33


speed countercurrent chromatography (HSCCC) units (~70g) significantly reducing separation time by a factor of 10 to between 20 to 40 minutes, while maintaining resolution.


HPCCC instruments have two bobbins that enable additional processing versatility where semi-continuous processing can be achieved by continuously injecting sample in between the two bobbins and intermittently switching the direction of flow between the aqueous mobile phase from one end of the column to the organic mobile phase from the other end of the column. In this processing configuration, the more hydrophilic compounds elute with the aqueous phase and the more hydrophobic compounds elute with the organic phase while a chosen target compound can be concentrated inside the column and harvested at regular intervals.


Why has CCC had such a low profile? CCC has had a relatively low profile in the separation science community with applications being largely confined to the preparative isolation of natural products [3] where its appeal is two fold; 1) the absence of a solid stationary phase to cause problems with irreversible adsorption and decomposition of sensitive compounds and 2) the provision of an alternative separation tool for difficult separation problems such as those arising from closely related structural isomers. It is only with the advent of HPCCC instruments relatively recently that commercial instrumentation capable of producing separations on a similar time scale to HPLC has been available. The introduction of high g-level instruments also enabled small bore columns requiring only milligrams of sample to be used for scouting purposes. Finally, previous generations of CCC instruments have traditionally not been marketed as integrated separation units, in contrast to the situation with HPLC, where over the last 20 years instrument integration under software control has revolutionised practice [4, 5]


. This


gap is now being addressed by integration of HPCCC instrumentation with conventional computer controlled HPLC systems, which can then be programmed to perform the analysis


automatically, including the automated proportioning and delivery of the mobile and stationary phases [6]


. A photograph of an integrated instrument is presented in Figure 2.


The key benefits that HPCCC instruments offer the chromatographer are:


• Higher sample loading per injection • Easy scale-up • Very high recoveries of injected samples


This is due to the high stationary phase volumes in the column, a single primary mechanism of separation and the lack of expensive solid stationary phase packing materials, which also clearly add considerable expense as the scale of the separation is increased. In addition, CCC is capable of handling relatively ‘dirty’ samples containing particulates as shown in Figure 3 [7, 8] which would normally require considerable sample preparation.


N required to give RS for given α values RS = 1.0 RS = 1.5


α


1.000 1.005 1.010 1.020 1.050 1.100 1.250 1.500 2.000


N -


650000 163000 42000 71000 1900 400 140 65


N -


1450000 367000 94000 16000 4400 900 320 145


Table 1- Relationship between selectivity,α, plate count, N and resolution, Rs


All CCC, including HPCCC instruments have relatively low efficiencies with plate counts per column amounting to only several hundred compared with the thousands or tens of thousands of plates (N) per column which are typically available when using HPLC. However, as shown in Table 1, baseline resolution (Rs ≥ 1.5) of two components in CCC is achievable with extremely modest


Figure 3: Two views of a sample loading tube reported in [8] showing particulate matter which was subsequently successfully processed


Separation capability At the outset of the project the consortium has focussed on generating a set of applications which illustrate the selectivity and versatility of CCC. The aim here was to allow an understanding to be gained of the capability of CCC and to use the resulting separations portfolio to influence the separation science community. A variety of purification challenges have been successfully overcome including the separation of isomers, purification of crude reaction products and recovery of product from mother liquors. Compounds spanned a range of polarity and structural types. The data that has been obtained to date [9]


illustrate that the


Figure 2: An integrated HPCCC system (Shimadzu(UK)/ Dynamic Extractions Spectrum)


technique has excellent applicability. Thirteen out of the fifteen mixtures studied were separated at loadings suitable for preparative use and of these nine were achieved with a simple heptane, ethyl acetate, methanol, water system, known as the HEMWat solvent system. This work has also shown that CCC has the potential to provide an alternative to solid phase chromatography and produce the quantities required to support the development of drug candidates.


efficiency values if selectivity, (α) can be sufficiently enhanced. 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. HPCCC instrumentation offers an alternative orthogonal approach to preparative chromatography.


This approach may appear counter intuitive to the chromatographer used to HPLC. Nonetheless, by focussing on selecting optimum partitioning conditions it is possible to achieve some very challenging separation objectives. This is illustrated by the example presented below.


Separation of a target from a complex mixture – purification of a waste stream The isolation of a target material from a complex mixture is a very good illustration of the capability of CCC. A crude mother liquor sample from a crystallisation has been reprocessed by CCC to yield a purified fraction (Figure 4 A, B & C) [10]


. This material has


subsequently been processed by further crystallisation to give pass quality product.


Where does CCC fit? There are two clear areas where CCC can add value. The first is as a complementary preparative technique to HPLC and other


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