‘THE ART OF SEPARATION’
CHROMATOGRAPHY – AN INTRODUCTION
Chromatography is a technique through which a mixture of chemical components are separated, identified and determined accurately. This technique while provides a way for analytical separations, also useful for preparative techniques by which pure compounds can be obtained.
Sample Injection + Mobile Phase
d fi d as a technique
t h i
hi h the
t off a mixture
t d based
upon the rates at which they are moved or carried over a stationary phase by a gaseous or liquid mobile phase” Based on the mobile phase this technique can be classified as: ‘Liquid
Chromatography’ and ‘Gas
With the basic two classifications as LC and GC, there are further classifications based on the type of stationary phase (column), mobile phase (eluent) and type of separation occurs within the column.
column The simplified version on various types is given as follow: Chromatography
Apart from normal liquids as the mobile phases, supercritical fluids such as liquid CO2 can also be used as mobile phase and the technique is designated as Supercritical Fluid Chromatography (SFC).
CHROMATOGRAPHY – HPLC PRINCIPLES AND MODES
As seen, in liquid chromatography the mobile phase used is a liquid solvent. In earlier days this was carried out in long packed glass columns with wide diameter. But the flow rates of the mobile phase was very slow and the separation times were sometimes took several hours. Nowadays, the speed of the flow rates has been improved by applying high pressure to the gp
p of various types
yp and the p
performance is improved,
, this along
reduction in the particle size of the packed materials down to 5-10microns (when compared to earlier 150-200microns) is achieved to give better separation and faster analysis ending within few minutes. With this development LC has been renamed as ‘High-Performance Liquid Chromatography’ or ‘High-Pressure
‘High Pressure Liquid Chromatography’ (HPLC).
In HPLC there are three main components that define good separation: pump, mobile phase and stationary phase (column) besides the migration and characteristics of solutes (analytes).
The three primary modes employed in HPLC can be explained briefly as follow:
In this process the analyte species (components to be separated and analyzed) are adsorbed onto the surface of a polar stationary packing. Surface adsorption involves electrostatic interactions such as H-bonding, dipole-dipole and dipole-induced dipole attractions. If the component mixture is eluted through the column with the mobile phase, different components present in the mixture adsorb to different degrees of strength and they become separated as the mobile phase steadily moves through the column. The stationary phase could be silica gel or alumina coated with polar particles.
This process is extensively used for the separations of relatively non-polar, water-insoluble organic compounds, since more polar analytes will be adsorbed strongly on to the surface of the stationary phase, which will be later removed by using high polar solvents. One particular application is in resolving isomeric mixtures such as m- and p-substituted benzene derivatives. An approximate order of increasing polarity among classes of organic analytes is: alkanes < alkenes < aromatics < ethers < esters < ketones < aldehydes < thiols < amines < amides < alcohols < phenols < acids
It is the most widely used...
References: 1. ‘Gas and Liquid Chromatography in Analytical Chemistry’, R M Smith, John Wiley & Sons,
2. ‘Chromatographic Methods’, A. Braithwaite and F. J. Smith, 5th Edition, Blackie Academic &
Professional, Glasgow, 1996.
3. ‘Principles of Instrumental Analysis’, D A Skoog, F J Holler and T A Nieman, 5th Edition,
Thomson Learning, Crawfordsville, 1998.
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