The preparative chromatographic technique called countercurrent chromatography (CCC) faces two major problems: first, to find the appropriate biphasic liquid system for the desired purification, and second, to retain enough liquid stationary phase inside the CCC column so that the preparative separation can be achieved. In this study, a small-volume (38 mL) hydrostatic CCC column with rotary seals working at pressures as high as 70 bar and rotor rotation speeds as high as 3000 rpm was used to quickly estimate the separation properties of a given biphasic liquid system. The usefulness of this technique was demonstrated in the direct measurement of octanol–water partition coefficients. This approach can be used to purify milligram quantities of materials, but more importantly, the results obtained (solute partition coefficients) can be directly transposed to methods using larger CCC columns — both hydrostatic and hydrodynamic — for larger loads using the same biphasic liquid system.

Countercurrent chromatography (CCC) is a separation technique that uses a biphasic liquid system to purify valuable materials. All chromatographic processes separate sample components according to their affinity for a stationary phase when eluted by a contacting mobile phase. In CCC, both the mobile and the stationary phase are liquid. The term countercurrent chromatography comes from a previous countercurrent distribution process (Craig machine) that also used biphasic liquid systems and countercurrent extractions to separate solutes. Although this term is not seriously challenged today, it is clearly a misnomer: There is no countercurrent liquid circulation in classical CCC, and a single pump can be used for the mobile phase during the run (1).

Modern CCC was recently presented in an LCGC Europe article (2). The main analytical use of CCC is the direct determination of octanol–water partition coefficients by working with an octanol stationary phase and an aqueous octanol saturated mobile phase; the solute peak positions provide the respective octanol–water coefficient without any extrapolation (1). Apart from that, CCC is a preparative technique used mainly for efficient purification of significant amounts of material. In the last decade, the CCC process has been gaining wider acceptance for preparative use as a result of the development of improved CCC columns that produce faster separations with increased loads as high as several hundredths of a gram injected onto a 5-L column (3). These modern columns allow for quick and easy scale-up from small- to large-volume CCC columns (3).

The consequences of this very simple retention equation are simple: In CCC, solute retention volumes and sample processing size are both directly related to column volume. Because CCC is mainly a preparative technique, the commercially available columns often have a significant volume, between 0.1 and 5 L (3). In the past, few small-volume CCC columns were commercially available. There was an 18-mL hydrodynamic column that could not work at flow rates higher than 1.5 mL/min (4) and a 50-mL hydrostatic column that had rotary seals unable to withstand pressures higher than 40 bars and rotation speed higher than 2000 rpm (5,6). Table I illustrates the problem: Because solute retention volumes are directly related to the respective distribution ratio and column volume, significant solvent volumes and working time are needed when working with large CCC columns.

Table I presents the calculated retention volumes obtained with six different CCC columns retaining the same relative amount of stationary phase, namely 70% of the CCC column volume, expressed by S _f = 70%. The selected mobile phase flow rates and column efficiencies were experimentally observed (1,5). For example, hypothetical solute F of Table I has a distribution ratio of 5 in the selected biphasic liquid system. This means that it will be the last eluted solute (equation 1). When injected into the small-volume 30-mL CCC column, it is eluted in 11.4 min and requires 114 mL of mobile phase. When injected in the large-scale, 20-L CCC column, the same solute F needs 13 h and 76 L of mobile phase to be eluted.

In the example in Table I, when the large-volume column was used, 200 g of solute were processed. So a rough calculation shows that this separation produced 1 g of solute in 4 min using 380 mL of solvent. The same calculation shows that when the small column was used, injecting 0.2 g of solute, the separation produced 1 g in 57 min (5 × 11.4 min, although in reality, five injections would surely need more time) using 570 mL of mobile phase.

Given these rough calculations, it is clear that purification of solute F should be performed using the large-volume CCC column. However, the purification conditions could be determined quickly by working with the small CCC column and using the experimental conditions shown in Table I (a flow rate of 10 mL/min with 70% stationary phase retention). However, at the time these examples were prepared, there were no small volume CCC columns able to work with the conditions listed in Table I. A good small-volume CCC column must be able to retain a significant amount of liquid stationary phase while working at flow rates corresponding to a complete column volume in a matter of minutes.