Go against the flow

Eddie Goodall and Mike Giles tell Laboratory News about an exciting technique for preparative scale separations – high performance counter current chromatography.

As the many scientists using reverse phase-HPLC or flash chromatography have learned – separations using these techniques depend on interactions between a solid stationary phase and a liquid mobile phase. In using such solid-liquid chromatographic techniques the ‘received wisdom’ is that greater efficiency leads to improved resolution. This works very well at an analytical scale with micrograms or less of a multi-component sample to separate, but at preparative scales where hundreds of milligrams to kilograms have to be separated, the approach of increasing efficiency is not an option and as loading increases, peak-width also increases resulting rapidly in reduced resolution.

High performance counter current chromatography (HPCCC) is an innovative, preparative-scale, liquid-liquid chromatographic technique that is generating considerable interest amongst pharmaceutical synthetic chemists, life scientists and natural product researchers involved with larger scale separations. HPCCC can be considered as a complementary and orthogonal technique to its solid-liquid counterparts – the technique has been proven to fit seamlessly into workflows and provide productivity improvements in many applications.

In HPCCC, the stationary and mobile phases comprise a pair of selected immiscible liquids. Using liquids for both stationary and mobile phases confers a number of unique features and advantages for the preparative scale chromatographer including selectivity driven high resolution purification, significantly higher mass throughput, simple and reliable scale-up and quick post-purification work up. HPCCC is a high recovery technique which is as easy as the chromatography you do today but produces much higher yields and requires between a fifth and a tenth of the solvent usage – reducing expenditure and making your lab greener.

An HPCCC instrument employs an alternative type of chromatography column that uses a liquid stationary phase that is ’held’ within the column, while a mobile phase, also a liquid and immiscible with the stationary phase, is pumped through it. When solutes with different physic-chemical properties have to be separated, they will have differential, relative affinities for each of the liquid phases. HPCCC exploits these different relative affinities (selectivity) to separate the solutes by partition chromatography which requires only modest and fixed chromatographic efficiency.

The central, hypothetical chromatogram in figure 1, shows a pair of inadequately resolved components. One way of improving the resolution of components in such a situation is by increasing the efficiency of the system (path B); equally the components can be resolved by increasing the selectivity of the system (path A). Path B is commonly used by solid-liquid chromatographers since the opportunities for enhancing the selectivity of solid phases are limited. Path A is how counter current chromatographers achieve resolution.

All Counter Current Chromatography (CCC), including HPCCC instruments and systems have low efficiencies with plate counts per column amounting to only several hundred compared with the thousands or tens of thousands of plates per column which are typically available when using HPLC. Table 1 shows how baseline resolution (R = 1.5) of two components is achievable with extremely modest efficiency values if selectivity can be sufficiently enhanced. In solid-liquid chromatography, improved resolution is usually driven by increased efficiency since changing media or solvent components usually offers only very limited scope for improving selectivity. Although the efficiency of CCC systems is modest, the options for enhancing selectivity are extensive: virtually any combination of solvents can be used as long as it can produce two (or more), readily separable, immiscible phases. This indicates that high resolution purification is possible, but other factors also need to be taken into consideration.

In the widely and most often used form of the Snyder resolution equation, resolution is expressed in terms of N, ? and k which in this form is very suitable for the expression of resolution for solid-liquid chromatographic systems. However, for liquid-liquid systems, re-expression with more suitable terms is appropriate:

In this equation, the ratio VM/VS – where VM is the mobile phase volume and VS the stationary phase volume – is the retention factor (k) equivalent and the relationships of D and D’ refer to selectivity. In liquid-liquid chromatography D, which is called the distribution or partition ratio or constant (and other variants of more or less correctness) is used synonymously with the expression KD, and is the ratio of the concentration of a solute in the stationary phase and its concentration in the mobile phase i.e. D = [SP]/[MP].

Consideration of figure 2 shows how the relative ratios of VM and VS differ between liquid-liquid and liquid-solid systems. The equation above is less useful for solid-liquid systems since the ratio, VM/VS, is constant and in bonded phase media, large whereas in liquid-liquid systems it is variable and often small.

The most important characteristic is that since resolution is inversely proportional to the ratio VM/VS, it is directly proportional to the ratio VS/VM, i.e. the higher the value of VS compared with VM, the higher is the resolution. Furthermore, in solid-liquid chromatographic systems, the ratio evaluates to 0.1 or less whereas in liquid-liquid systems, the expression typically evaluates to more than 3 and often approaches 20. It is for this reason that Counter Current Chromatography is a high resolution technique despite being low efficiency and is also the reason why the technique enjoys such high comparative dynamic capacity with working solute concentrations which are typically five to ten times those encountered in solid-liquid chromatography

In liquid-liquid chromatography, the factor which controls retention and hence selectivity, is the differential distribution (partition) of components between the two phases under consideration. In multi-component, biphasic solvent systems, modest changes in the relative volumes of one or more components can have profound effects on the relative distributions of solutes so altering selectivity.

The examples in figure 3 and 4 show consecutively, the normal and reversed phase separations of a mixture of??-blockers when stepping through solvent combinations from a HEMWat (Hexane, Ethyl Acetate, Methanol and Water) Solvent System Table.

Points of note when comparing the chromatograms:
• Selectivity and hence resolution changes significantly with the step-progression of solvent combinations used.
• The elution order of the components is completely reversed on switching from normal to reversed phase elution. Data of SS17 in the two panes should be considered.
• A process such as that shown i.e. considering the separation of a single mixture using different solvent combinations, is a significant part of the method development process described elsewhere on this website.
• For all of the chromatograms, following injection of the sample, the column was subjected to 20 minutes of ‘classical’ elution mode and the column contents were then extruded.
• These data were generated on a fully automated HPCCC system using a quaternary pump for blending the solvents (SP and MP) in the correct proportions and a switching valve to change between NP and RP elution modes.

HPCCC is an orthogonal chromatographic technique where separation is based on differential solubility of solutes in two immiscible solvent phases. Separation is based on enhanced selectivity, where the peaks are ‘moved apart’, rather than efficiency where the peaks are made taller and thinner. The advantage of improving selectivity rather than efficiency is that moving peaks further apart by alteration of selectivity allows you to load your sample at higher levels before resolution is compromised by increased peak-width. In solid phase chromatography the scope for large increases in selectivity is much less.