separation studies of organic and inorganic …ir.amu.ac.in/3652/1/t 5538.pdf · 2015. 7. 21. ·...
TRANSCRIPT
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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Applied Chemistry
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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)
Certificate
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Residence : "AL-RAIYAN" Iqra Colony, New Sir Syed Nagar, Aligarh-202002 (INDIA), 8 : 0571-408196
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
\-5\iL
10 - 15 cm
30 - 200 min
10
10 X 20 or 10 X 10 cm
5 ^mi
200 ^mi
12 nm
0.1-0.2 iL
3 - 6 c m
3 - 2 0 min
18 or 36
8
t 40-.
35- .
30-. VI e
.225' 19 O
320-a
^15
10-'
5--
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 '^In 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?'
< i 1 1 1
en ."H '3 08 O
a 08 en
>
<
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
REFERENCES
1. M. F. Borgerding, R. L. Williams, W. L. Hinze and F. H. Quina,
J. Liq. Chromatogr., 12 (1989) 1367.
2. G. M. Jain and J. Issaq. / Liq. Chromatogr., 15 (1992) 927.
3. M.G.Khaledi, Anal. Chem., 1 (1988) 293.
4. J. T. Ward and D. Karen, Surfactant Sci. Ser., 55,40 (1996) 517.
5. J. Sherma, Anal. Chem. JO (1998) 7R.
6. D. W. Armstrong and J. H. Fender, Biochem. Biophys. Acta, 75
(1977) 478.
7. D. W. Armstrong and G. Y. Stive, J.Am.Chem.Soc,105 (1983)
2962 and 6220.
^. M. Almgren and S. Swarup, J.Colloid Interface Sci., 91 (1983)
256.
9. A. S. Kord and M. G. Khaledi, Anal. Chem., 64 (1992) 1901.
10 D. W. Armstrong and K. H. Be, J.Liq.Chromatogr., 5 (1982)
1043.
11. K. Saitoh, Kagaku, 45 (1990) 884, Chm.,Abstr., 115 (1991)
63443k.
12. A. Mohammad and S. Tiwari, ^epi?. Sci. and Tech., 30 (1995)
3577.
13. A. Mohammad, M. Ajmal, S. Anwar and E. Iraqi, J. Planar
Chromatogr. - Mod. TLC, 9 (1996) 318.
14. B. Fried and J. Sherma (eds) Practical Thin Layer
Chromatography "A Multidisciplinary Approach", CRC Press,
Boca Raton, Fl (1996).
15. H.Wagner and S.Bladt (eds). Plant Drug Analysis , A Thin Layer
Chromatography Atlas, Springer, Berlin, Germany (1995).
16. R. Bhushan and V. Prashad, J.Chromatogr., A 736 (1996) 235.
88
17. R. Bhushan, 1. Ali and S. Sherma, Hiomcii. Chromalogr., 10
(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)
S5: Silica gel impregnated with a mixture of 2% aqueous CUSO4 and
3%Brij-35 (2:1, 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.
Preparation of TLC Plates:
(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.
3.3 RESULTS AND DISCUSSION
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)
S5: Silica gel impregnated with a mixture of 2% aqueous CUSO4 and
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.
Preparation of TLC Plates:
(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 (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.
3.3 RESULTS AND DISCUSSION
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
REFERENCES
1. C. C. Nolter, Chemistry of Organic Compounds, 3"* ed.; Co.
Philadelphia, (1965) 935.
2. S. Ruhemann, Trans. Chem. Soc, 99 (1911) 1486.
3. S. Moore and W. H. Stein, J. Biol. Chem., 176 (1948) 367.
4. E. Kawerau and T. Weiland, Nature, 168 (1951) 77.
5. D. W. Armstrong and Q. Terrill, Anal. Chem., 51 (1979) 2160.
6. M. Borgerding, W. L. Hinge, L. D. Stafford, G. W. Fulp and W.
C. Hamlin, Anal. Chem., 61 (1989) 1353.
7. J. G. Dorsey, M. T. De Elchegery and J. S. Landy, Anal. Chem.,
55 (1983)924.
8. A. Berthod and A. Roussel, J. Chromatogr., 449 (1988) 349.
9. H. L. Rosano and M. Clausse, Microemulsion Systems:
Surfactant Sci. Series. 24, Marcel Dekker: New York, (1987).
10. A. Berthod, D. Nicolas and M. Porthault, Anal. Chem., 62 (1990)
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.
4.3 RESULTS AND DISCUSSION
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
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116
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117
REFERENCES
1. D. K. Singh, and A. Darbari, Chromatographia, 23 (1987) 93.
2. R. S. Dhillon, J. Singh, V. K. Gautam and B. R. Chhabra,
J.Chromatogr., 435 (1988) 256.
3. K. C. Khulbe and R. S. Mann, Z. Anal. Chem.. 330 (1988) 642.
4. A. O. Kuhn and M. Lederer, J. Chromatogr., 440 (1988) 165.
5. R. B. Geerdink, J. Chromatogr., 445 (1988) 237.
6. M. C. Gennaro and E. Marlugb, Chromatographia, 25 (1988)
603.
7. R. Foster, Organic Charge Transfer Complexes, Academic Press
London, New York, (1969).
8. D. B. Parihar, S. P. Sharma and K. K. Verma, / Chromatogr.,
31(1967)120.
9. A. K. Dwivedy, D. B. Parihar, S. P. Sharma and K. K. Verma, J.
Chromatogr., 29 (1967) 120.
10. K. Yasuda, / Chromatogr., 87 (1973) 565.
11. K. Yasuda, J. Chromatogr., 60 (1971) 144.
12. K. Yasuda, J. Chromatogr., 72 (1972) 413.
13. A. Mohammad, M. Ajmal and S. Anwar, J. Planar
Chromatogr.-Mod. TLC, 4 (1991) 319.
14. M. Ajmal, A. Mohammad and S. Anwar, J.Planar Chromatogr.-
Mod. TLC,3 (1990)336.
15. F. Feigl Spot Test in Organic Analysis, Elsevier Scientific
Publiching, New York, 7*'' edn., 1966, 381 & 243.
16. S. A. Nabi, A. Mohammad and P. M. Qureshi, Bull Chem. Soc.
Jpn., 56 (1983) 1865.
118
17. M. R. Porter, Handbook of Surfactant, Published by Blackie
Academic and Professional, Glasgow G64 2NZ, 2"** edn., 1994,
37.
18. S. N. Shtykov, E. G. Sumia, E. V. Smushkina and N. V.
Tyurina, J. Planar Chromatogr.-Mod. TLC, 12 (1999) 129.
19. D. W. Armstrong and R. Q. Terrill, Anal. Chem., 51 (1979)
2160.
20. S. N. Shtykov and E. G. Sumina, J. Anal. Chem., 53 (1998)
446.
21. D. B. Parihar, S. P. Sharma and K. K. Verma, J.Chromatogr.,
31 (1967) 120.
22. R. T. Morrison and R. N. Boyd, Organic Chemistry Published
by Prentice-Hall of India, New Delhi, 6'*' ed., 1999, 824.
23. E. Papp and G. Vigh, J. Chromatogr., 282 (1983) 59.
24. C. Tanford, The Hydrophobic Effect, Wiley, New York, (1973).
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 (lO^iL) 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.
Procedure: For the determination of Rp value and limit of detection of
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.
For semiquantitative determination by spot-area measurement
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.
5.3 RESULTS AND DISCUSSION
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
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B 9
-VI
^nAm^ced S^efux/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
2 0.04 M Boric acid - 0.04 M phosphoric 50 : 50 : 14 7.0
acid - 0.24 M NaOH
3 0.04 M Boric acid - 0.04 M phosphoric 50 : 50 : 60 11.9
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.
For semiquantitative determination by spot-area measurement
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.
6.3 RESULTS AND DISCUSSION
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^\ Cd^Und 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 (Rj^OO)
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
«s
If)
I I
If)
o B O
I
3 B 03
.P
O • • •<
£
SD
(iUi3) lojB | o d s
153
REFERENCES
1. M.P Volynets, L.P. Kitaeva and A.P. Timerbaev, Zh. Anal. Khim., 41
(1986) 1989.
2. A. Mohammad and M. A. Majid Khan, J. Chromatogr., 642 (1993)
455.
3. Y. Tonogai and M. J. Iwaida, J. Food Prot., 44 (1981) 835.
4. A. Mohammad and J. P. S. Chahar, J. Chromatogr. A, 714 (1997) 373.
5. A. Mohammad and J. P. S. Chahar, J. AOAC International, 82 (1999)
172.
6. N. Fatima and A. Mohammad, Sepn. Sci. Technol., 19 (1984) 429.
7. J. K. Rozylo, D. Gwischamicz and I. Malinowska, Chem. Anal.
(Warsaw), 36(1991)279.
8. T. Shimizu, S. Jindo, N. Iwata and Y. Tamura, J. Planar Chromatogr.
Mod. - TLC, 7 (1994) 98.
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.
For semiquantitative determination by spot-area measurement
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.
7.3 RESULTS AND DISCUSSION
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)£e^i/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.
8.3 RESULTS AND DISCUSSION
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
T = Tailed spot (RL-RT> 0.3)
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 + H^O
(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
T = Tailed spot (RL-RT> 0.3)
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.
References
[1] A. Lohninger and F. Hamler, Drugs Exptl. Clin. Res. 18 (1992) 73-77.
[2] W. Gofkiewicz, M. Gadzikowska, J. Kuczynski, and L. Jiisiak, J. Planar Chromatogr. 6 (1993) 382-385.
[3] E. Taborska, H. Bochorakova, H. Paulova, and/ Dostal, Planta Med. 60 (1994) 380-381.
[4] C. Bugatti, M. L. Colombo, and F. Tome, J. Chromatogr. 393 (1987)312-316.
[5] J.A. Peny, T.H. Jupille, and L.J. Glanz, Anal. Chem. 47 (1975) 65A-74A.
[6] C.E Poole, S.K. Poole, W.PN. Fernando, T.A. Dean, H.D. Ahmed, and/.yl. Bemdt, i. Planar Chromatogr. 2 (1989) 336-345.
[7] E. Soczewimki and W. Markowski, J. Chromatogr. 370 (1996)
63-73.
[8] W. Markowski, J. Chromatogr. 635 (1993) 283-289. [9] P.V Colthup, J.A. Bell, and D.L. Gadsden, J. Planar
Chromatogr. 6 (1993) 386-393.
[10] W. Markowski, J. Chromatogr. 726 (1996) 185-192.
[11] H.D. Smolarz and M. Waksmundzka-Hajnos, J. Planar Chromatogr. 6 (1993) 278-281.
[12] T.H. Jupille and//I. Peny, J. Chromatogr. 99 (1974) 231-242.
[13] B. Szabady, M. Ruszinko, and Sz. Nyiredy, J. Planar
Chromatogr. 8 (1995) 279-283.
[14] T. Wawrzynowicz, K. L. Czapinska, and W. Markowski, J. Planar
Chromatogr. 11 (1998) 388-393.
[15] Sz. Nyiredy, C. A. J. Erdelmeier, and O. Sticker, in R.E. Kaiser (Ed.), Planar Chromatography, Vol. 1, Huthig, Heidelberg,
1986, p. 119.
[16] K. Glowniak, E. Soczewinski, and T. Wawrzynowicz, Chem.
Anal. (Warsaw) 32 (1989) 161-170.
[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.
212 VOL. 13. MAY/JUNE 2000 Journal of Planar Chromatography
TLC of Transition Metals with Surfactant-Modified Mobile Phases
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
Journal of Planar Chromatography VOL. 13. MAY/JUNE 2000 213
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
214 VOL. 13. MAY/JUNE 2000 Journal of Planar Chromatography
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
[i] U.A.Th. Brinkman. G. De Vries, and R. Kuroda, J. Chroma-togr. 85(1973) 187.
[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.
Journal of Planar Chromatography VOL. 13. MAY/JUNE 2000 215
TLC of Transition Metals with Surfactant-Modified Mobile Phases
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.
[4] A. Mohammad, M. Ajmal, S. Anwar, and E. Iraqi, J. Planar Chromatogr. 9(1996)318.
[5] J. Sherma, Anal. Chem. 70(12) (1998) 7R-26W.
[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 FH. Quina, J. Liq. Chromatogr. 12(8) (1977) 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, J. Chromatogr. 191 (1997) 780.
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[12] A. Szymanski and W. Szczepaniak, Chem. Anal. (Warsaw) 43
(1998) 617.
[13] T.A. Walker, J. Liq. Chromatogr. Relat. Technol. 19 (1996)
1715.
[14] J.S. Landy and/G. Dorsey, Anal. Chim. Acta 178 (1985) 179.
[15] A. Berthod, I. Girad, and C. Gonnet, Anal. Chem. 58 (1986)
1362.
[16] L.J. Cline-Love, J.G. Haharta, and/G. Dorsey, Anal. Chem. 56 (1984) 1132A.
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.
Journal of Planar Chromatography VOL. 13. SEPTEMBER/OCTOBER 2000 367
Micellar TLC of Amino Acids
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
Micellar TLC of Amino Acids
^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
Journal of Planar Chromatography VOL. 13. SEPTEMBER/OCTOBER 2000 369
Micellar TLC of 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
Micellar TLC of Amino Acids
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
Journal of Planar Chromatography VOL. 13. SEPTEMBER/OCTOBER 2000 371
Micellar TLC of Amino Acids
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
Micellar TLC of Amino Acids
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
Micellar TLC of Amino Acids
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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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