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Understanding High Performance Counter Current Chromatography

Having discovered the development path that has driven this technology, we would like to guide you in developing a better understanding of the fundamentals and some of the key parameters of the technique, as explained below.

Distribution Ratio (D)

All separations in counter current chromatography occur because sample components of a mixture have differing relative affinities for the two phases of the solvent system in use - solubility in a biphasic solvent system. This effect is measured by the distribution constant D (also (incorrectly) referred to and known as KD):

Distribution coeffiecient formula

Cs is the concentration of a sample component in the stationary phase (SP) and Cm is the concentration of the same component in the mobile phase (MP). Sample components with D > 1 will have a higher concentration in the SP than in the MP, and will elute later from the column. Components with constant < 1 will have a higher concentration in the MP, than in the SP, and will elute earlier. If a compound is equally distributed in the two phases (D = 1), it will elute after one column volume of MP has been pumped through the column regardless of which phase has been chosen as the MP.

Stationary Phase Retention (Sf)

NB Note in the discussion below we are, for the sake of simplicity, ignoring factors such as the system volume and 'dead' volume. Naturally in a real experiment, these factors must be included in the calculation of Sf and other parameters.

In a standard elution mode experiment, the column is filled with SP by pumping at high flow rate then rotation is started and mobile phase is pumped at the chosen flow rate. This process has the effect, over a brief time, of displacing a quantity of SP which is measured. When the displacement of SP ceases the column has reached the state which is known as hydrodynamic equilibrium i.e. the column is equilibrated and ready for injection. Since the displaced volume and the column volume are now known, it is possible to calculate Sf, the stationary phase retention factor by using the following formula:

Stationary Phase Retention (Sf) formula

Where VC is the known column volume and VDISP is the measured, displaced volume of SP

Larger Sf values mean that the column capacity available for the separation is greater and which enhances resolution.

Elution Time (t)

Schematic showing variables used in CCC formulas

Once both D for your target compound and Sf for the biphasic solvent system have been determined, you can calculate the peak elution time for any of your target compounds from the following equation:

Formula for calculating peak elution time for any compounds

where Vc is the column volume, F the MP flow rate, Sf the SP retention (expressed as a decimal) and D the distribution ratio. Your target peak will always have the same retention time from a given column under the same conditions. Once your system is developed, prediction of elution times is easy.

Component elution

Theoretical elution of a compound with a distribution coefficient D

This diagram, on the right, shows the theoretical elution of a compound with a distribution coefficient D. A component with a low distribution constant (i.e.<1) will have a higher concentration in the MP than in the SP and elutes quickly. A component of the sample with a high distribution constant (i.e.>1) will have a higher concentration in the SP than in the MP and elutes slowly. In the section that describes separation development, we shall use this property to show how elution time can be accurately predicted for any scale of separation.

Resolution

One form of the Snyder resolution equation tells us that chromatographic resolution (RS) is directly proportional to the ratio of SP volume (VSP) and MP volume (VMP) i.e. the higher the ratio, the better the resolution will be:

RS α VSP / VMP

Berthod et al demonstrated very well in the chromatograms of the figure below/left/right that chromatographic resolution declines dramatically as Sf decreases:

Chromatograph resolution declines with the decrease in Sf

High Sf values at high mobile phase flow rates are the principal reason for the high performance of this technology. As well as the benefits of high column capacity and resolution, the achievement of such high Sf values offers two other important benefits:

Separation science has been well served by RP-HPLC which achieves fast separations for samples ranging from medium to low polarity. However neither RP-HPLC nor NP- or RP- flash chromatography handle very polar compounds well.

The first, extra advantage of high performance counter current chromatography is found when polar biphasic solvent systems are used. When using counter current chromatography techniques, compounds are usually best separated using biphasic solvent systems whose polarity matches that of the sample i.e. polar compounds require polar solvent systems. It is well understood that as you move from non-polar biphasic solvent systems to polar ones, there is a steady fall in the maximum achievable Sf. High performance counter current chromatography produces relatively high stationary phase retention across the complete range of biphasic systems used.. In the past the poor SP retention of polar solvent systems has limited the technique's usefulness across the whole range of polarities that purification challenges present.

The second advantage, which is a consequence of the improved SP retention of HPCCC, is reduced cycle time and thence increased throughput.

As we have discussed, the best resolution is obtained with the highest chromatographic capacity and this is determined by the ratio of VSP/VMP within the column i.e. Sf.

Sf depends on the qualitative properties of the solvent system in use and is directly proportional to the gravitational field force to which the column is subjected. Sf is also highly dependent on the flow rate used in an experiment. For a given, fixed gravitational field force, as the flow rate increases, there is a progressive decline in Sf and hence chromatographic capacity until eventually this decline has a major detrimental effect on chromatographic resolution.

The higher SP retention of high performance counter current chromatography instruments allows higher MP flow rates and thence shorter cycle times to be used without loss of resolution for a given separation. Now that HPCCC machines are available, high throughput separations can be achieved across the whole range of polarity. The accompanying figure shows the impact of increasing flow rate on Sf and resolution.

In summary, using a fixed solvent system, resolution depends on Sf which depends on applied gravitational field force and flow rate. The ability of HPCCC instruments to run with much higher gravitational field force gives superior Sf at higher flow rates than other manufacturer's equipment thereby reducing run times and increasing throughput. Cycle times, measured in minutes or tens of minutes, are about one tenth of those required by other manufacturers' equipment.

The much higher gravitational field force gives superior Sf at higher flow rates

Effect of column length

Resolution of any separation is improved by increasing the length of the column; unlike solid phase chromatography techniques, high performance counter current chromatography is a low-pressure technique and that does not limit the length of the column within reason. When comparing the lengths of two columns, e.g. doubling, with the same internal diameter, the resolution improves by the square root of the ratio of the lengths of the columns i.e. the longer column will have 1.4 times the resolution of the shorter. Obviously doubling the length of the column doubles the cycle time if other experimental factors remain the same, however, throughput is unaffected because the sample load can also be doubled.

Due to this direct, linear, volumetric scale up relationship, separations can be developed on small columns, minimising solvent and sample usage before proceeding to larger 'off the shelf' or custom-made columns in order to maximise the most important process requirement (throughput, yield or purity).