CCC4LABPREP FOR THE QUATTRO CCC™ AND ADVANCED COUNTER CURRENT CHROMATOGRAPHY
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In HSCCC a biphasic
liquid / liquid solvent system is used ( See CCC Introduction on Home
Page ). The mobile phase may be either the more polar solvent ( reverse
phase ) or less polar solvent ( normal phase ) with their respective partner
phases as the stationary phase. Indeed it is possible to change from one
mode to the other at any stage during a run.
1 gives the structure of a
variety of bioactive natural product polyphenols, which are then shown
in Slide 2 resolved
in a preparative isocratic reverse phase application using a Quattro CCC
Dr Andrew Marston of the University of Lausanne ( Lecture at Rio 2001, Home Page ) has achieve compound loadings of as high as 20 grams on a 600 ml Quattro CCC. The author has found that approximate loadings of 1 gram per 100 ml are typical for Quattro CCC fitted with 1.6 mm ID tubing when used in gradient mode. The actual loading resolvable by HSCCC is both sample and solvent dependent and can hence vary considerably.
In HSCCC as in HPLC isocratic analysis only elutes a limited polarity window of compounds for each constant composition solvent choice, as shown in Slide 7. From this figure it will be noted that in isocratic analysis, increasing the eluent strength ( in this reverse phase application, a higher amount of methanol initially and then finally both methanol and isopropanol is added to the aqueous mobile phase ) to elute late eluting compounds causes early eluting compounds to co-elute and ultimately elute on the solvent front.
Progressively increasing the elution strength of the mobile phase relative to the stationary phase will in both gradient HSCCC and HPLC allow a much broader range of polarities to be examined in one chromatogram without the above problem. This is demonstrated in Slide 8, which compares the gradient elution of the same standards initially shown by isocratic elution with different strength mobile phases in Slide 7
The choice of gradient solvent in reverse phase HPLC is relatively easy, most chromatographers using either a water : methanol or a water : acetonitrile gradient with or without buffering. It would be relatively rare in HPLC to use multiple solvent gradients such as water : methanol : acetonitrile : THF and variation of the relative percentages of the solvents ( other than the water to organic ratio ) is almost unheard of in HPLC.
The situation is not so simplistic in HSCCC gradients as almost any immiscible solvent combinations can be used in the gradient, and 3, 4 or 5 + different solvents may be utilized, with on some occasions an additional solvent(s) being phased in part way through the gradient.
Slides 9 describes the context of the inter-comparison of gradient HSCCC to Flash Chromatography. Slide 10 shows a GC-FID trace of the commercial agrochemical being tested for trace impurities and the relative percentages of the major component and impurities. Slide 11 shows the gradient used for the HSCCC. It should be noted that a variable gradient both with regards to slope and solvent ( fifth solvent added to enhance selectivity ). Slide 12 Shows the relative percentage of each the compound in the start mixture ( shown in yellow, note that many of the percentages are so low as to be not noticeable ), plus the % of the compound in the isolated/purified Flash Chromatography separation ( shown in blue ) and the same for the Quattro gradient HSCCC separation ( shown in red ). It will be noted that in the vast majority of cases Quattro CCC purification yielded both higher recovery and higher purity than Flash Chromatography. This result is even more remarkable if one considers that the chromatographers doing the research had over 40 years experience with Flash Chromatography with this and similar samples and only a few months experience with the Quattro CCC after a training period with the author.
Gradients need not only be limited to changes in organic solvents but also can in addition or by themselves be pH gradients.
The variations possible in biphasic solvent immiscibility is shown below.
Slide 13 shows totally miscible solvents and totally immiscible solvents. Slide 14 shows partially immiscible solvents where no relative density differences between upper and lower layer occurs as gradient solvent modifier is added and an example of a chloroform gradient where an inversion of an upper aqueous layer to a lower aqueous layer occurs as the organic gradient modifier is progressively added.
Examples on Slide 13 have no application in CCC whilst the first example on Slide 14 is an usable, predictable gradient throughout the gradient. The second chloroform gradient of Slide 14 which has an inversion of its upper aqueous layer to a lower aqueous layer as the methanol gradient modifier is added, can only be used either before or after the inversion point, as the inversion would void the stationary phase in HSCC
Experimentation has shown that there is a workable correlation between the elution characteristics of a 0 to 100 % B Water ( A ) to Acetonitrile ( B ) reverse phase HPLC gradient to the polarity of components and hence potential generic HSCCC solvent systems as shown in Slide 15.
Section A :- Highly-polar components can usually be accommodated by :-
PEG : Water or Butanol : Methanol : Water gradients.
Section B :- Mid-polar compounds by :-
Hexane : Ethyl Acetate : Methanol : Water gradients.
Section C :- Non-polar components by non aqueous mixes such as :-
Hexane : Modifier : Methanol ( and / or Acetonitrile ) gradients.
Optimization within a Section can often be facilitated by using simple test tube shake partition tests ( with suitable analysis as appropriate, the partition / the relative concentrations in the upper and lower phase can be assessed ) to model potential CCC response to various solvent mixes.
The effect of pH on partitioning can on occasions be very considerable Slide 16.
The HSCCC researcher should note that on occasions successful test tube partitions fail in the HSCCC and that the reverse is also true. It can on these occasions be necessary to go straight to generic step gradient optimization strategies discussed below.
For this reason it is often easier to ignore test tube simulating a CCC response and instead use a mid-polarity generic CCC gradient as shown in the first lecture presentation :- that is a reverse phase gradient with stepped increases of the methanol content of the aqueous mobile phase used to first define the components polarity, and then begin optimization from the result, as required :-See Slide 17 and Slide 18.
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 )
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.
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.
1. If no retention
of a component in a specific stationary phase occurs then this component
will elute at solvent front.
Once the elution profile is established optimization of the best HSCCC system for any or all components becomes logical.
In gradient HSCCC it is possible to vary either the upper or lower phase modifier solvent in either a normal or reverse phase gradient.
Experimentation has shown in Section B gradient compositions Slide 15 that for reverse phase gradients the best results are usually obtained by varying the methanol ratio and in normal phase gradients the best results are obtained by varying the ethyl acetate ratio.
The radically different selectivity when doing the above whilst screening complex natural product samples, for novel bioactivity etc. sample is shown in Slide 21.
Whilst linear gradients
have been successfully used in many applications including one step isolation
of novel bio actives from untreated plant extracts as shown in Slide
22 a difficulty arises when trying to extrapolate from the linear
gradient elution position to a suitable isocratic elution system.
We should like to stress that online monitoring, especially by UV can be very misleading in CCC, and it is best to fraction collect and do offline tlc, hplc, hplc-ms, nmr or bioactivity assessment of these collected fractions in order to achieve a true picture of the resolving power of Quattro CCC's.
In hplc it is relatively easy to extrapolate an isocratic condition from a gradient because only the mobile phase is changing.
In HSCCC if one makes an isocratic mix based on the elution profile, then that isocratic mix will have a different stationary phase solvent ratio from that in the column. This occurs as both phases in HSCCC are interactive and inside a dynamically changing coil is a very different equilibrium environment from a static unchanging test tube.
This will happen for both linear and step gradients, but the constancy of the step isocratic portion, allows easier optimization than in the linear gradient situation.
In addition, with step gradients, the additional wash-off of stationary phase once stepped initiated rapidly equilibrates and stabilizes. In Slide 20 a typical complex wash-off profile of a linear gradient is shown. These complex wash-off profiles also cause considerable extra complication when trying to optimize an isocratic separation from a linear gradient.
When method developing a novel HSCCC separation where test tube partitioning prescreening has proven unsuccessful, it is recommended to first try a reverse phase gradient and fractionate the complex matrix into predictable polarity bands. It is possible that component(s) of interest may still be in the presence of other components ( sometimes a single CCC run can yield individual component purities of 98% + ).
The gradient composition of the fraction of interest is noted.
The HSCCC chromatographer can then utilize the different selectivity of reverse and normal phase gradients, see Slide 21 and run a new normal phase gradient, which brackets the organic modifier elution concentration for the collected fraction of interest, which requires addition purification.
The fraction of interest is dried and dissolved in the start mobile or mobile / stationary or stationary phase as required, and the new normal phase step gradient run. Usually adequate resolution is obtained by this two dimensional CCC. This approach can easily be modified and enhanced by adding a fifth solvent for enhanced selectivity. The author is presently researching two-dimensional CCC and two-dimensional CCC / Prep HPLC.
Collaboration with other researchers on these novel topics is always welcome.
It is believed the later orthogonal techniques will have considerable merit, as generic CCC gradients can separate complex natural products etc without fear of expensive Prep HPLC column poisoning, into predictable polarity fractions. The relatively low complexity CCC fractions of known polarity can then be easily resolved into individual constituents by chromatographers who will invariably have more experience in HPLC than CCC. The chromatographer will also have the added advantage of having far less loading to put onto the preparative HPLC column, therefore greatly reducing time and cost.
The detection technique utilized in this HSCCC gradient was the AECS Moving Belt LCI-FID/MS detector. LCI stands for the LIQUID CHROMATOGRAPH INTERFACE. This Interface allows any liquid stream ( be this HPLC, HSCCC or a liquid process stream etc ) containing solutes to be interfaced to any gas phase detectors such as FID, NPD, MS, and MPD etc. For researchers interested in this technology for compounds with little to no chromophores the following three slides detail information on its operation :-
1. The AECS moving
belt evaporative LCI-FID/MS operational principles are shown in Slide
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.
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 :
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.