Separation by High-Speed Countercurrent Chromatography

James McAlpine

INTRODUCTION

Modern high-speed countercurrent chromatography (HSCC) has arisen only over the last 15 or so years and offers the natural product chemist a new separation tool with many unique advantages.

Countercurrent methodology had its beginning in the 1950s with the Craig machine (1), a mechanical system of sequential separating cells in which one phase of a two-phase solvent system could be equilibrated with the other phase in successive cells, thereby carrying a solute along according to its partition coefficient between the two phases.

Current Instruments

Although several minor variants are available, instruments are basically of two types: the Centrifugal Partition Chromatography instrument, sold by Sanki Instruments; and the Coil Planet Centrifuge, designed by Yochiro Ito and sold by PC Inc.

A typical instrument would contain 12 such cartridges. A distinct advantage of this instrument is the ability to use solvent systems containing relatively viscous solvents, such as n-butanol at room temperature.

The commercial instrument has a ceramic-graphite spring-loaded seal with a specified 1500-h life expectancy.

The Coil Planet Centrifuge is just one of a large number of instruments that have been the life work of Yochiri Ito. His study of the movement of one phase of a two-phase solvent system with respect to the other under a variety of imposed vectors and the use of this behavior as a separatory tool is without equal.

OPERATION

The use of HSCC as a separation tool in natural product chemistry can have various aspects.

All bioactive extracts, while still at the crude extract stage, are subjected to HSCC on an Ito coil.

The retention time of the bioactivity eluted from the column can then be correlated with those of similar activities in the database, and the presumptive identity of the bioactive component can be checked spectoscopically.

1. Separation of Crude Mixtures

One can liken the course of an HSCC chromatography to a TLC. Analytes that strongly favor the stationary phase tend to behave as would those in a TLC that have an Rf of zero, whereas those that strongly favor the mobile phase behave like those with an Rf of 1.0. When running a TLC, the highest resolving power is usually obtained for those analytes with Rf in the vicinity of 0.4.

2. Separation of Two Closely Related Congeners

In the course of a natural product isolation, the chemist is often presented with mixtures containing very closely related biosynthetic relatives that may differ only by one or two methylenes, the placement of an olefin, or the stereochemistry of a nonpolar substituent.

If the molecules have strongly polar groups common to their structure, the difference in polarity associated with these structural difference can be insignificant and render an adsorption type chromatographic method useless.

In this situation, HSCC is often the separation method of choice, especially if the mixture has already been the subject of multiple chromatographic steps. Although the methods given above for choice of a solvent system may well work, this may be the time to employ more sophisticated analytical techniques to ensure success.

TLC or analytical HPLC of each phase in the two-phase system will work if the congeners are sufficiently separated in such a system to assay them; if not, it is worth examining the two phases by 1H-NMR or some other technique in which the relative partition coefficients of the congeners can be assessed.

3. Choosing and Tailoring the Solvent System

Carbon tetrachloride was a common component in solvent systems. It had several desirable properties, including low viscosity and high density. But, as use of it is effectively proscribed for health reasons.

Methylene chloride and diethyl ether can both be used, but the researcher should be aware that a vapor lock will force the stationary phase from the system and abort the chromatography. Hence these solvents need to be used only if the ambient temperature permits.

A common approach to a four component system such as the hexaneethyl acetate-methanol-water system is to assume that:

(1) for organics of medium polarity, hexane and water will be poor solvents and ethyl acetate and methanol good solvents;

(2) The lower phase will consist mainly of methanol and water; and

(3) The upper phase will consist mainly of hexane and ethyl acetate.

4. Physical Aspects of Operation

All HSCC instruments are effectively closed systems, and it is not necessary to locate the actual instrument in an exhaust hood.

The solvent will be pumped from reservoirs and the eluent is usually collected in a fraction collector; and, since almost all systems involve volatile organic solvents, it is advisable to locate these peripherals in a hood. The pump must be capable of delivering between 2 and 5 mL/min and should not produce large pulses. A typical three-way injection valve is required and the sample can be loaded in any volume from 1 to 10 mL.

It is of paramount importance that the sample be completely dissolved. To avoid any possibility of salting out, it is common to load the sample in a mixture of the two phases. The machine should be fully loaded, and at least the initial parts of the instrument should be equilibrated and rotating before the sample is loaded.

5. Use of the Ito Coil Planet Centrifuge

When using the Ito coil, the researcher is presented with making a choice of three twofold variables:

1. The question of which phase to select as the stationary phase, i.e., which phase to fill the column with.

2. The choice of the inlet tube, either the "head" or the "tail" of the column.

3. The question of which direction to spin the column, i.e., to have the Archimedean force directed to the inside or outside of the spiral.

Examples of the Use of HSCC for the Separation of Natural Products

Separation of PristinamycinsThe pristinamycins, produced by Streptomyces pristinaespiralis, are an unusual complex of antibiotics in that they consist of two pairs of peptolide antibiotics very closely related within the two pairs but with virtually no structural relationship from one pair to the other.Pristinamycins IA and IB were best separated with a system in which the same components were in the ratio 6:4:8:1 where the last component was formic acid ''to control the pH" but of otherwise unspecified strength.

Separation of Taxol and CephalomannineThe anticancer agent, taxol, is now obtained from a number of Taxus species but invariably occurs with with sizeable amounts of the congener, cephalomannine.

Separation of NiddamycinsThe 16-membered antibacterial macrolide niddamycin is produced by Streptomyces djakartensis as a mixture of aliphatic esters of the 3"-hydroxyl, the secondary alcohol on the neutral sugar mycarose.Chen and coworkers (7) achieved baseline separation of a 200-mg sample of all three niddamycins on an Ito coil in a system of carbon tetrachloride-methanol-0.01 M aqueous phosphate buffer at pH 7.0 with a ratio of 2:3:2. With the aqueous phase, mobile niddamycin F was eluted first followed by niddamycin B while niddamycin A1 was retained and recovered from fractions of the stationary phase when it was pumped from the column.