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13 COUNTER-CURRENT CHROMATOGRAPHY KA is a constant at any given temperature, and it is unaffected by the presence of other substances or by the concentration of the solute. Usually it is expressed as the amount of solute in the stationary phase divided by that of the mobile phase as in conventional liquid chromatography. For a better CCC separation, the K values of the target compounds should be in the range of 0.5 ≤ K ≤ 1.0. A smaller K value elutes the solute closer to the solvent front with lower resolution while a larger K value tends to give better resolution but broader, more dilute peaks due to a longer elution time. In CCC, one can choose either the upper or lower phase as the stationary phase, but before deciding which phase is to be used as the stationary phase, one should temporarily express the partition coeffi cient as K U/L = C U / C L , where C U is the solute concentration in the upper phase and C L is that of the lower phase. If K U/L = 2, the lower phase should be used as the stationary phase, which gives K = 0.5. It is important that this preliminary K U/L be clearly distinguished from K using the subscripts to avoid confusion. The measurement of K U/L values may be performed by the shake-fl ask method. Add a small amount (a few milligrams) of each target compound (one at a time) to the two mutually equilibrated solvent phases (1–2 ml each) in a stoppered test tube (13 mm × 100 mm). Thoroughly mix to equilibrate the contents. After settling, pipette and deliver an equal volume of the upper and the lower phases (100–200 μl) each into a separate test tube, dilute each with an equal volume (2 ml) of a suitable solvent such as methanol, and measure the absorbance with a spectrophotometer at a suitable wavelength to obtain the K U/L value. If the pure sample is not available, one can subject each phase to HPLC analysis to compare the peak heights (or areas under the peaks). If the sample does not absorb in the UV or visible wavelengths, the K U/L value may be determined by other ways such as thin layer chromatography (color reaction) or evaporative light scattering detection (ELSD). Thin layer chromatography combined with densitometry is effectively used for simultaneous determination of K U/L of multiple components from a sample mixture. 13.2.3 Droplet Counter-current Chromatography In the early 1970s, Ito and colleagues at the US National Institutes of Health introduced Droplet Counter-current Chromatography (DCCC). In DCCC, as with the Craig-post apparatus, the stationary phase is held in place only with unit gravity. The apparatus consists of a set of vertical straight tubes serially connected with narrow transfer tubing (Figure 3). The original CCC apparatus was equipped with 300 glass tubes, each 60 cm × 1.8 mm i.d. 212
EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS mobile phase stationary phase Figure 3: Droplet counter-current chromatography The total capacity is about 600 ml including the volume in the transfer tubing (about 15% of total capacity). The operation of DCCC is initiated by fi lling the entire column with the stationary phase of an equilibrated two-phase solvent system followed by injection of sample solution. Then, the other phase is introduced into the fi rst unit in such a way that the mobile phase travels through the column of the stationary phase by the effect of gravity, i.e. the mobile phase is introduced from the bottom if it is the lighter phase, and from the top if it is heavier. Consequently, the solutes are separated according to their partition coeffi cients. As with all types of chromatography, compounds more soluble in the mobile phase move more quickly, while those more soluble in the stationary phase lag behind. Under optimum fl ow rate, the mobile phase forms multiple droplets in the stationary phase to divide the column space into numerous partition units; this process is repeated in each partition unit. DCCC necessitates a proper choice of solvent systems for producing a droplet fl ow of the mobile phase in the column. The most popularly used solvent system is composed of chloroform, methanol and water at various volume ratios, although a sizable separation usually takes a few days. 213
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CONTRIBUTORS Chapter 6 Aqueous Alco
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