Selection of Two Phase Solvent System

Among the purification of natural products, the isolation of antibiotics is one of the most difficult tasks since the crude sample often contains, in addition to numerous impurities, a set of closely related components that tend to exhibit similar partition behaviour in a given solvent system. Consequently, successful separation necessitates a painstaking search for a suitable solvent system, which often requires days, weeks and even months of hard trial. Once a suitable solvent system is found, however, the separation is usually completed within several hours.

HSCCC utilizes two immiscible solvent phases, one as a stationary phase and the other as a mobile phase. Solutes are subjected to a continuous partition process between these two phases along the column space free of a solid support, hence the separation is almost entirely governed by the difference between their partition coefficients.

Table 1 Separation of antibiotics by HSCCC



Solvent system

Mobile phase

Daunorubicin derivatives

Gramicidins A, B, and C Siderochelin A Efrotomycin

Pentalenolactone Bu 2313B

A 201E

Tirandamycin A and B Actinomycin complex

Benzanthrins A and B (quinone antibiotics) Coloradocin

Candicidin (polyene macrolide antibiotics) 2-Norerythromycins (macrolide antibiotics)

Niddamycins (macrolide antibiotics)

Tiacumicins (macrolide antibiotics)

Coloradocin (macrolide antibiotics) Sporaviridin complex Dunaimycin (macrolide antibiotics)

Bacitracin complex Bacitracin complex Mycinamicins


Pristinamycins (macrolide antibiotics) Pristinamycins (macrolide antibiotics) Ivermectin Colistin

100 mg 400 mg 670 mg

50 mg 200 mg

350 mg

134 mg

83 mg 620 mg

400 mg 100 mg 500 mg

200 mg

200 mg

400 mg 100 mg

50 mg 50 mg

Analytical works Analytical works 1 mg 1 mg 25 mg 20 mg

Chloroform/ethylene chloride/hexane/methanol/ UP water (1 :1 :1 :3.5:1)

Benzene/chloroform/methanol/water (15: 15: 23: 7)UP Chloroform/methanol/water (7:13:8) UP

Carbon tetrachloride/chloroform/methanol/water UP (5:5:6:4)

Chloroform/methanol/water (1 : 1 : 1) UP

n-Hexane/dichloromethane/methanol/water LP (5:1:1:1)

Carbon tetrachloride/chloroform/methanol/water UP (2:5:5:5)

n-Hexane/ethyl acetate/methanol/water UP

Ether/hexane/methanol/water (5:1:4:5) UP

Carbon tetrachloride/chloroform/methanol/water UP (4:1:4:1)

Chloroform/methanol/water (1 : 1 : 1) UP

Chloroform/methanol/water (4:4:3) ?

n-Heptane/benzene/acetone/isopropanol/ UP

Carbon tetrachloride/methanol/0.01 mol L~1 UP potassium phosphate buffer (pH 7) (2:3:2) Carbon tetrachloride/chloroform/methanol/water UP (7:3:7:3)

Chloroform/methanol/water (1 : 1 : 1) UP

n-Butanol/diethylether/water (10:4:12) LP

n-Hexane/ethyl acetate/methanol/water UP

Chloroform/ethanol/water (5:4:3) LP

Chloroform/ethanol/methanol/water (5:3:3:4) LP n-Hexane/ethyl acetate/methanol/8%aq. LP

n-Butanol/0.04mol L~1 TFA (1:1) containing 1% LP glycerol

Chloroform/ethyl acetate/methanol/water UP

Chloroform/ethyl acetate/methanol/water UP

n-Hexane/ethyl acetate/methanol/water LP

Generally speaking, the two-phase solvent system should satisfy the following requirements:

1. Retention of the stationary phase. Since the system eliminates the solid support, the retention of the stationary phase in the separation column entirely depends upon the hydrodynamic interaction between the two solvent phases in the rotating column under a centrifugal force field. While the hydrodynamic motion of the two phases is highly complex, the retention of the stationary phase may be predicted by the following simple procedure to measure the settling time of the two phases under gravity: Place 2 mL of each phase of the equilibrated two-phase solvent system into a 5 mL capacity graduated cyclinder (alternatively, a 13 mm o.d. and 100 mm long glass test tube equipped with a plastic cap may also be used) which is then sealed with a stopper. Gently invert the cylinder five times to mix the contents and immediately place it on fiat surface to measure the time required for the mixture to settle into two layers. This settling time should be considerably less than 30 s for stable retention of the stationary phase.

2. Partition coefficient (K). The partition coefficient is the key parameter for HSCCC. It is usually expressed by the analyte concentration in the stationary phase divided by that of the mobile phase. For a successful separation, the K value of an analyte should be close to 1. If K ; 1, the analyte will elute close to the solvent front resulting in loss of peak resolution. On the other hand, if K < 1, the analyte will remain in the separation column for a long period of time, producing an excessively broad peak. In order to separate two components, the ratio between their partition coefficients, which is called separation factor (a), should be 1.5 or greater for a standard semipreparative multilayer coil HSCCC equipment providing a moderate partition efficiency of about 800 theoretical plates.

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