18November/ December 2009 difficulty of needing several different instruments, to validate scale- up is avoided. For Process Chromatography, the base module is of 3 litres. Bobbins are interchangeable, and can be exchanged for re- winding if PTFE tubing chosen and cGMP requires virgin material. Most would use stainless steel or titanium tubing and appropriate cleaning techniques, but renewing PTFE coils is an option. If different bore sizes are required, different bobbin sets may be used. Bobbins can be used in series, in parallel or in simulated moving bed operations. Clutches and switching valves allow operating mode changes. The Partitron CPC T is shown in Fig 3 ( sun centrifuge, with separation chambers and id restricted links between chambers, with 2 rotating seals). This model range has a single process- scale chassis unit. Most hydrostatic CPC are manufactured as chambers created by a sandwich of machined or etched chambers formed into a stainless steel disc, with layers of PTFE sealing the individual disc layers from other layers. The whole assembly is bolted together, but can be prone to leaks and blockages. Machining and in particular etching of any surface radically increases the surface area exposed. Viewed under a microscope the machined/ etched surfaces will appear as mountains and valleys relative to the same material before machining/ etching. As CPC units are particularly suited to aqueous/ aqueous chromatography of peptides and proteins, enzymes etc, all of which are prone to degradation, the choice of machined/ etched stainless steel for most hydrostatic L- LC is suspect. The Partitron CPC T was specifically designed for large scale, GMP process chromatography. A totally different construction is utilised. The whole rotor assembly ( Fig 3) is machined from a single titanium block. Titanium is well recognised in chromatography for its inertness. A variety of titanium rotors, with volumes from 5 to 25 litres, with either one or two volumes per rotor, may be fitted to this versatile, uniquely modular hydrostatic L- LC. Fig 4 shows an industrial sub & super- critical extraction plant which is used in conjunction with L- LC production and research Results & Discission All experiments in Case Studies completed with a Quattro CCC T . Discussions regarding Confusions 1 to 2 Unpublished Grant funded research (" The Industrial Scale up of Countercurrent Chromatography". BBSRC/ DTI LINK Award Ref: 100/ BCE08803. Feb 98 - Jan 00 (£ 322,668), a collaboration of AECS, Brunel University, University College of Swansea, GSK, Astra Zeneca & Shell Research) supported comments by CCC experts, that CCC of different designs or even a single concept, if one varies key parameters this can, on occasions, prohibit scale- up. Keeping all parameters the same, only changing tubing bore, certain scale- ups failed. AECS & Brunel University interpreted the implication of these results in radically different ways in their subsequent independent commercialisation of L- LC. AECS rationalised design to minimise variability and has spent 9 years increasing its understanding scale-up failures. Brunel University and staff developed a range of CCC with radically different sun & planet radii, speed ranges etc. and formed their own spin- off company ( DE Ltd) six years ago to exploit their research. Discussions regarding Confusions 3 to 7 Non- confidential research is detailed below along with confidential research ( concept only), plus on our website www. ccc4labprep. com and in publications. Case Study 1. Client had a complex extract, when target mix prepared by reverse phase HPLC, had desired bioactivity. When process transferred to industrial non- HPLC manufacture, target mix exhibited extreme cytotoxicity. L- LC was used in direct cross correlation to gradient prep HPLC ( a single multi gram injection of same matrix onto a custom packed 50 x 250 mm, 15um C18 column, poisoned column, yet multiple L- LC preparations could be run) showed that laboratory studies with end capped, C18 HPLC prep columns, removed the then unknown cytotoxic compounds, which L- LC methods found. Case Study 2. Client had complex mixture, which had taken Sequential Flash, MPLC & HPLC 3 different International Labs each 6 months to prepare target. Two contract laboratories refused to do repeat preparations. By Sequential L- LC and HPLC, target was prepared in 4 weeks for first preparation and was completed in less than one week in repeat separation. There was a massive; over ten- fold reduction in solvent usage, as well as the obvious huge time saving. Case Study 3. During LINK Grant project working with GSK the results shown in Fig 5 were obtained. Two HPLC gradient traces are shown. Top is original gradient HPLC. Below is the HPLC of a single 4 ml fraction from a 200+ ml gradient Quattro L- LC run. The insert shows the amount of target in fractions before and after the main fraction. Over 90% of target was in one single 4 ml fraction. The bars labelled F above top chromatograph show polarity range of L- LC fractions. Apart from solvent front, all show the very small polarity range of OT HPL- LC fractions. In addition an unknown bioactive was found. Case Study 4. Sequential L- LC plus HPLC. The NEEM tree is the Holy Tree of India; it produces such a variety of bioactive targets, that villages in India define it as their Pharmacy. Fig 6 shows collaborative research with the University of Vicosa, Brasil. Previous to installing the Quattro L- LC, Prof Gulab Jham took months to prepare just the required amounts of AzA, by Sequential L- LC & HPLC, AzA and six other key related compounds, never prepared in that laboratory before, were prepared in weeks with better than 95% recovery and better than 95% purity ( 4). An injection/ recovery mass balance was conducted, by weighing the dried residue in each L- LC fraction. Within the scope of the method, a full mass balance was obtained. That would be an extreme rarity in S- LC for a raw natural product injection. Case Study 5. Deguelin obtained from an Amazonian plant is very valuable (~$ 20,000 g), the contaminant rotenone is of little value, but contaminates extracts. Researchers with decades of historic Japanese CPC 1000 ml instrument experience for this separation achieved loading of 150 mg per 1000ml CPC capacity. On upgrade to a modern manufactured 1000 ml CPC they doubled loadings to 300 mg per 1000ml CPC capacity. Their method failed on the Quattro CCC. A method developed in less than a day increased loading to 1625 mg per 1000ml Quattro L- LC capacity; over ten times that of historic CPC. The client subsequently increased Figure 3 Figure 4Figure 5 Figure 6

19 the initial loading to closer to a typical 5 to 40g loading per 1000 ml. C ase Study Wine Research Fig 7, 8, 9 & 10 by wine researchers ( 5) shows the worth of gradientsin L- LC, and of L- LC in unravelling difficult identification issues. This research led to the targets sensory properties being determined and tentative structural elucidation of new unknown oligomeric anthocyanins. The chromatogram on HPLC revealed an absence of standard baseline hump seen once the monomers etc were separated by L- LC. This highlights the value of doing a L- L chromatography sample polarity screening. L- LC helped these wine researchersto identify a new class of compounds ( oligomeric anthocyanin species), and to study their influence to the colour and sensory properties in wine. Case Study 7 HTPrep/ Combinatorial. In 2007 we custom designed the World's first Quattro HTPrep T for a Pharmaceutical Company in the USA. The research was presented at CCC2008 and published in the proceedings ( 6). Conclusions Liquid- Liquid Chromatography has matured into a valid science, which after almost 60 years deserves to be integrated into mainstream laboratory and process chromatography. L- LC been shown to compliment HPLC, with narrow range polarity cutting and by helping to find peaks co- eluting in HPLC. One beauty of L- LC is that the separation is based largely on defining on the polarities of targets, therefore classes of compounds can be separated which can then be optimised without sample loss. These narrow polarity range classes can finally be passed through a HPLC, assuming sample losses can be tolerated. If not, Sequential L- LC to L- LC with different solvents may be utilised. L- LC is a low-pressure technique ( typically 100 to 500 psi) thus it can use lower price ancillary equipment than HPLC. L- LC usage has the potential for considerable solvent cost and timesavings. References 1) Leitao G. G., El- Adji S. S., Melo, W. A., Leitao, S. G., Brown, L., J. Liq. Chromatogr. Relat. Technol. ( 2005) 28, 2041- 2051 2) Leitao G. G., Souza, P. A., Moraes, A. A., Brown, L., J. Liq. Chromatogr. Relat. Technol. ( 2005) 28, 2053- 2060 3) Comprehensive Analytical Chemistry XXXVIII, A Berthod ( Ed) ( 2002), Countercurrent Chromatography, Chapter 2, Berthod A, Brown L, Leitao G. G, Leitao. S. G. 4) Silva J. C. T., Jham G. N., Oliveira, R. D. L., Brown, L., J of Chrom A ( 2007), 1151, 203- 210 5) Vidal S., Hayasaka, Y., Meudec, E., Cheyner, V., Skouroumounis, G., J. Agric. Food Chem. ( 2004), 52, 713- 719 6) Wangenaar F. I., Hochlowski, J. E., Pan, J. Y., Tu, N. P., Searle, P. A, J Chrom A, 1216 ( 2009) 4154 - 4160. Figure 7 Figure 8 Figure 9 Figure 10 The NEW Thermo Scientific Chromatography Columns and Consumables Catalog The 2010 - 2012 catalog showcases the broadest collection of chromatography columns and consumables. Over 500 pages of high quality sample preparation, separation and analytical tools, including: . Extensive range of HPLC, GC and SPE products . Reagents and standards . Vials and closures www. thermo. com/ catalog Porvair Sciences 2009/ 2010 Catalogue now available For all your SPE and Microplate Needs To request a copy, please call or email on:- + 44 ( 0) 1372 824290 int. sales@ porvair- sciences. com Or to download a copy, visit: www. microplates. com