Chromotography And Its Different Types Biology Essay


Analytical procedures form the back bone of research and development in science and technology. Pharmaceutical analysis is the branch of practical chemistry which deals with the resolution, separation, identification determination and purification of a given sample of medicine or a pharmaceutical, the detection and estimation of impurities that may be present therein is also included2. Pharmaceutical analysis derives its principles from various branches of sciences like chemistry, physics, microbiology, nuclear science, electronics etc.3Pharmaceutical analysis includes both qualitative and quantitative analysis of drugs and pharmaceutical substances starts from bulk drugs to the finished dosage forms.

Qualitative Analysis: is the identification of elements, species, and compounds present in a sample.

Quantitative Analysis: is the determination of the absolute or relative amount of elements, species, or compounds present in the sample.4


The quality of pharmaceuticals or drugs on being analyzed should reflect the standards related to potency, safety, and efficacy.2 Pharmaceutical analysis deals not only with the medicaments (drugs and formulations), but also with their precursor that is with the raw materials whose degree of purity, which in turn decides the quality of medicaments.

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The analytical chemist is often confronted with the difficulty of selecting the most suitable method for the required determination. To choose an appropriate analytical method, analysts must be familiar with practical deals of the various techniques and theoretical principles on which they are based. Whichever the method finally chosen for the required determination, it should ideally be a specific method. Analysts will also be concerned with accuracy and precision, time and costing8. Analytical methods are often classified as being either classical or instrumental. Now the classical methods are replaced by instrumental methods. An instrument for chemical analysis converts information about the physical or chemical characteristics of the analyte to information that can be manipulated and interpreted by a human. 18

The instrumental technique can be categorized as follows5,13

Spectroscopic methods:

The spectral methods are based on the absorption or emission characteristics of drugs.


UV â€" Visible spectroscopy

Fluorescence and Phosphorescence spectroscopy,

Atomic spectroscopy

Infra-red spectroscopy

X ray radiation technique

Nuclear magnetic resonance

Electron spin resonance


Nephloturbidimetrymetry, etc.,

Electrochemical methods:

The Electro chemical method based on interdependence of Electro chemical properties and composition of system can be classified as-





Electro gravimetry

Chromatographic methods:

High performance thin layer chromatography

Gas chromatography

Super critical chromatography

High performance liquid chromatography

Hyphenated methods:




Physical methods



Optical rotatory dispersion

Miscellaneous methods:

Thermal analysis

Mass spectrometry

Kinetic techniques


Nearly all the samples presented to the pharmaceutical analyst are mixtures, sometimes very complex ones. The analysis of one component in others presence may, however, be difficult or sometimes impossible because of interference by one substance in the assay of another1.There are very few methods for chemical analysis that are specific for a single chemical species .Consequently; the separation of analyte from potential interferences is quite often a vital step in analytical procedures18.

Amongst the most techniques available to the analyst chromatography became a powerful separation method that finds applications in all branches of science.skg Chromatography is essentially a group of techniques for the separation of the compounds

of mixtures by their continuous distribution between two phases, one of which is moving past the other7. One of the phases is affixed bed of large surface area, while the other is a fluid which moves through the surface of the fixed phase. The fixed phase is called stationary phase, and the other is termed as the mobile phase. It involves the methods that allow the separation, identification, and determination of closely related components of complex mixtures18..


According to the technique of chromatography

Adsorption chromatography

Partition chromatography

Ion exchange chromatography

Size exclusion or gel permeation chromatography.

Affinity chromatography

Adsorption chromatography

In adsorption chromatography stationary phase is a solid on which the sample components are adsorbed. The mobile phase may be a liquid or a gas, the sample components are distributed between the two phases through a combination of sorption and desorption process14. Usually silica or alumina is utilized as the adsorbent with relatively non polar solvents such as hexane as the mobile phase in normal phase adsorption whereas in reversed phase adsorption non polymer beds with relative polar solvents such as water, AcetoNitrile methanol as mobile phase.

Partition chromatography

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In partition chromatography an inert solid material such as silica gel or diatomaceous earth serves to support a thin layer of liquid which is the effective stationary phase. The mobile phase is a liquid or a gas. In normal mode of operations of liquid â€" liquid partition a polar stationary phase is used with nonpolar mobile phase. This favors the retention of polar compounds and elution of non polar compounds and is called as normal phase chromatography. If a non polar stationary phase is used along with a polar mobile phase then non polar solute is retained favoring elution of polar solute. This is called as reverse phase chromatography. 14

Ion exchange chromatography

In ion exchange chromatography the stationary phase consists of an ion exchange resin which is a polymer containing fixed charged groups and replicable counter ions. When the sample containing ions is passed in to the column the ions of the same charge as the counter ions displace the counter ions in to the mobile phase and are retained in the column. Cation and anion exchange resins are available he mobile phase used in this type is always an aqueous solution containing one or more electrolytes.7

Size exclusion chromatography

In size exclusion chromatography the stationary phase is an inert gel of dextran or other polymers which are cross-linked to give a porous three dimensional molecular structure. Solutes whose molecular size is sufficiently small will leave the mobile phase to diffuse into the pores. Large molecules which will not fit into the pores remain in the mobile phase and are not retained. Thus the substances were eluted in the order of decreasing molecular size. The mobile phase in this type are usually buffered aqueous solutions.7

Affinity chromatography

In affinity chromatography the stationary phase is a solid support to which an immobilized biochemical called as affinity ligands is covalently attached. When the sample is passed through the column, only the solute that selectively bind to the complimentary ligands is retained , the other sample components elute without retention. The retained solute is eluted by changing the mobile phase condition. The separation exploits lock and key binding which ensures tremendous specificity of the technique.

The modern instrumental techniques of GLC and HPLC provide excellent separation and allow accurate assay of very low concentrations of wide variety of substance in complex mixtures.5

According to the nature of the stationary and mobile phases.4,6







Gas chromatography

Gas liquid chromatography

Gas solid chromatography




(helium, nitrogen or hydrogen)



Liquid chromatography


Size exclutionn chromatography

Ion exchange chromatography

Chiral chromatography

Solid/bonded phase

Controlled porosity solid

Ion exchange resin

Solid chiral sector

Liquid water or organic solvents such as methanol, AcetoNitrile, propanol , hexane

Partition or adsorption


Ion exchange

Selective adsorption

Paper chromatography


Liquid water or organic solvents such as methanol, AcetoNitrile, propanol , hexane

Partition or adsorption

Thin layer chromatography

Silica, cellulose, alumina

Non polar organic solvents


High performance Thin Layer Chromatography (HPTLC)25

HPTLC is the improved version of thin layer chromatography (TLC). It is superior to TLC in their separation power, performance and in reproducibility. It is a sophisticated and automated form of adsorption chromatography. The main principle behind it is adsorption. Here separation takes place due to the differential migration of the components in the sample between the mobile phase and the stationary phase.

Features of HPTLC

Simultaneous processing of sample and standard with better analytical precision and accuracy

Several analysts can work simultaneously

Analysis time and cost per sample is less

Maintenance cost is less

Simple sample preparation

No prior solvent treatment

Mobile phase consumption per sample is low

As fresh stationary is used for each analysis there is no interference from previous analysis

Visual detection possible

post-chromatographic derivatization techniques are available for Non UV absorbing compounds

Steps involved in HPTLC

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Selection of HPTLC plate.

Layer prewashing.

Layer preconditioning or Activation of plate.

Sample and standard preparation.

Application of standard or sample solution.

Chromatographic development.

Evaluation of spot.


Selection of HPTLC plate

Precoated plates with different support materials, different sorbent layer, and of different thickness are available in the market.

Supporting materials --- Glass, plastic , aluminum

Layer thickness --- 100-200 µm

Particle size --- 4-8 µm

Plate size --- different size are available but 20cmx20cm is economical.

sorbent--- aluminium oxide- for alkaloids and steroids