Countercurrent Chromatographic Methods

Fig. 1 Chromatograms of hesperetin (1), kaempferol (2), and quercetin (3) by different countercurrent chromatographic methods. Solvent system chloroform-methanol-water (33 40 27). (A) DCCC (B) RLCC (C) multilayer coil CPC system and (D) cartridge CPC system. (Elsevier Science Ireland, Ltd.)...

Kohler, N. et al.. Development of a new preparative spiral-coil low speed rotary countercurrent chromatographic (spiral-coil LSRCCC) method, J. Liq. Chromatogr. Rel. TechnoL, 27, 2547, 2004. 

Various countercurrent chromatographic techniques have been successfully employed for the separation of flavonoids. Countercurrent chromatography is a separation technique that relies on the partition of a sample between two immiscible solvents, the relative proportions of solute passing into each of the two phases determined by the partition coefficients of the components of the solute. It is an all-liquid method that is characterized by the absence of a solid support, and thus has the following advantages over other chromatographic techniques ... 

Fig. 4a shows countercurrent chromatographic separation of four basic dyes, i.e., methyl green (MG), methylene blue (MB), neutral red (NR), and basic fuchsin using a two-phase solvent system composed of isoamyl alcohol-ethanol-acetic acid-distilled water (4 2 1 5, v/v). The first coil displays the separation of the mixture, and the other coils show distribution of individual dyes to demonstrate the reproducibility of the method. The separation was performed with 6 m of 0.35-mm-lD tubing (ca. 300 helical turns) at relative coil rotation of 0.25 rpm at 300 X g for 10 hr. 

Countercurrent chromatography (CCC) is a chromatographic method which separates solutes more or less retained in the column by a stationary phase (liquid in this case) and are eluted at the outlet of column by a mobile phase. Two treatments of column effluent have been used until now in CCC. Either the column outlet is directly connected to a detector commonly used in high-performance liquid chromatography (HPLC) (on-line detection) or fractions of the mobile phase are collected and analyzed by spec-trophotometric, electrophoretic, or chromatographic methods (off-line detection). 

Countercurrent chromatography overcomes all of the problems caused by solid support matrix present in most other chromatographic methods. Countercurrent chromatography can purify alkaloids with high recovery and reproducibility. 

Compared with chromatographic methods, this countercurrent distribution method is probably less efficient, the apparatus required is very much more complicated, and the labor involved is considerably greater nevertheless, it possesses several definite advantages over chromatographic methods. The chief of these is the absence of any solid phase, which may act as an adsorbent for the solutes. In partition chromatographic methods this adsorption may often lead to distortion of the bands and render fractionation very inefficient. The behavior of a solute on a countercurrent distribution depends only on its partition coefficient, which in most cases is constant, so that it is possible to calculate the exact theoretical distribution curve and this may be used as a very sensitive test for purity. 

Kitazume, E. High-speed countercurrent chromatograph device and analysis method. Jpn. Kokai Tokkyo Koho JP. 2005,315731 A. 

Sato, Y. Shinomiya, K. Kabasawa, Y. Aqueous two-phase partitioning method by using rotation locular countercurrent chromatograph-an apphcation to enantiomeric separation. J. Chem. Soc. Japan 1994, 1067-1071. 

In addition to chromatographic methods for the separation of urinary ketosteroids, countercurrent distribution is also applicable. Archibald (2) and Engel el major components of the keto-steroid mixture may be separated by coimtercurrent distribution and identified by examination of the infrared spectra. 

Techniques and applications All the operational techniques ordinarily used for adsorption are used also for ion exchange. Thus we have batch or stagewise treatment of solutions, fluidized- and fixed-bed operations, and continuous countercurrent operations. Fixed-bed percolations are most common. Chromatographic methods have been used for fractionation of multicomponent ionic mixtures. Applications have been made in the treatment of ore slurries ( resin-in-pulp ) for collection of metal values. 

High-speed countercurrent chromatography (HSCCC) is a relatively new technique. It is a liquid-liquid partitioning chromatographic method in which the stationary phase is immobilized by a centrifugal force. HSCCC is the most advanced form of CCC in terms of partition efficiency and separation time. As solvent system, a mixture in different ratio is usually used, depending on the flavonoids nature ethyl acetate/... 

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