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.