CHROMATOGRAPHY

INTRODUCTION
Chromatography is a combination of two words;
Chromo – Meaning color
Graphy – representation of something on paper

DEFINITION
It is a physical separation method in which the components of a mixture are separated by differences in
their distribution between two phases, one of which is stationary (stationary phase) while the other
(mobile phase) moves through it in a definite direction . The substances must interact with the stationary
phase to be retained and separated by it .

CHROMATOGRAPHY TERMS

* Chromatogram: It is the visual output of the chromatograph.

* Chromatograph: It is equipment that enables a sophisticated Separation.

* Stationary phase (bounded phase): It is a phase that is covalently bonded to the support

particles or to the inside wall of the column tubing.

* Mobile phase: It is the phase which moves in a definite direction.

* Analyte (Sample): It is the substance to be separated during chromatography.

* Eluate: It is the mobile phase leaving the column.

* Retention time: It is the characteristic time it takes for a particular analyte to pass through the

system (from the column inlet to the detector) under set conditions.

* Eluent: It is the solvent that will carry the analyte.

* Retardation factor ( R ): Fraction of an analyte in the mobile phase of a chromatographic

system.

Dept. of Pharmacognosy, SJCPS, Kattappana

CLASSIFICATION
According to mechanism of separation
1. Adsorption chromatography
2. Partition chromatography
3. Ion-exchange chromatography
4. Affinity chromatography
5. Size-exclusion chromatography

ADSORPTION CHROMATOGRAPHY
It is a type of chromatography in which a mobile liquid or gaseous phase is adsorbed onto the surface of a
stationary solid phase. The equilibration between the mobile and stationary phase accounts for the
separation of different solutes.

TYPES:

THIN-LAYER CHROMATOGRAPHY
Principle:
The principle of separation is adsorption. One or more compounds are spotted on thin layer of adsorbent
coated on a chromatographic plate. The mobile phase solvent flows through it because of capillary action.
The components move according to their affinities towards the adsorbent in such a manner that the
component with lesser affinity towards the stationary phase travel faster and the components with more
affinity towards the stationary phase travel slower. In this way the components are separated on TLC
plate.
Instrumentation:
1. Chromatography jar
2. Capillary tube
3. Thin layer chromatography plate
4. Stationary phase
5. Mobile phase

Dept. of Pharmacognosy, SJCPS, Kattappana

 1. Chromatography jar: It is made of glass and has a lid on it. Jar maintains proper environment
that is required for separation.

2. Capillary tube: It is used to apply sample mixture on TLC plate.

3. Stationary phase: Adsorbents
Adsorbents: An adsorbent is a substance, usually porous in nature and with a high surface area that can
adsorb substances onto its surface by intermolecular forces.”
An ideal adsorbent: The Ideal adsorbent must fulfill the following requirements:
 Insoluble in mobile phase
 Inert to solutes (adsorptive)
 Colorless especially when work with colored mixtures
 Suitable particle size enough to give good separation and reasonable flow rate
Common adsorbents:
 Hydrated silica gel
 Silica gel
 Modified silica gel
 Alumina
 Kieselghur (Diatomaceous earth)
 Cellulose MN 300
 Cellulose microcrystalline

4. TLC plate: Borosilicate glass plates are preferred. Most commonly used sizes are;
 20 X 20cm

Dept. of Pharmacognosy, SJCPS, Kattappana

  20 X 10cm
 20 X 5cm
 Microscopic slides are also used.
5. Mobile phase: Mobile phase may be a single liquid or a mixture of liquids. Commonly used
mobile phases are; Methanol, Ethanol, Ethyl acetate, Diethyl ether, Acetone, Chloroform
Procedure:
1. Clean and dried chromatography jar is taken.
2. A paper impregnated in the mobile phase is applied to the walls to ensure that atmosphere of the
jar is saturated with solvent vapors.
3. Mobile phase is added to the jar at a length of 0.5-1cm from the bottom.
4. Jar is closed.
5. Equilibrium is allowed to be maintained.
6. Base line is marked on adsorbent.
7. Sample is applied on TLC plate with help of capillary tube and is air dried.
8. TLC plate is put in the chromatography jar and lid is closed.
9. The system is allowed to be static until the solvent move to a proper distance from baseline.
10. TLC plate is taken out and dried.

Location of separated components:

If the sample is separated into colored components, then the location is dried in ordinary light but in case
of colorless components following are used;
 Uv lamp
 Iodine crystal
 Spraying agents

Dept. of Pharmacognosy, SJCPS, Kattappana

Documentation:
Storage of chromatogram for TLC is difficult. It is usually undesirable since plates are employed for
repeated use. Various methods for separation include;
 Rf value in TLC: Rf (Retardation factor) value can be alculated by using the formula:
Distance travelled by the solute
Rf = --------------------------------------------------
Distance travelled by the solvent front
Rf value is specific and constant for every compound in a particular combination of stationary and
mobile phase.
 Preservation of chromatogram by peeling off adsorbent.
 Graphical copying i.e. tracing on transparent paper.
 Photography
Advantages:
1. It is the simple method and cost of the equipment is also low.

2. Any type of compound can be analyzed.

3. The method is rapid and many separations can be completed within short period.

4. Separation of microgram of substance can be achieved.

5. The capacity of thin layer can be altered. So analytical and preparative separation can be made.

6. Corrosive spray reagents can be used without damaging the plate.

7. Need less solvent, stationary phase for every separation compared to column chromatography.

8. TLC is economical as the solvent (mobile phase) consumption is small.

9. Recovery of separated component is very simple in TLC.

10. It has a efficiency of separation. Very small particle size can be used which increases the
efficiency of separation.

Applications:
1. Separation of mixture of chemical or biological origin plant extract etc
2. Separaation of carbohydrates,vitamins,antibiotics,alkaloids,glycosides etc
3. To detect the presence of foreign substances in drugs.
4. It is used for separation and identification of Amino acids, Peptides and proteins, Alkaloids,
Carbohydrates, Fats and fatty acids, Antibiotics etc

Dept. of Pharmacognosy, SJCPS, Kattappana

HIGH PERFORMANCE THIN LAYER CHROMATOGRPHY (HPTLC)
HPTLC is sophisticated and automated form of TLC. It is used for the qualitative and quantitative
analysis of herbal products. Traditional Thin Layer Chromatography & its modern instrumental
quantitative analysis version HPTLC are very popular for many reasons such as visual chromatogram,
simplicity, multiple sample handling, low running and maintenance costs, disposable layer etc.
Principle:
Separation may result due to adsorption or partition or by both phenomenon depending upon the nature of
adsorbents used on plates and solvents system used for development.
Steps involved in HPTLC
1. Selection of chromatographic layer
2. Sample and standard preparation
3. Layer pre-washing
4. Layer pre-conditioning
5. Application of sample and standard
6. Chromatographic development
7. Detection of spots
8. Scanning
9. Documentation of chromatic plate
1. Selection of chromatographic layer
 Depends on the nature of material to be separated.
 The pre-coated plates are used and the particle size of stationary phase is less than 10μ in
diameter.
2. Sample and standard preparation
 For normal phase chromatography silica gel / alumina pre-coated plates are used and the solvents
are non polar.
 Reverse phase chromatography , usually polar solvents are used.
 Solvents used are Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1),
Chloroform: Methanol: Ammonia (90:10:1) etc.
3. Layer pre-washing :
 Layer pre-washing helps to remove water vapors volatile impurities which might get trapped in
the plates.
 To avoid this, plates are cleaned by using methanol as solvent by ascending or descending etc.
4. Layer pre-conditioning:

Dept. of Pharmacognosy, SJCPS, Kattappana

  Freshly open box of plates do not require activation.
 Plates exposed to high humidity or kept on hand for long time to be activated.
 By placing in an oven at 110-120ºc for 30minutes prior to spotting.
 Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for
15 minutes.
 Un- saturated chamber causes high Rf values · Saturated chamber by lining with filter paper for
30 minutes prior to development for uniform distribution of solvent vapours and less solvent for
the sample to travel - lower Rf values.
Selection of mobile phase:
Normal phase :
 Stationary phase is polar and Mobile phase is non polar
 Non-polar compounds eluted first because of lower affinity with stationary phase
 Polar compounds retained because of higher affinity with the stationary phase·
Reversed phase:
 Stationary phase is non polar and mobile phase is polar
 Polar compounds eluted first because of lower affinity with stationary phase non
 Polar compounds retained because of higher affinity with the stationary phase
 Twin trough chambers are used only 10 -15 ml of mobile phase is required.
 Components of mobile phase should be mixed introduced into the twin - trough chamber.
5. Application of sample and standard:
 Usual concentration range is 0.1-1µg / µl. Above this causes poor separation.
 Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe on
TLC plates as bands
 Band wise application allows better separation and high response to densitometer
6. Chromatographic development
 After development, remove the plate and mobile phase is removed from the plate to avoid
contamination of lab atmosphere
 Dry in vacuum desiccator - avoid hair drier - essential oil components may evaporate
7. Detection of spots
 Detection under UV light is first choice
 Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long
wave length)

Dept. of Pharmacognosy, SJCPS, Kattappana

  Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel
GF ·
 Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1%
iodine solution ·
8. Scanning and documentation
 HPTLC plates are scanned at selected UV regions
 Wavelength by the instrument & the detected spots are seen on computer in the form of peaks.
 The scanner converts band into peaks & peak height or area is related to the concentration of the
substance on the spot.
Advantage of HPTLC
1. Ability to analyze crude samples containing multi-components.
2. The separation process is easy to follow especially with colored compounds.
3. Several samples can be separated parallel to each other on the same plate resulting in a high
output, time saving, and a rapid low-cost analysis.
4. Choice of solvents for the HPTLC development is wide as the mobile phases are fully evaporated
before the detection step.
5. Two-dimensional separations are easy to perform. Stability during chromatography should be
tested using two-dimensional development.
6. Specific and sensitive colour reagents can be used to detect separated spots (Dragendroff
reagent/Kedde reagent).
7. HPTLC can combine and consequently be used for different modes of evaluation, allowing
identification of compounds having different light absorption characteristics or different colours.
8. Contact detection allows radio-labelled compounds to be monitored and microbial activity in
spots to be assessed.
9. HPTLC method may help to minimizes exposure risk of toxic organic effluents and significantly
reduces its disposal problems, consequently, reducing environment pollution.
Application of HPTLC
1. Standardization of herbal extrats and other pharmaceutical formulations.
2. The analytical profiles of alkaloids, carotenoids, anthracene glycosides, flavonoids, lipids etc.
have been developed.
3. Obtain finger print patterns of various herbal formulations and quatification of active ingredients.
4. Estimation of herbal constituents.
5. Cosmetic and environmental analysis.
6. Metallurgy, electroplating

Dept. of Pharmacognosy, SJCPS, Kattappana

 7. Toxicology and forensic analysis.

COLUMN CHROMATOGRAPHY
Colummn chromatography refers to the type of chromatography in which the stationary phase (solid) is
placed uniformly in a glass tubing (column) via which the solution whose components are to b either
separated or transported.
Principle:
The principle of separation is adsorption. When a mixture of component is dissolved in the mobile phase
is introduced in the column the individual components move with different rates depending upon their
relative affinity. The compounds with lesser affinity toward the stationary phase (adsorbent) moves faster
and it is eluted out first from the column. The one with greater affinity towards stationary phase moves
slower and is eluted out later. Hence the compounds are separated.
Instrumentation and working
The column chromatography requires a vertical column (preferably glass column) with a knob at the
bottom end. This is preferably a burette shaped cylindrical column without graduations or readings.
The stationary phase or adsorbent must be:
 Spherical in shape
 Mechanical stability must be high
 They shouldn’t react chemically
 It should be useful for separating for wide variety of compounds
 It should be freely available & inexpensive
The stationary phases used are: silica gel, activated alumina, activated magnesia, calcium carbonate,
magnesium carbonate, talc, starch etc.
Mobile phase preferably solvents of chromatography grade either a single solvent or a mixture of solvents
as required for the separation. Eg: petroleum ether, carbon disulphide, ether, benzene, toluene, water
organic acid, carbon tetrachloride etc.
Procedure:
The stationary phase material is suitably moistened with mobile phase and packed sufficiently in the
column with a cotton or asbestos pad at the bottom. The extract material or sample to be separated is
placed on the top of packed stationary phase with a second cotton or asbestos pad in between. The mobile
phase is poured into the column over the sample. A collecting beaker is placed at the bottom of column
near the end to collect the elute. The mobile phase percolates through entire stationary phase reaches the
bottom of the column. From there it is elutes out and gets collected in the beaker placed below. When the
mobile phase flows through, different components of the sample travel with different rates through the

Dept. of Pharmacognosy, SJCPS, Kattappana

silica gel. This rate of travel is decided by the adsorption and affinity of molecules towards the stationary
phase and mobile phase. The fractional components of the mixture with greater affinity to mobile phase
travels fast and reach the bottom early. Those with higher affinity to stationary phase travel slow and
reach bottom late. Thus the colored bands of the sample are formed. Each color is an indicator of one
particular set of compound in the sample mixture. This elution is drop by drop and the process may take
few hours to days based on the sample size, length of the column, mobile phase used and the packing
material used.

Advantages
1. Any type of mix. can be separated
2. Any quantity of mix. can be separated
3. Wider choice of M.P
4. Automation is possible
Disadvantages
1. Time consuming
2. Large quantity of M.P required
3. Automation makes the techniques more complicated & expensive
Applications:
1. Column chromatography is best suited to separate active principle from plant materials. Since
plants contain many ingredients like alkaloids, resins, glycosides, tannins, flavonoids and other
bio-molecules, the individual constituents are to be separated. Since the plant extract is bulk this
method is best to separate them.

Dept. of Pharmacognosy, SJCPS, Kattappana

 2. In separation of compounds after organic synthesis to obtain desired molecule.
3. To separate or purify natural compound mixtures like alkaloids, glycosides.
GAS CHROMATOGRAPHY
Gas chromatography relies on the separation of components of a volatile compound. Gas chromatography
is of two types:
1. Gas solid chromatography (GSC)
2. Gas liquid chromatography (GLC)
In both types gas is used as mobile phase and either solid or liquid is used as stationary phase. GSC is
not used widely because of limited number of stationary phases available.
Principle:
The principle of separation in GLC is partition. Gas is used as mobile phase. Liquid which is coated non
to a solid support is used as stationary phase. The mixture of components to be separated is converted to
vapour and mixed with gaseous mobile phase. The component which is less soluble in the stationary
phase travels faster and eluted out first and then component which is more soluble in the stationary phase
travels slower and eluted out later. Hence components are separated according to their partition
coefficient.
Instrumentation
The requirements of GC are :
1. Carrier gas
2. Flow regulators
3. Flow meters
4. Injection devices
5. Column
6. Temperature control devices
7. Detector
8. Recorder
Carrier gas determines the efficiency of chromatograhy separation. Eg; are helium, hydrogen, nitrogen
and argon. As carrier stored under high pressure flow regulators are used to deliver the gas with uniform
pressure. Flow meters are used to measure the flow rates of carrier gas. Different types of injection
devices are used to inject the sample (gas, solid or liquid) into the column. Column is one of the most
important part of GC which decides the efficiency of separation. Columns are made up of glass or
stainless steel. Preheaters are used to control the temperature and they convert the sample into its
vapour form and mix them with the carrier gas or mobile phase.
Detectors are also the most important part of GC instruments. The various detectors used are:

Dept. of Pharmacognosy, SJCPS, Kattappana

  Flame Ionization Detector (FID)
 Thermal Conductivity Detector (TCD)
 Electron Capture Detector (ECD)
 Argon ionization detector etc.
Recorders are used to record the response obtained from detectors after amplification.

Advantages
1. The technique has strong separation power and even complex mixture can be resolved into
constituents
2. The sensitivity of the method is quite high
3. It gives good precision and accuracy
4. The analysis is completed in a short time
5. The cost of instrument is relatively low and its life is generally long
6. The technique is relatively suitable for routine analysis
Applications
1. Quantitative analysis of volatile oils, official monograph gives chromatography profile for some
drugs. E.g. to aid distinction between anise oil from star anise and that from Pimpinelle anisum
2. Separation of fatty acids derived from fixed oils
3. Analysis of foods like carbohydrates, proteins, lipids, vitamins, steroids, drug and pesticides
residues, trace elements
4. Pollutants like formaldehyde, carbon monoxide, benzen, DDT etc
5. Dairy product analysis- rancidity
6. Separation and identification of volatile materials, plastics, natural and synthetic polymers, paints,
and microbiological samples
7. Inorganic compound analysis

Dept. of Pharmacognosy, SJCPS, Kattappana

 PARTITION CHROMATOGRAPHY
This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid
stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.
PAPER CHROMATOGRAPHY
Paper Chromatography Paper chromatography was first introduced by the German scientist, Christian
Friedrich Schonbein in 1865. It is a type of a planar chromatography. It is the simplest and widely used
type of chromatography procedures which runs on a specialized paper.
Principle:
The principle of separation is mainly partition rather than adsorption. Paper impregnated with silica or
alumina acts as the adsorbent (stationary phase) and solvent as the mobile phase. The moisture or water
present in the pores of the cellulose fibers present in the filter paper acts as the stationary phase and
another solvent as the mobile phase.
IN GENERAL, PAPER CHROMATOGRAPHY = PAPER PARTITION CHROMATOGRAPHY
Types:
Paper Chromatography has different types or modes:
Ascending chromatography: As the name indicates, the chromatogram ascends. Here the development
of paper occurs due the solvent movement or travel in upward direction on the paper.
Descending chromatography: Here the development of paper occurs due to solvent travel downwards
on the paper. Ascending- descending mode: Here solvent first travels upwards and then down wards on
the paper.

Radial mode: Here the solvent travels from center (midpoint) towards periphery of Circular
chromatography paper.

Dept. of Pharmacognosy, SJCPS, Kattappana

Two dimensional chromatography: Here the chromatogram development occurs in two directions at
right angles.

Instrumentation:
1. Chromatography jar
2. Capillary tube
3. Stationary phase (liquid impregnated paper)
4. Mobile phase
Chromatography jar: It is made of glass and has a lid on it. Jar maintains proper environment that is
required for separation.
Capillary tube: It is used to apply sample mixture.
Stationary phase: Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40,
No.42 etc are used.
Mobile phase: Mobile phase may be a single liquid or a mixture of liquids.
Commonly used mobile phases are; Methanol, Ethanol, Ethyl acetate, Diethyl ether, Acetone,
Chloroform.

Dept. of Pharmacognosy, SJCPS, Kattappana

Procedure:
 Clean and dried chromatography jar is taken.
 A paper impregnated in the mobile phase is applied to the walls to ensure that atmosphere of the
jar is saturated with solvent vapors.
 Mobile phase is added to the jar at a length of 0.5-1cm from the bottom.
 Jar is closed.
 Equilibrium is allowed to be maintained.
 Base line is marked on adsorbent.
 Sample is applied on Whatman filter paper with help of capillary tube.
 Sample spot is air dried.
 Paper is put in the chromatography jar and lid is closed.
 The system is allowed to be static until the solvent move to a proper distance from baseline.
 Paper is taken out and dried.
 Location of separated components:

 If the sample is separated into colored components, then the location is dried in ordinary light.
But in case ofcolorless components following are used;
 Uv lamp
 Iodine crystals
 Spraying agents
Documentation:
 Storage of chromatogram.
 Calculating Rf values
Advantages:
1. Simple ,rapid ,inexpensive ,excellent resolving power
Applications:
1. It is used for separation and identification of Amino acids, Carbohydrates, Tannins, Glycosides,
Alkaloids etc.
2. Useful in determining the purity and authenticity of a pure natural product.
3. Separation of mixtures of drugs
4. Identification of drugs
5. Identification of impurities
6. Analysis of metabolites of drugs in blood , urine etc.

Dept. of Pharmacognosy, SJCPS, Kattappana

ION-EXCHANGE CHROMATOGRAPHY
It is a process that allows the separation of ions and polar molecules based on their charge. In this type of
chromatography, a resin (the stationary solid phase) is used to covalently attach anions or cations onto it.
Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic
forces. Ion exchange chromatography is performed in columns but can also be useful in planar mode.
Principle:
Ion - exchange chromatography retains sample molecules on the column based on ionic interactions. The
surface of stationary phase displays ionic functional groups that interact with analyte ions of opposite
charge.
Mechanism:
Ion exchange chromatography uses a charged stationary phase to separate charged compounds including
anions, cations, amino acids, peptides, and proteins. In conventional methods the stationary phase is an
ion exchange resin that carries charged functional groups which interact with oppositely charged groups
of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins.

Types:
Cation exchange chromatography: Cation exchange chromatography retains positively charged cations
because the stationary phase displays a negatively charged functional group.
Anion exchange chromatography:Anion exchange chromatography retains anions using positively
charged functional group.
Applications:
1. It can be used for almost any kind of charged molecule including large proteins, small nucleotides
and amino acids.
2. Protein purification
3. Water analysis
4. Quality control

Dept. of Pharmacognosy, SJCPS, Kattappana

SIZE-EXCLUSION CHROMATOGRAPHY
It is also known as gel permeation or gel filtration chromatography. It is a chromatographic method in
which molecules in solution are separated by their size, and in some cases molecular weight.
This type of chromatography lacks an attractive interaction between the stationary phase and solute. The
liquid or gaseous phase passes through a porous gel which separates the molecules according to its size.
The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to
enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass
through the column at a faster rate than the smaller ones. It is usually applied to large molecules or
macromolecular complexes such as proteins and industrial polymers.
Principle:
Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and
removed from the flow of the mobile phase. The average residence time in the pores depends upon the
effective size of the analyte molecules. However, molecules that are larger than the average pore size of
the packing are excluded.

Applications:
1. Purification and analysis of synthetic and biological polymers, such as; Proteins, Polysaccharides,
Nucleic acids.
2. It is also useful for determining the tertiary structure and quaternary structure of purified proteins.
3. It is generally a low-resolution chromatography technique and thus it is often reserved for the
final, "polishing" step of purification.

Dept. of Pharmacognosy, SJCPS, Kattappana

AFFINITY CHROMATOGRAPHY
This is the most selective type of chromatography. It is a method of separating biochemical mixtures and
based on a highly specific biological interaction such as that between antigen and antibody, enzyme and
substrate, or receptor and ligand.
For example, the immobilized molecule may be an antibody to some specific protein. When solute
containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this
antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength
or pH.
Principle:
The stationary phase is typically a gel matrix (often agarose) .The molecule of interest has a known and
defined property. The process is an entrapment in which the target molecule becomes trapped on
stationary phase. The Stationary phase can then be removed from the mixture, washed and then target
molecule is released from the entrapment.

Applications:
1. Purify and concentrate an enzyme solution
2. Purification of recombinant proteins
3. Purification of antibodies

Dept. of Pharmacognosy, SJCPS, Kattappana