Chromatography refers to a group of techniquesused to separate complex mixtures on the basis ofdifferent physical interactions between theindividual compounds and the stationary phaseof the system. The basic components in anychromatography technique are the mobile phase(gas or liquid), which carries the complex mixture(sample); the stationary phase (solid or liquid),through which the mobile phase flows; the columnholding the stationary phase; and the separatedcomponents (eluate).
Modes of separationa. Adsorption chromatography (liquid-solidchromatography)
It is based on the competition between thesample and the mobile phase for adsorptive siteson the solid stationary phase. There is equilibriumof solute molecules being adsorbed to the solidsurface and desorbed and dissolved in the mobilephase. Those molecules, which are most soluble inthe mobile phase, move fastest, whereas those,which are least soluble, move slowest. Thestationary phase can be either acidic polar (eg, silicagel), basic polar (eg, alumina) or nonpolar (eg,charcoal). Commonly, the non-polar organiceluents for example hexane, dicholoromethane,and ethyl actetate are used. It can be used toseparate compounds, which are highly soluble inorganic solvents (eg., Fat-soluble vitamins).
b. Partition chromatography
Partition chromatography is also referred to asliquid-liquid chromatography. Separation of soluteis based on the relative solubility in an organic(nonpolar) solvent and an aqueous (polar) solvent.Modern partition chromatography uses pseudo-liquid stationary phases that are chemically bondedto the support or high-molecular-weight polymersthat are insoluble in the mobile phase. Partitionsystems are called
when the mobilephase is less polar than the stationary solvent andare termed
when the mobile solventis more polar. When the elution strength of themobile phase is constant throughout theseparation, it is called
elution and whenvaried is called as
elution.In liquid chromatography, the resolution isproportional to the column length (i.e., the numberof theoretical end plates/unit length). Increasingthe surface area leads to an increase in the numberof theoretical plates. An increase in the surface areacan be achieved by decreasing the particle size butcomes with the disadvantage of increasedresistance to flow, which impairs resolution becauseof backpressure. In the recent past, new stationaryphases with small particle size that can withstandhigh pressures and offer better resolution have beendeveloped. This has lead to the development of highperformance liquid chromatography (HPLC).
High Performance Liquid Chromatography(HPLC)
The term High Performance LiquidChromatography (HPLC) was coined to describethe separation of molecules under high pressurein a stainless steel column filled with a matrix andis used for the separation and determination oforganic and inorganic solutes (mol.wt.< 1000)(Figure 1).
Fundamentals of HPLC
1.Selectivity: It is the ability of a column toseparate two components depending on itsaffinity and retention.2.Capacity factor: It is the ability of a column toretain a particular compound.3.Resolution factor: It is the resolving power of acolumn to separate two structurally closelyrelated compounds.
ESTIMATION OF NEUROTRANSMITTERS IN THE BRAIN BYCHROMATOGRAPHIC METHODS
Deepti Nair, Ramkumar K, Srikumar BN, Raju TR and Shankaranarayana Rao BS
Brain and Behavior
. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.),National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:134-141.