Gradient Elution in Countercurrent Chromatography

Abstract

Countercurrent chromatography is a form of liquid-liquid partition chromatography in which the stationary liquid phase is retained in the apparatus without the use of a solid support. Gradient elution in countercurrent chromatography can be used in many different ways, such as linear gradients, stepwise elution gradients, pH gradients, etc. The main goal of using the gradient approach is to shorten the duration of the separation and improve resolution, especially when the retention range of the sample to be purified is broadening and, thus, the compounds cannot be purified by only one solvent system. The principle is based on modifying the mobile phase to increase the elution strength with no or minimum changes in the stationary phase, which can be a difficult task since both phases are in intimate contact all the time. The most common ways to perform gradients in countercurrent chromatography are changing the mobile's phase polarity, flow rate, and pH, what is called linear or step gradient, flow rate gradient, pH gradient, respectively.

Separation of epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as an additive

“…Countercurrent chromatographic techniques have been widely used for separation and purification of chemical components from natural products and synthetic mixtures due to its preparative capacity, nonirreversible adsorption properties, and flexible elution modes . Successful separations of various types of chemical components have been reported in the recent years .…”

Application of linear gradient elution in countercurrent chromatography for the separation of triterpenoid saponins from the roots of Pulsatilla koreana Nakai

“…The extracts of natural products are complex mixtures of several components that can be separated by the application of gradient elution in most chromatographic methods . CCC gradient elution is a powerful method for the separation of complex mixtures.…”

“…CCC gradient elution is a powerful method for the separation of complex mixtures. For example , the target compounds are strongly held in the stationary phase, the polarities of the compounds are considerably different, and the selectivity of the solvent system is not sufficient for separation. Several methods of gradient elution in CCC have been developed : pH‐related gradient elution, which affects the aqueous mobile phase rather than the organic stationary phase; salting‐out gradient elution; step‐gradient elution, which abruptly changes the mobile phase composition; and the linear gradient elution.…”

“…For example , the target compounds are strongly held in the stationary phase, the polarities of the compounds are considerably different, and the selectivity of the solvent system is not sufficient for separation. Several methods of gradient elution in CCC have been developed : pH‐related gradient elution, which affects the aqueous mobile phase rather than the organic stationary phase; salting‐out gradient elution; step‐gradient elution, which abruptly changes the mobile phase composition; and the linear gradient elution. However, gradient elution methods in CCC are not as easy as in HPLC, because changes to the mobile phase composition may affect the composition of the stationary phase, resulting in a possible loss of equilibrium and a lack of retention of the stationary phase .…”

“…Several methods of gradient elution in CCC have been developed : pH‐related gradient elution, which affects the aqueous mobile phase rather than the organic stationary phase; salting‐out gradient elution; step‐gradient elution, which abruptly changes the mobile phase composition; and the linear gradient elution. However, gradient elution methods in CCC are not as easy as in HPLC, because changes to the mobile phase composition may affect the composition of the stationary phase, resulting in a possible loss of equilibrium and a lack of retention of the stationary phase . Therefore, the investigation of various factors is vital for developing a successful gradient elution method for CCC .…”