ReviewGolden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography
Introduction
In the early 1970s, a new separation technique called counter-current chromatography1 (CCC) was developed. The method provides an advantage over the conventional column chromatography by eliminating the use of a solid support where an amount of stationary phase is limited and dangers of irreversible adsorption from the support are inevitably present. As usual in the development of new methods, the early models had various problems: the first model called helix CCC (now called toroidal coil CCC) [1], [2] had a rotary seal and the effluent was introduced from the rotating syringe. This analytical model yielded thousands of theoretical plates (TPs), but it required an overnight run. The second model called droplet CCC [2], [3] produced a preparative separation at near 1000 TPs, but separating 30 mg of a test sample required 3 days. Unfortunately, the performance of these early models produced a long-standing false image that CCC is a time-consuming technique. In the intervening years, the method has been radically improved in terms of resolution, separation time and sample loading capacity by the development of high-speed CCC (HSCCC) [4], [5], [6], [7], which yields a highly efficient separation of multigram quantities of samples in several hours. HSCCC, which is one form of CCC, is now accepted as an efficient preparative technique, and widely used for separation and purification of various natural and synthetic products.
The use of this instrumentation, however, requires some simple but special technical knowledge, since the selection of the experimental conditions and the practical separation procedure are quite different from those of conventional column chromatographic methods. During the past 30 years, a number of publications including monographs [8], [9], [10], [11], [12], [13], [14], encyclopedia [15], [16] and review articles [17] in addition to a great number of research articles on HSCCC in chromatographic journals have been issued. Unfortunately, while exploring these references, I have noticed that these publications are divided into two main categories, one explaining theories, principles and designs, and the other, focused on practical applications. What is lacking, however, are the technical details to be grouped in one place for beginners to follow, i.e., how to select a suitable solvent system, how to prepare the sample solution, how to determine the inlet and outlet of the column, how to select the direction and the rotation speed of the apparatus, how to monitor the effluent, etc. Although these procedures are obvious for scientists routinely working on CCC separations, a misuse of one facet may lead to unsuccessful separations and this might well discourage one from continued use of the method.
The purpose of this article is to explain comprehensively the practical CCC procedures for beginners so that they can quickly learn how to use the HSCCC instrument under optimum conditions. To ease understanding the techniques, the mechanisms of both standard HSCCC and pH-zone-refining CCC are briefly described with illustrations.
Section snippets
Mechanism
Fig. 1 schematically illustrates the type-J synchronous planetary motion of a multilayer coil separation column. The planetary motion is produced by engaging a planetary gear mounted on the column holder axis to an identical stationary sun gear rigidly fixed to the centrifuge framework. This 1:1 gear coupling produces a particular type of planetary motion of the column holder, i.e., the holder rotates about its own axis while revolving around the centrifuge axis at the same angular velocity
pH-zone-refining counter-current chromatography
pH-zone-refining CCC [29], [30], [31], [32], [33] is generally employed as a large-scale preparative technique for separating ionizable analytes. The method elutes highly concentrated rectangular peaks fused together with minimum overlapping while impurities are concentrated and eluted between the outside the major peaks according to their pKa and hydrophobicity. Although it somewhat resembles displacement chromatography, there are distinct differences between these two methods [30].
The
Conclusion
The author hopes that the instructions and advices presented above will be useful and encourage others interested in separations to use this technique which he feels, with admitted prejudice, has so much to offer them. If questions arise, the author can be reached at [email protected].
Acknowledgment
The author wishes to express many thanks to Professor Roger W. Giese for his kind invitation to write this article and a number of reviewers who have given invaluable comments and suggestions.
The author is also indebted to Dr. Henry M. Fales of National Institutes of Health for editing the manuscript with useful suggestions.
Glossary
- Archimedean screw effect (head and tail)
- when the coiled column filled with two solvent phases is subjected to a planetary motion (rotating centrifugal force field), both phases tend to advance toward one end (head) of an open coil determined by the Archimedean screw. However, in a closed coil type-J synchronous planetary motion gives an advantage for the lighter phase to occupy the head end while the heavier phase is pushed back toward the other end called the tail. This phenomenon is
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