Chromatographic separation performed with the stationary phase packed in a column as against being planar is column chromatography. The sorbents used in CC are taken in a column and the mobile phase instead of moving up by capillary action comes down due to gravity against the resistance of the sorbent. The mixture to be fractionated is introduced on the column in a small volume of organic solvent. While in TLC the solvent run is terminated and the compounds separated on the plate identified in their positions on the planar phase, in CC, the solvent is allowed to run out of the column (elution) from the bottom, till the compounds are carried along separated from one another.

One of the oldest forms of chromatographic techniques, compound separation is effected by partition and the stationary phase is held onto a sorbent as the mobile phase moves down. Solute resolution is controlled by changing the mobile phase, the interaction between the mobile and stationary phase overall effecting solute separation. The separation of compounds is influenced by the displacement effect of one compound with another and partitioning between phases is independent of concentration and presence of other solutes.

Sorbents of high polarity such as silica gel are used with others like reversed phase silica, ion-exchange resins and size exclusion chromatography phases being used for specific separations. Mobile phase is a miscible solvent mixture of constant ratio-isocratic elution, or of different changing proportions— gradient elution—as the elution continues. In the mobile phase, resolved compounds are collected in fractions. These upon concentration are analyzed by TLC to identify the separated compound(s). Choice of stationary phase and mobile phase for partition chromatography by column is made from a preliminary TLC analysis of the mixture to be resolved. In general, the solvents should be pure, non-viscous, and depending on the nature of the mixture, mobile phase may be selected from a whole range of nonpolar to polar solvents. Polar compounds may be separated using polar stationary phase and nonpolar mobile phase. Conversely, nonpolar solutes may be best separated using a nonpolar stationary phase and a polar mobile phase. In practice, after the range of solvents to be used for separation is selected, the mobile phase composition is adjusted as required, while the elution progresses. Purpose of chromatography may be fractionation of a total extract, further resolution of separated fractions, purification of isolated compound mixtures, or resolution of closely similar solutes.

Several modified forms of CC, used today include:

 

– Gel filtration chromatography, which is based on molecular size separation

– Ion-exchange chromatography, which separates compounds based on their charge and partitioning effect

– Affinity chromatography, which separates compounds based on the molecular shape due to the functional groups they carry

– Vacuum Liquid Chromatography in which a slight negative pressure is applied at the point of eluent collection to speed up elution

– Flash chromatography, which uses mobile phase passing down the column quickly due to applied positive pressure over it

– High performance liquid chromatography (HPLC) in which the conventional cylindrical column is replaced by narrow columns. Here the stationary phase is bonded to a porous polymer held in a capillary sized stainless steel column and the mobile phase is forced through under pressure.

 

Of all these forms, HPLC has become one of the most extensively used chromatographic techniques for all types of compound (that are soluble and nonvolatile) separations including phytochemicals. It is a highly sensitive and efficient separation technique especially applicable to analysis of nonvolatile compounds such as alkaloids, lipids, sugars, higher terpenoids, and phenolic compounds. The eluate is closely monitored by a range of detecting systems. UV/VIS detection is most commonly employed. HPLC is a fully automated technique employing highly efficient pre-packed columns and it is one of the latest chromatographic techniques of wide applicability in plant drug identification and analyses. However, it is of limited applicability to preparative separations.

Despite the availability of several sophisticated modifications of CC, the conventional CC has still not been replaced by any other technique for large-scale isolations of compounds. It is however time-consuming and much larger quantities of sorbents and solvents are needed. Mobile phase flow is to be optimized for efficient separation of compounds because very slow and very fast elution does not bring about optimal separations.

With solvent ratios, nature of elution, sorbent selection optimized, CC still forms a reliable and practical option for routine separations of carotenoids, mixtures of alkaloids, and in general for the separation of virtually any category of compounds from their mixtures.