28 gives example of the LCI-FID for gradient hplc chromatographs
for pesticide / alkanes / ethoxylated detergents etc.
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 Unpublished research by the UK Consortium showed that engine oil additives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
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
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
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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