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3.  Slide 28  gives example of the LCI-FID for gradient hplc chromatographs for pesticide / alkanes / ethoxylated detergents etc.
4. 
 Slide 25 gives examples of the LCI-FID working with gradient elution from a Quattro CCC.


Summary of the use of isocratic, step and linear gradients in HSCCC


Brief descriptions and practical examples of the relative merits of isocratic, step and linear gradients in are given below.

1. In general isocratic HSCCC as in HPLC will allow a relatively narrow polarity window of compounds to be examined. Although in HSCC the unique option exists to stop rotation and elute the stationary phase with its retained components. This procedure is very valuable in isocratic, step and linear Brief descriptions and practical examples of the relative merits of isocratic, step and linear gradients in HSCCC / CPC are given above.
2. With isocratic analysis, it is highly recommended to do a literature search for similar compounds / matrices prior to beginning CCC. This option is more limited in gradient CCC, as relatively few methods have been published, but gradients based around proven isocratic conditions are often successful.
3. Step and linear gradient HSCCC have an infinitely greater potential to cover a wider polarity range of compounds in a single run than HPLC.
4. Unpublished research by the UK Consortium showed that engine oil additives ranging from low molecular weight highly polar surfactants ( few 100's molecular weight ) to very non- polar high molecular weight ( several 100,000's ) viscosity index modifiers could all be examined in a single HSCCC gradient with good resolution of these and all intermediate polarities. Such a separation is not possible by any single HPLC column technology presently available.
5. Solvent choice in step and linear gradients can be facilitated by defining the polarity of components of interest by linear water to acetonitrile reverse phase hplc gradient. Based on this polarity definition appropriate generic solvent systems can be chosen.
6. Test tube and to a lesser extent tlc partition tests can help with defining the best possible gradient. Two examples of very successful additional phase optimization by test tube partition testing have been detailed in the first slide show in Applications.
7. It is the authors experience that caution has to be taken with this approach as on many occasions no correlation between these test tube / tlc partitions and the HSCCC results may result.
8. The easiest way to proceed with novel CCC method development on such occasions is to use gradient HPLC to define polarity and generic choice of gradient system.

9. A standard reverse phase step gradient ( the gradient in  Slide 17  +      Slide 18 )   is an excellent trial reverse phase step gradient for Section B polarity solvents with stop rotation and collection of stationary phase will allow definition of the polarity for the total polarity range of compounds present in the sample.

10. If inadequate resolution is achieved several logical options exist :


I. Repeat the separation using a normal phase gradient and exploit the different selectivity of reverse and normal phase gradients.
II. Take the fraction of interest, which is now defined for HSCCC solvent elution, and re-inject this relatively pure ( and probably far lower mass ) sample into an appropriate normal phase HSCCC gradient, Section V-C. Alternatively use known polarity to choose an preparative HPLC column ( if active compound not adversely affected by solid phase support ). The advantage of the later, if appropriate, is the matrix will be relatively pure, and the mass injected far less, both making this fraction far more suitable for preparative HPLC than, for example a crude plant extract.
III. Examine the literature to see if any similar compounds / matrices separated by isocratic HSCCC and evolve step / linear gradients based on these.
IV. Examine the chemistry of components of interest and there relative solubility's in various solvents, and applying the standard like to like, best separates criteria common to most forms of chromatography, devise novel gradients based on the near infinite variety of immiscible solvent systems available

In summary the Quattro HSCCC offer separation scientists unique options in analytical, laboratory, pilot plant and process scale chromatography, which can be readily assessed by gradient method development strategies.


Acknowledgements : Part of the Quattro HSCCC research above represents a summary of an initial collaborative program between Dr Les Brown ( author ) of AECS, exclusive worldwide distributors of the QUATTRO CCC, University College of Swansea, UK ( Prof David Games, David Wheatley, Dr Rachel Ann-Whiteside, Dr Huw Kidwell ) and University College of London, UK ( Dr Gary Lye, Andrew Booth ), BIB, UK plus Shell Research, UK ( Dr Stuart Forbes and Euan McKerrel ), Astra Zeneca, UK ( Colin Strawson, Dr Ian McConvey, Antony Graham, Phillip Shering ), Syngenta, UK ( Brian Kemp, Dr Peter Massey, Linda Russel ) and GlaxoSmithKline, UK ( Dr Mike Dawson, Dr Martin Hayes, Stephen Jackson, Dr Tracey Shoulder, Dr Chris Preston ). This project was partially funded by all the companies involved and by an UK LINK - BBSRC Grant plus UK EPSRC PhD Grant funding

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