IntroductionThere are very many fundamentally differentmodes of L-LC instrumentation design. The twomajor design modes in present use are planetarycentrifuges (usually referred to as CCC, HSCCC orHPCCC) and sun centrifuges (often referred to assun or droplet CPC), Recently the InternationalCCC Committee voted to define planetary CCCas hydrodynamic CCC, and sun or droplet CPC ashydrostatic CCC, whilst acknowledging that bothare Centrifugal Partition Chromatographs (CPC).

The industry wide use of CCC nomenclature haslead to much confusion in the mode ofoperation, as to non L-LC chromatographers;Counter Current modes would involve twoliquids moving in different directions. WithL-LC/CCC despite the fact they can readily beused with liquids moving in two opposingdirections, in reality over 99.9+% of usage cases,only one phase is stationary and one phase ismobile. For this reason we have chosen to referto this science as L-LC rather than CCC or CPC.In this publication we will refer to planetary CCC,HSCCC and HPCCC as hydrodynamic L-LC andsun or droplet CPC as hydrostatic L-LC.

Solid Liquid Chromatography (S-LC) techniqueswould include Open Tubular, Flash, MediumPressure Liquid Chromatography (MPLC) andHigh Pressure (Performance) LiquidChromatography (HPLC).

In S-LC one phase is stationary and one phasemobile. The stationary phase is often an

immobilised liquid, which has been immobilised bybonding to a solid substrate. Liquid-LiquidChromatography is therefore in many ways directlyanalogous to Solid Liquid Chromatography.

The main difference between L-LC and S-LC inmany cases is L-LC maintains one of the pair ofimmiscible liquids stationary, through itsphysical mechanical/electrical instrumentdesign, rather than adsorption onto a solidparticle. Why then is not L-LC the equalscientifically/commercially of S-LC?

A Review of CCC/CPC Historic Confusions,To Enable L-LC Become MainstreamChromatography

Confusion 1. Nomenclature.In the Introduction the major confusionsassociated with the nomenclature and mode ofoperation of CCC/CPC is discussed. We proposedin this paper, as we have for several years to theInternational CCC Committee, that considerationbe given to more appropriate nomenclature.

Confusion 2. L-LC is a laboratory scale curiosity.Historically yes, now no.

Modern L-LC instrumentation can range from 7ml for L-LC MS studies of trace amounts, tosingle units of 10 to 25 litres, or modular processunits in any number/configuration of multiplesof 3 litres capable of multiple tonnes/annumproduction. Research applications includenatural products, nutriceuticals, agrochemicals,pharmaceuticals, and food/beverage etc. Large-

scale production (20 litre + units) is mainlylimited to natural product based products.

Confusion 3. Different concepts of L-LC caninterchange methodology. True on occasions,but still a misleading statement.

The reality is that the equivalence of differentL-LC modes to other L-LC modes is notcomparable in SLC to one manufacturer’s C18or silica column to another’s C18 or silicarespectively (although we all know significantdifferences can occur even in S-LC). It may noteven be equivalent to comparing in chiral S-LC, a cellulose to an amylose carbamate. Itcould be more like comparing these to a Pirklechiral column. The reality is, L-LC’s withfundamentally different design principles maydo the same separation, but each might do ina radically different way. Transfer of methodbetween two different L-LC design conceptswith the same solvent, target and matrix, is asmuch luck as science.

The above can even be applied within a singlemanufacturer’s product range, if themanufacturer varies key L-LC design factors. Themore factors they vary, the greater thelikelihood of failure during Process Scale-up.Rationalisation of design by using modularity ofdesign in both modern forms of L-LCinstrumentation can reduce the problem ofscale-up to make them no more difficult than inreverse phase S-LC.

Applications of Liquid-LiquidChromatography Instrumentationfor Laboratory Preparative &Process ChemistryBrown, Leslie & Luu, Trinh Anh of AECS-QuikPrep Ltd, PO Box 80, Bridgend, S. Wales, UK. CF31 4XZ ( aecs@gmx.com )

Author for correspondence

Hill, Colin & Flockhart, Ian of Botanical Developments Ltd, of UK

Garret, Pierre-Henri & Margraff, Rodolph of ETS-Couturier, of France

Skouroumounis, George of University of Adelaide, of Australia

Liquid-Liquid Chromatography instrumentation (L-LC), also referred to as Counter Current Chromatography (CCC), andCentrifugal Partition Chromatography (CPC), design has been instigated and, championed largely by Academics at Universityand Research Institutes in USA, Japan, Europe and China. To date instrumental L-LC has not been universally adopted bychromatographers in many commercial sectors. Discussed is a scientific treatise from a commercial viewpoint, of usage anddesign advantages and disadvantages of instrumental L-LC.

Keywords: Liquid-Liquid Chromatography, Counter Current / Countercurrent Chromatography, Centrifugal PartitionChromatography, Laboratory / Process Chromatography, High Throughput / Combinatorial Preparation.

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The key design factors in hydrodynamic L-LCare sun & planet radii, beta values (even ifcompensation of changing rotation speed ismade), coil winding technique, tubing boreetc. For hydrostatic L-LC the key designparameters are chambershape/design/insertions, chamber volume,sun radii etc. For both L-LC principles as morefactors are changed, the percentage successof Process Scale-up and cross compatibilitybetween different L-LC fundamentallydifferent design modes will reduce.

Confusion 4. Solvent selection in L-LC is notscientific. Incorrect, it is governed by sameprinciples as S-LC

From discussions, non L-LC chromatographershave repeatedly said that they see L-LC biphasicsolvent choice in published methods, as avariety of abstract solvent mixes or quote,“Witches Brews”. At best, they see the Arizonaetc methods as “semi-defined scales of setratios, in supposed sequences, for which L-LCresearchers chose convenient, unrealisticstandards to argue their validity”.

Our biphasic solvent selection research(presently being prepared for publication)shows this does not need to be so. Biphasicsolvent selection in L-LC is governed by exactlythe same principles as S-LC, with target &matrix solubility, functionalities, polarities,molecular weights, dipole moments, shapeconfigurations, complex and micelle formation,pH etc all taken into account and utilised incomparison to known solvent properties, asdefined by the Synder Triangle and solventpolarity series.

Confusion 5. At Big Prep 5 it was confirmed, aCompany has over one million bioactivecompounds in its library, and all only required S-LC. One could ask do we need L-LC? Theanswer is definitely yes. To assist this statement,we would add that a Quattro CCC was recentlycustom designed for a USA PharmaceuticalCompany for High Throughput Preparationapplication. This custom build shows that notonly is L-LC’s maintenance of compoundintegrity applicable in searching for unknownbio-actives/taste/colour/nutriceuticals etc innatural products, but it may possibly one dayhave a place in mainstream Pharmaceuticals aswell (see Results & Discussion).

Why is the answer above yes? The answer aboveis yes in part because the above initial statementis self-fulfilling prophecy. If research only everused S-LC to define a library, by definition anycompound that would be absorbed by thephase, or would have been degraded by thephase would not be in the library. Perhaps itwould be well for us all to remember that silica isused as a catalyst for certain hydrolysis reactions.The question that should be asked is how manybioactive targets or cytotoxic contaminants maybe missed owing to use of only S-LC? (RefResults & Discussion). The polarity limit of RP-C18/ NP-silica etc in HPLC etc is alarmingly small,compared to L-LC. With stop rotation-wash-off orelution-extrusion each L-LC run can go from

infinitely polar, to infinitely non-polar (or viceversa). If using a switching valve, Head to Tail maybe reversed during a run, and even change froma reverse phase to normal phase run (or viceversa) at any time of the users choosing.

Confusion 6. L-LC always has low plate counts.Why bother with L-LC? The reply is, L-LC hasmassively higher stationary phase retentionthan S-LC, thus achieves resolution with lowplate count, plus L-LC can achieve selectivitythrough its vast stationary phase options. L-LCcan use half to less than a tenth of solvent toprepare the same mass of target in same matrixwhen cross-compared to SLC. L-LC has a veryhigh loading capacity (5 to 15% of coil volume),plus only requires low cost, low-pressure liquidpumps. All these factors make furtherconsideration of L-LC important as cheapermore “Green” techniques are researched.

Regarding low plate per metre (p/m) counts, itshould be noted that the percentage ofstationary phase is fundamental to fullresolution equation, though this factor is oftendeleted in HPLC texts. The reason HPLC etcrequires high plate counts is in part that it has avery low percentage of stationary phase relativeto the total content of the containing vessel.

In Flash and HPLC the numbers of stationaryphases are extremely limited compared to L-LC.Therefore resolution by massive changes inselectivity is limited in S-LC.

L-LC traditionally uses 70 to 98% stationary phase.In a reverse phase C18 column, the C18 might be10 to 21% of the stationary phase, which is itself asmall percentage of total container voidvolume/void mass. In L-LC logical biphasicsolvent selection, with options of isocratic (1,2),linear or step gradients (3), ionic liquids, pHbased frontal chromatography (called pH Zonerefining in CCC), micelles, reverse micelles,aqueous/aqueous, aqueous/organic,organic/organic, stop rotation-wash-off, elution-extrusion are all usable using the same L-LCsystem. All these possibilities are created only bychoice of different biphasic or even triphasicsolvents. Inorganic cations (inclusive preciousmetals, transition metals, radioactive isotopes)and anions, plus organic compounds, evencertain shape orientations/chiral compounds, canbe resolved with a single L-LC instrument.

Confusion 7. L-LC is mainly a stand-alonechromatography technique. Incorrect,Sequential L-LC and HPLC has been for thelast 10 years our preferred operation mode inContract Preparations of targets.

Almost every difficult application at the AECS-QuikPrep Ltd laboratory uses L-LC to polarityfractionate highly complex matrices (that wouldpoison an HPLC column with one injection), intonarrow polarity bands. We would stress themutual benefit of Sequential L-LC and HPLC,and never view L-LC and HPLC as mutuallyexclusive. The L-LC fractions are so extremelyrestricted in polarity, that they only requireisocratic HPLC to complete our standardSequential L-LC to HPLC runs. 95 to 99+% pure

target can be obtained after a single GenericGradient L-LC run and Sequential HPLC Prepcolumn (see below). This Sequential L-LC andHPLC principle can be repeatedly successful,even when starting from unknown, and totallyuncharacterised, raw natural product or crudesynthesis materials. Sequential L-LC to L-LC,utilising different biphasic solvents for thesecond L-LC run, is only used if targetsirreversibly adsorb or degrade on 5um, endcapped C18 etc HPLC phases.

Context

Fig 1 & 2 show the chassis and coil/volumeoptions of the Quattro CCC™ model range.(“J” Type Planetary Centrifuge, open, constantid tubing, wound on a planetary bobbin, withno rotating seals). The bobbins (planetaryrotating body, holds the coiled columns) canhave tubing with different material choice.Options include PTFE, Stainless Steel or Titanium.Tubing bore for id can vary from 0.5 to 12.5 mm,and volumes from 7 to 3000 ml for a single rotorassembly. A single bobbin can have two coils. Allmodels except the entry IntroPrep™ have twodynamically balanced bobbins, with up to 4 coilsas an option. Each coil can be usedindependently for same or different preparations,or used in any combination, in series with any coilor multiple of coils of the same id. Uniquely forhydrodynamic L-LC model ranges, all modelsshare the same key L-LC design parameters,inclusive of the same sun & planet radii, speedranges, beta values, winding techniques and onlytubing bore is varied. This model range is also theonly one that allows even the largest bore to betested on a laboratory based unit, prior tointroduction to process based preparation.Hybrid coil winding, that is multiple id.’s in thesame instrument or bobbin, can be manufacturedproduced. Multiple bobbin sets for a singlechassis are available. In this way the major

Figure 1

Figure 2

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difficulty of needing several different instruments,to validate scale-up is avoided.

For Process Chromatography, the base moduleis of 3 litres. Bobbins are interchangeable, andcan be exchanged for re-winding if PTFE tubingchosen and cGMP requires virgin material. Mostwould use stainless steel or titanium tubing andappropriate cleaning techniques, but renewingPTFE coils is an option. If different bore sizes arerequired, different bobbin sets may be used.Bobbins can be used in series, in parallel or insimulated moving bed operations. Clutches andswitching valves allow operating mode changes.

The Partitron CPC™ is shown in Fig 3 (suncentrifuge, with separation chambers and idrestricted links between chambers, with 2rotating seals). This model range has a singleprocess-scale chassis unit. Most hydrostaticCPC are manufactured as chambers createdby a sandwich of machined or etchedchambers formed into a stainless steel disc,with layers of PTFE sealing the individual disclayers from other layers. The whole assembly isbolted together, but can be prone to leaksand blockages. Machining and in particularetching of any surface radically increases thesurface area exposed. Viewed under amicroscope the machined/ etched surfaces willappear as mountains and valleys relative to thesame material before machining/etching. AsCPC units are particularly suited toaqueous/aqueous chromatography ofpeptides and proteins, enzymes etc, all ofwhich are prone to degradation, the choice ofmachined/etched stainless steel for mosthydrostatic L-LC is suspect.

The Partitron CPC™ was specifically designedfor large scale, GMP process chromatography.A totally different construction is utilised. Thewhole rotor assembly (Fig 3) is machined froma single titanium block. Titanium is wellrecognised in chromatography for itsinertness. A variety of titanium rotors, withvolumes from 5 to 25 litres, with either one ortwo volumes per rotor, may be fitted to thisversatile, uniquely modular hydrostatic L-LC.

Fig 4 shows an industrial sub & super-criticalextraction plant which is used in conjunctionwith L-LC production and research

Results & DiscissionAll experiments in Case Studies completed witha Quattro CCC™.

Discussions regarding Confusions 1 to 2Unpublished Grant funded research (“The

Industrial Scale up of CountercurrentChromatography”. BBSRC/DTI LINK Award Ref:100/BCE08803. Feb 98 - Jan 00 (£322,668), acollaboration of AECS, Brunel University,University College of Swansea, GSK, Astra Zeneca& Shell Research) supported comments by CCCexperts, that CCC of different designs or even asingle concept, if one varies key parameters thiscan, on occasions, prohibit scale-up. Keeping allparameters the same, only changing tubing bore,certain scale-ups failed. AECS & Brunel Universityinterpreted the implication of these results inradically different ways in their subsequentindependent commercialisation of L-LC. AECSrationalised design to minimise variability and hasspent 9 years increasing its understanding scale-up failures. Brunel University and staff developed arange of CCC with radically different sun & planetradii, speed ranges etc. and formed their ownspin-off company (DE Ltd) six years ago to exploittheir research.

Discussions regarding Confusions 3 to 7Non-confidential research is detailed belowalong with confidential research (concept only),plus on our website www.ccc4labprep.com andin publications.

Case Study 1. Client had a complex extract,when target mix prepared by reverse phaseHPLC, had desired bioactivity. When processtransferred to industrial non-HPLC manufacture,target mix exhibited extreme cytotoxicity. L-LCwas used in direct cross correlation to gradientprep HPLC (a single multi gram injection ofsame matrix onto a custom packed 50 x 250mm, 15um C18 column, poisoned column, yetmultiple L-LC preparations could be run)showed that laboratory studies with endcapped, C18 HPLC prep columns, removed thethen unknown cytotoxic compounds, which L-LCmethods found.

Case Study 2. Client had complex mixture,which had taken Sequential Flash, MPLC &HPLC 3 different International Labs each 6months to prepare target. Two contractlaboratories refused to do repeat preparations.By Sequential L-LC and HPLC, target wasprepared in 4 weeks for first preparation andwas completed in less than one week in repeatseparation. There was a massive; over ten-foldreduction in solvent usage, as well as theobvious huge time saving.

Case Study 3. During LINK Grant projectworking with GSK the results shown in Fig 5 wereobtained. Two HPLC gradient traces are shown.

Top is original gradient HPLC. Below is the HPLCof a single 4 ml fraction from a 200+ ml gradientQuattro L-LC run. The insert shows the amountof target in fractions before and after the mainfraction. Over 90% of target was in one single 4ml fraction. The bars labelled F above topchromatograph show polarity range of L-LCfractions. Apart from solvent front, all show thevery small polarity range of OT HPL-LC fractions.In addition an unknown bioactive was found.

Case Study 4. Sequential L-LC plus HPLC. TheNEEM tree is the Holy Tree of India; it producessuch a variety of bioactive targets, that villagesin India define it as their Pharmacy. Fig 6 showscollaborative research with the University ofVicosa, Brasil. Previous to installing the QuattroL-LC, Prof Gulab Jham took months to preparejust the required amounts of AzA, by SequentialL-LC & HPLC, AzA and six other key relatedcompounds, never prepared in that laboratorybefore, were prepared in weeks with betterthan 95% recovery and better than 95% purity(4). An injection/recovery mass balance wasconducted, by weighing the dried residue ineach L-LC fraction. Within the scope of themethod, a full mass balance was obtained. Thatwould be an extreme rarity in S-LC for a rawnatural product injection.

Case Study 5. Deguelin obtained from anAmazonian plant is very valuable (~$20,000 g),the contaminant rotenone is of little value, butcontaminates extracts. Researchers withdecades of historic Japanese CPC 1000 mlinstrument experience for this separationachieved loading of 150 mg per 1000ml CPCcapacity. On upgrade to a modern manufactured1000 ml CPC they doubled loadings to 300 mgper 1000ml CPC capacity. Their method failed onthe Quattro CCC. A method developed in lessthan a day increased loading to 1625 mg per1000ml Quattro L-LC capacity; over ten times thatof historic CPC. The client subsequently increased

Figure 3

Figure 4 Figure 5

Figure 6

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the initial loading to closer to a typical 5 to 40gloading per 1000 ml.

Case Study Wine Research Fig 7, 8, 9 & 10 bywine researchers (5) shows the worth ofgradients in L-LC, and of L-LC in unravellingdifficult identification issues. This research ledto the targets sensory properties beingdetermined and tentative structuralelucidation of new unknown oligomericanthocyanins. The chromatogram on HPLCrevealed an absence of standard baselinehump seen once the monomers etc were

separated by L-LC. This highlights the value ofdoing a L-L chromatography sample polarityscreening. L-LC helped these wineresearchers to identify a new class ofcompounds (oligomeric anthocyanin species),and to study their influence to the colour andsensory properties in wine.

Case Study 7 HTPrep/Combinatorial. In 2007 wecustom designed the World’s first QuattroHTPrep™ for a Pharmaceutical Company in theUSA. The research was presented at CCC2008and published in the proceedings (6).

ConclusionsLiquid-Liquid Chromatography has matured intoa valid science, which after almost 60 yearsdeserves to be integrated into mainstreamlaboratory and process chromatography. L-LCbeen shown to compliment HPLC, with narrowrange polarity cutting and by helping to findpeaks co-eluting in HPLC. One beauty of L-LC isthat the separation is based largely on definingon the polarities of targets, therefore classes ofcompounds can be separated which can thenbe optimised without sample loss. These narrowpolarity range classes can finally be passedthrough a HPLC, assuming sample losses can betolerated. If not, Sequential L-LC to L-LC withdifferent solvents may be utilised. L-LC is a low-pressure technique (typically 100 to 500 psi) thusit can use lower price ancillary equipment thanHPLC. L-LC usage has the potential forconsiderable solvent cost and timesavings.

References

1) Leitao G.G., El-Adji S.S., Melo, W.A., Leitao, S.G.,

Brown, L., J. Liq. Chromatogr. Relat. Technol.

(2005) 28, 2041-2051

2) Leitao G.G., Souza, P.A., Moraes, A.A., Brown, L.,

J. Liq. Chromatogr. Relat. Technol. (2005) 28, 2053-2060

3) Comprehensive Analytical Chemistry XXXVIII, A Berthod

(Ed) (2002), Countercurrent Chromatography, Chapter 2,

Berthod A, Brown L, Leitao G.G, Leitao. S.G.

4) Silva J.C.T., Jham G.N., Oliveira, R.D. L., Brown, L.,

J of Chrom A (2007), 1151, 203-210

5) Vidal S., Hayasaka, Y., Meudec, E., Cheyner,

V., Skouroumounis, G., J.Agric. Food Chem.

(2004), 52, 713-719

6) Wangenaar F.I., Hochlowski, J.E., Pan, J.Y., Tu, N.P.,

Searle, P.A, J Chrom A, 1216 (2009) 4154 – 4160.

Figure 7

Figure 8

Figure 9

Figure 10

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