Chromatography Focus
Figure 4. Modes of LC as used in NEEM research
Different design modes of hydrodynamic L-LC (in main only different coil winding techniques) are unable to transfer methodology. Quattro L-LC™ utilisation benefits from Sequential L-LC plus HPLC usage:
The NEEM tree is the Holy Tree of India; it produces such a variety of bioactive targets, that villages in India define it as their Pharmacy. A published method on a competitor’s hydrodynamic L-LC could not be transfer to a Quattro L-LC™. The competitor’s CCC utilised a different coil-winding mode, but majority of other parameters were comparable. Rapid re-optimisation of biphasic solvent choice for Quattro L-LC™ [5] allowed signification improvement in achieved results over previous methodology. Figures 4, 5 & 6 show collaborative research with the University of Vicosa, Brasil. Previous to installing the Quattro L-LC™, Professor Gulab Jham took months to prepare just the required amounts of AzA by wet chemistry and S-LC. By Sequential L-LC and HPLC, AzA and six other key related compounds, never prepared in that laboratory before, were prepared in weeks with better than 95% recovery and better than 95% purity [5]. An injection/recovery mass balance was researched, by weighing the dried residue in each L-LC fraction. Within the scope of the method, a full mass balance was obtained. A full mass balance would be an extreme rarity in S-LC for a relatively raw natural product injection; often 20 to 70+ % of the column loading of the total mass injected can be retained if a silica Open Column or Flash Column or HPLC Column is used. Reverse phase columns (ref below) can also irreversibly adsorb or degrade targets. Can it be a surprise that many potentially interesting target compounds are never seen when only S-LC chromatography is utilised, and why L-LC is so often utilised in Natural Product research in preference to S-LC. More exposure of the problems of ‘on-column adsorption and degradation’ in S-LC could be detailed
Figure 5. Structures of compounds isolated from NEEM trees
in the literature to remind researchers of the need for vigilance and open mindedness, in considering the relative merits of S-LC and L-LC.
Different hydrostatic L-LC’s (CPC’s) design modes having different abilities, and methods used on both are incompatible with a hydrodynamic L-LC, resulting in a method not being transferable from either CPC to Quattro L-LC™. Benefits of Quattro L-LC™ design to significantly enhanced loading relative to CPC’s after method re-optimisation:
Deguelin obtained from an Amazonian plant is very valuable (~$20,000/g). Researchers with decades of historic Japanese CPC 1000 ml instrument experience for this separation achieved loading of 150mg per 1000ml CPC capacity. On upgrade to a modern French manufactured 1000 ml CPC using the same method they doubled loadings to 300mg per 1000ml CPC capacity. Their historic method failed on the Quattro CCC. A method developed in less than a day increased loading to 1625mg per 1000ml Quattro L-LC capacity. The client subsequently increased the initial loading to closer to a typical 5 to 40g loading per 1000ml.
Reverse Phase C18 HPLC irreversibly adsorbs/degrades cytotoxic which Quattro L-LC™ prepares:
A Client had a complex bioactive mixture. When it was prepared by reverse phase, end capped C18, preparative HPLC, it had the desired bioactivity. But when the process was transferred to industrial non-HPLC manufacture, the new target mix exhibited extreme cytotoxicity, but by S-LC assay appeared identical. L-LC was used in direct cross correlation to gradient preparative C18, HPLC showed that laboratory studies with end capped, C18 HPLC preparative columns, removed the then unknown cytotoxic compounds, which L-LC methods found. A single multi gram injection onto a 50 x 250mm, 15um
Figure 6. Fractions collected from NEEM seed extract purification
C18 column; poisoned the very expensive preparative HPLC column. Multiple L-LC preparations could be run for as many times as required, simply by refilling with liquid stationary phase and reconditioning with mobile phase, plus new injections.
Examples of Wine Research on a Quattro L-LC™ (CCC2006) [6], and HTPrep/Combinatorial research (CCC2008) [7] are available in the literature.
CONCLUSIONS
L-LC compliments HPLC, with narrow range polarity cutting and by helping to find peaks co-eluting in HPLC. L-LC separation are based largely on defining on the polarities of targets, therefore classes of compounds can be separated which can then be optimised without sample loss. These narrow polarity range classes can finally be passed through a HPLC, assuming sample losses can be tolerated. If not, Sequential L-LC to L- LC with different solvents may be utilised. L-LC is a low-pressure technique (typically 100 to 500 psi) thus it can use lower price ancillary equipment than HPLC. L-LC usage has the potential for considerable solvent cost and time savings.
REFERENCES
[1] Conway W.D, Countercurrent Chromatography, Apparatus, Theory & Applications VCH Publishers Inc, 1990
[2] Leitao G.G, El-Adji S.S, Melo, W.A., Leitao, S.G, Brown, L, J. Liq. Chromatogr. Relat. Technol. (2005) 28, 2041-2051
[3] Leitao G.G, Souza, P.A, Moraes, A.A, Brown, L, J. Liq. Chromatogr. Relat. Technol. (2005) 28, 2053-2060
[4] Comprehensive Analytical Chemistry XXXVIII, A Berthod (Ed) (2002), Countercurrent Chromatography, Chapter 2, Berthod A, Brown L, Leitao G.G, Leitao. S.G.
[5] Silva J.C.T, Jham G.N., Oliveira, R.D. L., Brown, L, J of Chrom A (2007), 1151, 203-210
[6] Vidal S, Hayasaka, Y, Meudec, E., Cheyner, V, Skouroumounis, G,
J.Agric. Food Chem. (2004), 52, 713-719
[7] Wangenaar F.I, Hochlowski, J.E, Pan, J.Y, Tu, N.P, Searle, P.A, J Chrom A, 1216 (2009) 4154 – 4160.
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