Paper No.

: 03 Chromatographic Techniques

Module : 02 Chromatographic Techniques- TLC, HPTLC, IEC

Principal Investigator: Dr Nutan Kaushik, Senior Fellow,

The Energy and Resources Institute (TERI), New Delhi

Co-Principal Investigator: Dr Mohammad Amir, Professor of Pharm. Chemistry,

Jamia Hamdard University, New Delhi

Paper Coordinator: Dr Abhilasha Shourie, Professor & HOD- Biotechnology, Manav

Rachna International Institute of Research and Studies, Faridabad

Content Writer: Dr. Nidhi Didwania, Associate Professor, Biotechnology, Manav

Rachna International Institute of Research and Studies, Faridabad

Content Reviwer: Dr Abhilasha Shourie, Professor & HOD- Biotechnology, Manav

Rachna International Institute of Research and Studies, Faridabad

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 Description of Module
Subject Name Analytical Chemistry / Instrumentation

Paper Name Chromatographic Techniques

Module Name/Title Chromatographic Techniques- TLC, HPTLC, IEC

Module Id 02

Pre-requisites Knowledge of Fundmental Chemistry

Objectives 1. To understand the fundamental concepts, principle, method, applications,

advantages and disadvantages of Thin Layer Chromatography.

2. To learn the fundamental concepts, principle, method, applications,

advantages and disadvantages of High Performance Thin Layer
Chromatography.

3. To understand the fundamental concepts, principle, method, applications,

advantages and disadvantages of Ion Exchange Chromatography.

Keywords Thin Layer Chromatography , High Performance Thin Layer Chromatography, Ion
Exchange Chromatography, Chromatogram, mobile phase,stationary phase

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 List of Contents:

 Introduction to Thin Layer Chromatography

 Concept, principle and method of Thin Layer Chromatography
 Applications of Thin Layer Chromatography
 Advantages and disadvantages of Thin Layer Chromatography

 Introduction to High Performance Thin Layer Chromatography

 Concept, principle and method of High Performance Thin Layer Chromatography
 Applications of High Performance Thin Layer Chromatography
 Advantages and disadvantages of High Performance Thin Layer Chromatography

 Introduction to Ion Exchange Chromatography

 Concept, principle and method of Ion Exchange Chromatography
 Applications of Ion Exchange Chromatography
 Advantages and disadvantages of Ion Exchange Chromatography

Introduction
Chromatography is used in analytical chemistry for analyzing and separating the compounds
from a sample mixture. Chromatography in general has two phases, a stationary phase and a
mobile phase. Confiding upon the character of material utilized as mobile phase and
stationary phase, chromatography can be of various types like thin layer chromatography,
adsorption chromatography, ion exchange chromatography, gas chromatography and affinity
chromatography etc.Planar Chromatography for instance TLC (Thin layer Chromatography)
and HPTLC (High Performance Liquid Chromatography) uses a flat (planar) stationary phase
for segregationas antithetical to column chromatography (e.g. GC, HPLC).

THIN LAYER CHROMATOGRAPHY

Thin layer chromatography is analogous to paper chromatography for identification,

segregation and purification of constituents of a mixture. Here ascending technique is used
like the paper chromatography.

Principle:

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 The chromatography technique involves the partition of components of a mixture to be
separated between the two phases which move with respect to each other. The two phases are
a fixed phase (solid/liquid) and a mobile phase (liquid). This technique is useful for
separation of lipids, amino acids and sugars etc.

Adsorbants:
Silica gel with binder gypsum, Alumina, cellulose powder, polyacrylamide etc.

Procedure:
Preparation of TLC plate (stationary phase): Generally glass plates of 5 x 20 cm and 20 x 20
cm are used. Plates are immaculate with chromic acid, detergents and water and then dried
completely. Silica gel paste (stationary phase) is disseminated on the plate by means of rod
made up of glass and then heated to evacuate moisture.

Solvent System (Mobile Phase):

Choice of the solvent system depends upon the nature of the mixture to be analysed.

For non-polar lipids following solvents are used:

For polar lipids following solvents are used:

About 100 ml of solvent is sufficient and it is put in a solvent chamber.

Application of the Mixture:

With the help of capillary tube, mixture to be analyzed is applied like a spot near one edge of
the plate.

Development of the Plate:

The plate is then dipped in the chamber of solvent and the spot containing the mixture
gradually starts moving up the plate and spreads near the top.

Marking of Spots:
Majority of the substances are not visible on the plate. For this purpose staining agents like
iodine or 50% analar grade sulphuric acid or minhydrin is sprayed on the plate. A fluorescent
compound is added to the adsortant that glows in ultraviolet light for easy identification.

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 Ninhydrin reacts with amino acids to give brown or purple colour. Iodine crystals added
release iodine vapours which make some substances brown. The spots become coloured and
are marked on the backside of the plate with the help of marking pen.

Identification:
It can be done by calculating the Rf value of different spots.

Rf value for a compound is constant using the same solvent. Normally a standard experiment
can be run along-with the unknown compound to locate its presence. It is used in evaluating
fatty acids, analyzing pesticides in water and food, in forensics, in recognition of medicinal
plants and their components.
Earliest definitive work in the field was done by Izmailar and Schraiber which involved the
separation of alkaloids. Though TLC is not very efficient, it has advantages of speed,
versatility and simplicity.

Chromatogram
m
Solvent Front
Distance travelled

Spots

Chromatogram
m

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 Figure 1: TLC plate

Applications, Advantages & Limitations:

In an organic chemistry, the reactions are qualitatively audited with Thin Layer
Chromatography (TLC). Sampling of the spotsis done by a capillary tube and spots are
locatedon the plate: a spot of originating material, a spot from the reaction concoction
(mixture), and a “co-spot” with the pair.

The analysis is qualitative, and it will show how many products are generated,if product has
appeared and starting material has disappeared.Thin Layer Chromatography’s unfortunately
from lower-temperature reactions may provide inaccurate conclusions, as the sample is
cautioned to room temperature inside the capillary. DIBALH reduction of ester to aldehyde is
one such reaction.

TLC is used in analysing fatty acids, disclosure of pesticides in contents such as in food and
water, in forensics, in identification of medicinal plants and their constituents. Though TLC is
not very efficient, it has advantages of speed, versatility and simplicity.

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

High performance thin-layer chromatography (HPTLC)is solitary approach based on the
adequate potential of thin layer chromatography (TLC). Thus it is the ultimate advanced
mode of instrumental TLC.HPTLC uses authentic methods for quantitative and qualitative
analysis and is regulated by an integrated software scaffold. It has the advantages of
thereliability, reproducibility and usefulness of generated data. Thus even in a fully regulated
environment, HPTLC is a method that accommodates entire quality requirements of present
analytical labs.
HPTLC maintains all advantages of the planar separation principle and initial costs for the
system and its maintenance further as the cost per sample still endures comparatively
low.One of the most valuable aspects in TLCis the possibility of visualization and analysis of
separated samples on the plate. In HPTLC modern techniques gives new dimension
generating and evaluating digital images.
PRINCIPLE
It involves the same theoretical principle of thin layer chromatography. The segregation may
proceed due to adsorption or partition or both phenomenon confiding upon nature of
adsorbents on the plates and solvent systems utilized for establishment.
METHODOLOGY
HPTLC may be used for both quantitative and qualitative analysis, optimization of analysis
timeand for segregation of two constituents as well as multicomponent natured mixtures.

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 HPTLC insists elementary knowledge about the sample nature, namely, polarity, structure,
stability, volatility and the solubility criteriafor analysis of drugs in multicomponent dosage
forms. Extensive trial and error operations are involved in the development of the method.
The most difficult problem is the choice of mobile phase and the point to start in
multicomponent analysis.
Silica gel is the most suitable and reasonable choice for all kind of drugs. Thus option of
stationary phase is entirely simple.For mobile phase optimization three level techniques are
carried out. In the first level we utilize precise solvents and then find some such solvents
having moderate segregation power for the drugs to be assessed. Second level
includesincreasing or decreasing the solvent strength using water or hexane for various
functions. In the third level inspite of neat solvents mixtures from the selected solvents of
first and second level are tried and these can be further optimized by the use of modifiers like
acids or bases.
Analytes are evaluated utilizingabsorbance mode or fluorescence mode. But,there is a need of
change of mobile phase or stationary phase or needs the aid of pre or post chromatographic
derivatization if the analytes are not detected completely. Meagerchange in mobile-phase
composition directs to a reasonable chromatogram having entire aspire peaks in symmetry
and well segregated and helps in optimization.The capacity of spot (analogous to peak
capacity in HPLC) can be enhanced by establishing the plate with two distinct solvents,
utilizing two-dimensional chromatography.Automation is effective in conquering the size
uncertainty and position of droplet when the sample is enforced to the TLC plate by hand.
Recentlypiezoelectric devices and inkjet printers are used for implementing the sample
approach to automation.
HPTLC is the utmost elementary separation approach today and can be considered a time
machine that can accelerate your work and permits to do variable things at a time usually
impossible with other analytical techniques.
The three main steps in HPTLC:
1. For sample analysis chromatogram layer volume precision and suitable position are
acquired by use of suitable instrument.
2. The mobile phase(solvent) immigrates the planned distance in stationary layer by
capillary action in this procedure sample separated in its components.
3. Separation trails are examined in densitometer with light beam in visible or ultra
violet region.

Features of HPTLC
1. Contemporary processing of standard and sample

2. Superioraccuracy and analytical precision and minimum demand for internal standard
3. Several analysts can work concurrently
4. Estimation of lower time and minimum cost per assessment
5. Lesser cost ofmaintenance
6. Elementary sample preparationand can shaft diverse natured samples
7. No previous treatmentas filtration and degassingis needed for solvents

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 8. Per samplelow consumption of mobile phase
9. Requirement of no intrusion from prior analysis - fresh stationary and mobile phases
for exclusive assessment - no contagion commenced
10. It is an open system and so visual detection is possible.
11. With the cooperation of post-chromatographic derivatization, non UV absorbing
compounds can be detected easily.

Applications of HPTLC
HPTLC is one of the convenient chromatographic techniques, which is utilized for the
recognition of constituents, identification and detection of impurities, and quantitative
evaluation of active substances.
HPTLC remains one of the most extensile, steady, and cost-efficient segregation approach
ideally suitable for the evaluation of botanicals and herbal drugs. Utilized with standardized
processes, it guarantees reproducible conclusions, an important element in the routine
identification of complicated fingerprints of pharmaceutical and plant extract products.
HPTLC rapidly gaining importance in several fields of science like cosmetics, forensic
analysis,environmental analysis, biochemistry, toxicology, pharmaceuticals analysisand
Pharmacokinetics studies, etc.

Advantage of HPTLC :
High-performance thin layer chromatography (HPTLC), an extension of TLC issimplest,
robust, efficient and rapid, device in quantitative analysis of compounds. HPTLC is a
interpretive approach based on TLC, but with improvementslike the use of higher aspect TLC
plates with exquisite particle sizes in the stationary phase,intended to allow quantitative
analysis of the compounds and enhance the resolution of the compounds to be segregated.
The segregation can be enhanced further by the imitated plate development, utilizing a multi
development device. As a result, HPTLC provideslesser LODs(Limit of Detection) alongwith
better resolution.
As analytes, herbs are extremely complex.and their analysis is very challenging for several
reasons. Even herbal developments such as extracts contain various compounds in
concentration that can cover numerous forms of magnitude. For example, chemical
composition of the herb is not absolutely known. For several of the Ayurvedic and Chinese
herbs, there are no entrenched methods of analysis available. The requisite of a fingerprint
determination can be effectively distinct from those for a quantitative evaluation of marker or
key compounds, although the herbal preparation segregated for fingerprints, but for
quantitative evaluation of maker compounds. It is essential to completely segregate those
compounds from all others.Herbal components that belong to appropriate distinct classes of
chemical compounds can generally create crisis in detection. With this in mind, TLC and
especially HPTLC can provide various advantages.

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 ION EXCHANGE CHROMATOGRAPHY

INTRODUCTION
In ion exchange chromatography (IEC)ionizable molecules are separated on the basis of their
total charge. The analogous form of molecules that would be crucial to segregate by other
approaches due to the charge possessed by the molecule of interest can be easily segregated
by changing buffer pH. IEC (Ion exchange chromatography) process is an important
analytical approach for the refinement and separation of ionic compounds, in combination
with partition, ion interaction and ion exclusion chromatography. It employs on charged
molecules viz. proteins, amino acids and nucleotides and related biopolymers of different
molecular sizes and nature. Ion chromatography segregation is based on electrostatic
interactions between polar and ionic compounds that are attached to support, ions etc. present
in the running solventused in chromatography.

Two distinct processes in the separation of ion chromatography are: exchange of ions due to
attractionand exclusion of ions due to repulsion betweencharged ionsin analyte.

Principle

Ion exchange has been considered as an important mode of ion chromatography. The
separation principle depends on thedevelopment of electrostatic bonds between the ion
exchange support sustaining the opposite charges and charged molecules. Bio molecules
exhibit various level of interactions with theelectrostatically charged mediumin
chromatography owed totheir divergence in characteristics ofcharge.

The widespread presenceof IEC (Ion exchange chromatography) has been raised in
contemporary period as the approach results in evaluating a variety of analytes in agriculture,
biotechnology,environment, pharmaceuticsetc.

SCOPE

Ion exchange chromatography envelops liquid chromatographic techniques and has been
defined by the range of solutes instead of specific combinations of detection and separation
modes. It is devised explicitly for the segregation of disparately charged compounds from
stationary and mobile phases. The development of IEC is prompt as it provides a reliable and
authentic method for the investigation of cations, anions, metal ions, amino acids, phenols,

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 nucleotides and alternative polar molecules. The technique has been strongly enforced
successfully for the evaluation of raw materials, conflicting impurities, ions and decadent
products, analysis of cleaning solutions of production equipments and various other
applications. Immense pertinence including average cost, tremendous performance,
influentialdetermining ability, hugesample handling properties and affluent range of conjoint
automation has contrived ion exchange chromatography as specific, significant and broadly
used among all liquid chromatographic technique. IEC is considered as mature and well
established approach for chemical, petrochemical industries, food and beverage industries,
pharmaceutical, clinical industries and metallurgical applications.

TYPES OF ANALYSIS
Fractionation of liable biological substances is done with the method of ion exchange and has
determined to be the prime suitable methods. From the establishment of the approach in the
1960s´ to the development of modern high performance media, ion exchange
chromatography has rejoiced a vital role in thepurification and separation of biomolecules
and assigned extremely to our perception of biological processes.

IEC can be used for many types of analysis.

Qualitative analysis of cations, anions,halides etc.

Quantitative analysis of cations, anions,halides etc.

Trace analysis and ultra trace analysis of heavy metals etc.

Ion exchange can be combined with other advanced techniques which work on different
aspects and parameters such as size (known as gel filtration), hydrophobic property (termed
as hydrophobic interaction chromatography or RPC) or biological activity (known as affinity
chromatography), etc.

The chromatography is subdivided further into cation exchange chromatography and

anion exchange chromatography:
i. The stationary phase exhibits a negatively charged functional group and therefore cation
exchange chromatography contains positively charged cationsviz, Phosphoric acid.

ii. Anion exchange chromatography exhibits negatively charged anions utilizing positively
charged functional group like quaternary ammonium cation.

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 Figure: 2Chromatogram depicting the 4 main stages of Ion Exchange Chromatography.
Protein A carries the same charge as the stationary phase and does not bind. Protein B
binds and is later eluted from the stationary phase

Applications of Ion-exchange Chromatography:

1. Ion exchange technique was used by Chargaff to determine the base composition of
nucleic acids.
2. Ion exchange technique is used extensively in water purification. Water is completely
deionised by exchanging hydrogen and hydroxyl ions using anion and cation exchangers.
Water purification machines work on this principle.
3. Ion exchange is extensively used in analysis of amino acids. Amino acid composition of a
protein can be determined with a machine called amino acid analyser which works on ion
exchange principle using a strong cation exchanger.
4. Ion exchange is used for the separation of many vitamins and organic acids.
5. It is generally used as a basic stride in protein purification.

Advantages of Ion exchange chromatography

One of the advantages of IEC is evaluation of both cationsand anions concerning poly
hydroxyl compounds, peptides, organic acids, metal ions, nucleotides, alcohols,
aminoglycosides etc. The technique has been auspiciously used in the analysis of deteriorated
products, impurities; active components etc at various stages of production steps in addition
with the evaluation of the cleaning solutions of production apparatus, desolated streams,
container compatibility including other utilizations. Ion exchange chromatography has

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 become one of the chief chromatography as compared to other liquid chromatographic
approaches:

 Average cost.
 Huge sample handling capability.
 Easy automation as well as scale-up.
 Dynamic resolving ability.
 Extensive applicability in addition with high through put application scheme and
broad applicability.
 It can be used at all the phases and proportions of purification.
 It is a non denaturing process.
 It can be maintained by altering the salt concentration, pH and the ion exchange
media.
 It provides high selectivity and can resolute molecules with little variations in charge.
 It can serve as intensifying step. A huge volume of diluted sample can be enforced to
a media and the absorbed proteins afterwards eluted in petite volume.
 Eco friendly technique as it deals with substances existing in water.
 Longer life of resin.
 Economical maintenance.

Disadvantages

Despite of broad use of IEC the mechanism of segregation has not completely been
enlightened. An extensive effort has been made to depict the ion exchange process
more theoretically. One of the major disadvantages of IEC technique is that it presents
no direct information on the situation occurring at the stationary phase surface as the
ion exchange symmetry is always resolute by the balance between the eluent and
solute interaction with the active site resins.

One of the essential disadvantages of ion exchange chromatography is that ion exchange
columns required to be loaded in low-salt buffers as binding to ion exchange resins depends
on electrostatic interactions between the stationary phase and proteins of interest. A buffer
exchange step is needed antecedent to ion exchange chromatography to carry out some of the
applications.Weak ion exchangers highly depend on pH and these resins rapidly lose capacity

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC
 and resolution although working out of their optimal pH range which is a limitation of ion
exchange chromatography

Major disadvantages are depicted as:

 Properties and nature of ion exchange resins.

 Chemicals & equipments are expensive.
 Turbidity should be maintained below 10ppm.
 Nature of ions exchanged.
 Substances viz. organic matter or resins can filthy effect to resin.

Analytical Chromatographic Techniques

Chemistry Chromatographic Techniques- TLC, HPTLC, IEC