Countercurrent chromatography partition coefficients

Berthod, A., Carda-Broch, S., and Garcia-Alvarez-Coque, M.C. Hydrophobicity of ionizable compounds. A theoretical study and measurements of diuretic octanol-water partition coefficients by countercurrent chromatography. Anal. Chem., 71(4) 879-888, 1999. 

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 ... 

Table 7.5 Solute Partition Coefficients Measured by Countercurrent Chromatography in Two Ionic Liquid-Containing Diphasic Liquid Systems...

Ilchmann, A., Wienke, G., Meyer, T., GmehUng, J. (1993) Countercurrent Uquid/Uquid chromatography— A reliable method for the determination of partition coefficients. Chem.-Ing.-Tech., 65(1), 72-75. 

Countercurrent chromatography has been mainly developed and used for preparative and analytical separations of organic and bio-organic substances [1], The studies of the last several years have shown that the technique can be apphed to analytical and radiochemical separation, preconcentration, and purification of inorganic substances in solutions on a laboratory scale by the use of various two-phase liquid systems [2], Success in CCC separation depends on choosing a two-phase solvent system that provides the proper partition coefficient values for the compounds to be separated and satisfactory retention of the stationary phase. The number of potentially suitable CCC solvent systems can be so great that it may be difficult to select the most proper one. 

Countercurrent chromatography utilizes a pair of immiscible solvent phases which have been preequilibrated in a separatory funnel One phase is used as the stationary phase and the other as the mobile phase. A solvent system composed of 12.5% or 16.0% (w/w) PEG 1000 and 12.5% (w/w) potassium phosphate was usually used for the type XL and XLL cross-axis CPCs. These solutions form two layers the upper layer is rich in PEG and the lower layer is rich in potassium phosphate. The ratio of monobasic to dibasic potassium phosphates determines the pH of the solvent system this effect can be used for optimizing the partition coefficient of proteins. 

Countercurrent chromatography is a two-phase procedure where the separation is based on the difference in partition coefficients of solutes within the phases. To achieve efficient separation of lipoproteins from human serum, it is essential to optimize the partition coefficient of each component by selecting an appropriate pH for the polymer phase system. 

The decisive advantage of countercurrent chromatography (CCC) in Po/w measurement is that there is no correlation at all. Water, saturated with octanol, is the mobile phase. Octanol saturated with water is the stationary phase. The octanol-water partition coefficient of a given solute is the only physicochemical parameter responsible for the solute retention. If the solute is not highly pure, it is likely that the impurities will have differing Po/w values. This means that if the impurities have differing retention volumes, they are separated during the measurement from the solute of interest. The Po/w value is easily derived from the CCC retention equation ... 

High speed countercurrent chromatography (HSCCC) produces highly efficient chromatographic separations of solutes without the use of solid supports Thus the method eliminates all complications caused by the solid support, such as adsorptive loss and deactivation of samples, tailing of solute peaks, contamination, etc. As with other CCC schemes, HSCCC utilizes two immiscible solvent phases, one as a stationary phase and the other as a mobile phase, and the separation is highly dependent on the partition coefficient values of the solutes, i.e., the ratio of the solute concentration between the mobile and stationary phases. Therefore the successful separation necessitates a careful search for the suitable two-phase solvent system that provides an ideal range of the partition coefficient values for the applied sample. 

Tsai, R.-S., Carrupt, P.-A. and Testa, B. (1995). Measurement of Partition Coefficient Using Centrifugal Partition Chromatography. Method Development and Application to the Determination of Solute Properties. In Modern Countercurrent Chromatography (Conway, WD. and Pet-roski, R.J., eds.), American Chemical Society, New York (NY), pp. 143-154. 

Liquid-partition chromatography was a hybrid of Tswett s column-adsorption chromatography and a technique widely employed in industry called countercurrent extraction. The latter involved passing two immiscible liquids moving in opposite directions in contact with each other to separate components differing in their relative solubilities (partition coefficients) in the two solvents. Martin and Synge discovered that a solid support such as silica gel can be wetted by water to form, in essence, a stationary aqueous phase. The surface of the wetted silica gel has, surprisingly, the same solvent properties as pure water. When the stationary... 

The combination of preparative high-speed countercurrent chromatography with other separation methods, such as HPLC, and TLC, will enable chemists to isolate minor components of complex alkaloid mixtures more efficiently. This technique is not limited to alkaloid separations and, in theory, other complex mixtures of compounds having only minor differences in their partition coefficients should be efficiently separated by high-speed countercurrent chromatography. 

The octanol-water partition coefficient (/ o/w) >f a compound is the most universally accepted physicochemical parameter related to its polarity and/or hydrophobicity. Pq/w can be measured directly without any approximation by countercurrent chromatography (CCC). CCC uses two bquid phases to separate mixtures. When the stationary phase is octanol and the mobile phase is water, the retention volume of all injected components is directly proportional to their respective coefficient. 

Partition chromatography originated with the work of Archer John Porter Martin (6. 1910) in the 1930s. He devised a technique known as countercurrent distribution. The early experiments involved a line of separating funnels containing two immiscible solvents. After shaking the funnels, one of the liquid layers fi-om each funnel was transferred to the next in the line, and the process was repeated. Two solutes travelled at different rates if their partition coefficients for the two phases were different. Subsequently the separating funnels were replaced by special tubes, which meant that the process could be automated. 

Countercurrent distribution is the principle behind all types of partition chromatography. It consists of many repetitive partitions of a mixture of solutes between two immiscible solvents (Fig.1). Consider two solvents A, and B, which are immiscible in each other. Solute X is present initially in B only. Suppose that the partition coefficient, K = X /X is equal to 1.0, i.e., the solute distributes equally between solvent A and B. Tube 1 in Fig. 1 contains 32 units of solute X in one volume of solvent B. Tubes 2 to 7 contain only one volume each of the solvent B.. Now, if one volume of solvent A is brought in contact with tube 1, and the two solvents shaken to produce an equilibrium, a partition of X between solvents A and B will result. Since partition coefficient of X is 1.0 exactly 16 units will remain in solvent B while 16 units will be transferred to solvent A. Now, if one transfers solvent A (top layer) from tube 1 to tube 2 and adds fresh solvent A to the first tube and shakes both the tubes to equilibrium, the following results would be obtained in both the tubes each of the solvent layer will contain 8 units of X. After each partition, the top layer of the solvent can be removed and moved to the next tube while each time a fresh aliquot of solvent A can be added to tube 1. Fig. 1 shows result of six such partition steps. Actually hundreds of such partition steps may be employed to achieve separation of one solute from another. 

Conway W D, Ito Y 1984 Solvent selection for countercurrent chromatography by rapid estimation of partition coefficients and application to polar conjugates of /7-nitrophenol. J Liq Chromatogr 7 275-289... 

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