <m-CAL < m-NAL;

AL < p- CAL < p- NAL;

0- CAL < m- CAL;

0- NAL < m- NAL

0- NAL < p- NAL and

DMAL < AL

was just opposite to the basicity order of amines (22). DMAL being

stronger base than aniline or substituted aniline has the minimum

mobility.

The results presented in (Figure 4.2) show that the mobility of

amines is significantly influenced by the presence of electrolyte (NaCl

or NaBr) and non-electrolyte (urea) in the hybrid (CTAB-H20-MeOH)

mobile phase. Generally the presence of salt or urea in the mobile

phase. (M24-M26) leads in reduction of mobility of amines as a result of

their enhanced affinity to stationary phase. However the decrease in Rp

value is more pronounced in case of urea compared to NaCl or NaBr.

Several factors such as greater affinity of silanols for CTA^ than for

Na* (23), capability of urea to decrease hydrophobicity effect (24), and

the decrease in CMC value and increase in micelle size (25) on the

addition of these additives are responsible to alter the retention pattern

of amines on silica layer. The same behaviour is observed in pure

aqueous mobile phase, 5 - 95% v/v methanol - water phases, and O.IM

NaCl phases on both stationary phases. Minor changes in mobility of

individual amines were noticed on substitution of NaCl by NaBr in the

mobile phase. This effect may be attributed to common ion effect.

I l l

An attempt has also been made for the semiquantitative

determination of p-anisidine by spot-area measurement method. For

this purpose, 0.01 niL of standard solutions of p-anisidine (0.05-2%)

were spotted on silica thin layer. The plates were developed and

anisidine spot was detected on TLC plate. The spots obtained were

copied directly on tracing paper from the chromatoplates and the spot-

area was measured. A relationship between the spot-area and

microgram quantities of p-anisidine follows the empirical equation, <^

= k m where ^ is the spot area, m is the spotted amount and k is

constant. The linearity is maintained up to 200 jig/spot of p-anisidine.

At higher concentration, a negative deviation from linear law was

noticed. The precision and accuracy was ± 15%.

The lowest possible detectable microgram amounts alongwith

dilution limits of amines (given in parenthesis) on silica gel layer were

p-DAB (0.05, 1: 2 X 10^), ID (0.14, 1: 7.1 x 10^), o-CAL (0.10, 1 : 1 x

10^), m-CAL (0.08, 1 : 1.2 x 10^), oc-NPA (0.11, 1 : 9.09 x lO ) and p-

TLD (0.07, 1 : 1.4 x 10^). The present method is superior for sensitive

detection of p-DAB and indole compared to the reported method (26)

The effect of some heavy metal cation and phenol additives on

the separation efficacy of indole from p-DAB was examined. Heavy

metal cations do not offer deleterious effect on the separation of indole

from p-DAB. In fact, an improved separation of indole from p-DAB

was realized in the presence of Cd " . Interestingly, the separation of

indole from p-DAB is not hampered by the presence of resorcinol,

orcinol or phloroglucinol (Rp * 0.98) despite the fact that these phenols

move with the solvent front ahead to indole during the development of

TLC plates. It is an important observation in the light of the fact that

these phenols interfere in the detection of indole with most of

chromogenic reagents in solution phase (27).

112

Table 4.1 Mobility (i.e. Rp) of Amines in the Presence and Absence of Surfactant in the Mobile Phase

Amine

DPA

o-CAL

m-CAL

p-CAL

a-NPA

p-BAL

p-ASD

p-PND

AL

DMAL

o-TLD

m-TLD

p-TLD

o-NAL

m-NAL

p-NAL

ID

p-DAB

DL-TTP

M,

0.44

0.82

0.75

0.67

0.67

0.75

0.77

0.70

0.77

0.03

0.80

0.85

0.72

0.65

0.70

0.82

0.67

0.49

0.88

R F

M2

0.55

0.62

0.77

0.68

0.70

0.72

0.70

0.74

0.77

0.03

0.75

0.80

0.75

0.75

0.86

0.85

0.78

0.67

0.92

Value

M3

0.61

0.67

0.90

0.84

0.80

0.87

0.75

0.75

0.82

0.07

0.75

0.72

0.75

0.67

0.78

.0.85

0.80

0.58

0.81

Ms

0.59

0.73

0.85

0.73

0.70

0.80

0.78

0.70

0.82

0.08

0.63

0.67

0.67

0.81

0.78

0.80

0.90

0.59

0.89

113

Table 4.2 Mobility of Amines on Silica Layer Developed with CTAB - MeOH - H2O Mobile Phase

Amine

DPA

o-CAL

m-CAL

p-CAL

a-NPA

p-BAL p-ASD

p-PND

AL

DMAL

o-TLD

m-TLD

p-TLD

o-NAL

m-NAL

p-NAL

ID

p-DAB

DL-TTP

M4

0.55

0.70

0.85

0.74

0.74

0.78 0.75

0.70

0.75

0.10

0.70

0.67

0.68

0.80

0.60

0.75

0.75

0.59

0.87

Ms

0.59

0.73

0.85

0.73

0.70

0.80 0.78

0.70

0.82

0.08

0.63

0.67

0.67

0.81

0.78

0.80

0.90

0.59

0.89

RF

Mfi

0.57

0.65

0.77

0.66

0.73

0.72 0.69

0.77

0.65

0.04

0.81

0.75

0.70

0.68

0.78

0.78

0.78

0.55

0.90

Value

M7

0.60

0.75

0.77

0.77

0.80

0.75 0.75

0.80

0.72

0.05

0.77

0.72

0.77

0.78

0.79

0.80

0.80

0.54

0.89

Ms

0.59

00.76

0.75

0.73

0.75

0.82 0.75

0.74

0.70

0.05

0.78

0.77

0.74

0.78

0.76

0.75

0.65

0.48

0.88

M9

0.56

0.80

0.74

0.77

0.75

0.82 0.82

0.75

0.80

0.07

0.76

0.70

0.79

0.78

0.75

0.75

0.76

0.48

0.85

U4

Table 4.3 Mobility of Amines as a Function of their Basicity'

RF Value

Mobile Phase AL DMAL o-CAL o-NAL m-CAL m-NAL p-CAL p-NAL

Mi 0.65 0.04 0.67 0.68 0.77 0.78 0.66 0.75

M, 0.72 0.05 0.75 0.78 0.77 0.79 0.77 0.80

Mg 0.70 0.05 0.76 0.78 0.75 0.76 0.73 0.75

M,o 0.60 0.03 0.64 0.66 0.72 0.80 0.62 0.72

Ml, 0.77 0.08 0.77 0.80 0.78 0.79 ' 0.75 0.78

M,2 0.78 0.07 0.81 0.82 0.79 0.79 0,79 0.80

Mi3 0.78 0.05 0.80 0.80 0.82 0.85 0.78 0.83

MM 0.67 0.07 0.75 0.76 0.76 0,78 075 0.80

M,6 0.75 0,05 0.76 0.78 0.77 0.80 080 0.87

M|7 0.73 0.12 0.74 0.76 0.75 0.79 0 76 0.79

Mi8 0.65 0.10 0.68 0.72 0.75 0.81 0.72 0.80

M,9 0.70 0.07 0.70 0.79 0.70 0.75 0.70 0.70

M20 0.75 0.05 0.76 0.78 0.80 0.80 0.77 0.81

M2, 0.75 0.07 0.76 0.82 0.77 0.78 0.76 0.87

^ Basicity (kb x lo'" value) of amines given in parenthesis are: AL (4.2), DMAL

(1L7), o-CAL (0.05), o-NAL (6 x 10"), m-CAL (0.3), m-NAL (0.03), p-CAL (LO) and

P'NAL (JO'). Data from (ref21)

115

dii-ia

' BVa-d

[ CD

- TVH-d

- IVN-m

TVN-o

aii-d

aii-m

aii-o

- Tma

TV

QNd-d

asv-aj

TVO-0

Vda

S

.«s ® • / ) 1-1

• ^ 1 F ^

00 1 ^

§s ss 1 1 t l

1 1

? .

^

• (ft

e E

o •**

o E

S o 08

a

'.Q

o E

o o

"oB

o a >>

o

^ i ^

•

4 u 3 bjD

116

Tma 2 s

1 <

«r> f s f s

u fj

I B o

B

B es

/'•'^ u CQ

CQ

z B 03

U e» Z

^ .

2 « ^ B ^ 'Z ^ S 0) CO

o o 9. >> w •*-

B =

V O

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s s :? s 0 H

B _ O

Tt a

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117

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25. A. Berthod, I. Girard and C. Gonnet, Anal. Chem., 58 (1986)

1362.

26. F. Feigl, Spot Test in Organic Analysis, Elsevier Scientific

Publishing, New York, 7*'' edn., (1966) 238.

27. F. Feigl, Spot Test in Organic Analysis, Elsevier Scientific

Publishing, New York, 1'^ edn., (1966) 382.

119

-V

5.1 INTRODUCTION

The separation of heavy metal ions has attracted considerable attention

in recent years because of their environmental importance in aqueous

media. Amongst separation techniques, thin layer chromatography (TLC)

is considered to be more application oriented because of its versatility,

simplicity, low cost and reasonable sensitivity. The separation of ions on

thin layers is mainly influenced by the ion-exchange properties of the

adsorbent and the coordinative properties of the solvent. As a general

practice the composition of mobile phase is usually altered to achieve a

desired separation on a particular adsorbent. From the literature of

inorganic TLC (1-5), various solvent systems including (a) inorganic

solvents (acids, bases, salt solutions mixtures of acids and bases or their

salts) prepared in distilled water or water-methanol mixtures, (b)

organic solvents (acids, bases, alcohols, aldehydes, ketones, esters,

phenols and their mixtures), (c) mixed aqueous-organic solvents (organic

solvents mixed with mineral acids, inorganic base, salt solutions or

water) and (d) complex forming solvents (EDTA, DMSO etc.) have been

identified as mobile phase are currently in use. Amongst these systems,

mixtures of organic solvents containing aqueous acid, base or buffer

have been considered most suitable for the separation of ionic species.

Surfactant-mediated mobile phases have received considerable

attention in liquid chromatographic separations of organic and inorganic

substances since the first report by Armstrong and Fendler (6) who

coupled favorable features of micellar systems with chromatographic

technique. The use of surfactant ions below their critical micelle

concentration (CMC) in ion pair chromatography (IPC) and above their

CMC value in micellar liquid chromatography (MLC) as mobile phase

components has been the focus of numerous studies (7-13). In both IPC

and MLC surfactant provides electrostatic sites of interaction and thus

influence the migration behaviour of charged species. IPC has been

considered as an alternative to ion-exchange chromatography and it

120

allows simultaneous separations of ionic and non-ionic compounds.

However, the use of aqueous surfactant phases may not always lead to

the desired separations and under such situations organic additives

provide better separations by modifying the retention behaviour of

solutes. (14,15)

Surfactant-mediated systems being inexpensive, non-toxic and non-

inflammable have been extensively used in reverse-phase liquid

chromatographic separations of biologically active organic compounds

(16-19). On the other hand, a few studies have been reported upon the

use of these systems as mobile phase in chromatographic separations of

metal cations (20-23). Micellar chromatography of inorganics has been

reviewed by Okada (24) and Manowski. (25)

The use of aqueous solutions of sodium dodecyl sulfate and Triton

X-100 as mobile phases to separate cobalt (III)-l-(2-pyridyl)-2-

naphthoi complexes on polyamide layer have also been reported .Our

recent studies . on TLC of anions (26) and amines (27) with

microemulsion eluent point to the necessity for further investigations

in order to fully explore the potential of surfactant-mediated mobile

phases in inorganic TLC. It is therefore, amenable to develop a

effective TLC system for achieving analytically important separations.

As a result, a reliable identification and fast separation of Zn^", Cd ,

Hg^" or/and Co^^ ions using cetyl trimethyl ammonium bromide

(CTAB)-ethanol-H20 mobile phase have been achieved. The

semiquantitative determination of Zn^" and Ni " has been attempted.

The proposed method has been successfully employed for

identification and separation of Hg^*, Cd ^ and Zn^" from the spiked

samples of river and industrial waste water. To demonstrate further

usefulness of the method, it has been applied for identification of Zn^",

Cd , Hg , Ni and Pb in synthetically prepared metal hydroxide

sludge in addition to the identification of Hg^^ Zn^^ and Cd^^ from

cinnabar (HgS), zinc blende (ZnS) and greenoekite (CdS) ores with

preliminary separation from their mixture.

121

5.2 EXPERIMENTAL

Chemicals and Reagents: Silica gel 'G', n-butanol and dimethyl

glyoxime were of [Qualigens, India] N-cetyl-N,N,N-trimethyl

ammonium bromide (CTAB), potassium ferrocyanide,

1,10-phenanthroline, methanol, ethanol and 2-propanol were of [CDH,

India]. All other chemicals were also of Analar Reagent grade.

Metal Ions Studied: Fe^\ Fe'*, Cu^\ Ni^\ Co^\ UOj^', VO^', Cd^\

Zn % Ag\ ?h^\ TV, B i ' \ Hg^ and Al'^

Test Solutions: Chromatography were performed on standard aqueous

solutions (1%) of the chloride, nitrate or sulfate salts of the above

mentioned metal ions except Fe " for which 1% ferrous ammonium

sulfate salt solution with 3 mL of H2SO4 was used.

Detection: ¥t^^ was detected with 0.5% solution of 1,10-

phenanthroline; Fe^^, Cu^\ V0^\ U02^* with 1% aqueous potassium

ferrocyanide; Ni^^ and Co^^ with 1% solution of alcoholic dimethyl

glyoxime; Zn^\ Cd^', Ag\ Pb^\ Tl', Bi ' ' and Hg^' with 0.5%

dithizone in CCI4 and A\^^ with 1% aqueous aluminon solution were

detected.

Stationary Phase: Silica gel 'G'

Mobile Phases:

Symbol Composition

Ml 0 . 3 3 % aqueous CTAB

M: ethanol + water (1:99)

M5 0.5 mL[CTAB + methanol, 1:2 w/v]* + 99.5 ml water

M4 1.0 mL[CTAB + methanol] + 99.0 mL water

M5 1.5 mL[CTAB + methanol] + 98.5 mL water

Mfi 0.5 mL[CTAB + ethanol] + 99.5 mL water

M7 1.0 mL[CTAB + ethanol] + 99.0 mL water

1.5 mL[CTAB + ethanol] + 98.5 mL water

5.0 mLlCTAB + ethanol] + 95.0 mL water

M,o 10.0 mL[CTAB + ethanol] + 90.0 mL water

122

M,

M9

Mn 15.0 mLlCTAB + ethanol] + 85.0 mL water

M,2 20.0 mLlCTAB + ethanol] + 80.0 mL water

Mij 0.5 mLlCTAB + propanol] + 99.5 mL water

M,4 1.0 mLjCTAB + propanol] + 99.0 mL water

M,5 1.5 mLlCTAB + propanol] + 98.5 mL water

Mi6 0.5 mLlCTAB + butanol] + 99.5 mL water

Mi7 1.0 mL(CTAB + butanol] + 99.0 mL water

M,8 1.5 mL[CTAB + butanol] + 98.5 mL water

M,9 1.0 mLlCTAB + ethanol] + 99.0 mL aq. NaCl (0.01-0.2M)

M20 1.0 mLlCTAB + ethanol] + 99.0 mL aq. HCl (0.01-0.2M)

M2, 1.0 mLlCTAB + ethanol] + 99.0 mL aq. MgCl. (0.01-0.2M)

M22 1.0 mLlCTAB + ethanol] + 99.0 mL aq. CaCh (0.01-0.2M)

M23 1.0 mLlCTAB + ethanol] + 99.0 mL aq. LiCl (0.01-0.2M)

M24 1.0 mLlCTAB + ethanol] + 99.0 mL aq. KCl (0.01-0.2M)

M25 1.0 mLlCTAB + ethanol] + 99.0 mL aq. NaBr (0.0I-0.2M)

M26 1.0 mLlCTAB + ethanol] + 99.0 mL aq. urea (O.IM)

M27 1.0 mLlCTAB + ethanol] + 99.0 mL aq. KBr (O.IM)

M28 1.0 mLlCTAB + ethanol] + 99.0 mL aq. NaNOj (O.IM)

M29 1.0 mLlCTAB + ethanol] + 99.0 mL aq. oxalic acid (O.IM)

M30 10 mLlCTAB + ethanol] + 99.0 mL aq. tartaric acid (O.IM)

M31 1.0 niLlCTAB + ethanol] + 99.0 mL aq. KOH (O.IM)

M32 1.0 mLlCTAB + ethanol] + 99.0 mL aq. NaOH (O.IM)

M33 1.0 mLlCTAB + DMF] + 99.0 mL water

M34 1.0 mLlCTAB + DMSO] + 99.0 mL water

M35 1.0 mLlCTAB + acetone] + 99.0 mL water

* CTAB + organic solvents were taken as 1:2, w/v in all cases. Salt solutions were prepared in distilled water.

(a) Preparation of Plain TLC Plates: As described in chapter 2.

(b) Preparation of Spiked, Industrial Wastewater and River Water: A

50 mL volume of industrial wastewater (pH 2.98) collected from lock

industries, Aligarh, India or river water (pH 7.48) obtained from

Ganga river at Naraura, India was spiked with lOO ig each of Zn, Cd

123

and Hg salts. About 30 niL of 0.5% thioacetamidc solution was added

into the spiked sample. The resultant precipitate of Zn, Cd and Hg

sulfides was washed with distilled water, centrifuged and dissolved in

minimum possible volume of concentrated HCl. The acid was

completely removed by evaporation and the residue was dissolved in

5mL of distilled water. An aliquot (lOîL) of each sample was applied

on TLC plate and chromatography was performed as done for the

standard samples.

(c) Preparation of Synthetic Ores: Cinnabar (HgS), zinc blende (ZnS)

and greenoekite (CdS) were synthetically prepared by spiking 50 mL

of distilled water (pH 5.6) with Zn, Cd and Hg salt solutions following

the same procedure as described above in (b).

(d) Preparation of Heavy Metal Hydroxide Sludge: Synthetic heavy

metal sludge of Zn, Cd, Hg, Pb and Ni were prepared by adding

sufficient volume of 1% NaOH solution into a mixture containing 1%

solution of these metal salts in equal volumes. The metal hydroxide

precipitate so obtained was filtered, dried and dissolved in minimum

volume of concentrated hydrochloric acid. The acid was completely

evaporated, the residue was dissolved in 5 mL of distilled water and

TLC was performed using 10|iL sample.


metal ions as described in chapter 2 for amino acids.

For the separation, equal amounts of metal ions to be separated

were mixed and lOjiL of the resultant mixture was loaded on the TLC

plate. The plates were developed, the spots were detected and the Rp

values of the separated metal ions were determined.


method, 0.01 mL from a series of standard solutions of Ni ^ & Zn^^

(0.05-0.20%) were spotted on silica layers. The plates were developed

with M7. After detection the spots were copied onto tracing paper from

the chromatoplates and then the area of each spot was calculated.

124

Semiquantitative Determination by Visual Comparison: Aliquot (0.01

mL) of standard solutions of different concentrations (0.2-1%) of

nickel chloride were spotted on TLC plates alongwith the spotting of

0.01 mL of industrial wastewater sample. After completing the

chromatographic process, the colour intensity and the RF value of

analyzed industrial wastewater sample were matched with the coloured

spots of standard reference solutions of nickel chloride. The amount of

nickel present in the industrial sample was determined according to

colour intensity of its spot on TLC plate after visual comparison with

the colour intensities of standard solutions.


Mobility of 15 transition metal ions was determined on silica gel layer

using surfactant mediated mobile phases. In order to examine the

effect of surfactant, the mobility of metal ions were determined using

(a) aqueous CTAB (0.33%), (b) ethanolic-aqueous CTAB (0.33 and

0.5%) and hydro-organic solvent (ethanoNwater mixture) as mobile

phases. The results are compared in Figure 5.1. It is evident from this

figure that aqueous CTAB and hydro-organic solvent systems behave

in almost identical manner to influence the mobility of all metal ions.

However, Hg^ shows much faster mobility in aqueous CTAB.

Interestingly, all metal ions show differential migrational tendency

causing enhanced separation possibilities with ethanolic-aqueous

CTAB as mobile phase. Thus, the presence of ethanol in aqueous

CTAB modifies the mobility trend of metal ions on silica gel layer.

These results clearly demonstrate that the effectiveness of hydro-

organic solvent systems (e.g. ethanol-water mixture) can be improved

by the addition of CTAB, a cationic surfactant. From figure 5.1, the

separation of Hg " from Cd^ , Zn^^ and Co^^ is possible with ethanolic-

aqueous CTAB mobile phase whereas this separation can not be

achieved with ethanol-water (zero CTAB concentration) solvent

system. The increase in the degree of ionization of a cationic

surfactant upon addition of alcohols has been reported by Zana (28).

125

Therefore the enhanced ionization of CTAB in the presence of organic

modifiers results in improved separations of metal ions. The

concentration of CTAB has been taken as 0.33% in mobile phase M7

which is below its CMC value (0.46%) and hence the surfactant in M7

is present as cationic monomer which can be considered simply as a

strong electrolyte. On increasing surfactant concentration to 0.5%

(Mg), just above its CMC value where the surfactant monomers are

expected to be in equilibrium with CTAB micelles, the mobility of

metal ions either remains the same or decreases slightly compared to

their mobility in M7 ( i.e. CTAB, 0.33%). The highest mobility of Hg^"

in M7 facilitates its clear separation from almost all other metal ions.

Separation of mixtures of Zn^*, Cd " , and Hg^^ ions is

analytically very important because of their similar physical and

chemical properties. Zinc(-d^°,4s^), cadmium(-d'^5s^) and mercury

(-d^°,6s^) with the electronic configuration (n-1) d'%s^ belongs to the

same group (IIB) of the periodic table and Cd is generally associated

with Zn-minerals, for example zinc blende ore contains about 3% Cd.

On the other hand, mixtures of zinc, cobalt and cadmium compounds

are of technical importance and frequently one metal contains small

quantities of the others, which has a deleterious effect on performance.

Separation of Cd from Zn bears biochemical importance also as in

some metalloenzymes Zn^^ is substituted by Cd^^ leading to cadmium

toxicity.

From literature (29), organic solvent additives have been the

choice of chemists and physicist for modifying the properties of

micellar mobile phase in order to get better efficiency. Keeping this in

mind we replaced ethanol by other protic as well as aprotic organic

solvents (methanol, 2-propanol, n-butanol, acetone, DMF, DMSO) in

the CTAB mobile phase and the mobility of all metal ions were

determined. The development time for 10 cm ascent was 20-22 min.

for all mobile phases except methanol, for which the development time

was 35 min. All metal ions were clearly detected as compact spots in

TDA

all mobile phases. With' these mobile phases (M^-Mg, Mi.i-Mig and

M33-M35) following trends in regards to the mobility of metal ions

were noticed.

(a). Fe - Fe^\ Cu^\ Zr^^\ Ag\ W\ B i ' \ Al^\ \}Q^^ and VO^

show little mobili^ and remained near the point of application

(RF < 0.05).

(b). Cd ^ and Tl* show mid Rp value (Rp 0.4-0.6) and hence can be

separated from all metal ions having higher and lower Rp values.

(c). Hg^ goes with the solvent front showing high Rp value (Rp =

0.9-0.98)

(d). Ni " and Co^* show variable mobility, the Rp values given in

parenthesis for Ni^" and Co^' in M3(0.70,0.85), M4(0.95,0.75),

M5(0.82,0.70), M6(0.57,0.80), M7(0.80,0.72), M8(0.60,0.60),

M,3(0.82,0.70), M,4(0.62,0.65), M,5(0.72,0.58), Mi6(0.62,0.65),

Mi7(0.72,0.60),Mi8(G.60,0.57), M33(0.47,0.55), M34 (0.60,0.45)

and M35 (0.65,0.35) respectively were observed.

From above presented results, it is clear that surfactant

containing mobile phases with added organic modifiers can be

successfully exploited for achieving novel separations.

The detection of spots was sharper in the case of alcohols

compared to acetone, DMSO and DMF. Contrary to the present case,

acetone having the tendency to suppress hydrolysis of metal ions and

DMSO (aprotic dipolar solvent), a stronger solvating agent for cations

in preference to anions have been found most suitable for TLC analysis

of metal ions with non-surfactant mediated acidic mobile

phases(30,31). Thus clearer detection of metal ions in surfactant-

mediated system can be obtained using ethanol or propanol (proton

donor) compared to acetone (proton acceptor) as one of the

components of mobile phase.

127

Amongst alcohols (called as co-surfactants), ethanol and propanol

were found more useful compared to methanol and butanol for

achieving separations from multicomponent mixtures of metal ions.

Though Zn^^- Cd^*- Hg^^- Co^" separation can be realized in the

presence of any one of the alcohols but more compact spots were

noticed with ethanol or propanol (Figure 5.2). Thus in our case both

ethanol and propanol show the identical efficiency. These alcohols

improve the separation possibilities by reducing the binding of cationic

surfactant (CTAB with positive charge residing on the quaternary

nitrogen atom) on the porous silica surface. The silanol groups (=Si-

OH) of silica gel being weakly acidic (32) provides the opportunity to

silica gel to acquire negative charge in contact with water and thus the

hydrated silica gel captures the positively charged species. Therefore,

mobile phases M7 and M14 containing ethanol and propanol were used

for detailed study. With M7, a linear relationship between Rp value and

ionic potential (ionic potential = charge of ion / ionic radius) exists for

Zn^\ Cd^\ Hg^" and Ni^", Co^', Bi^" in (Figure 5.3). It show that the

mobility of these metal ions is also influenced by their ionic radii.

In order to widen the applicability of CTAB containing systems

as mobile phase, distilled water in M7 was substituted by 0.01-0.2 M

NaCl (saline water), 0.01-0.2 M HCl, 0.1 M oxalic or tartaric acid

(acidic water), 0.01-0.2 M chlorides of calcium, magnesium, lithium or

potassium, 0.01-0.2M bromide and O.IM nitrate of sodium (salt

solutions), 0.1 M NaOH or KOH (alkaline water) and 0.1 M urea

solution. The effect of these added impurities was investigate on Zn " -

Cd^* - Hg^* separation. From the results presented in Table 5.1, it is

evident that these impurities have a detrimental effect on the

separation, by influencing water-surfactant interactions, because these

ionic additives interact with the surfactant molecules. The separation

of Cd^^(RF=0.48) from Zn^'(RF=0.30) deteriorates in the presence of

0.2 M HCl, and could not be achieved because of the increase in

mobility of Zn^^. The separation of Hg ^ from Zn " is however, always

128

possible but its separation from Cd ^ could not be realized in the

presence of complexing carboxylic acids (oxalic or tartaric acid). Urea,

a water structure breaker does not hurt Zn^^ - Cd ^ - Hg * separation.

The aqueous solution of KSCN and Kl (O.IM) produce white

precipitate on addition of 1 mL of ethanolic-aqueous CTAB solution

and hence the effect of I' and SCN" on the separation of Zn ^ from Cd^*

and Hg " could not be examined. The results of Table 5.1 also indicate

that the anions (Cr, NO3") and cations (U\ Na \ K\ C&\ Mg^') do not

hamper a three-component, Zn ^ - Cd " - Hg^* separation over the

entire concentration range of the added impurities. Ca " and K" are the

exceptions which influence this separation adversely by increasing the

mobility (lower k ) of Cd^^ at 0.2M concentration. The variation in k

value of Cd^^ in the presence of different concentrations of inorganic

cationic and anionic impurities is summarized in (Figure 5.4). The k

values for Hg and Zn remain almost unchanged showing only minor

effect on their mobility due the presence of inorganic ionic impurities

at all concentration levels. The minor changes in the Rp value of Zn^^,

Cd^* and Hg " without effecting the separation in the presence of

impurities may be attributed to comptetitive adsorptive interactions

among positively charged species (alkali and alkaline earth metal ions)

and surfactant cation of the mobile phase with the negatively charged

silica gel surface. The interaction between silica gel and alkali/alkaline

earth metal ions in aqueous mobile phases have been reported earlier

(33,34). Following cation-exchange reaction takes place when silica

gel comes in contact of an electrolyte

sSi-OH' + Na' ;?=i =Si-ONa" + H^

In spite of the fact that both ethanol and propanol work in

identical fashion to resolve the mixture of metal ions, the sensitivity is

better in the case of propanol (M14) compared to ethanol (M7) as evident

from Table 5.2. The minimum possible amount detectable is 2 to 3

times lower in the case of propanol. Alternatively, better sensitivity can

129

also be obtained by substituting laboratory made silica gel plates with

commercially available HPTLC plates. Therefore, it is concluded from

present study that for better sensitivity (or lower detection limit) either

propanol may be preferred over ethanol or HPTLC plates may be used

instead of laboratory made silica gel layers.

In addition to the qualitative analysis a quantitative evaluation of

the metal ions is often required to ascertain the level of toxic metals in

environmental samples. A relatively less accurate but the simplest

method for quantitation is based on the measurement of the size of the

spot by drawing the outline of the spot on a piece of tracing paper.

Therefore, an attempt was made to achieve semiquantitative

determination of metal ions by measuring the spot area. A linear

relation obtained when the amount of the sample spotted was plotted

against the area of the spot (Figure 5.5) follows the empirical equation

^^ = km, here <| is the area of the spot, m is the amount of the solute

and k is a constant. The linearity is maintained up to 200 \xg I spot of

Ni^^ or Zn^ . At higher concentration, a negative deviation from linear

law in both cases was observed. The standard curve constructed for

semiquantitative determination of Ni^^ was used to find out the amount

of nickel present in water samples. The accuracy and precision were

below ± 15%. The results of semiquantitative determination by visual

comparison method were applied for estimating the nickel present in

industrial wastewater. The analysed industrial wastewater samples were

found to contain nickel content in the range 3.5-4 g/L.

Applications:

The proposed method was applied for identification and separation of

heavy metal ions in spiked river and industrial wastewater samples, in

synthetic metal hydroxide sludge and in metal sulfide ore samples. The

results listed in Table 5.3 clearly demonstrate the applicability of the

method for identification of Zn, Cd, Hg, Ni and Pb as well as the

mutual separation of Hg from Cd and Zn present in a variety of

130

environmental and geological samples. These results of the Table 5.3

clearly indicate the applicability of the method for detection and

separation of Hg in cinnabar (HgS), Cd in greenoekite (CdS) and Zn in

zinc blende (ZnS).

131

Table 5.1 Separation of Mixtures of Zn % Cd^* and Hg * Ions on Silica Gel G Developed with Different CTAB-Containing Mobile Phase Mobile Phase

Svmbol Composition

z«'* 0.05

0.03

0.02

0.02

0.02

0.15

0.22

0..30

0.05

0.02

0.03

0.05

0.0.5

0.05

0.05

0.04

0.05

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.05

0.10

0.52

0.58

0.05

0.05

Cd *

0.22

0.34

0.56

0.68

0.25

0.40

0.43

0.48

0.32

0.37

0.50

0.87

0.29

0.37

0.47

0.57

0.32

0.50

0.67

0.76

0.16

0.35

0.55

0.70

0.48

0.42

0.92

0.90

0.25

0.25

Hg ^

0.92

0.95

0.98

0.98

0.82

0.92

0.95

0.95

0.87

0.92

0.87

0.90

0.8,5

0.95

0.97

0.95

0.76

0.90

0.95

0.92

0.95

0.97

0.95

0.97

0.97

0.92

0.95

0.95

0.81

0.85

M,,

M„

Mr

M,

\h

M„

M,«

M,,

Mxo

M.M

M„

l.OmL (CTAB - ethanoj, 1:2 w/v)" + 99.0 mL 0.0IM aq NaCI

l.OmL (CT.\B - ethanol) + 99.0 mL 0.05M aq. NaCl

l.OmL (CT.'^ * ethanol) + 99.0 mL O.IM aq. NaCl

1 OmL (CTAB ^ ethanol) + 99.0 mL 0. 2M aq. NaCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. OlM aq. HCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. 05M aq. HCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. HCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. 2M aq. HCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. OlM aq. CaClz*

l.OmL (CTAB + ethanol) + 99.0 mL 0. 05M aq. CaCl,

l.OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. CaClj

l.OmL (CTAB + ethanol) + 99.0 mL 0. 2M aq. CaClj

l.OmL (CTAB + ethanol) + 99.0 mL 0. OlM aq. LiCI

l.OmL (CTAB + ethanol) + 99.0 mL 0.05M aq. LiCl

1 .OmL (CTAB + ethanol) + 99.0 mL 0.1M aq. LiCl

1 .OmL (CTAB + ethanol) + 99.0 mL 0. 2M aq. LiCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. OlM aq. KCl

l.OmL (CTAB + ethanol) + 99.0 mL 0.05M aq. KCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. KCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. 02M aq. KCl

l.OmL (CTAB + ethanol) + 99.0 mL 0. OlM aq. NaBr

l.OmL (CTAB + ethanol) + 99.0 mL 0. 05M aq. NaBr

l.OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. NaBr

l.OmL (CTAB + ethanol) + 99.0 mL 0. 2M aq. NaBr

l.OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. urea

l.OmL (CTAB + ethanol)+ 99.0 mL 0. IM aq. NaNO,

l.OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. oxalic acid

1 .OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. tartaric acid

l.OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. KOH

l.OmL (CTAB + ethanol) + 99.0 mL 0. IM aq. NaOH

^'The ratio of CTAB to organic solvent was always 1:2, w/v.

^'Sali solutions were prepared in distilled water.

Table 5.2 Limit of Detection of Metal Ions on Silica Gel G Developed with CTAB-Ethanol-HzO (M-) and CTAB-Propanol-HzO (M14) Mobile Phases

Metal Ion Limit of Detection (fig)

M7 M,4

Zn * 0.024(0.014)"^ 0.013

Cd * 0.041 (0.016) 0.016

Hg^' 0.033 (0.014) 0.014

Bi'" 0.028 0.014

Pb^' 0.030 0.013

"^The values in parenthesis refer to the lower limit of detection on

HPTC plates (silica gel 6OF254: Merck, Darmstadt, Germany).

Table 5.3 Separation of Mixtures of Zn % Cd^^ and Hg^^ Ions from Spiked Water and Synthetically Prepared Metal Ores and Heavy Metal Sludge Samples

Spiked/Synthetic Samples Ri

Zn^*

0.02

0.05

0.05

0.02

0.05

Cd '"

0.37

0.35

0.46

0.34

0.43

Hg^^

0.90

0.91

0.92

0.96

0.95

River water

Industrial wastewater

Sulfides

Sludge "

Distilled water

c)\Ti2+ / D _ n c/11 J DA^ + Ni-" (Rp = 0.50) andPb'^ (Rp = 0.02) were also detected.

133

1 -1

0.8

0.6 J

0.4

0.2 -

0 ^

/o - -a

" * 4 A

LI »A

tr^—^^^^"^"^ i ffi* Fe^ Cu Ni Co UO VO Cd Zn Ag Pb Tl Bi Hg Al

Metal ions

Ml M2 M7 o- M8

Figure 5.1 Mobility of metal ions on silica gel G developed with different mobile phase

1

0.8

0.6

0.4

0.2

0

Methanol Ethanol Propanol Alcohols

Butanol

Zn A Cd Hg ^—Co

Figure 5.2 Mobility of Zn, Cd, Co and Hg on silica gel G developed with mobile phases M4, M7, M14 and Ml7 containing alcohols of different chain length

134

1.0

0.9.

0.8-

0.7-

0.6

at 0.5-

0.4-

0.3-

0.2-

0.1

0.0

1.0

• Ni»*

Cd'"

• I

2.0 3.0 4.0 5.0

IONIC POTENTIAL

Figure 5.3 Plot of ionic potential against RF

135

0.01

o-

0.05 0.1 Molar concentration of salt solutions

a NaCl -A-NaBr LiCI

0.2

KCI

6 5 4

2

1

0

0.01 0.05 0.1 Molar concentration of salt solutions

0.2

CaCh A- MgCb

Figure 5.4 Mobility of Cd ^ on silica gel G developed with CTAB-ethanol-aqueous mobile phases containing different concentrations of salts of alkali and alkaline earth metals.

136

0 50 100

Amount of Ni * (jig)

150 200

0 SO 100

.2+ Amount of Zn (^g)

150 20O

Figure 5.5 Dependence of spot area, ^, on amount of (a) Ni and (b) Zn 2+

137

REFERNCES

1. U. A. Th. Brinkman, G. De Vries and R. Kuroda, J. Chromaiogr.,

85(1973) ]87.

2. R. Kuroda and M. P. Volynets, CRC Handbook of

Chromatography: Inorganics, Vol. 1 (M. Quareshi, eds), CRC

Press, Boca Roton, Gla, (1987) 89.

3. A. Mohammad and K. G. Varshney, Handbook of Thin Layer

Chromatography, (Joseph Sherma and Bernard Fried, eds). Marcel

Dekker, Inc, 71 (1996)507.

4. A. Mohammad, M. Ajmal, S. Anwar and E. Iraqi, J. of Planar


5. J. Sherma, Anal. Chem., 70 (1998) 7R-26R.

6. D. W. Armstrong and J. H. Fendler, Biochem. Biophys. Acta, 75

(1977)478.

7. M. F, Borgerding, R. W. Williams, W. L. Hinze and F. H. Quina, J.

Liq. Chromatogr, 12 (1989) 1367.

8. A. S. Kord and M. G. Khaledi, Anal. Chem., 64 (1992) 1901.

9. M. G. Khaledi, J. K. Strasters, A. H. Rodgers and E. D. Breyer,

Anal. Chem., 62 (1990) 130.

10. A. Berthod, / Chromatogr., 191 (1997) 780.

11. M. G. Khaledi, J. Chromatogr, 3 (1997) 780.

12. A. Szymanski and W. Szczepaniak, Chem. Anal. (Warsaw), 43

(1998)617.

13. T. A. Walker, J. Liq. Chromatogr., 19 (1996) 1715.

14. J. S. Landy and J. G. Dorsey, Anal. Chem. Acta., 178 (1985) 179.

15. A. Berthod, I. Girad and C. Gonnet, Anal. Chem., 58 (1986) 1362.

16. L. J. Cline-Love, J. G. Habarta and J. G. Dorsey., Anal. Chem., 56

(1984) 1132A.

17. M. G. Khaledi, Trends Anal. Chem., 7 (1988) 293.

138

18. M. Amin, K. Harrington and R. Von Wandruszka. Anal. Chem.

(Warsaw), 65 (1996) 2346.

19. R. W. Williams, Jr., Z. Fu and W. L. Hinze, J. Chromatogr. Sci.,

28(1990)292.

20. T. Okada, Anal. Chem., 60 (1988) 2116.

21. T. Okada, Anal. Chem., 64 (1992) 5899.

22. A. Berthod, X. Jun, A Serge and C. Gonnet-Collet, Can. J. Chem.,

74(1996)227.

23. T. Okada, J. Chromatogr., 607 (1992) 135.

24. T. Okada, J. Chromatogr. A., 343 (1997) 780.

25. J. Manowski, Chem. Abstr., 122 (1991) 219028m.

26. A. Mohammad, S. Tiwari, J. P. S. Chahar and S. Kumar, J. Am.

Oil. Chem. Soc, 72 (1995) 1533.

27. A. Mohammad and E. Iraqi, J. of Surfactant and Detergents., 2

(1999)85.

28. R. Zana, in D. O. Shah (ed.), Surface Phenomena in Enhanced Oil

Recovery, Plenum, New York, 1980.

29. M. F. Borgerding and W. L. Hinze, Anal. Chem., 57 (1985) 2183.

30. A. Mohammad andN. Fatima, Chromatographia., 22 (1986) 109.

31. M. Qureshi, K. G. Varshney and N. Fatima, Sepn. Sci. TechnoL, 13

(1978)321.

32. D. L. Dugger, J. H. Stanton, B. N. Irby, B. L. Mcconnell, W. W.

Commings and R. W. Maatman, J. Phys. Chem., 68 (1964) 757

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211.

34 R. K. Ray and G. B. Kauffman, J. Chromatogr., 464 (1990) 504.

B 9

-VI

^nAm^ced Sêfux/UjUicn SPelecHmlu <)£

6.1 INTRODUCTION

Thin layer chromatography (TLC) is more efficient than column and

paper chromatography for the separation of metal ions because it gives

faster and more accurate results. Some interesting applications of TLC,

such as identification of total heavy metals in industrial wastewaters

(1), transition metals in seawater and wastewater (2), polyphosphates

in soft drinks (3), recovery of thiocynate from photogenic wastewater

(4) and determination of Ag (I) from synthetically prepared horn silver

(5) have demonstrated its utility as an effective and rapid analytical

technique.

Various solvent systems (6-8) and chromatographic techniques

have been used for the separation of coexisting transition metal

cations. Most of the reported studies involve the use of non-micellar

solvent systems e.g. mineral or carboxylic acids, alkali salt solutions,

lower aliphatic alcohols and ketones as mobile phases. On the

contrary, the use of micellar mobile phases in inorganic TLC has been

limited (9-12). Because of their unique physico - chemical properties,

micellar solutions have much to offer to the chemists concerned with

synthesis, reaction mechanism, photochemical process, chemical

analysis and petroleum exploration. It is therefore, not surprising that

interest in micellar systems is growing and these systems have

experienced a dramatic surge in recent years due to their non-toxic

effect, low cost and capability to solubilize the reagents which are

sparingly soluble in water.

Separation of heavy metal cations by micellar exclusion

chromatography involving use of micellar mobile phase and a size

exclusion column (13), reversed phase column chromatography with

SDS micellar mobile phases (14) and normal - phase TLC with Brij-35

eluents (11) have been attempted. As far as we are aware, no work is

reported on the separation of coexisting Zn^*, Ni^^, Hg^^ and Mn ^ or

Cd by micellar TLC. The present communication reports a reliable

140

micellar TLC method for quick separation and identification of

coexisting Zn^% Ni^\ Hg^* and Cd^' or Mn^^ on silica layers using

aqueous solutions of N-cetyl-N,N,N-trimethyl ammonium bromide

(CTAB) as mobile phase. In addition to semiquantitative determination

of Ni^\ the proposed method was applied for separation and detection

of Zn, Cd, Hg, Ni and Mn from drag out nickel plating solution as well

as from their hydroxide sludge sample.

6.2 EXPERIMENTAL

Chemicals and Reagents: Silica gel 'G', and dimethyl glyoxime were

of Qualigens, India; N-Cetyl-N,N,N-trimethyl ammonium bromide

(CTAB), potassium ferrocyanide and 1,10-phenanthroline were of

CDH, India. All other chemicals were also of Analar Reagent grade.

Test Solutions: Standard aqueous solutions (1%) of the chloride,

nitrate or sulfate salts of Fe^\ Cu^\ Ni^', Co^\ CT^\ W0^\ Cd^\ Zn^\

Mn^ , and Hg^^ were used. Fe^^ was taken as 1% ferrous ammonium

sulfate salt solutions containing 3 mL of H2SO4.

Buffer Solutions:

S.No Composition Volume Ratio pH

1 0.04 M Boric acid - 0.04 M phosphoric 50 : 50 : 8 3.4

acid - 0.24 M NaOH


acid - 0.24 M NaOH


acid - 0.24 M NaOH

Detection: Conventional Chromogenic reagents as mentioned in

chapter 5 were used for detection purposes of metal ions. Mn^* was

detected with 2M sodium hydroxide mixed with 30% H2O2 in 1:1 ratio.

Stationary Phase: Silica gel 'G'

141

Mobile Phases: The following solvent systems were used as mobile

phase

Symbol Composition

M, H:0

M; 0.1 -300 mM CTAB in H:0

M, Borate - phosphate buffered 50 mM aqueous CTAB (pH 11.9. 7.0 and 3.4)

M:i Aqueous urea solution (0.05 - 0.5 M) containing 50 mM of CTAB

M5 Aqueous thiourea solution (0.05-0.5 M) containing 50 mM of CTAB

Ms Aqueous NaCl solution (0.05 - 0.5 M) containing 50 mM of CTAB

M, Aqueous KCl solution (0.05 - 0.5 M) containing 50 mM of CTAB

Mg Aqueous NaBr solution (0.05 - 0.5 M) containing 50 mM of CTAB

Ms Aqueous KBr solution (0.05 - 0.5 M) containing 50 mM of CTAB

(a) Preparation of Plain TLC Plates: TLC plates were prepared as

described in chapter 2

(b) Preparation of Spiked Drag out Nickel Plating Solution: A 50 mL

volume of nickel plating solution collected from Progressive Platers,

Basaverswaranagas, Banglore, India was spiked with lOOng each of

Zn, Cd, Ni, Mn and Hg salts. About 30 mL of 0.5% thioacetamide

solution was added into the spiked sample. The resultant precipitate of

Zn, Cd, Ni, Mn and Hg sulfides was washed with distilled water,

centrifuged and dissolved in minimum possible volume of concentrated

HCl. The acid was completely removed by evaporation and the residue

was dissolved in 5 mL of distilled water. An aliquot (lOjaL) was

applied on TLC plate and chromatography was performed as for the

standard samples.

(c) Preparation of Heavy Metal Hydroxide Sludge: Synthetic heavy

metal sludge of Zn, Cd, Mn, Hg, and Ni was prepared by adding

sufficient volume of 1% NaOH solution into a mixture containing 1%

solution of these metal salts in equal volumes. The metal hydroxide

precipitate so obtained was filtered, dried and dissolved in minimum

142

volume of concentrated hydrochloric acid. The acid was completely

evaporated the residue was dissolved in 5 mL of distilled water and

TLC was performed using lO iL sample.

Procedure: The chromatography of heavy metal cations was performed

following the procedure as described for amino acids in chapter 2.

For the separation, equal amounts of metal ions to be separated

were mixed and lO iL of the resultant mixture was spotted on the

activated TLC plate which was then dried in air. The plates were

developed to a distance of 10 cm, the spots were detected and the

separated metal cations were identified on the basis of Rp values.


method, 0.01 mL from a series of standard solutions of Ni (50 - 200

Hg) were spotted on silica layers. The plates were developed with 50

mM aqueous CTAB and dried in air. After detection, the spots were

copied onto tracing paper from the chromatoplates and then the area of

each spot was calculated.


This study presents a novel chromatographic system for the analysis of

heavy metal cations. The important features include (a) the use of

commercially available silica gel as stationary phase in combination

with micellar mobile phases which are capable of dynamic

modification to silica gel surface, (b) application of a cationic

surfactant (CTAB) for the separation of cationic species and (c) the

examination of effects of pH of micellar media, presence of chloride

ion and nonionic organic compound (urea) in the mobile phase (or

micelles) on the mobility (or retention) of metallic cations.

The literature survey (1974 - 1999) on TLC of metal ions

reveals, the following pattern on the use of different materials as

stationary phase : silica gel > cellulose > inorganic ion exchangers

>alumina > chitin and chitosan > diatomite soil. For the analysis of

143

metal complexes and inorganic anions, silica gel has also been the

most preferred layer material. The selective adsorptive power of silica

gel has proved to be one of the essential qualifications for making it a

useful support material in the area of TLC of inorganics and

organometallics. On the other hand, the surfactant micelles have been

used to remove cationic or anionic multivalent ions from water. The

micelles are highly charged and the ions of opposite charge (i.e.

counterions) to the surfactant ( cationic to anionic surfactant and vice

versa) are easily adsorbed onto the surface of micelles via electrostatic

attractive forces. This approach has been common in practice.

However, deviating from the generally acceptable practice, we have

used cationic (CTAB) surfactant micelles to analyse cationic species

(i.e. metal cations). Another interesting aspect of this study is that

silica gel (the stationary phase) also contains on its surface localized

positive charges, originating from protons of silanol groups ( =Si - O -

r). Thus, in the present investigation, we are dealing with a system

where stationary phase, mobile phase and the analyte are all positively

charged species. It is assumed that during the chromatography of

cations with aqueous micellar solution certain processes such as (i) the

cation exchange between analyte and the proton of silanol groups, (ii)

the analyte - micelles interaction and (iii) the adsorption of surfactant

to silica gel surface are operative which influence the mobility of

metal cations in a peculiar manner to provide novel separation pattern.

The results obtained have been summarized in Tables 6.1- 6.5 and

Figure 6.1. From the data listed in Table 6.1, following conclusions

are drawn:

(a) In pure water (zero surfactant), Hg^*, Zn^*, Mn^ and Cd " can

not be separated from their mixtures.

(b) At low surfactant concentration (0.1 - 1.0 mM) in the mobile

phase i.e. below or near CMC of the surfactant (CMC of CTAB,

144

0.92 mM,) mutual separation of metal ions is hampered due to

the formation of diffused (or tailed, RL - RT > 0-3) spots for

N i ' , Co^\ CdÛnd Hg^\

(c) At higher surfactant concentration (50 - 300 mM) i.e. much

above the CMC value, excellent separation possibilities of metal

ions from their multicomponent mixtures are arised as a result of

modified interactions of metal cations with the mobile phase or

the micelles. For example mutual separations such as Hg " -

Mn^" - Zn^^ - Ni^' and Hg^ - Cd^' - Zn^' - Ni^' are always

possible with eluents containing surfactant concentration above

50 mM.

Taking into consideration the shorter development time and

sharp detection of spots, we used mobile phase consisting of 50 mM

CTAB to achieve separation of coexisting Hg " , Zn " , Cd^^ or Mn "

and Ni ions. The order of mobility (Rp value) was Hg^' > Ni^' > Cd^'

or Mn " > Zn " . This sequence shows that Hg " is partitioned to

micelles and easily eluted by the mobile phase whereas Zn " is

strongly attached with the stationary phase and requires stronger

mobile phase for elution. Ni^^, Cd " and Mn " show intermediate

mobilities.

The Rp value of metal ions, when chromatographed as mixture

differs to a magnitude of about ± 0.05 from their individual Rp value

obtained on being chromatographed alone. The separation of Hg^ ,

Cd ^ or Mn " , Zn " and Ni " from their mixture is a happy consequence

because of the following important features.

(a) Zinc (soZn), cadmium (48Cd) and mercury (goHg) constitute II B

group of the periodic table and Zn has strong chemical

similarities to Cd.

(b) Mn( 3d^ 4s^), Ni (3d\ 4s^) and Zn (3d'°, 4s^) belong to first

transition series of the same period of the periodic table.

145

(c) Easy separation of Mn (paramagnetic), Zn (diamagnetic) and Ni

(ferromagnetic) along with the separation of Cd (diamagnetic)

from diamagnetic Zn.

(d) Separation of Zn^* from Cd^' is biochemically important as in

some metalloenzymes it is substituted by Cd resulting m

cadmium toxicity.

(e) Cd is generally associated with Zn in several zinc ores.

An important feature of this study is that the proposed method

can be used for mutual separation of metal ions over a wider pH range

without hampering the separation efficiency. We have successfully

separated Hg " from Zn " , Cd " or Mn^* and Ni^^ is acidic (pH, 3.4),

neutral (pH, 7.0) and alkaline (pH, 11.9) pH range of the mobile phase

(Table 6.2). The differences between Rp values and compactness of the

separated chromatographic zones were almost the same at all pH

levels.

The microenvironment of micellar system is greatly influenced

by the presence of added organic molecular substance and inorganic

electrolytes. To examine this aspect, the mixtures of Hg^ , Zn ^ Cd ^

or Mn " and Ni^" were chromatographed using aqueous CTAB at (50

mM concentration level and) containing 0.05 - 0.5 moles of urea,

thiourea, NaCl, KCl, NaBr or KBr as mobile phase. From data listed in

Table 6.3 following trends are noticeable:

(i) No change in the mobility of Zn^^ (Rp « 0.05)

(ii) With the increase in concentration of additives, Rp values of

Cd^ (except thiourea), Ni ^ and Mn " are increased this increase

in Rp value of these metal ions from their synthetic mixtures at

higher concentrations (0.2 or 0.5 M) of additives.

(iii) The mobility of Hg is not affected by the nature or the

concentration level of the additives. Thiourea is the only

exception. Similar to Cd^ , the mobility (i.e. Rp) of Hg^^ tends to

146

decrease with increasing thiourea concentration in the mobile

phase. The lower Rj. ( or mobility) value of Cd and Hg" with

thiourea containing mobile phases compared to their Rj values

with urea containing mobile phases may be attributed to strong

binding of mercury and cadmium to the sulfur atom of thiourea

molecle.

From Table 6.4, it is clear that the presence of amines and phenolic

impurities influence the mobility of cations by lowering their Rp

values in some cases without hampering the separation of Zn, Mn or

Cd, Ni and Hg from their mixtures.

We have also attempted the semiquantitative determination of

metal ions following spot - area measurement method. A linear

relationship was obtained for Ni when the spotted amount of the

sample (50 - 200 |ag) was plotted against the area of the spot

(Figure 6.1). The accuracy and precision was around ± 12%.

Application: The method was used for the separation and identification

of heavy metal ions in synthetic hydroxide sludge and drag out of

nickel plating solution samples. The results listed in Table 6.5 clearly

demonstrate the applicability of the proposed method for the mutual

separation of Zn, Ni, Hg, and Cd or Mn from a variety of

environmental and geological samples.

147

Table 6.1 Effect of CTAB Concentration on the Mobility of Metal Cations*

Concentration or CTAB(mM)

Metal Ion HiO 0.1 1.0 10 SO 100 150 200 300

Ni-' 0.47 0.35T 0.55T 0.50 0.62 0.61 0.58 0.56 0.60

Co^* 0.35 0.32T 0.43T 0.32 0.42 0.47 0.50 0.43 0.52

Cd-* 0.12 0.07 0.15T 0.15T 0.35 0.32 0.37 0.43 0.45

Zn'* 00 0.06 00 0.05 00 0.03 0.05 0.05 0.07

Mn-* 0.20T 0.20 0.20 0.27 0.31 0.30 0.35 0.40 0.40

Hg^* 0.21T 0.30T 0.55T 0.95 0.97 0.95 0.95 0.97 0.95

T = Tailed spot (RL-RT> 0.3)

* Fe^^, Fe^^, Cu^*, Cr^* and VCf^ remained at the point of application (RjÔO)

148

Table 6.2 Separation of Coexisting Zn % Mn ^ or Cd^\ Ni ^ and Hg * on Silica Layer Developed with Non-Buffered and Buffered CTAB (50 mlM) Solutions

Mobile Phase Separation (Rp) Separation (RF)

Zn * Mn'* Ni'* Hg'* TM^* Cd'* Ni'* Hg'*

Non-Buffered aq. CTAB 0.04 0.36 0.64 0.95 0.04 0.27 0.55 0.98 (50 mM)

Buffer* PH3.4 00 0.50 0.70 0,99 00 0.42 0.70 0.99

PH7.0 00 0.47 0.70 0.97 0.05 0.35 0.62 0.99

PH11,9 0.04 0.36 0.64 0.95 00 0.37 0.67 0.99

* Mixture of metal cations got precipitated at pH 7.0 and 11.9. The precipitated was centrifuged, washed with water and dissolved in minimum volume ofHCl. The HCl was completely removed by evaporation, the residue was dissolved in 1 mL of distilled water and 0.01 mL of it was spotted for separation.

149

Table 6.3 Effect of Organic and Inorganic Additives in the Micellar Mobile Phase (CTAB) on the Separation of Zn^\ Ni % Hg ^ and Cd ^ or Mn ^ from their Mixture

Aqueous Solution of Different Impurity

Urea

Thiourea

NaCl

KCl

NaBr

KBr

Without additives

Molarity

0.05

0.1

0.2

0.5

0.05

0.1

0.2

0.5

0.05

0.1

0.2

0.5

0.05

0.1

0.2

0.5

0.05

0.1

0.2

0.5

0.05

0.1

0.2

0.5

Zn'*

0.02

0.03

0.04

0.05

0.02

0.02

0.05

0.02

0.03

0.02

0.02

0.05

0.02

0.05

0.03

0.03

0.05

0.01

0.02

0.02

00

00

00

0.02

0.02

Separation

Cd**

0.22

0.24

0.35

0.38

0.17

0.16

0.18

0.17

0.37

0.60

0.85

0.93

0.40

0.55

0.65

0.67

0.30

0.45

0.55

0.67

0.40

0.67

0.72

0.80

0.27

Ni'*

0.55

0.55

0.67

0.67

0.43

0.50

0.57

0.57

0.77

0.82

0.85

0.85

0.70

0.76

0.75

0.88

0.55

0.65

0.75

0.87

0.65

0.75

0.87

0.90

0.55

Added CTAB (50

(RF)

Hg'^

0.98

0.98

0.95

0.95

0.50

0.37

0.47

0.45

0.97

0.97

0.97

0.97

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.98

Zn^*

0.05

0.05

0.05

0.05

0.02

0.02

0.02

0.02

0.03

0.02

0.05

0.05

0.05

0.05

0.03

0.03

0.05

0.04

0.02

00

0.02

00

00

00

0.04

mlM)

Separation (RF)

Mn'*

0.30

0.27

0.27

0.26

0.27

0.28

0.35

0.35

0.50

0.50

0.57

0.72

0.56

0.55

0.60

0.70

0.30

0.37

0.42

0.52

0.42

0.47

0.68

0.75

0.36

Ni**

0.55

0.51

0.65

0.65

0.55

0.55

0.62

0.55

0.78

0.77

0.85

0.88

0.76

0.77

0.77

0.88

0.55

0.62

0.77

0.80

0.65

0.75

0.78

0.85

0.64

Hg^^

0.95

0.98

0.98

0.98

0.52

0.40

0.45

0.42

0.97

0.97

0.97

0.97

0.99

0.99

0.99

0.98

0.99

0.99

0.99

0.99

0.99

0.98

0.99

0.99

0.95

150

Table 6.4 Separation of Coexisting Zn % Ni % Hg ^ and Cd'* or Mn * in the Presence of Amines and Phenolic Impurities in the Sample

Added Impurities 50 mM Aqueous CTAB

Separation (Ry) Separation (RF)

Txf Mn Nl 1+ Hg'* Zn^ Cd Ni' Hg'^

o-Nitrophenol 0.02 0.27

o-Nitroaniline 0.02 0.28

m-Nitroaniline 0.05 0.30

o-Chloroaniline 0.02 0.26

p-Chloroaniline 0.05 0.28

m-Toluidine

Indole

Picric acid 0.02 0.25

Oricinol 0.02 0.27

0.42 0.%

0.53 0.99

0.52 0.92

0.47 0.98

0.50 0.99

0.05 0.25

0.02 0.20

0.02 0.20

0.05 0.27

0.02 0.27

0.05 0.25

0.05 0.30

0.55 0.98 0.05 0.25

0.55 0.99 0.02 0.25

0.42 0.94

0.51 0.95

0.05 0.27

0.02 0.30

Ammonium oxalate 0.01

Potassium iodate 0.02

0.27

0.25

0.60 0.99 0.05 0.23

0.52 0.99 0.02 0.22

0.55

0.52

0.55

0.57

0.56

0.55

0.97

0.97

0.95

0.95

0.95

0.47 0.95

0.57 0.95

0.97

0.55 0.92

0.58 0.99

0.56 0.98

Sodium molybdatc 0.02 0.25 0.53 0.99 0.02 0.20 0.58 0.99

Without impurity 0.04 0.36 0.64 0.95 0.02 0.27 0.55 0.98

151

Table 6.5 Separation of Mixtures of Zn^^ Ki^\ Hg ^ and Cd ^ or Mn * Ions from Metal Sludge Sample

Spiked / Synthetic Sample Separation (Rr) Separation (Rr)

Zn'* Ctf* Ni'* Hg** Zn'* Mn** Ni"* Hg'*

Sludge 0.02 0.32 0.55 0.99 0.05 0.38 0.65 0.99

Nickel plating solution 0.02 0.37 0.61 0.99 0.05 0.37 0.63 0.95

Distilled water 0.02 0.27 0.55 0.98 0.04 0.36 0.64 0.95

152

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9. Z. Ge and H. Lin, Fenxi Huaxua, 20 (1992) 1369.

10. S. N. Shtykov and E. G. Sumina, Zh. Anal. Khim., 53 (1998) 508

11. A. Mohammad, E. Iraqi and I. A. Khan, J. Surfactant and Detergent, 2

fl999) 523.

12. A. Mohammad and S. Tiwari, J. Am. Oil. Chem. Soc, 72 (1995) 1533

13. T. Okada, Anal. Chem., 60 (1998) 2116.

14. T. Okada, Anal. Chem., 64 (199 ) 589.

154

-VII

7.1 INTRODUCTION

An extensive survey of literature of last forty years on thin layer

chromatography (TLC) of anions (1-10) reveals that the single and

multicomponent eluents belonging to the following main groups have

been in use for TLC separation of anions.

(i) Organic solvents (carboxylic acids, bases, phenols, alcohols,

ketones, ethers, benzene and their mixtures)

(ii) Inorganic solvents (mineral acids, bases, salt solutions, mixtures

of acids and bases or'their salts)

(iii) Mixed aqueous-organic solvents (organic solvents mixed with

inorganic bases, salt solutions, water, mineral or carboxylic

acids)

Of these eluents, mixed aqueous-organic solvent systems have been

found most useful for separation of inorganic species on silica layers.

Salting-out planar chromatography that involves the use of aqueous

salt solutions as mobile phase has also been used to separate certain

anions on cellulose layers (11). Interesting studies on the use of

distilled water as an eluent with mixed adsorbent layers consisting of

silica gel 'G' and alumina or cellulose (12) and the water-in-oil

microemulsion mobile phase with kieselguhr as stationary phase (13)

for the TLC separations of anions have been reported from this

laboratory. In most of cases, the ternary separation of all three iodine

anions (iodide, iodate and periodate) was not satisfactory. The aim of

the work reported here was to develop a simple, inexpensive and rapid

TLC method for reliable separation of coexisting iodine anions in the

presence of interfering species (i.e. metal cations, amines and phenols)

that are frequently encountered in the aqueous environment using

relatively non-toxic micellar mobile phases. The separation of

coexisting iodine anions assumes significance, as these anions are

interconvertible. In alkaline medium, iodine is capable to react with

OH' ions to produce I' and \0i whereas in solutions of moderate

155

acidity (0.1-2.0 M HCl), IO3' can be reduced to iodine. The proposed

method expands the scope of using micellar eluents for difficult

separations of inorganic species.

7.2 EXPERIMENTAL

Apparatus: A thin layer chromatographic apparatus (Toshniwal India),

20 X 3 cm glass plates and 24 x 6 cm glass jars were used. Thermostate

and the Red wood viscometer No. 1 were used for the determination of

Krafft point and relative viscosity of mobile phases respectively.

Chemicals and Reagents: Silica gel 'G', sodium dodecyl sulfate (SDS)

and n-butanol, [Qualigens, India]; N-cetyl-N,N,N-trimethyl ammonium

bromide (CTAB), 2-propanol, and aluminium oxide (alumina) [CDH,

India] ; pentanol [ Fluka, AG, Switzerland] and Triton X-100 [Loba

Chemie, India] were used. All reagents were of Analar Reagent grade.

Test Solutions: The test solutions (1 %, w/v) were sodium salts of

nitrate, nitrite; and potassium salts of iodide, iodate, periodate,

perinangnate, chromate, dichromate, ferricyanide, ferrocyanide.

Phosphate and thiocyanate were taken as their ammonium salts.

Double distilled water with a specific conductivity (K = 2 x 10^ ohm"'

cm'') at 25^C was used for the preparation of salt solutions. Similarly,

1% aqueous solutions of metal ions (1%) and ethanolic solutions of

phenols and amines (1%) were used to examine their effect on mutual

separation of I' ,103' and IO4".

Detection Reagents: For the detection of various anions, the following

reagents were used;

(a) Saturated AgNOa solution in methanol for V, CT20J^\ Cr04^"

and P04^'

(b) Diphenylamine (0.2-0.5%) in 4M H2SO4 for NO2', NO3', lOa",

IO4' and Mn04".

(c) Ferric chloride (10%) in 2.0 M HCl for SCN, Fe(CN)6^' and

Fe(CN)6'-.

156

Stationary Phases: The sorbent layers used were: silica gel 'G' and

alumina

Mobile Piiases: The mobile phases used include:

(a) Distilled water.

(b) Aqueous solutions of [i] SDS (1.0 - 100 mM), [ii] CTAB (0.5 -

250 mM) and [iii] Triton X-100 (15.0 - 200 mM).

(c) Aqueous solutions (0.2 - 5,0 M) of acetone, NaCl, NaBr,

CHsCOONa, urea, propanol, butanol and pentanol with added

100 and 300 mM of SDS.

Preparation of Plain TLC Plates: TLC plates were prepared as

described in chapter 2.

Procedure: For the measurement of Rp values and determination of

limit of detection of anions, procedures as described in chapter 2 were

followed.

For the separation, equal volumes of anions to be separated were

mixed and 10 iL of the resultant mixture was loaded on the TLC plate.

The plates were developed, the spots were detected and the Rp values

of the separated anions were determined.


method, 0.01 mL from a series of standard solution of Fe(CN)6'*" (50 -

200p.g) were spotted on alumina layers. The plates were developed

with aqueous SDS (100 mM). After detection, the spots were copied

onto tracing paper from the chromatoplates and then the area of each

spot was calculated.

For examining the effect of impurities, one-drop (5nL) of each I'

, lOa', IO4' and impurity solution was spotted successively at the same

spot on the line of application of TLC plate. The spot was completely

dried after each spotting. After final drying, TLC plate was developed

157

with 100 mM aqueous SDS and the Rj. values of the resolved spots

were determined.


Thin-layer chromatography of anions on silica gel G and alumina

layers was performed with aqueous solutions of SDS and CTAB. The

comparative data with eluents containing SDS (1-100 mM) and CTAB

(0.5-100 mM) show better separation possibilities alongwith clearer

detection of anions on alumina. On silica layers, all the anions

examined migrate with the mobile phase giving Rp value equal to 0.95

± 0.02 and hence no separation was possible. Therefore, alumina was

selected for separation studies of anions. In fact, alumina has many

desirable properties such as rigid structure, little swelling in water or

solutions containing electrolyte and organic modifiers, stability under

mild acidic/ basic conditions and reasonable resistance to strong

oxidizing as well as reducing agents. Alumina is capable to retain

anions by following anion exchange mechanism.

A r O H " + X- ^?=^ A r x + O H '

The following aspects of TLC of anions with surfactant - mediated

mobile phases investigated.

(a) Effect of Type and Concentration of Surfactant on Mobility: The

Rp values of anions obtained on alumina layers with eluents containing

different concentrations of an anionic (SDS), cationic (CTAB) and

non-ionic (Triton X-100) surfactants have been listed in Table 7.1.

The surfactant concentrations as shown in Table 7.1 were taken below

and above the CMC values of SDS (CMC, 8mM), CTAB (CMC,

0.98mM) and Triton X-100 (CMC, 28mM) to examine the mobility of

anions. Below CMC value, surfactants are mostly in the (anionic,

cationic or non-ionic) monomeric form whereas above CMC micelles

(spherical or possibly ellipsoidal association colloids of surfactant) are

in dynamic equilibrium with a smaller number of free monomers in

solution. As evident from this Table, the mobility (i.e. Rp value) of

158

anions is slightly modified in the presence of surfactants compared to

their mobility in non-surfactant (pure water) mobile phase. For

example, slight increase in mobility (Rj. = 0.25) of IO3" in 100 mM

SDS facilitates the mutual separation of V , lO^' and IO4". The order of

mobility was I" > IO3" > IO4". It appears that I" (high Rp) is easily

eluted by the mobile phase (or specifically by the micelles) whereas

IO4" is completely excluded and IO3" showing little mobility (low Rp)

is weakly eluted. SDS was found to be better eluent compared to

CTAB and Triton X-100 for separation of anions and it was selected

for detailed study using alumina as stationary phase. To understand the

effect of increasing concentration of surfactant (or micelles) in the

mobile phase, anions were chromatographed with SDS micellar

systems containing surfactant upto 600 mM (about 75 times higher

than CMC) and the Rp values on alumina layer were recorded. The

difference in Rp values, ARp (ARp = Rp in 600 mM SDS minus Rp in

150 mM SDS) were plotted (Figure. 7.1). Three basic trends are

distinguishable from Figure. 7.1

(a) Increase in retention (lowering in Rp value) with the increase in

surfactant (or micelle) concentration for anions such as NO2",

NO3', Mn04", r , SCN",I03' and Fe(CN)6^' is indicative by

negative ARp values.

(b) Decrease in retention (increasing in Rp value) upon increasing

the micelle content in the mobile phase for certain anions such

as Cr04^", Cr207 ' and Fe(CN)6''' is indicative by positive values

of ARp.

(c) No change in Rp value of anions (IO4" and P04^') upon altering

the micelle concentration in the mobile phase as evident from

ARp value equal to zero.

The observation mentioned above under (a) can not be explained on

the basis of ion-pairing mechanism as the charges of solutes (or

anions) and surfactant (SDS) are usually the same and the stationary

159

phase has been saturated with surfactant (14). It appears that anions

with negative ARi value are strongly repelled by the micelles in the

mobile phase or show pronounced antibinding to SDS micelles.

Conversely, anions with positive ARi values likely bind to micelles.

Anions with zero ARp values appear to be nonbinding to micelles and

in such a case either there are no interactions between anions and

micelles or these forces (attractive and repulsive) are approximately

equal.

(b) Effect of Additives on Mobility: To examine the effect of organic

compounds (urea, acetone, alcohols etc.) and inorganic electrolytes

(sodium salts) as additives in the micellar mobile phases on the

separation of I', IO3' and IO4' from their mixtures, aqueous solutions of

different molarities of these additives (Table 7.2) were prepared by

dissolving the required amount / volume in distilled water and these

solutions were used as solvent for preparing SDS micellar solutions

(100 or 300 mM). The Rp values (average of triplicate determinations)

of resolved spots of I', IO3' and 104' from their mixtures are listed in

Table 7.2.

In addition to influence other micellar properties, additives

(organic or inorganic) alter the Krafft point ( The temperature at which

the CMC equals to the solubility and below the Krafft point the

surfactant exists as hydrated crystals). To examine this parameter on

the mobility, we determined the cloud points of some aqueous

solutions containing 100 mM SDS. The values, given in bracket, were

for 0.5 M acetone or propanol (0°C.), 1.0 and 5.0 M acetone (-1 and -

6°C), 0.5 and 1.0 M urea (-2 and -3°C) and 0.5 M NaCl (+13.5°C).

Since the Krafft point of 100 mM aqueous SDS was +3°C, the organic

additives were found to decrease the Krafft point whereas inorganic

additive (NaCl) was responsible to increase the Krafft point of micellar

system. The added NaCl, by screening the electrostatic repulsions

between headgroups makes a surfactant effectively more hydrophobic.

The relative viscosity of all these mobile phases varies within 1.15 -

160

1.63. All the additives except NaCl (0.5 M) and acetone (5.0 M) did

not offer any substantial influence on the R^ values and 1' - lOj' - IO4"

separation. The Rp value of V is dropped to 0.77 from 0.90 in 0.5 M

NaCl and that of IO3' is decreased to 0.02 from 0.27 in 5.0 M acetone.

The increase in viscosity of the medium increases the development

time of TLC plate.

It is clear from Table 7.2 that the mobility of 1" and lOs' are

influenced, though to little extent, by the altered microenvironment of

micellar mobile phase due to the presence of additives. However, IO4"

firmly located at the point of application (Rp = 00) irrespective of the

nature of mobile phase. The change in Rp values of I' and IO3" leads to

better mutual separation of I", IO3" and IO4' at 100 mM SDS

concentration level specially in the presence of NaCl, urea or propanol

(0.2 M), butanol (1.0 M) and pentanol (0.5 M). At 300 mM

concentration level of SDS, the enhanced mobility of I' (Rp > 0.8)

compared to its mobility in the absence of additives (Rp = 0.75)

improves the separation. However, IO3" and 104' could not be resolved

with 5 M acetone. The added organic solvents and salts are supposed

to affect CMC, the aggregation number, the partial specific volume of

the surfactant and the actual structure of the micellar assembly (15,16)

Alcohols (or co-surfactant) improves the separation possibilities by

virtue of their tendency of reducing the binding of surfactant to the

stationary phase. However, higher concentration of propanol or butanol

(5%) hampers the separation of IO3" from IO4' (Table 7.2). Thus, for

improved mutual separation of I", IO3' and IO4" one should prefer either

SDS micellar system (100 mM) containing organic or electrolyte

additives or SDS micelles (300 mM) without additives.

(c) Effect of Impurities on Separation: Results summarized in

Table 7.3 indicate that I" - IO3" - 104' separation can be successfully

performed in the presence of heavy metal cations, amino and phenolic

impurities. In fact Ni *, m-nitrophenol and toluidine improve the

161

separation efficiency by promoting a slight increase in the mobility of

103-.

(d) Limit of Detection: Data presented in Table 7.4 show that the

proposed method is highly sensitive for the detection of anions. The

sensitivity obtained with alumina - aqueous SDS (100 mM) TLC

system is about ten times higher than the sensitivity achieved by

earlier reported kieselguhr - water-in-oil microemulsion system (13).

Excellent sensitivity may be attributed to the fact that a significant

fraction of these anions resides close to the micellar - water interface.

(e) Semiquantitative Determination: In addition to qualitative

separations, semiquantitative determination of Fe(CN)6'*" has also been

attempted. A plot of the area of chromatographic zones versus the

amount of anion present in the range 50 - 200 )ig, gave a straight line

(Figure 7.2). The procedure developed has been tested to determine

the concentration of Fe(CN)6''' in synthetic mixtures with the precision

and accuracy of about 16%.

162

Table 7.1 Rj. Values (or Mobility) of Anions Obtained on Alumina Layer Developed with Water (Purely Aqueous) and Surfactant -Mediated Mobile Phases

Anion *

NO3

NO2

Mn04

lOj-

IO4"

Cr04'

CrjO/

r

PO4'

SCN-

Fe(CN)6'-

Fe(CN)6'-

Water

0.97

0.97

0.15T

0.17

0.07

0.08

0.10

0.98

00 •

0.98

0.02

0.95

1

SmM

0.97

0.98

0.05

0.16

00

0.05

0.07

0.99

00

0.87

00

0.97

SDS

lOOmM

0.97

0.96

00

0.25

00

0,17

0.05

0.92

00

0.85

0.05

0.90

Surfactant

CTAB

0.5mM

0.92

0.93

00

0.20

00

0.07

0.08

0.92

00

0.92

0.03

0.90

lOOmM

0.95

0.92

00

0.22

00

0.12

0.15

0.95

00

0.92

0.02

0.97

Tritoi

ISmM

0.97

0.97

00

0.20

0.04

0.07

0.06

0.92

00

0.92

0.02

0.90

1 X-100

lOOmM

0.92

0.90

00

0.18

00

0.02

0.03

0.82

00

0.82

0.03

0.90

* All anions show Rf about 0.95 ± 0.02 on silica gel layer with CTAB or SDS (100 mM)

163

Table 7.2 Effect of Organic and Inorganic Additives in the Micellar Mobile Phase (SDS) on the Separation of 1, IO3 and IO4 from their Mixture

Aqueous Different

Acetone

NaCl

NaBr

Solution of Impurit}'

Molarit>'

0.2

0.5 1.0

5.0

0.2

0.5

0.2

0.5

1.0

CHjCOONa 0.2

Urea

Propanol

Butanol

Pentanol

Without additives

0.5

0.2

0.5

1.0

5.0

0.2

0.5

1.0

5.0

0.2 0.5 1.0

5.0

0.2

0.5 1.0

0.0

Added SDS * 100 mM SDS 300mM SDS

Separation (Rf N'alue) Separation (Rf Value)

I

0.85

0.85 0.85

0.85

0.92

0.77

0.85

0.85

0.84

0.90 0.85

0.85

0.85

0.85

0.85

0.92

0,95

0.95

0.85

0.85 0,84 0.85

0.85

0.92

0.87 0.85

0.90

10,

0.23

0.23 0.24

0.02

0.34

0.23

0.17

0.20

0.25

0.28 0.25

0.30

0.30

0.27

0.30

0.32

0.31

0.27

0.10

0.31 0.32

0.31

0.15

0.27

0.31 0.29

0.27

10/

00

00 00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00 00

00

00

00

00 00

00

I

0.85

0.75 0.85

0.80

-

-

0.71

0.75

0.77

0.75 -

0.77

0.85

0.85

0.92

0.80

0.85

0.87

0.85

0.85 0.85 0.90

0.85

0.80

0.85 -

0.75

IO3

0.30

0.32 0.37

0.05

-

-

0.30

0.30

0.31

0.35 -

0.31

0.32

0.35

0.32

0.35

0.37

0,31

0.10

0.28 0.37 0.37

0.17

0.32

0,37 -

0.37

10,

00

00 00

00

-

-

00

00

00

00 -

00

00

00

00

00

00

00

00

00 00 00

00

00

00 -

00

* Clear solution could not be obtained when 100 mM of SDS were added into aqueous solution of NaCl (1.0 or 5.0 M). NaBr (5.0 M), CHiCOONa or pentanol (5.0 M). Similarly, clear solution could not be obtained when 300 mM of SDS were added into aqueous solution of NaCl (0.2 or 0.5 M). NaBr (5.0 M). CHiCOONa (0.5- 5.0 M) or pentanol (J - 5M).

164

Table 7.3 Separation of Coexisting 1", IO3' and IO4 in the Presence of Organic and Inorganic Impurities in the Sample

Separation (RF Value)

Impurities IO3 IO4

Phenol

Phloroglucinol

m-Nitrophenol

m-Hydroxyacetophenon

o-Toluidine

m-Toluidine

Aniline

Cu ^

Zn ^

Ni'^

Co ^

VO "

Fe ^

Fe ^

Ti'^

0.75

0.75

0.85

0.75

0.75

0.85

0.87

0.82

0.85

0.85

0.90

0.87

0.86

0.87

0.85

0.25

0.25

0.32

0.28

0.24

0.30

0.30

0.25

0.22

0.32

0.22

0.24

0.25

0.26

0.27

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

165

Table 7.4 Detection and Dilution Limits of Anions on Alumina

Layer with 100 mM SDS as Mobile Phase

Anion Limit of Detection (^g) Dilution Limit"

103-

IO4"

4-Fe(CN)6

Fe(CN)6'-

CrO. "

2.00

0.50

1.00

0.10

0.20

0.10

0.06

1:10000

1:40000

1:10000

1:100000

1:100000

1:100000

1:300000

*Dilution limit = 1: volume of test solution x l(f /limit of detection

166

Anions

Fe(CNiTe^CN),

Figure 7.1 Plot of A RF = (RF in 600 mM SDS minus RF in 150 mM SDS) Vs anions

as

% a6

^ a2

0 50 100 150

Amount (^g / spot) of Fe(CN)6''"

200

Figure 7.2 Calibration curve for semiquantitative determination of Fe(CN)6*-

167

REFERENCES

1. T. Baba, K. Sakushima and H. Yoneda, Bull. Chem. Soc, 43 (1970)

931.

2. R. Kuroda and M. P. Volynets, in : M. Qureshi (ed.) CRC Handbook

of Chromatography : Inorganics, Vol I, CRC Press, Boca, Raton, Fla

U.S.A, 1987.

3. A. Mohammad and S. Tiwari, J. Planar Chromatogr.- Mod. TLC,A

(1991)485.

4. A. Mohammad and S. Tiwari, Chromatographia, 30 (1990) 405

5. A. Mohammad and S.Tiwari, Microchem, J., 44 (1991) 39.

6. A. Mohammad and S. Tiwari, Sepn. Sci. TechnoL, 30 (1995) 3577.

7. A. Mohammad M. Ajmal, S. Anwar and E. Iraqi, / Planar

Chromatogr.- Mod TLC, 9R (1996) 318.

8. A. Mohammad, Thin - Layer Chromatography of Inorganics and

organometallics, chapter 19 pp. 507-617 in Handbook of Thin Layer

Chromatography (B. Fried and J. Sherma eds.). Marcel Dekker Inc.,

U.S.A, 71 (1996).

9. J. Sherma, Anal. Chem., 70 (1998) 7R.

10. J. Sherma, Anal. Chem., 72 (2000) 9R.

11. A. O. Kuhn and M. Lederer, J. Chromatogr., 406 (1987) 389.

12. A. Mohammad and S. Tiwari, Microchem. J., Al (1993) 379.

13. A. Mohammed, S. Tiwari, J. P. S. Chahar and S. Kumar, J. Am.

Oil Chem. Soc, 72 (1995) 1533.

14. D. W. Armstrong and F. Nome, Anal. Chem., 53 (1981) 1662.

15. D. W. Armstrong and G. Y. Stive, J. Am. Chem. Soc, 105 (1983)

6220.

16. J. S. Landy and J. G. Dorsey, Anal. Chim. Acta, 178 (1985) 179.

168

-VIII

(11)^ ^ve<x)(x<m(]i/m)£eî/iA]Ue ( I I I ) a/nd

8.1 INTRODUCTION

The literature survey of inorganic thin-layer chromatography (TLC) as

documented in various reviews (1-6) and books (7-10) reveals that

little work on TLC of anions has been performed as compared to that

on cations and organometallics. The selective separation of anionic

species by TLC has traditionally been realized on silica gel, alumina

and cellulose layers using mixed aqueous- organic systems as mobile

phase. Whereas extensive data are available on the use of hydro-

organic systems containing ketone (acetone or ethyl methyl ketone),

alcohol (methanol, propanol, butanol etc.), dioxane or dimethyl

sulphoxide (DMSO) in TLC of anions. The use of corresponding

mobile phases containing acetonitrile have been completely neglected.

In fact the latest review by Mohammad and Tiwari, on TLC of

inorganic anions (6) published in 1995 and the subsequent reviews by

J. Sherma (3-5) on TLC of inorganics and organometallics did not refer

to the use of water - acetonitrile or water - acetonitrile - organic

systems as mobile phase. However, acetonitrile containing mobile

phases have been found satisfactory eluents for TLC separation of

metal complexes (11-14).

The separation of coexisting thiocyanate, hexacyanoferrate(II)

and hexacyanoferrate (III) is important from analytical point of view.

The alkali salts of hexacyanoferrates find application in photography,

electroplating, dyeing, textile printing and pigment industry.

Hexacyanoferrate (III) has been a popular redox reagent. These anions

form complexes with Fe^* and hence the presence of one anion offers

detrimental effect on the detection (15,16) or determination (17) of

another anion .The TLC methods reported till date (6,18-20) record

good mutual separation of hexacyanoferrate (II), hexacyanoferrate (III)

and thiocyanate from their binary mixtures but none of these

procedures claim the separation of these anions from their ternary

mixtures on silica or alumina layers. However, Haworth et al. (21) has

169

reported a method for the separation of coexisting Fe(CN)6 ", Fe(CN)6 '

and SCN' on microcrystalline cellulose layer with acetone + ethyl

acetate + water (6+1+3) mobile phase.

This paper describes the use of a new mobile phase comprising

of distilled water, acetonitrile and carbon tetrachloride in TLC of

anions. The proposed method is reliable, fast and capable to separate

hexacyanoferrate (II), hexacyanoferrate (III) and thiocyanate from their

mixture within a short period of about 15 min. It has advantage in

respect of being rapid and capable to separate coexisting Fe(CN)6^',

Fe(CN)6'*" and SCN" in the presence of heavy metal cations and amino

acids compared to the method of Haworth. Furthermore, the present

method can be utilized for separation of these anions from a variety of

water samples such as hard water, industrial wastewater and saline

water.

8.2 EXPERIMENTAL

Chemicals and Reagents: Silica gel 'G', acetone, ferric chloride,

methanol, acetonitrile (Qualigens, India); carbon tetrachloride (Merck,

India) and DMSO and propanol (CDH, India) were used. All other

chemicals were also of Analytical Reagent grade.

Test Solutions: The test solutions (1%, w/v) were sodium salts of

nitrate, nitrite and acetate; potassium salts of iodide, iodate, periodate,

bromide, fluoride, permanganate, chromate, dichromate, ferricyanide,

ferrocyanide and phosphate. Thiocyanate, tungstate and molybdate

were taken as their ammonium salts. Double distilled water was used

for the preparation of salt solutions.

Detection Reagents: For the detection of various anions, the following

reagents were used:

(a) Saturated AgNOa solution in methanol for I", Br", F", Cr207^",

Cr04^" and P04^"

170

(b) Diphenylamine (0.2-0.5%) in 4.0 M H2SO4 for NOj", NO3", IO4".

IO3", MnOj" and W04^"

(c) Ferric chloride (10%) in 2.0 M HCl for SCN, Fe(CN)6^' and

Fe(CN)6'-

(d) Alcoholic pyrogallol (0.5%) for Mo04^' and Mo7024^'

Stationary Phase: Silica gel 'G '

Mobile Phases: The following solvent systems were investigated

Symbol Solvent Composition

(% volume)

M,

M2

M3

M4

Ms

Me

M7

Mg

M9

Mio

M„ M,2

Mn

MM

M,5

M,6

M,7

Mu

Mia_

Water

Acetonitrile

Carbon tetrachloride

Methanol

Dimethyl sulphoxide

Propanol

Acetone

Acetonitrile + water

Acetone + water

Acetonitrile + water + carbon tetrachloride

Acetone + water + carbon tetrachloride

80 + 20

50 + 50

20 + 80

80 + 20

50 + 50

20 + 80

80 + 5 + 5

80 + 10+5

80+15 + 5

80 + 5 + 5

8 0 + 1 0 + 5

80+15 + 5

171

Preparation of Plain TLC Plates: As described in chapter 2.

Procedure: For determination of Rp value of anions procedure

described in chapter 2 for amino acids was followed.

For separation, equal volumes of anions to be separated were

mixed and 10 ^L of the resultant mixture was spotted on the activated

TLC plate which was then dried in air. The plates were developed to a

distance of 10 cm, the spots were detected and the separated anions

were identified on the basis of Rp values.

In order to investigate the effect of metal cations, anions and

amino acids on the separation of coexisting Fe(CN)6 ', Fe(CN)6*' and

SCN", the impurities were added to the mixture of anions to be

separated in volume ratio of 1:1 and 10 ^L of the resulted synthetic

mixture was spotted on the TLC plate. The chromatography was

performed as done for the individual anions.


The results of this study has been summarized in Tables 8.1-8.6 and Figure 8.1. The chromatography of 18 anions was performed on silica

gel layer using distilled water and pure organic solvents (acetonitrile,

CCI4, MeOH, DMSO, propanol and acetone) as mobile phases. The

mobility data (i.e. Rp value) obtained for anions are summarized in

Table 8.1. Following trends are noticeable.

(a) All anions remain at the point of application (i.e. Rp = 0.0) with

CCI4 mobile phase. Conversely, all anions except F', P04^', IO4',

Mn04' and W04 * migrate with water, methanol or DMSO as

mobile phase.

(b) Anions such as (Mn04", IO4", P04 ", W04 " and F' do not show

any mobility with any mobile phase used.

(c) SCN' is migrated with all the solvent systems showing high Rp

value (Rp » 0.9). Thus, SCN' can be selectively separated from all

those anions which are showing low Rp.

172

(d) Interestingly, all anions show almost identical mobility pattern in

acetonitrile and acetone mobile phases.

(e) The mobility trend of Fe(CN)6^" and Fe(CN)6''" is reversed on

substituting MeOH by a less polar alcohol (i.e propanol) as

mobile phase. Both anions migrate with the solvent front when

MeOH was taken as mobile phase whereas they remain near the

point of application in case of propanol. Thus, the polarity of

alcohol seems to be an important factor for controlling the

retention behaviour of hexacyanoferrates.

(f) Binary separation of SCN" (high Rp) from hexacyanoferrates (low

Rp) is only possible with acetonitrile (15 min), acetone (15 min)

and propanol (30 min). The values in bracket refer to the

development time.

(g) All the pure solvent systems fail to resolve Fe(CN)6^' from

Fe(CN)6''' because both the anions either co-migrated with the

solvent front or retained near the point of application, depending

upon the nature of the mobile phase used.

With the aim of resolving hexacyanoferrates, the nature of the

mobile phase was changed and mixed mobile phases (water plus

acetone or acetonitrile) were used instead of pure single-phase

solvents. The results are shown in Table 8.2. It is evident from Table

8.2 that mixed aqueous-acetonitrile or acetone systems containing 20 %

water (by volume) are capable to resolve hexacyanoferrates from their

mixture. With this mobile phase, though SCN' can also be separated

from Fe(CN)6''' but its separation from Fe(CN)6^' is not possible. Figs.

8.1a and b provide more clear picture regarding the effect of

concentration of acetone or acetonitrile in acetone/acetonitrile - water

mixture on the separation efficacy of Fe(CN)6^' from SCN' or

Fe(CN)6^'. It is also evident from these Figures that for the binary

separation of Fe(CN)6^', the aqueous - organic mobile phase must

173

contain the organic solvent component (acetone/acetonitrile)in the

range 75-95%.

To achieve the separation of coexisting hexacyanoferrates and

thiocyanate anions, CCI4 was added as third component into a mixture

of water and acetonitrile or acetone. The resultant mobile phases (listed

in Table 8.3) were used as developer. The data given in Table 8.3

clearly indicate that SCN" - Fe(CN)6^' - Fe(CN)6'*" separation can be

easily achieved with mobile phases consisting of acetonitrile, CCI4 and

water. However, the best mobile phase providing sharp detection and

better separation was acetonitrile - CCI4 - water (80 + 5 + 10). It is

also evident from this Table that acetone - water mobile phases with

added CCI4 (M17 - M19) are incapable to resolve coexisting SCN' and

hexacyanoferrates as both Fe(CN)6 ' and Fe(CN)6 " are strongly

retained (Rp « 0.1) by the stationary phase.

From experimentally achieved binary and ternary separations

(Table 8.4), it may safely be concluded (on the basis of A Rp value)

that aqueous acetone systems (Mn - Mi3) are better mobile phase for

separating Fe(CN)6^" from Fe(CN)6'*" compared to aqueous - acetonitrile

mobile phases (Mg - Mio). Conversely, CCI4 added aqueous -

acetonitrile systems (M14 - Mje) are better mobile phases for the

separation of coexisting SCN", Fe(CN)6^" and Fe(CN)6'''. The Rp value

for these anions decreases in the order SCN' > Fe(CN)6 ' > Fe(CN)6'*'

which is analogous to the results obtained on microcrystalline cellulose

layer with acetone - ethyl acetate - water eluent (21). The lower

mobility of Fe(CN)6^' compared to Fe(CN)6^" may be attributed to the

following two factors:

(a) Ion-pair formation of hexacyanoferrate (II) with Ca " of gypsum

binder is stronger than with hexacyanoferrate (III) as evident

from their formation constants (22,23). Thus, weakly bound

ferrate (III) migrates faster through silica gel 'G' showing higher

Rp value compared to ferrate (II).

174

(b) The greater hydrogen bond formation tendency of ferrate (II)

with the surface hydroxy! groups of silica gel compared to

ferrate (III) which results in the stronger affinity of the former to

silica (24,25).

To widen the applicability of proposed method, the separation of

SCN" from Fe(CN)6 ' and Fe(CN)6^- was carried out from a variety of

water samples. The results presented in Table 8.5 show that the

presence of sulfates, chlorides and bicarbonates of calcium and

magnesium (hard water), NaCl (saline water) and nickel (industrial

wastewater) does not hamper the separation.

Table 8.6 summarizes the effect of various additives on SCN' -

Fe(CN)6'*' -Fe(CN)6^' separation. Heavy metals and amino acids do not

influence the separation though the Rp value of Fe(CN)6^" is slightly

changed from its standard value (0.55) in the presence of impurities in

the sample. A significant lowering in Rp value of Fe(CN)6' " was

noticed in the presence of nitrate and nitrite ions. The oxyanions of

iodine (IO3", IO4) influence the mobility of both SCN' and Fe(CN)6^'

resulting in the formation of tailed spots that adversely affect the

separation efficiency.

175

Table 8.1 Mobility (i.e. Rp) of Anions Chromatographed on Silica Gel Layer Using Pure Mono-Component Solvent Systems as Mobile Phase

Anion

N03"

NO2

Mn04"

103-

104*

Cr04^"

Cr207'-

r P04^-

SCN

Fe(CN)6'-

Fe(CN)6'-

Mo04^-

M07024^"

W04^'

Br-

COO

F

M,

0.95

0.92

0.00

0.95

0.10

0.85T

0.87T

0.90

0.00

0.98

0.98

0.98

0.99

0.92

0.00

0.91

0.90

0.02

Mono-

M2

0.10

0.17T

0.00

0.05

0.00

0.25T

0.27T

0.65

0.00

0.82

0.10

0.15T

0.00

0.00

0.00

0.00

0.00

0.00

- Component Mobile Phase

M3

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

M4

0.97

0.97

0.04

0.97

0.07

0.97T

0.98T

0.98

0.05

0.98

0.98

0.98

0.97

0.97

0.00

0.97

0.95

0.05

Ms

0.97

0.97

0.02

0.97

0.00

0.97T

0.98T

0.97

0.00

0.97

0.97

0.97

0.97

0.98

0.00

0.96

0.92

0.02

Mfi

0.92

0.95

0.00

0.97

0.05

0.97T

0.95T

0.97

0.00

0.95

0.05

0.12

0.97

0.95

0.00

0.97

0.87

0.00

M7

0.10

0.25T

0.00

0.05

0.05

0.22T

0.20T

0.75

0.00

0.92

0.12

0.12

0.10

0.10

0.00

0.00

0.00

0.00

T = Tailed spot (RI-RT^ 0.3)

176

Table 8.2 Mobility (i.e. RF) of Anions Chromatographed on Silica Gel Layer Developed with Two-Component Aqueous-Organic Solvent Systems

Anion

N 0 3 '

N02"

MnOV

103-

104'

Cr04^'

Cr207^-

r P04^-

SCN-

Fe(CN)6'*"

Fe(CN)6^-

Mo04^"

M07024^"

W04^'

Br-

COO-

F-

Bi

Ms

0.55

0.82

0.00

0.65

0.00

0.40T

0.63T

0.87

0.00

0.96

0.40

0.90

0.05

0.17

0.00

0.62

0.27

0.00

nary Aqueous-Organic Mobile Phase

M„ 0.85

0.85

0.00

0.95

0.07

0.87T

0.85T

0.97

0.00

0.95

0.10

0.95

0.00

0.15T

0.00

0.95

0.20

0.05

M9

0.97

0.97

0.00

0.97

0.80

0.38T

0.30T

0.90

0.00

0.95

0.87

0.97

0.92

0.88

0.00

0.82

0.60

0.05

Mn

0.97

0.95

0.00

0.95

0.05

0.80T

0.75T

0.95

0.00

0.95

0.95

0.95

0.95

0.95

0.00

0.95

0.72

0.00

M i o

0.95

0.96

0.07

0.98

0.80

0.50T

0.40T

0.90

0.00

0.95

0.97

0.97

0.98

0.98

0.00

0.80

0.85

0.00

M,3

0.97

0.95

0.00

0.97

0.05

0.95T

0.95T

0.97

0.00

0.95

0.95

0.97

0.95

0.97

0.00

0.95

0.80

0.05

T = Tailed spot (RL-RT^ 0.3)

177

Table 8.3 Mobility (i.e. RK) of Anions on Silica Gel Layer Using Three-Component Mixed Aqueous - Organic Solvent Systems Containing CCI4 as Mobile Phase

Anion

NO3'

NO2

Mn04'

103-

l O /

Cr04^-

CrjOv'-

r P04^-

SCN-

Fe(CN)6'-

Fe(CN)6'-

Mo04^"

M07024'"

W04^"

Br"

coop-

Ternary Aqi

M,4

0.15

0.45T

0.00

0.38

0.00

0.30T

0.22T

0.40

0.00

0.78

0.02

0.35

0.00

0.00

0.00

0.20

0.00

0.00

M,7

0.65

0.75

0.00

0.07

0.05

0.25T

0.23T

0.85

0.00

0.85

0.05

0.12

0.05

0.05

0.00

0.40

0.00

0.00

ueous -

M,5

0.15

0.62

0.00

0.45

0.02

0.35T

0.32T

0.72

0.00

0.96

0.05

0.58

0.02

0.10

0.00

0.30

0.20

0.00

Organic

M,8

0.85

0.80

0.00

0.05

0.06

0.37T

0.35T

0.95

0.00

0.95 .

0.02

0.15T

0.05

0.05

0.00

0.45

0.00

0.00

Mobile Phase

M,6

0.47

0.77

0.00

0.57

0.00

0.38T

0.35T

0.82

0.00

0.96

0.07

0.72

0.05

012

0.00

0.45

0.25

0.00

M,9

0.85

0.81

0.00

0.10

0.05

0.40T

0.37T

0.95

0.00

0.95

0.00

0.15T

0.00

0.00

0.00

0.50

0.00

0.00


178

Table 8.4 Separation of Coexisting Hexacyanoferrate (II), Hexacyanoferrate (III) and Thiocyanate on Silica Layer Developed with Mixed Aqueous - Organic Solvent Systems

Sample

Fe(CN)6'- - Fe(CN)6'*-

Mobile Phase

Acetonitrile + H;0

(9:1)

(8 :2)

Acetone + HÔ

(9:1)

(8:2)

Separation (RF)

(0.75)-(0.05)

(0.85)-(0.30)

(0.92) - (00)

(0.90)-(0.05)

ARF

0.70

0.55

0.92

0.85

SCN" - Fe(CN)6 - - Fe(CN)6^- Acetonitrile + CCI4 + U.O

( 8 : 5 : 1 ) (0.95)-(0.50)-(0.05)

( 8 : 5 : 0.5) (0.70)-(0.30)-(00)

179

Table 8.5 Separation of SCN , Fe(CN)6'' and Fe(CN)6' Present as Mixture in Different Water Samples Stationary Phase: Silica Gel 'G* Mobile Phase: M15

Sample *

Distilled water

Hard water

Saline water

Industrial wastewater

SCN

0.96

0.95

0.85

0.85

Separation (Rp)

Fe (CN)6'-

0.07

00

00

00

Fe(CN)6'-

0.55

0.55

0.48

0.50

The solutions (1%) of thiocyanate of ammonium; ferricyanide and ferrocyanide of potassium were directly prepared in hard water, saline water and industrial wastewater.

180

Table 8.6 Separation on Silica Layer of Coexisting SCN", Fe(CN)6'*' and Fe(CN)6 " in the Presence of Amino Acids, Heavy Metal Cations and Anions Mobile phase: Mis

Impurities

DL-valine

DL-tryptophan

DL-methionine

DL-alanine

DL-phenylalanine

3 -(3,4-diphenyl)DL-alanine

DL-aspartic acid

Cu ^

Hg^^

Cd^^

m'\ Co^^

NOs'

NO3"

Mn04'

IO4"

103-

Mo04^"

Without impurity

SCN"

0.90

0.90

0.91

0.87

0.87

0.90

0.97

0.95

0.92

0.92

0.91

0.90

0.95

0.95

0.92

0.85T

0.87T

0.90

0.96

Separation

Fe(CN)6'-

0.05

0.07

0.05

0.07

0.06

0.05

0.05

00

00

00

00

00

00

00

00

010

0.10

0.07

0.07

( R F )

Fe(CN)6'-

0.45

0.45

0.46

0.42

0.46

0.45

0.44

0.52

0.42

0.51

0.42

0.42

0.34

0.30

0.50

0.52T

0.45T

0.52

0.55


181

1 0.8 0.6 0.4

0.2 H

0 1 1 1 — — I 1 1 1 — — I 1 1

0 10 20 30 40 50 60 70 80 90 100 Volume of acetone (%)

-<^SCN~ -*-Fe(CN)e^--^Fe(CN)3-

Figure 8.1a Plot of RF VS concentration (% volume) of acetone in a mixture of acetone - water

0 10 20 30 40 50 60 70 80 90 100

- o - SCN ~ Volume of acetonitrile ("/©)

^ F e ( C N ) e ^

- ^ Fe(CNy

Figure 8.1b Plot of Rp Vs concentration (% volume) o acetonitrile in a mixture of acetonitrile - water

182

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184

Applicability of Surfactant-Modified Mobile Phases in Thin-Layer Chromatography of Transition Metal Cations: Identification and Separation of Zinc(ll), Cadmium(ll), and Mercury(ll) in their Mixtures

AM M o h a m m a d * and Vineeta Agrawal

Key Words:

Thin-layer chromatography

Transition metal ions

Surfactant-modified mobile phase

N-cetyl-N,N,N-trimethylammonlum bromide (CTAB)

Separation

Detection

Summary Thin-layer chromatography of fifteen metal ions has been performed on silica gel layers with solutions of the surfactant cetyltrimethylammonium bromide (CTAB) as mobile phase. The effect of surfactant concentration below and above the critical micellar concentration (CMC) on the retention behavior of the metal cations was examined. The effect of protic and aprotic organic modifiers and of alkali and alkaline earth metal salts on the mobility and separation efficiency of the metal ions was also assessed. A novel mobile phase comprising (1:2 (w/v) CTAB-ethanol)-water, 1 + 99, was identified as the best mobile phase for rapid separation of mixtures of Zn-*, Cd^*, Co"*, and Hg"*. Separation was better in the presence of propanol and ethanol than with other organic additives and the best sensitivity (i.e. lowest detection limit) was obtained with propanol. Semiquantitative determination of Ni'* and Zn'* by visual comparison of color intensities on TLC plates and by measurement of spot area was also attempted. The method was applied to the identification and TLC separation of zinc, cadmium, and mercury present in river water and industrial waste water and samples of hydroxide sludge. The method is well suited to identification and separation of Hg, Zn, and Cd from synthetically prepared ores such as cinnabar (HgS), zinc blende (ZnS), and greenoekite (CdS).

1 Introduction

The separation of heavy metal ions has attracted considerable attention in recent years, because of their environmental importance in aqueous media. Among separation techniques, thin-layer chromatography (TLC) is considered to be more application-oriented because of its versatility, sim-

A. Mohammad and V. Agrawal, Analytical Research Laboratory. Department of Applied Chemistry. Faculty of Engineering, Aligarh Mush'm University, Aligarh 202(102, India.'

plicity, low cost, and reasonable sensitivity. The separation of ions on thin layers is mainly influenced by the ion-exchange properties of the adsorbent and the coordinative properties of the solvent. In general practice the composition of mobile phase is altered to achieve a desired separation on a particular adsorbent. According to the literature on inorganic TLC [1-5] several mobile phases are currently in use, including: (i) inorganic solvents (acids, bases, salt solutions, mixtures of acids and bases or their salts) in distilled water or water-methanol mixtures; (ii) organic solvents (acids, bases, alcohols, aldehydes, ketones, esters, phenols, and their mixtures); (iii) mixed aqueous-organic solvents (organic solvents mixed with mineral acids, inorganic base, salt solutions, or water); and (iv) complex-forming solvents (e.g. EDTA, DMSO, etc.). Those containing aqueous acid, base or buffer have been considered most suitable for the separation of ionic species.

Surfactant-modified mobile phases have received considerable attention for liquid chromatographic separation of organic and inorganic substances since the first report by Armstrong and Fendler [6] who exploited the favorable features of micellar systems in chromatography. The use of surfactant ions as mobile phase components either below their critical micellar concentration (CMC), in ion-pair chromatography (IPC), or above their CMC, in micellar liquid chromatography (MLC), has been the focus of numerous studies [7-13]. In both IPC and MLC the surfactant provides electrostatic sites of interaction and thus influences the migration behavior of charged species. Although IPC has been regarded as an alternative to ion-exchange chromatography, because it enables simultaneous separation of ionic and nonionic compounds, the use of aqueous surfactant solutions does not always result in the desired separations and in such circum-•stances organic additives might afford better separations by modifying the retention behavior of the solutes [14,15].

210 VOL. 13. MAY/JUNE 2000 Journal of Planar Chromatography

TLC of Chelidonium majus L. Alkaloids

Figure 13

Densitogram obtained from micropreparative zonal chromatography of a ternary alkaloid mixture on an alumina plate with CHjClj - MeOH, 98 + 2, as mobile phase. Detection by UV at A = UV 254 nm.

achieved with horizontal DS chambers (frontal + elution chromatography) results in better separations of larger samples owing to partial separation during the application of the sample.

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[17] T.H. Dzido and E. Soczewinski, J. Chromatogr. 516 (1990) 461^66.

• 1 lUMt B

B

Figure 14

Photoscans (obtained by use of the Desaga Video - Scan ProViDoc V3.01) of some plates presented above as densltograms: A. Detection by UV at A = 366 nm, conditions as for Figure 4. B. Detection by UV at A = 366 nm, conditions as for Figure 6. C. Detection by UV at A = 366 nm, conditions as for Figure 7. D. Detection by UV at A = 366 nm, conditions as for Figure 8. E. Detection by UV at X. = 254 nm, conditions as for Figure 9.

[18] TH. Dzido, J. Planar Chromatogr. 6 (1993) 78-81.

[19] T. Wawrzynowicz, E. Soczewinski, and K. Czapinska, Chromatographia 20 (1985) 223-227.

[20] TH. Dzido, J. Planar Chromatogr. 3 (1990) 199-200.

[21] E. Soczewinski, and T. Wawrzynowicz, J. Chromatogr. 218 (1981) 729-732.

[22] G. Matysik, E. Soczewinski, and B. Polak, Chromatographia 39 (1994) 497-504.

[23] W. Gokiewicz and M. Gadzikowska, Chromatographia, 50 (1999) 52-56.

[24] J.J de Stefano and J.J Kirkland, Anal. Chem. 47 (1975) 1103.

Ms received: March 10, 2000 Accepted by SN: March 28, 2000

/ Journal of Planar Chromatography VOL. 13. MAY/JUNE 2000 209

TLC of Transition Metals with Surfactant-Modified Mobile Pfiases

Although, because surfactant-modified mobile phases are inexpensive, nontoxic, and nonflammable, they have been used extensively in reversed-phase liquid chromatographic separations of biologically active organic compounds [16-19], there have been few reports of studies on the use of these systems as mobile phases for chromatographic separation of metal cations [20-23]. Micellar chromatography of inorganic species has been reviewed by Okada [24] and by Manowski [25]. The use of aqueous solutions of sodium dodecyl sulfate and Triton X-100 as mobile phases to separate cobalt (III)-l-(2-pyridyl)-2-naphthol complexes on polyamide layers has been reported. Our recent studies on the TLC of anions [26] and amines [27] with microemulsion mobile phases point to the need for further investigations to explore fully the potential of surfactant-modified mobile phases in inorganic TLC.

We have, therefore, investigated TLC with surfactant-modified mobile phases as a means of accomplishing analytically important separations and have achieved rapid separation and reliable identification of Zn-+, Cd-*, Hg-+, and/or Co-* ions by use of a mobile phase containing cetyltri-methylammonium bromide (CTAB), ethanol, and water. The semi-quantitative determination of Zn-* and Ni-* has been attempted and the method has been used successfully for the separation and identification of Hg-*, Cd-*, and Zn-* from spiked samples of river and industrial waste water. To demonstrate further the usefulness of the method, it has been applied to the identification of Zn-+, Cd-+, Hg-*, Ni-*, and Pb-* in synthetically prepared metal hydroxide sludge and to the identification of Hg-*, Zn-+, and Cd-* from cinnabar (HgS), zinc blende (ZnS), and greenoekite (CdS) ores with preliminary separation from their mixture.

2 Experimental

2.1 Chemicals and Reagents

Silica gel G, n-butanol, and dimethylglyoxime were from Qualigens (India) and n-cetyl-A',A'-trimethyl ammonium bromide (CTAB), potassium ferrocyanide, 1,10-phenan-throline, methanol, ethanol, and 2-propanol were from CDH (India). Other chemicals were of analytical-reagent grade.

The metal ions studied were Fe'% Fe '+, Cu'% Ni'% Co'+, UOr% VO=% Cd-% Zn-% Ag% ?h\ JW W\ Hg-\ and AV\ Chromatography was performed on standard aqueous solutions (1%) of the chloride, nitrate, or sulfate salts of these ions, except for Fe-* for which a 1% solution of the ferrous ammonium sulfate salt in 3 mL H,S04 was used.

2.1.1 Preparation of Spiked Industrial Wastewater and River Water

Industrial wastewater (pH 2.98) collected from local industry, Aligarh, India, or river water (pH 7.48), from the Ganga

river at Naraura, India, (50 mL) was spiked with Zn, Cd, and Hg salts (100 |ag of each). Thioacetamide solution (0.5%, ca 30 mL) was added to the spiked sample and the resulting precipitate of Zn, Cd, and Hg sulfides was washed with distilled water, centrifuged, and dissolved in minimum possible volume of concentrated HCl. The acid was completely removed by evaporation and the residue was dissolved in distilled water (5 mL). An aliquot (10 |iL) of each sample was applied to TLC plate and chromatographed as described in Section 2.2.

2.7.2 Preparation of Synthetic Ores

Cinnabar (HgS), zinc blende (ZnS), and greenoekite (CdS) were prepared synthetically by spiking distilled water (pH 5.6, 50 mL) with Zn, Cd and Hg salts and following the procedure described in Section 2.1.1.

2.1.3 Preparation of Heavy Metal Hydroxide Sludge

Synthetic heavy metal sludge containing Zn, Cd, Hg, Pb, and Ni was prepared by adding sufficient NaOH solution (1%) to a solution of the metal salts (1% of each). The metal hydroxide precipitate was filtered, dried, and dissolved in a minimum volume of concentrated hydrochloric acid. The acid was completely evaporated, the residue was dissolved in distilled water (5 mL) and TLC was performed on a 10-|iL sample.

2.2 Chromatography

Chromatography was performed on silica gel G. A 1:3 {w/w) mixture of silica gel and demineralized water was shaken continuously to give a homogeneous slurry which was applied as 0.25-mm layers to 20 cm x 3 cm glass plates by means of a Toshniwal (India) applicator. The plates were first dried at room temperature and then activated at 100 ± 5°C for 1 h in an electrically controlled oven. The activated plates were stored in a closed chamber at room temperature until used.

Test solution (ca 5 |iL) or other samples (Sections 2.1.1-2.1.3, 10 |iL) were spotted ca 2.0 cm above the lower edge of thin-layer plates, by means of a micropipet, and the spots were left to dry in air. The plates were then developed, to a distance of 10 cm, by the one-dimensional ascending technique, in 24 cm X 6 cm glass jars, with the mobile phases listed in Table 1. After development the plates were dried in air and the spots were visualized. Fe-* was detected with 1,10-phenanthroline solution (0.5%), Fe'*, Cu-*, VO-*, and UO,-* with aqueous potassium ferrocyanide solution (1%), Ni-* and Co-* with ethanolic dimethylglyoxime solution (1%), Zn-*, Cd-*, Ag*, Pb-*, Tl*, Bi'*, and Hg-* with dithizone in CCI, (0.5%), and AF* with aqueous aluminon solution (1 %). The limits of detection of the metal ions were determined by spotting different amounts of the ions, developing the plates, and detecting the spots. The method was repeated with successive reduction of the amount of the metal until no spot was detected.

Journal of Planar Chromatography VOL. 13. MAY/JUNE 2000 211

TLC of Transition Metals with Surfactant-Modified Mobile Phases

Table 1

The mobile phases used.

Symbol Composition

Ml M2 M3 M4 M5 M6 M7 M8 M9 MIO Mil M12 M13 M14 M15 M16 M17 M18

M19

M20

M21

M22

M23

M24

M25

M26

M27 M23 M29 M30

M31 M32 M33 M34 M35

0.33% Aqueous CTAB Ethanol-water (1 + 99) 0.5 mL (CTAB + methanol, 1:2 iv/y)"' + 99.5 mL water 1.0 mL (CTAB + methanol) + 99.0 mL water 1.5 mL (CTAB + methanol) + 98.5 mL water 0.5 mL (CTAB + ethanol) + 99.5 mL water 1.0 mL (CTAB + ethanol) + 99.0 mL water 1.5 mL (CTAB + ethanol) + 98.5 mL water 5.0 mL (CTAB + ethanol) + 95.0 mL water 10.0 mL (CTAB + ethanol) + 90.0 mL water 15.0 mL (CTAB + ethanol) + 85.0 mL water 20.0 mL (CTAB + ethanol) + 80.0 mL water 0.5 mL (CTAB + propanol) + 99.5 mL water 1.0 mL (CTAB + propanol) + 99.0 mL water 1.5 mL (CTAB + propanol) + 98.5 mL water 0.5 mL (CTAB + butanol) + 99.5 mL water 1.0 mL (CTAB + butanol) + 99.0 mL water 1.5 mL (CTAB + butanol) + 98.5 mL water 1.0 mL (CTAB + ethanol) + 99.0 mL aq. NaCl"' (0.01-0.2 M)

+ 99.0 mL aq. HCl

99.0 mL aq. MgCl,

99.0 mL aq. CaCL

1.0 mL (CTAB + ethanol) (0.01-0.2 M) 1.0 mL (CTAB + ethanol) (0.01-0.2 M) 1.0 mL (CTAB + ethanol) (0.01-0.2 M) 1.0 mL (CTAB + ethanol) + 99.0 mL aq. LiCI (0.01-0.2 M) 1.0 mL (CTAB + ethanol) + 99.0 mL aq. KCI (0.01-0.2 M) 1.0 mL (CTAB + ethanol) + 99.0 mL aq. NaBr (0.01-0.2 M) 1.0 mL (CTAB + ethanol) + 99.0 mL aq. urea (0. 1 M) 1.0 mL (CTAB + ethanol) + 99.0 mL aq. KBr (0.1 M) 1.0 mL (CTAB + ethanol) + 99.0 mL aq. NaNO, (0.1 M) 1.0 mL (CTAB + ethanol) + 99.0 mL aq. oxalic acid (0.1 M)

1.0 mL (CTAB + ethanol) + 99.0 mL aq. tartaric acid (0.1 M )

1.0 mL (CTAB + ethanol) + 99.0 mL aq. KOH (0.1 M) 1.0 mL (CTAB + ethanol) + 99.0 mL aq. NaOH (0.1 M) 1.0 mL (CTAB + DMF) + 99.0 mL water 1.0 mL (CTAB + DMSO) + 99.0 mL water 1.0 mL (CTAB + acetone) + 99.0 mL water

"'The ratio of CTAB to organic solvent was always 1:2, wjv. '''Salt solutions were prepared in distilled water.

The smallest amount of metal detected on the plate was taken as the limit of detection.

R^ {Rf, of the leading front of the spot) and 7? {Rf of the trailing front) were measured for the detected spots and R^ values were calculated from Rf = {R^ + Rj)/2. Capacity factors (k) were calculated from the 7?F values by use of the formula A: = (l-/?p)//?p

For separation of the metals equal amounts of the ions to be separated were mixed and the resulting mixture (10 |iL) was

applied to the plates. The plates were developed, the spots were detected, and the R^ values of the separated metal ions were determined. For semiquantitative determination by spot-area measurement, standard solutions of Ni-* and Zn-* (0.05-0.20%, 10 \iL) were spotted on the plates and these were developed with mobile phase M7 (Table 1). After detection the spots were copied from the plates on to tracing paper and the area of each spot was calculated. For semiquantitative determination by visual comparison, standard solutions of nickel chloride of different concentrations (0.2-1%, 10 |iL) were spotted on to TLC plates with the extract obtained from the synthetic industrial wastewater sample (10 |uL). After chromatography the color intensity and Rf of the spot obtained from the industrial wastewater sample were matched visually with those of the spots obtained from standard reference solutions of nickel chloride.

3 Results and Discussion

The mobilities of fifteen transition metal ions were determined on silica gel layers developed with surfactant-modified mobile phases. To examine the effect of the surfactant the mobilities of the metal ions were determined with (i) aqueous CTAB (0.33%), (ii) ethanolic aqueous CTAB (0.33 and 0.5%), and (iii) ethanol-water mixtures as mobile phases. The results are compared in Figure 1, from which it is evident that the mobilities of most of the metal ions are very similar when aqueous CTAB and ethanol-water mixtures are used as mobile phases, although Hg-* is much more mobile when aqueous CTAB is used. Interestingly, use of ethanoUc-aque-ous CTAB as mobile phase results in differential migration of all the metal ions, i.e. the presence of ethanol in aqueous CTAB modifies the mobilities of the metal ions on silica gel, resulting in enhanced possibilities of separation. These results clearly show that the effectiveness of water-alcohol mobile phases (e.g. ethanol-water) can be improved by addition of CTAB, a cationic surfactant. Figure 1 shows that separation of Hg-+ from Cd'^, Zn-+, and Co^* is possible with CTAB-modified ethanolic-water mobile phases but not with unmodified ethanol-water (zero CTAB concentration).

Increased ionization of cationic surfactants in the presence of alcohols has been reported by Zana [28]. The enhanced ionization of CTAB in the presence of organic alcohol modifiers has thus resulted in improved separation of the metal ions. Because the concentration of CTAB in mobile phase M7 was 0.33%, below its critical micellar concentration (CMC; 0.46%), the surfactant in M7 is present as the cationic monomer and can be considered simply as a strong electrolyte. When the concentration of surfactant is increased to 0.5% (M8), just above its CMC, and surfactant monomers are expected to be in equilibrium with CTAB micelles, the mobility of the metal ions is either the same or slightly lower than when M7 is used. The highest mobility of Hg'+ when M7 is used facilitates its clear separation from almost all the other metal ions.



Table 2

Mobility of Ni ^ and Co ^ with different mobile phases.

Hg Al

METAL IONS

Figure 1

Mobility of metai ions on silica gei G developed with ethanoi-water (-A-), aqueous CTAB (-4 -), and CTAB-ettianoi-water (-•-) mobile ptiases.

Separation of mixtures of Zn-^, Cd-*, and Hg-* ions is analytically very important because of their similar physical and chemical properties. Zinc (d"',4s-), cadmium (d"',5s-), and mercury (d"',6s-) with the electronic configuration (« - 1) d"',ns- belong to the same group (IIB) of the periodic table and Cd is generally associated with Zn minerals - for example zinc blende ore contains ca 3% Cd. Mixtures of zinc, cobalt, and cadmium compounds are also of technical importance and frequently one metal contains small quantities of the others, which has a deleterious effect on performance. Separation of Cd-* from Zn-+ is also of biochemical importance because substitution of Zn-* by Cd-* in some metalloenzymes leads to cadmium toxicity.

It is apparent from the literature [29] that organic solvent additives have been the choice of chemists and physicist for modification of the properties of micellar mobile phases to obtain better efficiency. With this in mind we replaced ethanol by other protic and aprotic organic solvents (methanol, 2-propanol, rt-butanol, acetone, DMF, DMSO) in the CTAB mobile phase and determined the mobility of the metal ions. The development time for 10 cm ascent was 20-22 min for all mobile phases except methanol, for which the development time was 35 min. All the metal ions were clearly detected as compact spots irrespective of mobile phase. With these mobile phases (M3-M8, M13-M18, and M33-M35) several trends in metal-ion mobility were observed.

(i) The mobilities of Fe=*, Fe'*, Cu-*, Zn-*, Ag*, Pb-*, Bi'*, Al'*, UO,'*, and VO-* are low and the metals remained near the origin (Ry < 0.05).

(ii) Cd-* and Tl* have intermediate Ry values (0.4-0.6) and can thus be separated from all metal ions with higher and lower /?,. values.

(iii) Hg-* has a high R^ value (0.9-0.98) owing to migration with the mobile phase front.

(iv) The mobilities of Ni-* and Co-* vary; Rf values of Ni-* and Co^* for different mobile phases are given in Table 2.

From these results it is clear that surfactant-containing mobile phases with added organic modifiers can be successfully exploited to achieve novel separations.

The detected spots were sharper for alcohol (ethanol or propanol, proton donor)-containing mobile phases for

Mobile phase

M3 M4 M5 M6 M7 M8 M13 M14 M15 M16 M17 M18 M33 M34 M35

R, Ni=*

0.70 0.95 0.82 0.57 0.80 0.60 0.82 0.62 0.72 0.62 0.72 0.60 0.47 0.60 0.65

R, Co-*

0.85 0.75 0.70 0.80 0.72 0.60 0.70 0.65 0.58 0.65 0.60 0.57 0.55 0.45 0.35

those containing acetone (proton acceptor), DMSO, or DMF. In contrast with this work, acetone, which tends to suppress the hydrolysis of metal ions, and DMSO, an aprotic dipolar solvent and a stronger solvating agent for cations in preference to anions, have been found most suitable for TLC analysis of metal ions with nonsurfactant-modified acidic mobile phases [30,31].

Among the alcohols (known as co-surfactants), ethanol and propanol were found more useful than methanol and butanol for achieving separations of multicomponent mixtures of metal ions. Although the separation of Zn-*, Cd-*, Hg-*, and Co-* from each other can be realized in the presence of any of the alcohols, more compact spots were obtained with ethanol or propanol (Figure 2) and identical efficiency was obtained with both. These alcohols improve separation possibilities by reducing the binding of the cationic surfactant (CTAB, with a positive charge residing on the quaternary nitrogen atom) to the porous silica surface. The silanol groups (=Si-OH) of silica gel, being weakly acidic [32], enable the silica gel to acquire a negative charge when in contact with water and thus the hydrated silica gel captures the positively charged species. Mobile phases M7 and M14 containing ethanol and propanol were, therefore, studied in detail.

With M7, a linear relationship was observed between 7? and ionic potential (= ionic charge/ionic radius) for Zn-*, Cd-*, and Hg-*, and for Ni-*, Co-*, and Bi'* (Figure 3). This shows that the mobility of these metal ions is also influenced by their ionic radii.

To widen the applicability of CTAB-containing systems as mobile phases, distilled water in M7 was substituted by 0.01-0.2 M NaCl, 0.01-0.2 M HCl, 0.1 M oxalic or tartaric acid, 0.01-0.2 M chlorides of calcium, magnesium, lithium, or potassium, 0.01-0.2 M sodium bromide, 0.1 M sodium nitrate, 0.1 M NaOH or KOH, or 0.1 M urea and the effect of these additions on the separation of Zn-*, Cd-*, and Hg-* was investigated. From the results presented in Table 3, it is


TLC of Transition IVIetals with Surfactant-Modified IVIobile Pliases

!^0.5

Pntpanol

Figure 2

Mobility of Zn (-•-), Cd (-A-), Co {-•-), and Hg {- -•- -) on silica gel G developed witti mobile phases M4, M7, M14, and M17 containing alcohols of different chain length.

evident that these impurities have a detrimental effect on the separation, by influencing water-surfactant interactions, because these ionic additives interact with the surfactant molecules. The separation of Cd-* (/?, = 0.48) from Zn-* (R, = 0.30) deteriorates in the presence of 0.2 M HCl, and could not be achieved because of the increase in the mobility of Zn-*. The separation of Hg-* from Zn-* is, however, always possible but its separation from Cd-* could not be realized in the presence of complexing carboxylic acids (oxalic or tartaric acid). Urea, which breaks down the structure of water does not adversely affect the separation of Zn=*, Cd=*, and Hg-*.

Aqueous solutions (0.1 M) of KSCN and KI produce white precipitates on addition of 1 mL ethanolic-aqueous CTAB solution and so the effect of \ and SCN" on the separation of Zn-* from Cd-* and Hg-* could not be examined. The results in Table 3 also indicate that the anions C\ and NO,-and the cations Li*, Na*, and Mg-* do not hamper the separation of Zn-*, Cd-*, and Hg-*, irrespective of the concentrations of the ions. At a concentration of 0.2 M, however, Ca-* and K* adversely effect the separation by increasing the mobility (lower A:) of Cd-*. The variation in the k value of Cd'* in the presence of different concentrations of inorganic cationic and anionic impurities is summarized in (Figure 4). The k values for Hg-* and Zn-* are almost unchanged, showing that the presence of inorganic ionic impurities, irrespective of concentration, has little effect on their mobility. The minor changes in the R^ values of Zn-*, Cd-*, and Hg-*, without effect on the separation, as a result of the presence of impurities, might be attributable to competitive adsorptive interactions between positively charged species (alkali and alkaline earth metal ions), and the surfactant cation of the mobile phase, and the negatively charged silica gel surface. Interactions between silica gel and alkali and alkaline earth metal ions in aqueous mobile phases have already been reported [33,34]. A cation-exchange reaction occurs when silica gel comes into contact with an electrolyte:

=Si-0-H* -I- Na* ^ =Si-ONa* + H*

Although both ethanol and propanol work identically to resolve the mixture of metal ions, the sensitivity is better with propanol (Ml4) than with ethanol (M7), as is evident from Table 4. The minimum amount detectable is a factor of 2 to 3 lower with propanol. Better sensitivity can also be

BT

1.0-

0 .9-

0.8-

0.7-

0.6-

0.5-

0.4-

0.3-

0.2-

O.I-

0.0

\ * H g "

\ i« Ni'*

\ ^°' A

\ \ Cd=** \ \

Zn=*. \ ••Bi'* 1 ' - • 1 1 1

1.0 2.0 3.0 4.0 5.0

IONIC POTENTIAL

Figure 3

Plot of Ionic potential against R^.

• • • S 1.1

MOLAK CONCENTRATION OF SALT SOLUTIONS

J

1.5

MOLAK C0NCENT1IAT10N 0 » SALT SOLimONS

Figure 4

Mobility of Cd^* on silica gel G developed with CTAB-ethanol-aqueous mobile phases containing different concentrations of salts of alkali and aiitaline earth metals: (- -•- -) NaCI, (-A-) NaBr, (-•-) LiCI, ( - • -) KCI, {-m-) CaCij, (- -A- -) MgClj.

obtained by substituting laboratory-made silica gel plates with commercially available HPTLC plates.

In addition to qualitative analysis, quantitative evaluation of the metal ions is often required for determination of the amounts of toxic metals in environmental samples. A simple, but relatively inaccurate method of quantitation is based on measurement of the size of the spot by drawing the outline of the spot on a piece of tracing paper. An attempt was made to achieve semiquantitative determination of metal ions by measuring


Table 3

Separation of mixtures of Zn '* , Cd^ ,

Mobile phase Symbol Composition

and Hg2*

1

ions on

TLC Of Transition Metals with Surfactant-Modified Mobile Phases

silica gel G developed with different CTAB-containing mobile phases.

Zn-* Cd-* Hg-*

M19

M20

M22

M23

M24

M25

M26 M28 M29 M30 M31 M32

1.0 mL (CTAB + ethanol, 1:2 wlvf + 99.0 mL O.OIM aq. NaCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.05 M aq. NaCI 1.0 mL (CTAB + ethanol) + 99.0 mL 0. 1 M aq. NaCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.2 M aq. NaCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.01 M aq. HCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.05 M aq. HCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. HCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.2 M aq. HCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.01 M aq. CaCL"' 1.0 mL (CTAB + ethanol) + 99.0 mL 0.05 M aq. CaCL 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. CaCL 1.0 mL (CTAB + ethanol) + 99.0 mL 0.2 M aq. CaCl, 1.0 mL (CTAB + ethanol) + 99.0 mL 0,01 M aq. LiCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.05 M aq. LiCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. LiCl LO mL (CTAB + ethanol) + 99.0 mL 0.2 M aq. LiCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.01 M aq. KCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.05 M aq. KCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. KCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.2 M aq. KCl 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. NaBr 1.0 mL (CTAB + ethanol) + 99.0 mL 0.05 M aq. NaBr 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. NaBr 1.0 mL (CTAB + ethanol) + 99.0 mL 0.2 M aq. NaBr 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. Urea 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. NaNO, 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. oxalic acid 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. tartaric acid 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. KOH 1.0 mL (CTAB + ethanol) + 99.0 mL 0.1 M aq. NaOH

0.05 0.03 0.02 0.02 0.02 0.15 0.22 0.30 0.05 0.02 0.03 0.05 0.05 0.05 0.05 0.04 0.05 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.05 0.10 0.52 0.58 0.05 0.05

0.22 0.34 0.56 0.68 0.25 0.40 0.43 0.48 0.32 0.37 0.50 0.87 0.29 0.37 0.47 0.57 0.32 0.50 0.67 0.76 0.16 0.35 0.55 0.70 0.48 0.42 0.92 0.90 0.25 0.25

0.92 0.95 0.98 0.98 0.82 0.92 0.95 0.95 0.87 0.92 0.87 0.90 0.85 0.95 0.97 0.95 0.76 0.90 0.95 0.92 0.95 0.97 0.95 0.97 0.97 0.92 0.95 0.95 0.81 0.85

•"The ratio of CTAB to organic solvent was always 1:2, wiv. '''Salt solutions were prepared in distilled water.

spot area. A linear relationship was obtained when the amount of the sample spotted was plotted against the area of the spot (Figure 5). The empirical equation expressing the relationship ht,- = kxm, where ^ is the area of the spot, m the amount of solute, and A: is a constant. The linearity is maintained up to 200 Hg Ni-* or Zn-* spot '. At higher concentrations a negative deviation from linearity was observed for both ions. The standard curve constructed for semiquantitative determination of Ni- was used to determine the amount of nickel in water samples. The accuracy and precision were below ±\5%. When semi quantitative determination by visual comparison was used to estimate the nickel content of industrial wastewater the nickel content, was found to be in the range 3.5^ g L"'.

Zn, Cd, Hg, Ni, and Pb and the mutual separation of Hg, Cd, and Zn from a variety of environmental and geological samples. The results in Table 5 clearly indicate the applicability of the method for the separation and detection of Hg in cinnabar (HgS), Cd in greenoekite (CdS) and Zn in zinc blende (ZnS).

Acknowledgment

The authors thank Professor K.G. Varshney, Chairman, Department of Applied Chemistiy, for providing research facilities. The All-India Council for Technical Education, New Delhi (India) is thanked for financial assistance.

3.1 Applications

The method was used for the separation and identification of heavy metal ions in spiked river and industrial wastewater samples, in synthetic metal hydroxide sludge, and in metal sulfide ore samples. The results listed in Table 5 clearly demonstrate the applicability of the method for the identification of

References

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[2] R. Kuroda and M.R Volynets, in: M. Qiiareshi (Ed.) CRC Handbook of Chromatography: Inorganics, Vol. 1, CRC Press, Boca Raton, 1987, p. 89.



100 150

AMOUNT OF NI"(ng)

100 150 200 AMOUNT OF Zn-*(ng)

Figure 5

Dependence of spot area, |, on amount of (a) Ni ^ and (b) Zn^

[3] A. Mohammad and K. G. Varshney, in: /. Sherma and B. Fried (Eds) Handbook of Thin-Layer Chromatography, Vol. 71, Marcel Dekker, 1996, p. 507.

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[10] A. Berthod, J. Chromatogr. 191 (1997) 780.

[11] M.G. Khaledi, J. Chromatogr. 3 (1997) 780.

[12] A. Szymanski and W. Szczepaniak, Chem. Anal. (Warsaw) 43

(1998) 617.

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1715.

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1362.

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Table 4

Limit of detection of metal ions on silica gel G developed with

CTAB-ethanol-HjO (M7) and CTAB-propanol-HjO (Ml 4) mobile phases.

Metal ion Limit of detection [fjg] M7 M14

Zn + Cd-^ Hg^-Bi'+ Pb-*

0.024 (0.014)"' 0.041 (0.016) 0.033 (0.014) 0.028 0.030

0.013 0.016 0.014 0.014 0.013

"'The values in parentheses refer to the lower limit of detection on HPTLC plates (silica gel 6OF254; Merck, Darmstadt, Germany).

Table 5

Separation of mixtures of Zri'*, Cti'*, and Hg^* ions from spiked water

and synthetically prepared metal ores and heavy metal sludge samples.

Spiked/synthetic sample Zn-* Cd Hg^

River water Industrial wastewater Sulfides' Sludge" Distilled water

0.02 0.05 0.05 0.02 0.05

0.37 0.35 0.46 0.34 0.43

0.90 0.91 0.92 0.96 0.95

"'Ni=* (Rf = 0.50) and Pb'* (R^ = 0.03) were also detected.

[17] M.G. Khaledi, Trends Anal. Chem. 7 (1988) 293.

[18] M. Amin, K. Harrington, and/?. Von Wandruszka, Anal. Chem. 65(1996)2346.

[19] R.W WilliamsJr, Z. Fu, and W.L. Hinze, J. Chromatogr. Sci. 28

(1990) 292.

[20] T. Okada, Anal. Chem. 60 (1988) 2116.

[21] T. Okada, Anal. Chem. 64 (1992) 5899.

[22] A. Berthod, X. Jun, A Serge, and C. Gonnet-Collet, Can. J. Chem. 74(3) (1996) 227.

[23] T. Okada, J. Chromatogr. 607(1) (1992) 135.

[24] T. okada, J. Chromatogr. A. 343 (1997) 780.

[25] / Manowski, Chem. Abstr. 122(18) (1991) 219028m.

[26] A. Mohammad, S. Tiwari, J.P.S. Chahar, and S. Kumar, J. Am.

Oil Chem. Soc. 72 (1995) 1533.

[27] A. Mohammad and E. Iraqi, J, Surf. Det. 2 (1999) 85. [28] R. Zana, in: D.O. Shah (Ed.), Surface Phenomena in

Enhanced Oil Recovery, Plenum, New York, 1980.

[29] M.F Borgerding and W.L. Hinze, Anal. Chem. 57 (1985) 2183.

[30] A. Mohammad and N. Fatima, Chromatographia 22 (1986) 109.

[31] M. Qureshi, K. G. Varshney, and A. Fatima, Sep. Sci. Technol. 13(4) (1978) 321.

[32] D.L. Dagger, J.H. Stanton, B.N. Irby B.L. McConnell, W.W. Commings, and R. W. Maatman, J. Phys. Chem. 68 (1964) 757.

[33] K. Kovdcs-Hadady, J. Planar, Chromatogr. 2 (1989) 211.

[34] R.K. Ray and G.B. Kanlfman, J. Chromatogr. 464 (1990) 504.

Ms received: June 13, 2000 Accepted by SN: June 15, 2000

2 1 6 VOL. 13. MAY/JUNE 2000 Journal of Planar Chromatography

Micellar Thin-Layer Chromatographic Separation and Identification of Amino Acids: Separation of L-Proline from other Aliphatic and Aromatic Amino Acids All Mohammad* and Vineeta Agrawal

Key Words:

Micellar TLC

Amino acids

L-Prollne

Summary Cationic and anionic surfactant-mediated systems have been used as mobile phases for thin-layer chromatographic separation of aliphatic and aromatic amino acids on alumina and on Li' -impregnated alumina layers. The results obtained with nonmicel-lar (i.e. water) and micellar (1% aqueous sodium dodecyl sulfate and cetyl trimethyl ammonium bromide) mobile phases have been compared. Addition of alcohol to micellar solutions improves the separation of amino acids. The mobility sequence of amino acids was almost identical in buffered and nonbuffered micellar mobile phases. The best TLC system for rapid separation of amino acids was plain alumina as stationary phase and 1% cetyl trimethyl ammonium bromide in water-butanol, 95 + 5, as mobile phase. With this system, L-proline was selectively separated from other aliphatic and aromatic amino acids. The lower limit of detection of amino acids has been determined, and the semiquantitative determination of DL-aspartic acid has been attempted. A linear relationship was established between amino acid Rf value and the number of carbon atoms in the amino acid molecule. The proposed method is suitable for identification of L-proline, L-hydroxyproline, DL-alanine and oL-phenylalanine in spiked samples of liver, stomach, and human blood.

1 Introduction

Micellar liquid chromatography with mobile phases containing surfactant ions above their critical micellar concentration (CMC) to control the retention of a solute has been the focus of numerous studies [1-5] since it was first pro-

A. Mohammad and V. Agrawal, Analytical Laboratory, Department of Applied Chemistry, Faculty of Engineering, Aligarh Muslim University, Aligarh 202002, India.

posed hy Armstrong and coworkers in 1977 [6]. The multiplicity of interactions (hydrophobic, electrostatic, and hydrogen-bonding) associated with micellar systems provide unique possibilities of separation of structurally similar solutes. Micelles are capable differentially solubilizing and binding a variety of solutes leading to their potential usefulness for achieving several new separations, including the resolution of optical isomers. The preferential binding (or partitioning) of compounds to micelles has been investigated by several chromatographers wishing to understand solute-micelle interactions in the mobile phase [7-11]. The solubilization properties of a micellar system for a specific solute can further be modified by addition of appropriate additives, for example organic solvents, ionic salts, cyclodex-trins, and ion-pairing and complexing agents. Organic additives (alcohols, ketones, acetonitrile) are used to modify solvent strength and, thereby, influence solute retention behavior.

Thin layer chromatography (TLC), an efficient and inexpensive technique, has been widely used for the separation of organic and inorganic substances with a variety of combinations of nonmicellar mobile phases and stationary phases of different polarities [5,12-15]. Because of the biological, medicinal, and pharmaceutical importance of amino acids their analysis has been a subject of importance for analytical scientists. Amino acids such as lysine, threonine, phenylalanine, tryptophan, etc. are used extensively in medical practice and are also used as food additives in poultry farming and cattle breeding. Several workers have resolved complex mixtures of amino acids on silica gel [16-18], alumina [19-21], cellulose and cellulose derivatives [22,23], polyamide [24], chitin and chitosan [25,26] and C,g-bonded [27] layers by use of mixed aqueous-organic mobile phases containing, usually, alcohols, ketones, amines, acetic acid, or acetonitrile as one of the components. Fewer studies

Journal of Planar Chromatography VOL. 13. SEPTEMBER/OCTOBER 2000 365

Mice((ar TLC of Amino Acids

have, however, been performed on the separation of amino acids by use of micellar systems. It is, therefore, worth exploring the possibihty of utihzation of micellar mobile phases in TLC analysis of amino acids.

This report is an attempt in this direction. We have developed a simple and rapid TLC method for some important separations, including that of proline from hydroxyproline on alumina layers developed with the hybrid mobile phase micelle-water-butanol. Proline and hydroxyproline have previously been separated by TLC [28] on silica-coated glass fiber sheets with 2-propanol-water, 7 -I- 3, as mobile phase. The separated spots were detected by spraying with ethano-lic ninhydrin reagent and by autoradiography. As far as we are aware, TLC on alumina with micellar mobile phases has not been investigated for the separation of amino acids; this study thus has practical and theoretical importance.

2 Experimental

2.1 Chemicals and Reagents

The amino acids studied were: glycine (Al), L-hydroxypro-line (A2), DL-alanine (A3), DL-serine (A4), L-proline (A5), DL-valine (A6), oL-threonine (A7), L-cysteine hydrochloride (A8), L-leucine (A9), DL-isoleucine (AlO), DL-nor-leucine (All), DL-methionine (A12), L-tyrosine (A13), DL.-tryptophan (A14), t-cystine (A15), DL-phenylalanine (A16), 3-(3,4-dihydroxyphenyl)-DL-alanine (A17), L-ornithine monohydrochloride (A18), L-lysine monohydrochloride (A19), L-histidine monohydrochloride (A20), L-arginine monohydrochloride (A21), DL-aspartic acid (A22), L-glu-tamic acid (A23) and DL-2-amino-A'-butyric acid (A24).

The amino acids were obtained from CDH (India), as were aluminum oxide 'G', kieselguhr 'G', propanol, ninhydrin, and cetyl trimethylammonium bromide. Sodium dodecyl sulfate, methanol, butanol, and silica gel 'G' were from Qualigens (India), and pentanol from Fluka (Buchs, Switzerland ). All reagents were analytical-grade.

Test solutions (1%) of the amino acids were prepared in double distilled water.

2.2 Chromatography

The adsorbents investigated were aluminum oxide 'G' (S,), alumina 'G' impregnated with different concentrations (1%, 3%, 5%, 10%) of aqueous LiCl (S,), silica gel 'G' (S,), cellulose (S4), and kieselguhr 'G' (S,). The surfactant containing mobile phases used are listed in Table 1.

2.2.1 Preparation of TLC Plates

Plain Thin-Layer Plates

Adsorbent and water in the ratio 1:3 were mixed with constant shaking until homogeneous slurries were obtained. Slurries were applied as 0.25 mm layers to 20 cm x 3 cm glass plates by means of a Toshniwal (India) applicator. The plates were first dried at room temperature and then activated at 100 ± 5°C by heating in an electrically controlled oven for 1 h. The activated plates were stored in a closed chamber at room temperature until used.

Impregnated Alumina Thin-Layer Plates

Alumina and 1-10% aqueous LiCl solution in the ratio 1:3 were mixed with constant shaking for 5-10 min. Plates were prepared from the resulting slurry as described above.

2.2.2 Procedure

Test solutions (ca 10 |aL) were spotted on the plates by means of a micropipet and the plates were developed by the ascending technique in 24 cm x 6 cm glass jars; mobile phase ascent was 10 cm for all developments. When development was complete the plate was withdrawn from the glass jar, dried at room temperature, and sprayed (glass sprayer) with a freshly prepared solution (0.4%) of ninhydrin in acetone. All the amino acids except L-proline appeared as dark violet spots on heating the TLC plates for 15-20 min at 90-100°C. The spot of L-proline was yellow. The spot

Table 1 The mobile phases used.

Code Composition

M,

M;

M, M„ M, M, M,

MM

1 % aqueous CTAB-methanol 1% aqueous CTAB-propanol 1 % aqueous CTAB-butanol 1% aqueous SDS-methanol 1% aqueous SDS-propanol 1% aqueous SDS-butanol 1% aqueous SDS-pentanol 1% CTAB in pH 2.3 buffer'"-butanol 1% CTAB in pH 5.7 buffer'-'-butanol 1% CTAB in pH 9.9 buffer"-butanol \% CTAB in pH 11.8 buffer'-'-butanol

20 + 80, 50 -I- 50, 80 + 20, 95 + 5 50 -I- 50, 70 + 30, 80 + 20, 95 -t- 5 95 + 5, 98 + 2 20 -I- 80, 50 + 50, 80 + 20 50 -1- 50. 70 + 30, 80 + 20 95 + 5, 98-1-2 95 + 5, 98 + 2 95 -I- 5 95 -H 5 95 + 5 95 -H 5

' Piiosphate-boric acid buffer.

3 6 6 VOL. 13. SEPTEIVIBER/OCTOBER 2000 Journal of Planar Chromatography

Micellar TLC of Amino Acids

of DL-phenylalanine appeared as a blue fluorescent spot on inspection under UV light. /?L (^F of leading front) and Rj (Rf: of trailing front) values for each spot were determined and the 7?p value was calculated from /?p = (7?L + ^T) /2 .

The limits of detection of the amino acids were determined by successively reducing the amounts of amino acids applied to the plates until no spot was detected. The minimum amounts of amino acids detectable on the plates were taken as the limits of detection.

To investigate the separation of L-proline, L-hydroxyproline, and impurities, equal amounts were mixed and 20 ^L of the resulting mixture was loaded on the plates. These were then developed, the spots were detected, and the R^ values of the separated amino acids were determined.

To separate and identify amino acids in biological samples human blood, liver, and stomach were separately spiked with mixtures of L-proline and L-hydroxyproline, of L-proline and DL-alanine, and of L-proline and DL-phenylalanine. TLC was performed with 10 |iL sample and the separated amino acids were detected on TLC plates. The samples were prepared as follows.

Liver and Stomach

The tissues (10 g) were submerged in cone. H2SO4 overnight and then digested by addition of cone. HNO3 until the solution became clear. The contents were washed thrice with distilled water and saturated ammonium oxalate solution (25 mL) was added. The solution was diluted with water to the required volume.

Blood

The sample was treated with a mixture of 10% aqueous NaOH and sodium tungstate, and a small amount of H2SO4 was added to destroy the precipitate. The contents were filtered and the filtrate was used for analysis.

Semiquantitative Estimation of DL-Aspartic Acid

Standard solutions of oL-aspartic acid (0.5-2%, 0.01 mL) were spotted on alumina layers and the plates were developed with 1% aqueous CTAB-butanol, 95 -I- 5. After detection, the spots were copied from the plates on to tracing paper and the area of each spot was calculated.

3 Results and Discussion

Alumina was selected for TLC separation of aliphatic or aromatic amino acids because: (i) The pH of the alumina slurry was ca 7.0 and at this pH most of the aliphatic amino acids exist as zwitterions; (ii) The hydroxyl (OH') and oxide (O"') groups both act as active sites on the surface, responsible for selective chromatographic separations; (iii) alumina undergoes a cation-exchange reaction:

AI2-O-H+ -I- Li+ ^=^ Al2-0-Li+ -I- H+

when treated with aqueous solution of lithium chloride; and (iv) the rigid structure of alumina undergoes little swelling

or shrinking in water or solutions containing electrolyte and organic modifiers.

To select the most appropriate surfactant we used 0.1-2% aqueous solutions of SDS (anionic) and CTAB (cationic) surfactants. This concentration range was selected to keep the concentrations of the surfactants below, near, or above the critical micellar concentrations (CMC)-reported to be 0.24% and 0.46% for SDS and CTAB, respectively [29]. Results summarizing the effects of surfactant concentration on the mobility of the amino acids on alumina layers are shown in (Figures la, b). It is apparent that SDS and CTAB have different effects on the mobility of amino acids. The mobility of amino acids when SDS was used was always higher than for use of CTAB, possibly because of the micellar structural differences resulting from charge, head-group size, and steric requirements. The positive charge residing on the quaternary nitrogen atom of CTAB interacts strongly with the carboxylate ion of amino acids and hence amino acids are more strongly retained by CTAB adsorbed on alumina surface. Because use of CTAB resulted in faster development than use of SDS-the time for 10 cm ascending development with 1% CTAB was 5 min compared with 16 min for 1 % SDS, the former was preferred for detailed study.

It is assumed that at this concentration CTAB is mostly present as micelles. To examine the role of mobile phase pH, amino acids were chromatographed with 1% CTAB solution prepared in buffered solutions of different pH (2.3, 5.7, 9.9, and 11.8). This range was selected because cationic surfactants are generally stable at both acidic and alkaline pH [30]. The results obtained with 1% CTAB at pH 5.7 were better in terms of detection clarity and spot compactness than those obtained at other pH values, and comparable with those obtained with 1% nonbuffered aqueous CTAB (pH « 6.9). 1% aqueous CTAB (nonbuffered) was, therefore, used as mobile phase in subsequent examination of the suitability of alumina as an adsorbent for the TLC separation of amino acids.

TLC of amino acids on alumina with water and aqueous surfactant (1% SDS or CTAB) solution result in dramatically different retention sequences of the amino acids and this provide new opportunities for separating these compounds with micellar mobile phases. The results obtained with nonmicellar and micellar (1% SDS or CTAB) aqueous mobile phases are compared in Figure 2, in which AR^ values, = 7?p with 1% SDS or CTAB as mobile phase - R^ with water as mobile phase, have been plotted. The positive and negative A/?p values are indicative of the significant influence of the micellar systems on the mobility of almost all the amino acids investigated. Positive ARj: values (faster mobility) for amino acids A,, A,, A,, A,5, and A|s and negative ARp values (slower mobility) for A4, A„ A^, and A-,, when micellar mobile phases are used show that alumina is more selective (more strongly sorbing) for amino acids A4, A5, A|i„ and A23 when micellar mobile phases are used, irrespective of the nature of the surfactant. The mobility of some neutral amino acids (A,,, A7, Ai,„ A,j, A,,, and A14) is.



Q1

• Glycine L-hydroxyproline - 4 - DL-threonine - • - L-arginine monohydro-

1 ': chloride " DL-2-amino-W-butyric acid

Q5

I T

Q8

Q1

• L-glutamic acid

- L-ornithine mono-hydrochloride

- DL-tryptophan

Q5 0+-Q1

L-proline DL-methionine DL-norleucine

Q5

1-

Q8

L-histidine mono-- hydrochloride

• L-cysteine hydrochloride

0 ^

Q1 Q5

L-phenyl alanine

3-(3,4-dihydroxyphenyl)-DL--alanine

1

1 -

Q8^

L-hlslidine mono-hydrochloride

• L-cysteine hydrochloride

Q1

• DL-valine-#- L-leucine-^ L-lysine monohydro-chloride

05 1

CTAB concentration (%)

• DL-phenyl alanine

• 3-(3,4-dihydroxyphenyl)-DL-alanine

0 » =

Q1 Q5

Figure 1a

Effect of the concentration of CTAB on the mobility of amino acids on alumina.

3 6 8 VOL. 13. SEPTEMBER/OCTOBER 2000 Journal of Planar Chromatography


^l -•-DL-alanine - i -L-pro l ine -*-DL-isoleucine

02\

08

- t -

06*'

02

n' '

DL-aspartic -•-L-glutamic -A- DL-2-amino-W-butyric acid acid acid

r——• • 01 05 01 05

1

QB4

DL-meth ionine • L-ornithine m o n o hydrochloride

DL-phenylalanine

01

• DL-phenyl alanine

L-ornithine monohydro-chloride

05

DL-serine - * - DL-threonine

« •

'' ^ -'*- DL-valine

! . 08 L-cysleine hydrochloride

• L-histidine monohydrochloride

•3-(3,4-dihydroxyphenyl)-DL-alanine

05 1

SDS concentration (%)

• Glycine L-leucine DL-norleucine

05 1 SDS concentration (%)

Rgure lb

Ettect ot the concentration of SDS on the mobility ot amino acids on alumina.

however, strongly influenced by the nature of the surfactant used. For these amino acids A/?p is positive when SDS is used and negative A/?p when CTAB is used. Ajo, a basic amino acid, behaves identically in both type of micellar

mobile phase and the acidic amino acids (A22, A23, and A24) have similar ARp values in mobile phases containing both SDS and CTAB, i.e. are least effected by the nature of the surfactant. The differential migration of the amino acids



•a*

-06

-as

Figure 2

AninDackb

Plots of ABf values for the amino acids: • • • • • , A«p = (Bp witti 1 % CTAB - R^ with water); — • — , Aflf = (Rp with 1% SDS - flp with water).

depends on the extent of their partitioning between the (micellar or nonmicellar) mobile phase and the stationary phase. Of the basic amino acids, the effect of the surfactant was moderate for A^ and A,,,; the mobilities of Anj and A2, were almost identical for mobile phases containing SDS or CTAB.

To modify the retention of amino acids on the alumina layer, hybrid mobile phases comprising micelle {1 % CTAB)-water-alcohol (methanol, propanol, or butanol) were also used, and found to result in better chromatographic performance than aqueous micellar mobile phases. The /?p values of amino acids were determined for micelle-water-alcohol mobile phases containing 1% aqueous CTAB and 5, 20, 50, or 80% (v/v) methanol, 5, 20, 30, or 50% (v/v) propanol, or 2 or 5% (v/v) butanol. With all these mobile phases R^ decreased (i.e. k, the capacity factor, increased) as the concentration of alcohol was increased. Representative plots are shown in Figure 3. The best separations were usually obtained by use of mobile phases containing 1% CTAB in 95:5 water-alcohol. It seems that alcohols modify the retention of amino acids on alumina by reducing the adsorption of the surfactants by the alumina. The micelle-water-alcohol mobile phase containing 5% butanol proved the best. Thus, moderately polar alcohols (e.g. butanol) enable better separations of amino acids in micellar systems because their association with the core of the micelles is stronger than that of more polar alcohols (e.g. MeOH), which are assumed to be present in the outer regions of the core.

Differences between Rf values measured for 1 % CTAB in water-butanol, 95 + 5, as mobile phase and /?p values mea-

A 2 A 3 A 4 A 5 « A ; A B « A 1 0 II A12 A13 A14 A15 AW A17 A18 Alfl A20 A21 A22 A23 A34

ArriiovadB

A1 A2 A3 A4 A5 * e A7 A9 AlO Al l A12 A13 A14 A15 Ate A17 AIB A18 A20 A21 A22 A23 A24

Amino KXIS

Figure 3

IMobllity of amino acids on alumina developed with 1 % nonbuffered CTAB containing different concentrations of methanol (A: —•— = 5%, - * - - = 20%, — • — = 50%, - - = 80%), propanol (B: —m— =5%, -•*•• = 20%, — • — = 50%, - - = 80%), and butanol (C: —•— = 2%, • • • • • = 5%).

sured for water or water-butanol, 95 + 5 are plotted as A/?p values in Figure 4a. Amino acids A,, A,,, A,,, and A24 are more strongly retained (positive A/?p values) by alumina when water is used as mobile phase than when surfactant is added to the water-alcohol mixture; for all the other amino acids except A,, the opposite retention behavior (negative A/?p values), indicating higher mobility when water is used as mobile phase. An remained at the origin (i.e. did not migrate, Rp = 0.0), irrespective of the nature of mobile phase (micellar, nonmicellar, water-alcohol or water-alcohol with added surfactant). Thus A,, can be selectively separated from several amino acids. It is evident from Figure 4b, a plot of the differences (ARp) between R^ values measured with water-butanol, 95 -I- 5, as mobile phase and

A) B)

i M /k' « - , « S ^ « ^ e ' A H Ai-Mi/M/^5 MS'A17 MS M9 A » A?1 ^ , A 2 3 A34

-0.4 J

Figure 4

Plots of AHp values for the amino acids. A: _ , _ , Aflp = (RpWith 1 % CTAB in water-butanol, 95 + 5 - flp with water-butanol, 95 + 5); - - * • - , Aflp = (Rp with 1 % CTAB

in water-butanol, 95 -i- 5 - fl^ with water). B: — • — , AHp = (Rp with water-butanol, 95 + 5 - Rp with water).

3 7 0 VOL. 13. SEPTEMBER/OCTOBER 2000 Journal of Planar Chromatography


Rf values measured with water alone, that the presence of butanol in water-alcohol mobile phases reduces the mobility of most of amino acids on alumina layers (negative A/?F values).

TLC of amino acids with 1% CTAB in water-butanol, 95 + 5, at pH 5.7 was comparable with that obtained with unbuffered mobile phase (Figure 5); this has been used to achieve important separations of L-proline from aliphatic (glycine, L-hydroxyproline, and DL-alanine) and aromatic (oL-trypto-phan and DL-phenylalanine) amino acids. It is evident from Table 2 that separation of L-proline from other amino acids with by use of a selective micellar mobile phase can be achieved only on alumina layers and not on silica, cellulose, and kieselguhr. The TLC data listed in Table 3 show that all such separations could be achieved in the presence of acidic (carboxylic acids) and basic (aromatic amines) impurities. Compared with the R¥ values measured for the pure single amino acids, small changes were observed when mixtures of amino acids were chromatographed with or without impurities. L-proline always moved faster (k = 0.36-0.42) than glycine, hydroxyproline, alanine, phenylalanine, or tryptophan {k = L0-L5). Similarly, DL-norleucine, a neutral amino acid was successfully separated from acidic (oL-aspartic acid) and basic (L-arginine monohydrochloride) amino acids in the presence of heavy metal ions (Table 4). Cu'" is the only metal ion with an adverse effect on the separation.

The linear relationship between R^ and the number of carbon atoms in glycine (C2H5NO2), DL-alanine (C3H7NO2), DL-valine (CsHuNOj), and L-leucine (CsH^NOj) (Figure 6) shows that mobility increases as the number of carbons, or the size of the molecule, increases.

To demonstrate the utility of Li'^-impregnated alumina, amino acids were also chromatographed on LiCl impreg-

A1 A2 M A4 A5 A8 A7 Aa Aa AIO A l l A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A2<

Amino adds

Figure 5

Mobility (Rp values) of amino acids on alumina developed with buffered and non-buffered mieelle-water-butanol mobile phases: » , 1% CTAB In water-butanol, 95 + 5; • • • • • 1% CTAB in pH 5.7 buffer-butanol, 95 + 5.

Noof caibon atoflns

Figure 6

Effect on amino acid mobility of the number of carbon atoms in the molecule: C , glycine (CjHsNOj); Cj, DL-alanine (C3H,N0j); C5, oL-valine (CsHiiNOz); Cj, L-leuclne (CH.jNG:).

nated layers with 1% CTAB in water-butanol, 95 + 5, as mobile phase. From the LiCl concentration range investigated (0.01-10%), the optimum concentration affording compact amino acid spots was 3% LiCl. The A/?p values (= Rf on plain alumina minus R^ on alumina impregnated with 3% LiCl) presented in Figure 7 show how the selectivity of alumina is altered by impregnation with LiCl. The positive A/?p values obtained for most of the amino acids

Table 2

Mobility (RF value) of amino acids on different stationary phases with 1% CTAB in water-butanol, 95 + 5, as mobile phase.

Amino acid ^ F

Silica Cellulose Kieselguhr Alumina

DL-Aspartic acid L-Glutamic acid DL-2-Aminobutyric acid L-Histidine monohydrochloride L-Lysine monohydrochloride L-Ornithine monohydrochloride L-Cystine L-Proline DL-Alanine DL-Phenylalanine DL-Valine L-Cysteine monohydrochloride DL-Threonine DL-Isoleucine DL-Serine Glycine

' ' Not detected.

0.97 0.95 0.97 0.77 0.48 0.53 ND"' 0.75 0.90 0.82 0.92 0.90 0.97 0.87 0.97 0.92

0.97 0.95 6.95 0.97 0.97 0.95 ND 0.92 0.95 0.95 0.92 0.95 0.95 0.90 0.92 0.92

0.95 0.92 0.95 0.95 0.95 0.95 ND 0.97 0.92 0.90 0.95 .0.92 0.92 0.95 0.95 0.92

0.08 0.14 0.64 0.30 0.35 0.27 ND 0.86 0.58 0.54 0.62 0.17 0.22 0.70 0.31 0.34



Table 3

TLC data (separation factors, a"', resolution, f?s'', and differences between Hp values, Aflp"') for the separation of L-proline from OL-tryptophan,

from DL-phenylalanine, from glycine, from L-hydroxyproline, and from oL-alanine on alumina with 1 % CTAB In water-butanol, 95 + 5, as mobile

phase, in the presence of acidic and basic organic impurities.

Separation

L-proline-DL-tryptophan

L-proline-DL-phenylalanine

L-proline-glycine

L-proline-L-hydroxyproline

L-proline-DL-alanine

Data

a

Rs ARy a

Rs ARf a

Rs ARf a

R. ARf a

Rs A/?p

Without

4.9 2.9 0.37 3.3 2.4 0.27 4.2 3.0 0.34 3.6 2.3 0.24 2.6 2.4 0.22

impurity Acetic acid

3.09 2.1 0.27 2.5 2.1 0.21 4.8 2.8 0.35 3.7 1.7 0.21 2.42 1.4 0.20

Formic acid

4.2 3.0 0.34 2.2 1.6 0.20 3.2 3.0 0.34 2.7 1.9 0.19 2.3 2.0 0.20

Added Aniline

3.8 3.1 0.32 2.2 2.0 0.20 3.0 3.3 0.33 3.6 1.8 0.21 2.3 2.0 0.20

impurity: 3-Chloro aniline

4.2 3.0 0.35 2.5 1.6 0.22 3.4 2.0 0.29 3.5 1.7 0.22 2.3 1.7 0.21

a-Naphthyl-amine

3.03 3.3 0.35 2.6 1.6 0.23 4.1 2.7 0.33 2.7 1.8 0.20 2.4 1.4 0.20

"' ARf is the difference between the Rj, value of L-proline and that of the amino acid from which proline is being separated.''' a = kjk„ where fc, is the capacity factor of proline and k^ is the capacity factor of the amino acid from which proline is being separated ( = (1 - /?F)//?P). • /?s = AA7[0.5(rf, + t/,)], where AA" is the center-to-center distance between the separated spots, d^ is the diameter of the proline spot, and rfi is the diameter of the spot of the amino acid from which proline is being separated.

Table 4

Rp values for the separation of oL-norleuclne, cL-aspartlc acid, and L-arginine monohydrochlorlde from their mixture in the absence and pres

ence of heavy metal cations on alumina developed with 1 % CTAB in water-butanol, 95 + 5.

DL-aspartic acid L-arginine monohydrochloride DL-norleucine

Without impurity Ag* Cu2+ II 2+

Or \&* W* Co^* Cd" Pb'* Hg^-Zn^^ Fe'+

Cd + Zn -

0.08 0.06 0.06 0.07 0.06 0.06 0.07 0.07 0.06 0.07 0.05 0.05

0.010 0.014

0.40 0.37 0.30 0.31 0.31 0.38 0.36 0.34 0.36 0.35 0.32 0.38

SD of Revalue (n = 5) 0.010 0.002

0,64 0.58 0.39 0.60 0.51 0.61 0.59 0.62 0.61 0.59 0.51 0.58

0.014 0.075

indicate that Li-impregnated alumina is more selective (more strongly adsorbing) for amino acids than plain alumina. Thus, impregnated alumina can be used to achieve additional separations (Table 5). Because longer development times were required for the impregnated layers, however, we subsequently focused our attention on the use of nonim-pregnated alumina layers to achieve more rapid separations.

The data listed in Table 6 show that separation on alumina layers can be successfully used to identify L-proline, L-hydroxyproline, DL-phenylalanine, and DL-alanine in biological samples.

The limits of detection of some of the amino acids on alumina layers were determined using ninhydrin as chromo-genic reagent. The lowest amount detectable [|ig] of some

372 VOL. 13. SEPTEMBER/OCTOBER 2000 Journal of Planar Chromatograptiy


amino acids is (in parentheses): oL-aspartic acid (16.0), L-arginine monohydrochloride (32.0), DL-isoleucine (24.0), L-cysteine hydrochloride (32.0), DL-threonine (24.0), L-histi-dine monohydrochloride (48.0), oL-serine (32.0), glycine (48.0), L-glutamic acid (32.0) and oL-phenylalanine (16.0). When the ninhydrin-amino acid complexes were inspected under UV illumination the phenylalanine-ninhydrin complex was found to have light blue fluorescence; this phenomenon was utilized to detect phenylalanine (14 |ag), The limit of detection of phenylalanine under UV light in the presence of ca fourfold excess of other amino acids was (in parentheses): DL-aspartic acid (48 |ig), L-arginine monohydrochloride, L-cysteine hydrochloride, or DL-methionine (56 |ig), and DL-threonine (55 ng).

In addition to qualitative analysis, quantitative evaluation

of amino acids is often required. We attempted semiquantitative determination of amino acids by measurement of the spot area and obtained a linear relationship (i.e. analogous with y = mx + c) between spot area and amount spotted for DL-aspartic acid in range 50-200 \ig (Figure 8). The accuracy and precision were ca ±15%. a-Tocopherol [31], cations [32], and anions [33] have previously been semi-quantitatively determined by use of similar relationships.

Although the more efficient and highly instrumental GC and HPLC are available for analysis of amino acids, TLC is especially suitable for economical routine analysis, because it enables the monitoring of several compounds simultaneously on a single plate and enables side-by-side comparison of substances of identical nature.

Figure 7

Plot of ARp, = Rp on plain alumina - R^ on alumina Impregnated wHh 3% LICI, for the amino acids.

Aapanic add ooootBtntiaa (%)

Figure 8

Calibration plot of spot area against amount of oL-aspartic acid In spot.

Table 5

Experimentally achieved separations of amino adds on plain alumina and on Impregnated alumina layers developed with 1 % CTAB in

water-butanol, 95 + 5, as mobile phase.

Stationary phase Separation {R^)

Plain alumina plates

Impregnated alumina plates^'

L-Hydroxyproline (0.66) from DL-serine (0.23), glycine (0.36), L-arginine monohydrochloride (0.31), L-histidine (0.30), and L-lysine (0.3) L-arginine monohydrochloride (0.31) from DL-2-amino-«-butyric acid (0.68), and DL-valine (0.65) L-proline (0.80) from glycine (0.34), DL-aspartic acid (0.10), glutamic acid (0.13), L-ornithine monohydrochloride (0.28), DL-methionine (0.48), and DL-serine (0.31) L-leucine (0.77) from glycine (0.34) and L-histidine monohydrochloride (0.30) DL-2-amino-n-butyric acid (0.64) from DL-aspartic acid (0.10) and L-ornithine monohydrochloride (0.28)

' These separations are not possible on plain alumina layers.

Table 6

Identification and separation of L-prollne from OL-aianine, of L-proline from oL-phenyiaianine, and of L-proline from L-hydroxyproiine from spiked

biological samples.

Sample L-proline-DL-alanine

Separation of amino acid pair (Rp) L-proline-DL-phenylalanine L-proline-L-hydroxyproline

Liver Stomach Blood

(0.80)-(0.59) (0.83)-(0.62) (0.80)-(0.59)

(0.80)-(G.56) (0.81)-(0.59) (0.82)-(0.59)

(0.84)-(0.63) (0.86)-(0.66) (0.85)-(0.65)

Journal of Planar Chromatography VOL, 13. SEPTEMBER/OCTOBER 2000 373


References

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Ms received: September 21, 2000 Accepted by SN: September 29, 2000

374 VOL. 13. SEPTEMBER/OCTOBER 2000 Journal of Planar Chromatography

Separation Science and Technology An Interdisciplinary Journal of Methods and Underlying Processes

Editor: STEVEN M. CRAMER, iserwann Department of Chemical Engineering. Rensselaer Polytechnic Institute. Troy. NV 12t80 phone fax: 518-377-0131 e-mail: [email protected]

March 26, 2001

Dr. Ali Mohammad Analytical Research Laboratory Dept. of Applied Chemistry Faculty of Engineering Aligarh Muslim University Aligarh 202002 INDIA

Manuscript Number 1156 Title: Thin-Layer Chromatography of Aromatic Amines With Hybrid CTAB-Alcohol-Water Mobile Phase Separation of Indole from Diphenylamine andp-Dimethylaminobenzaldehyde Author (s): Mohammad, Agrawal Date Received: August 16,2000

Dear Dr. Mohammad:

I am pleased to inform you that your revised manuscript has been accepted for publication as a technical note in Separation Science and Technology.

Galley proofs wiD be sent directly to you as soon as they are available. Your prompt review and return of these page proofs will facilitate publication of your work.

Many thanks for contributing to Separation Science and Technology. I have enjoyed working with you and I look forward to receiving your next manuscript.

Sincerely vours. reiy vours, ^

Steven M. Cramer Editor

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