HSCCC and natural food pigments
High-speed countercurrent chromatography solves an absorbing separation problem.
Harrowing tales of chromatographic separations undoubtedly fill the annals of frustrating laboratory experiences. Although significant developments in techniques like HPLC now preclude a fair amount of grief, there are still those difficult compounds that just dont separate as expected. These problem children are often highly polar molecules, which tend to absorb strongly onto typical column packing material. As the amount of material is increased beyond the very small (analytical) scale, this behavior becomes more consequential. Subsequently, less predictable separations result. Peak tailing occurs and there are often significant amounts of unrecoverable material. Furthermore, somewhat severe conditions (e.g., acidic eluents) used to counteract these effects often lead to irreversible analyte degradation.
The French Paradox
A particularly frustrating group of compounds is the natural pigments found in fruits, vegetables, wines, and teas. These polyphenol molecules, such as the highly colored anthocyanins (in fruits, vegetables, and red wines) and the more subtle catechins (in green teas), have generated great interest for their possible health benefits (Figure 1). Numerous studies linking substances such as green tea or red winethe so-called French paradoxwith a healthy heart and reduced cancer risk have brought the potential antioxidant role of these compounds to the public forefront. Research into their nutritional effects and possible applications (e.g., nutraceuticals) requires an efficient and affordable means of obtaining pure quantities of individual material, particularly for use as reference standards. Separating such compounds has remained a challenge, however, due to the adverse sorbent interactions that are commonplace with these species. Recently, an assortment of promising isolations has been demonstrated for such polyphenol pigments using a sophisticated form of pure liquidliquid partitioning, called high-speed countercurrent chromatography (HSCCC).
|Figure 1. Antioxidants Waiting To Happen.
Investigations into diets high in red wine or green tea have suggested that anthocyanins (left) and catechins may act as powerful antioxidants, preventing or modulating chronic disorders such as heart disease.
The most significant difference and advantage of liquidliquid partition chromatography in generaland of HSCCC in particularcompared to other chromatographic methods is the fact that the separation occurs between two nonmiscible liquid phases, says Matthias Hamburger, a natural product researcher at Friedrich-Schiller-Universität Jena in Germany. Thus, separations are based solely on the partition coefficients of the analytes with respect to the two solvent phases, and no absorption or degradation effects are possible.
In general, CCC involves the elution of a mobile solvent phase through a column filled with a stationary, immiscible liquid phase. To make this process an effective chromatographic technique in terms of good resolution and reasonable retention times, several parameters are crucial. A rapid mobile phase must be used, and at the same time there must be significant stationary phase retention within the column. Stationary phase retention is critical because it directly controls the level of resolution that can be attained. In addition, effective mixing between solvents (as can be accomplished in a standard separatory funnel) must be achieved to afford effective analyte partitioning.
These requirements are nicely satisfied by modern HSCCC, developed in the early 1980s by Yoichiro Ito and collaborators at the National Institutes of Health (Bethesda, MD). This technique is based on the effects of gravitational and centrifugal force on solvent flow behavior in helical-shaped tubing. When a coil containing two immiscible solvents is rotated within a certain critical speed range (based on the helical measurements), an asymmetrical force distribution is created around the width of the coil. This causes preferential bilateral distribution, in which the heavier phase occupies the space closer to the head of the coil (defined by direction of rotation), and, subsequently, the lighter phase occupies the tail end. Therefore, for example, if a mobile phase is pumped through the tail end into a rotating coil filled with a less dense stationary phase, a better-than-expected stationary phase can be maintained because of its predilection for the column inlet. With the addition of a centrifugal field, the forces directed on the column become more complicated, but the right setup can greatly strengthen the propensity toward bilateral distribution.
These ideas led Ito to develop the coil planet centrifuge. This device included a coiled tube column (made of inert plastic) wrapped around a rod-shaped holder that could be simultaneously rotated on its central axis (parallel to the direction of solvent flow) and revolved around a fixed axis outside of its circumference. Ito and his associates arrived at a particular setup called type-J, in which the column holder rotates around its own axis while revolving around the centrifugal axis of the centrifuge in the same direction and same angular velocity. The two axes are parallel to each other in this type of motion. The J designation indicates the shape in which the flow tubes (extensions of the columns inlet and outlet) are positioned in order to prevent tube twisting without the use of rotary seals (Figure 2).
|Figure 2. Schematic of a coil planet HSCCC unit.
The typical modern HSCCC apparatus uses type-J motion with a column mounted coaxially around the holder in a multilayer fashion. With this setup, a powerful force for bilateral distribution is created, in which the mobile phase can be pumped at high flow rates (up to 5 mL/min), with significant stationary phase retention (well over 50%, depending on the phase density difference and helical measurements). In addition, a fortuitous set of forces produces a highly efficient environment for interphase mixing. At points close to the centrifuge axis, the phases undergo violent mixing, but farther from the axis, the phases settle into separate layers that accelerate toward opposite ends of the coil. This allows the mobile phase to steadily pass through the stationary phase with sufficient interaction for analyte partitioning.
Hamburger sees such an HSCCC device as a very valuable tool for work in his laboratory. We use HSCCC, says Hamburger, whenever we have a preparative separation problem that cannot be adequately solved by liquidsolid chromatography. Recently, his lab was interested in obtaining multigram quantities of two catechins found in green tea for use in pharmacological studies and as pure analytical standards. While the separation was not feasible with HPLC, they were able to design a simple and efficient HSCCC method to procure what was needed within a short time (components from about 600 mg of tea extract could be separated in a little over an hour). The effectiveness of HSCCC for such troublesome systems is further supplemented, Hamburger points out, by an economical advantage: The method requires the purchase of only inexpensive solvents and not the more expensive sorbent materials. Furthermore, solutes can be readily recovered simply by evaporating off the solvents. If something goes wrong with the separation, you dont lose your precious sample, says Hamburger.
Other reports of HSCCC isolations of pure pigment material that were previously unavailable or only available at exorbitant costs have appeared in a string of papers during the past year or two from Peter Winterhalters lab at the Technische Universität Braunschweig in Germany. Generally, pigments in amounts of several hundred milligrams to grams have been retrieved in one working day from food materials, including red wines, chokeberry juice, red cabbage, and black and green teas.
The Bigger the Better
An additional bonus of using HSCCC with such natural pigment systems is its amenability to scale-up. Because HSCCC separations are based purely on partition coefficients, as long as the solvent ratios remain constant, larger-scale runs should give, relatively, very similar results to those with less material. The largest [commercial] preparative column for high-speed CCC, says Ito, is marketed from Pharma-Tech Research Corporation [Baltimore]. The column volume of this instrument is 800 mL, and it separates about 510 g of sample in the standard high-speed CCC system and 50 g by pH-zone-refining CCC, a technique that couples selective neutralization with typical chromatographic partitioning. In his own lab, Ito has a unit with double this capacity.
The one limitation of HSCCC is equipment-related. The strong centrifugal force becomes less manageable and more dangerous with increasingly larger equipment. However, an efficient industrial-scale HSCCC apparatus would be of great use to natural pigment research. A greater sample loading capacity, up to multikilogram . . . may be feasible using slowly rotating CCC instruments, says Ito. The record preparative-scale separation has been recently achieved by a slowly rotating multilayer coil . . . which successfully separated 150 g of tea extracts. This was accomplished using spiral ridged, or convoluted, tubing. The tube shape helps maintain a good proportion of the stationary phase retention and phase mixing that usually occurs as a result of the centrifugal field.
Thus far, HSCCC has remained confined to a niche market composed of individual laboratories with particular separation problems, and it hasnt broken out into wider commercial use. This, perhaps, has limited the speed of technical developments. Hamburger thinks that a key issue for the user is the still somewhat unsatisfactory reliability and robustness of the equipment. In addition, methodological strategies, such as those for choosing a solvent system, are still in the early stages.
There is an ongoing effort, however, to bring HSCCC (and CCC in general) into wider use. This was evidenced by the First International Conference on Countercurrent Chromatography, which convened last September in the United Kingdom. A major goal of the meeting (www.ccc2000.co.uk) was to draw up an international consensus on the priority areas for research and technological development with the aim of securing reliable and sustainable CCC instrumentation for industry. Such international collaboration could lead to continued improvements in HSCCCs effectiveness for resolving chromatographic separations that typically prove frustrating, such as those for natural food pigments.
- Baumann, D.; Adler, S.; Hamburger, M. J. Nat. Prod. 2001, 64, 353355.
- Countercurrent Chromatography: Theory and Practice; Chromatographic Science Series, 44; Mandava, N. B., Ito, Y., Eds.; Marcel Dekker: New York, 1988.
- Degenhardt, A.; Engelhardt, U. H.; Lakenbrink, C.; Winterhalter, P. J. Agric. Food Chem. 2000, 48, 34253430.
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- Degenhardt, A.; Knapp, H.; Winterhalter, P. J. Agric. Food Chem. 2000, 48, 338343.
- Du, Q.; Wu, P.; Ito, Y. Anal. Chem. 2000, 72, 33633365.
- High-Speed Countercurrent Chromatography; Chemical Analysis, 132; Ito, Y., Conway, W. D., Eds.; John Wiley & Sons: New York, 1996.
David Filmore is a staff editor with Todays Chemist at Work Send your comments or questions regarding this article to email@example.com or the Editorial Office 1155 16th St N.W., Washington, DC 20036.