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Alternatively a literature search could be utilized if looking for known components to see if published HSCCC methods are available for separations with a similar matrix to your sample. By choosing solvent initial and final gradient strengths to bracket the strength of the organic mobile phase for normal phase gradients and the aqueous phase in reverse phase gradients it is highly likely that a successful separation will be achieved.

Affect of HSCCC design on isocratic or gradient choice.

In commercially available HSCCC there are many variations in design :

i) preferred direction of coil winding same as rotation ( PC Inc, CentriChrom, Quattro ) or wound opposite to rotation ( PharmaTech )
ii) the gravity force differential can be obtained by a small sun and planet radius but high speed ( PharmTech Analytical ), or larger sun and planet radius but slower speed ( PC Inc, CentriChrom, Quattro, PharmaTech Prep )
iii) the internal diameter of tubing / rotation speed.
iv) temperature control ( Quattro ) no temperature control ( P C inc, CentriChrom, PharmaTech )

All of the above differences can affect isocratic solvent choice, but can become critical when trying to do repeated analyses with same gradient on different days, or trying to transfer a method used on one design of HSCCC to another design.

Experimentation even within a single instrument with all parameters set, including temperature ( Quattro ), have shown that gradient and even isocratic methods cannot always tolerate differences in internal bore for certain solvent systems.

Users must therefore specify all parameters in HSCCC and acknowledge that even change of a seemingly minor parameter may prevent reproduction of results, but if all parameters are controlled, no difficulty in reproducing gradient results have been experienced in the author's five years experience of gradient HSCCC applications.


Why step gradients preferable to linear gradients in HSCCC

In HSCCC the maintenance of stationary phase in the coil is facilitated by rotation.

Switching off rotation will, if either the stationary phase or mobile phase continues to be pumped, void both the mobile and stationary phase.

Experimentation has shown that very little band spreading occurs during this voiding of the coil, so that collection of the coil contents will allow the accurate prediction of the retention characteristics of the retained components to be obtained.

Thus :-     

1. If no retention of a component in a specific stationary phase occurs then this component will elute at solvent front.
2. Intermediate retentions can be correlated to elution position on the gradient.
3. Infinitely retained components void in the last drop of flushed out stationary phase.

Once the elution profile is established optimization of the best HSCCC system for any or all components becomes logical.

A typical linear gradient in HSCCC is shown in  Slide 19  plus the typical affect of such a gradient on an ultra violet detector response and on the stationary phase bleed, is shown in  Slide 20.

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