As per dictionary definition Chromatography is a method for separating the constituents of a solution(gas or liquid) by exploiting the different properties of different molecules. The technique employs a mobile phase(gas or liquid)to transport the solution to be analyzed through the stationary phase (solid or liquid)which absorbs or impedes different components of the solution to different degrees and thus causes their separation as different layers. It is an invaluable tool in the hands of analytical scientists for the separation and quantification of components in a mixture of organic compounds.
The technique had its origins in the pioneering work of Mikhail Tswett who separated plant pigments in 1900 using a packed glass column. As the separated pigments were differently coloured and separated as distinct bands the technique was coined as chromatography meaning separation by colours.
Over the years chromatography has evolved and advanced into versions such as HPLC, GC, HPTLC and SFC all of which to this day are based on the separation of mixture components through selective physico-chemical interactions and partitioning between the stationary and mobile phases.
Why it is used?
Since its evolution chromatography has found wide use in separation of components of mixtures ranging from simplest gases to most complex hydrocarbon mixtures containing hundreds of different compounds. Samples can be gaseous, liquids or even solids which are readily soluble in suitable solvents. Due to its versatility the applications cover complete range of chemical compounds having diverse characteristics such as boiling range, molecular weights, volatilities, and thermal stabilities. Gas chromatography, Liquid chromatography, Thin-layer chromatography, Super critical fluid chromatography and hyphenated techniques such as GC – MS, and LC – MS cover a vast number of applications in diverse areas such as pharmaceuticals, material development, foods, petroleum products and forensic investigations requiring very high resolution and detection limits.
Separation of sample components before detection is the essence of chromatographic techniques. A chromatographic system uses a column to achieve the desired separation. A column comprises of a tube packed with a stationary phase on which separation takes place based on physico – chemical interactions of separating compounds with the stationary phase. The mobile phase or the carrier gas elutes less weakly retained components first followed by more strongly retained components.The column dimensions and composition is based on the chromatographic technique selected and the degree of required separation. In general HPLC columns are shorter and wider than GC columns.
After separation the individual components reach the detector which provides response in terms of the area or peak height depending on the amount of the eluting compound. Each chromatographic separation technique offers a range of detectors depending on the nature of eluting compounds. Detection can be specific for a particular compound or a range of compounds or it can depend on some physical properties such as reflective index of the mobile phase. Such detectors are referred to as bulk property detectors in comparison to selective or specific detectors.
Chromatographic separations appear as peaks in the chromatogram except for thin-layer chromatography for which separate zones are seen on the plate. Chromatographic peaks are separated in time in the chromatogram depending on the nature of separating compounds and the separation efficiency of the column. Sharp well resolved peaks indicate high degree of column resolution whereas broad or overlapping peaks are indicative of poor resolution.
Each peak represents a sample component and is characterized by its retention time under defined operating conditions. For purposes of quantification the peak height or more accurately the area under the peak defines the amount of the component present in the mixture.
Quantitation methods require percentage composition of a particular component in the mixture and such estimations are made on the presumption that the detector responds to all the components equally and peaks are generated for each compound.
%of analyte = X100
Quite often the amount of analyte is specified in relation to another compound which is referred to as a reference standard having established traceability. Such compounds should be available in pure form and have chemical identity which is same or close to the identity of the analyte to be determined.
Modern-day analytical systems come equipped with sophisticated application softwares which are capable of operational control, data interpolation and calculations to achieve the desired results.
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