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    • ABSTRACTCountercurrent chromatography (CCC) is a generic term covering all forms of liquid–liquidchromatography that use a support-free liquid stationary phase held in place by a simplecentrifugal or complex centrifugal force field. Biphasic liquid systems are used with oneliquid phase being the stationary phase and the other being the mobile phase. Althoughinitiated almost 30 years ago, CCC lacked reliable columns. This is changing now, and thenewly designed centrifuges appearing on the market make excellent CCC columns. Thisreview focuses on the advantages of a liquid stationary phase and addresses thechromatographic theory of CCC. The main difference with classical liquid chromatography(LC) is the variable volume of the stationary phase. There are mainly two different ways toobtain a liquid stationary phase using centrifugal forces, the hydrostatic way and thehydrodynamic way. These two kinds of CCC columns are described and compared. Thereported applications of CCC in analytical chemistry and comparison with other separationand enrichment methods show that the technique can be successfully used in the analysis ofplants and other natural products, for the separation of biochemicals and pharmaceuticals,for the separation of alkaloids from medical herbs, in food analysis, etc. On the basis of thestudies of the last two decades, recommendations are also given for the application of CCCin trace inorganic analysis and in radio analytical chemistry. 1
    • INTRODUCTIONCounter current chromatography is a method of multiple liquid liquid extraction technique whereseparation of components’ having variable solubility in two immisible liquid is achievedIn a conventional liquid liquid extraction, 2 components example a and b are distributed between 2immisible liquids; according to their partition coefficient still pure a and b are not present in these 2liquids even after reaching equilibriumIn the counter current chromatography, two immisible solvents flow in an opposite direction inmultiple stages equilibrium is established and after several stages pure a’ and b can be obtained.Chromatography may be define as a method of separating a mixture of components into individualcomponents through equilibrium distribution between two phases. Essentially, the technique ofchromatography is based on the differences in the rate at which the components of a mixture movethrough a porous medium called stationary phase under the influence of some solvent or gas calledmobile phase 2
    • PRINCIPLEIn counter current chromatography when 2 components’ a and b having varying affinity or partionco-efficient, is distributed between 2 immisible solvents eg. X and Y which are allowed to flow inopposite direction separation of pure a and b takes places in multiple stagesIn the first stage when equilibrium is achieved in container 1, solvent X lighter or upper phase andsolvent Y heavier or lower phase will have both components a and b based on their distributioncoefficient ,let us say a is present more in X and b is present more in Y. the upper phase solvent x istransferred to next container 2, with similar composition of solvents. Fresh solvents X is added tocontainer 1.After achievement of equilibrium in container 2, now the upper phase will contains less of b, due toits low solubility in X and more of a. this upper phase is then transferred to container 3 with similarcomposition of solvents now, the upper layer of container 1 is then transferred to container 2 andfresh solvent is added to container 1. The above steps are repeated till the upper layer contains purea in the n container, where n is the last container the lower phase solvent Y of container 1 containsthe pure component of b . The value of n depends upon various factors describe.The number of steps required to separate a and b depends upon the difference in their distributioncoefficient when the difference between is more few steps are required. But when the difference indistribution coefficient between a and b is less then more steps are required. 3
    • 4
    • ADVANTAGES OF COUNTER CURRENT CHROMATOGRAPHYModern CCC technology has many advantages over traditional preparative techniques:FastCCC provides high throughput preparative separations.InexpensiveAfter the purchase of the machine the only running costs are that of the solvent. Furthermore,solvent usage in CCC is significantly lower (by 10-50%) than that of other preparativechromatography techniques, such as HPLC.Gentle TechniqueCCC is a very gentle technique in which the sample is only in contact with solvents and teflon (orother inert material). Therefore CCC provides the least chance for sampledegradation/decomposition.Versatile SelectivitySeparation of virtually every compound class has been demonstrated with CCC. Selectivity over afull range of polarities is achieved through the use of appropriate CCC Solvent Systems.ScaleableCCC is able to range from milligrams to tens of grams on the same instrument. Furthermore, manyManufacturers provide instruments that are capable of producing tons of pure product per year.When GC or HPLC is carried out with large sample loading, resolution is lost due to issues withsurface-to-volume ratios and flow dynamics; this is avoided when both phases are liquid. 5
    • 100% Sample RecoverySince CCC does not use a solid support, permanent adsorption of analyte onto the column isavoided, and a 100% recovery of the analyte can be achieved in practice TYPES OF COUNTER CURRENT CHROMATOGRAPHYINTRODUCTION  LIQUID LIQUID CHROMATOGRAPHY  SOLID LIQUID CHROMATOGRAPHYCountercurrent chromatography (CCC) is a liquid chromatography (LC) technique that uses twoimmiscible liquid phases without any solid support. As an LC technique, CCC uses many termsalready defined for chromatography [1]. This article will give the fundamentals of the CCCtechnique and briefly describe the special chromatographic columns capable of maintaining a staticliquid phase using centrifugal fields. A rapid approach to selecting solvent systems that can be usedin CCCLiquid–liquid extraction, also known as solvent extraction and partitioning, is a method toseparate compounds based on their relative solubilities in two different immiscible liquids, usuallywater and an organic solvent. It is an extraction of a substance from one liquid phase into anotherliquid phase. Liquid–liquid extraction is a basic technique in chemical laboratories, where it isperformed using a separatory funnel. This type of process is commonly performed after a chemicalreaction as part . 6
    • The term partitioning is commonly used to refer to the underlying chemical and physical processesinvolved in liquid–liquid extraction but may be fully synonymous. The term solvent extraction canalso refer to the separation of a substance from a mixture by preferentially dissolving that substancein a suitable solvent. In that case, a soluble compound is separated from an insoluble compound or acomplex matrix.Solvent extraction is used in nuclear reprocessing, ore processing, the production of fine organiccompounds, the processing of perfumes, the production of vegetable oils and biodiesel, and otherindustries.Liquid–liquid extraction is possible in non-aqueous systems: In a system consisting of a moltenmetal in contact with molten salts, metals can be extracted from one phase to the other. This isrelated to a mercury electrode where a metal can be reduced, the metal will often then dissolve inthe mercury to form an amalgam that modifies its electrochemistry greatly. For example, it ispossible for sodium cations to be reduced at a mercury cathode to form sodium amalgam, while atan inert electrode (such as platinum) the sodium cations are not reduced. Instead, water is reduced tohydrogen. A detergent or fine solid can be used to stabilize an emulsion, or third phaseTechniquesBatchwise single stage extractionsThis is commonly used on the small scale in chemical labs. It is normal to use a separating funnel.For instance, if a chemist were to extract anisole from a mixture of water and 5% acetic acid usingether, then the anisole will enter the organic phase. The two phases would then be separated.The acetic acid can then be scrubbed (removed) from the organic phase by shaking the organicextract with sodium bicarbonate. The acetic acid reacts with the sodium bicarbonate to form sodiumacetate, carbon dioxide, and water. 7
    • Multistage countercurrent continuous processesThese are commonly used in industry for the processing of metals such as the lanthanides; becausethe separation factors between the lanthanides are so small many extraction stages are needed. In themultistage processes, the aqueous raffinate from one extraction unit is fed to the next unit as theaqueous feed, while the organic phase is moved in the opposite direction. Hence, in this way, even ifthe separation between two metals in each stage is small, the overall system can have a higherdecontamination factor.Multistage countercurrent arrays have been used for the separation of lanthanides. For the design ofa good process, the distribution ratio should be not too high (>100) or too low (<0.1) in theextraction portion of the process. It is often the case that the process will have a section forscrubbing unwanted metals from the organic phase, and finally a stripping section to obtain themetal back from the organic phase.Multistage Podbielniak contactor centrifuges produce three to five stages of theoretical extraction ina single countercurrent pass, and are used in fermentation-based pharmaceutical and food additiveproduction facilities.Centrifugal extractors mix and separate in one unit. Two liquids will be intensively mixed betweenthe spinning rotor and the stationary housing at speeds up to 6000 RPM. This develops greatsurfaces for an ideal mass transfer from the aqueous phase into the organic phase. At 200 – 2000 gboth phases will be separated again. Centrifugal extractors minimize the solvent in the process,optimize the product load in the solvent and extract the aqueous phase completely. Counter currentand cross current extractions are easily established. 8
    • Extraction without chemical changeSome solutes such as noble gases can be extracted from one phase to another without the need for achemical reaction . This is the simplest type of solvent extraction. When a solvent is extracted, twoimmiscible liquids are shaken together. The more polar solutes dissolve preferentially in the morepolar solvent, and the less polar solutes in the less polar solvent. Some solutes that do not at firstsight appear to undergo a reaction during the extraction process do not have distribution ratio that isindependent of concentration. A classic example is the extraction of carboxylic acids into non polarmedia such as benzene. Here, it is often the case that the carboxylic acid will form a dimer in theorganic layer so the distribution ratio will change as a function of the acid concentration (measuredin either phase).Solvation MechanismUsing solvent extraction it is possible to extract uranium, plutonium, or thorium from acid solutions.One solvent used for this purpose is the organophosphate tri-n-butyl phosphate. The PUREXprocess that is commonly used in nuclear reprocessing uses a mixture of tri-n-butyl phosphate andan inert hydrocarbon (kerosene), the uranium(VI) are extracted from strong nitric acid and are back-extracted (stripped) using weak nitric acid. An organic soluble uranium complex[UO2(TBP)2(NO3)2] is formed, then the organic layer bearing the uranium is brought into contactwith a dilute nitric acid solution; the equilibrium is shifted away from the organic soluble uraniumcomplex and towards the free TBP and uranyl nitrate in dilute nitric acid. The plutonium(IV) formsa similar complex to the uranium(VI), but it is possible to strip the plutonium in more than one way;a reducing agent that converts the plutonium to the trivalent oxidation state can be added. 9
    • This oxidation state does not form a stable complex with TBP and nitrate unless the nitrateconcentration is very high (circa 10 mol/L nitrate is required in the aqueous phase). Another methodis to simply use dilute nitric acid as a stripping agent for the plutonium. This PUREX chemistry is aclassic example of a solvation extractionGeneral Procedure for Experiments on a Centrifugal LLC InstrumentPerforming an LLC experiment on a centrifugal instrument usually follows most of the stepsoutlined below:1. Find a suitable solvent system The HEMWat system is well tried and tested and proves adequatefor most separations2. Perform partition studies with different steps in the HEMWat series, with and without, acidic orbasic modifiers if there are ionisable species present in the sample to be purified. Determinepartition coefficient/distribution ratio (D and 1/D) for phases, examine the D and 1/D values toascertain whether resolution is feasible. D ≥ 0.5 usually produces a satisfactory separation3. The results of step 2 show whether the experiment should be performed in NP or RP mode. D ≈ 1i.e. in the range of approximately 0.5 – 2.5 (the sweetspot), for a component of interest is desirableNP mode: the more polar phase is designated as SP. For the HEMWat series this is always the lowerphase4. Prepare the solvent mixture chosen for the separation and separate the phases5. Perform scouting experiments and refine the experimental conditions or preparative runs asdescribed in steps 6 - 9 10
    • 6. Set up the instrument with respect to the choice for SP and fill the column with the chosen SP atthe highest usable flow rate7. Equilibrate the column with MP at the chosen elution flow rate. This step will displace somequantity of SP and from the displaced volume and the system and column volumes the initial SPretention can be calculated. These data allow prediction of elution volumes and times for thecomponents of interest8. Dissolve the appropriate quantity in a volume, equal to or less than 5 – 10% of the columnvolume, of either phase or preferably a mixture of the phases9. Inject the sample, perform the elution, collect fractions and analyze then work up the requiredfractionsControl of CCC selectivity is effected when using a stepped polarity solvent combination seriessuch as Solvent combinations in the series are formed by mixing hexane or heptanes, ethyl acetate,methanol and water in different proportions to produce biphasic mixtures. The figure shows howretention and selectivity change as the test mixture.Benefits of using LLC  No expensive, fragile solid phase used and a ‘fresh’ column for every experiment.  Does not require dedication of particular columns to particular separations to avoid the risks of cross-contamination  As long as a chosen solvent combination forms two, readily separable, approximately equal volume layers there are few restrictions on solvent and additive choices 11
    •  When compared with the widely used high performance technique of RP-SLC, solute capacities are high  The technique is readily scalable from mg directly to kg separations without the attendant problems often encountered when scaling up SLC separations  Tolerant of viscous and particulate-containing samples and require little or no sample preparation.  Separations of ‘dirty’ matrices do not usually require pre-chromatography prior to the high resolution step  Experiments can usually be run in NP so that fractions are collected in essentially organic solvent solution and so are easily worked up  There are no unpredictable and/or difficult to control, secondary chromatographic interactions to interfere with good resolution.Applications 12
    • • DNA purification: The ability to purify DNA from a sample is important for many modern biotechnology processes. However, samples often contain nucleases that degrade the target DNA before it can be purified. It has been shown that DNA fragments will partition into the light phase of a polymer–salt separation system. If ligands known to bind and deactivate nucleases are incorporated into the polymer phase, the nucleases will then partition into the heavy phase and be deactivated. Thus, this polymer–salt system is a useful tool for purifying DNA from a sample while simultaneously protecting it from nucleases. • Food Industry: The PEG–NaCl system has been shown to be effective at partitioning small molecules, such as peptides and nucleic acids. These compounds are often flavorants or odorants. The system could then be used by the food industry to isolate or eliminate particular flavorsLiquid-Solid Chromatography (LSC)This type of chromatographic technique is also called adsorption chromatography since themechanism of separation depends on adsorption of solutes on the stationary phase. The stationaryphase is a solid which is usually silica or alumina with the former being most widely used. Theretention times of some compound categories are as follows:Carboxylic acids > amides > amines ~alcohols > ketones ~ aldehydes ~ esters >nitrocompounds > halides > hydrocarbons.Mobile Phase SelectionThe only factor that is used to optimize α and k’ is the mobile phase composition since thestationary phase is a solid. Great variations in α and k’ can be obtained by variations in nature andcomposition of the mobile phase. The polarity index of solvents can be used as a guide forestimating the polarity index of the mobile phase. However, a better scale is optimally dependentupon the adsorption energy per unit area of the solvent which is called the eluent strength, εo. Thevalues of the eluentstrength are related to the polarity index.In selecting a mobile phase, two miscible solvents are used one with high eluent strength while theother with low eluent strength. k’ is then optimized by variation of the volume ratio of the twosolvents. A small increase in εo value will significantly change k’. Therefore, large variations in k’are possible by variation in mobile phase 13
    • Composition.In case of getting overlapping peaks but acceptable k’, the type of mobile phase constituents mustbe changed in order to change α. It is usually possible to carry a thin layer chromatographicseparation to optimize the mobile phase composition with regards to both eluent strength andcompositionApplications of Adsorption ChromatographyLSC is best suited for the separation of non polar compounds with molecular weights below 5000.Solutes must be soluble in non polar solvents and should have a limited solubility in aqueoussolvents. It should be remembered that the mobile phase in LSC should be non polar modified witha polar solvent. However, the solvent polarity must not be very large since irreversible adsorptionon the stationary phase can occur precluding the use of LSC. Therefore, water is usually excludedfrom mobile phases to be used in LSC. Separations of difficult to separate isomers were possiblewith LSC.Ion-Exchange Chromatography (IEC or ICSeparation of ionic species is efficiently done using ion-exchange chromatography (IEC) or simplyion chromatography (IC). Anions can be separated on an anionic exchange resin while cations canbe separated on a cationic exchange resin.Separation of ionic species is efficiently done using ion-exchange chromatography (IEC) or simplyion chromatography (IC). Anions can be separated on an anionic exchange resin while cat ions canbe separated on a cationic exchange resin.Ion-Exchange Resins and EquilibriaCationic exchange resins are mainly of two types: 14
    • a. Sulfonic acid group like ~SO3 H+b. Carboxylic acid group, ~COOH+Anionic exchange resins are mainly of the tertiary amine type, ~(CH3)3N+ OH The cationicexchange equilibria can be represented by the equationn RSO3 - H+ (solid) + Mn+ = (RSO3 -)n Mn+ (solid) + n H+ For singly charged cations like B+,we may write:RSO3- H+(s) + B+(aq) = RSO3- B+(s) + H+ (aq) kex = [RSO3Ion-Exchange Resins and EquilibriaCationic exchange resins are mainly of two types:a. Sulfonic acid group like ~SO3Hb. Carboxylic acid group, ~COO-H+Anionic exchange resins are mainly of the tertiary amine type, ~(CH3)3N+ OH Thecationicexchange equilibria can be represented by the equation:n RSO3- H+ (solid) + Mn+ = (RSO3-)n Mn+ (solid) + n H+For singly charged cations like B+, we may write:RSO3 H+ (s) + B+(aq) = RSO3- B+(s) + H+ (aq) kex = [RSO3- B+]s[H+]aq/[ RSO3 - H+]s[B+]aq- B+]s[H+]aq/[ RSO3 - H+]s[B+]aqIon-Exchange Resins and EquilibriaCationic exchange resins are mainly of two types:a. Sulfonic acid group like ~SO3 H+ 15
    • b. Carboxylic acid group, ~COOH+Anionic exchange resins are mainly of the tertiary amine type, ~(CH3)3N+ OH The cationicexchange equilibria can be represented by the equation:n RSO3- H+ (solid) + Mn+ = (RSO3-)n Mn+ (solid) + n H+For singly charged cations like B+, we may write:RSO3- H+(s) + B+(aq) = RSO3- B+(s) + H+ (aq)kex = [RSO3- B+]s[H+]aq/[ RSO3- H+]s[B+]aqkex = [RSO3- B+]s[H+]aq/[ RSO3- H+]s[B+]aqK = Cs/CM = [RSO3- B+]s/[B+]aqWhere, K is the distribution constant. Therefore, kex represents the affinity of the resin RSO3 - H+to cation B+ relative to cation H+. The affinity or kex is different for different ions and will dependon size and charge of ions .The anionic exchange equilibria can be represented by the equation:n R(CH3)nN+ OH- (solid) + An- = (R(CH3)nN+)n An- (solid) + n OH.Eluent Suppressor ColumnsUnfortunately, detectors available for use with IC are rather limited. Conductivity detectors are mostcommon where simply ions will increase the conductivity. A major drawback to using conductivitydetectors is the high salt concentration in the mobile phase which may make it very difficult todetermine differences in conductivity, especially at low solute concentrations. The problem of highsalt content of the mobile phase was solved by the use of what is called suppressor columns. Thesuppressor column is packed with a second ion exchange resin that converts the ions in themobile phase to molecular species of limited ionization. For example, for the separation of cations,HCl is used as the eluent and the suppressor column in this case is packed with a hydroxide anionexchange packing. The following equilibrium takes place in the suppressor column: 16
    • H+aq + Claq+ Resin+ OH- (solid) = Resin+ Cl- (solid) + H2O For separations of anions, thesuppressor column contains a hydrogen ion cationic exchange resin and the eluent is acarbonate/bicarbonate mobile phase. The following equilibrium takes place: Na+aq + HCO3-aq +Resin- H+ (solid) = Resin+ Na+ (solid) + H2CO3 (aqueous) H2CO3 (aqueous) is a weak electrolytewhich will contribute very little to conductivity. Therefore, in both cases the conductivity of themobile phase was eliminated using suppressor .Parameter Effect on retention in anion ICThe Mobile PhaseThe mobile phase transports the sample through the system and affects both retention and selectivityof the separation. The mobile phase is usually a solution of a salt in water, which works as a buffer,providing a stable pH. Several considerations govern the choice of a mobile phase. The first factor isthe kind of sample ions that will be separated, but the type of separation column is also important.For catinic separations, HCl is a most common mobile phase while the two most common mobilephases for anionic separations are based on hydroxide or carbonate as eluting anion. In carbonatebased mobile phases, the eluent is an aqueous solution of carbonate and hydrogen carbonate salts,where the ionic strength of the mobile phase and the ratio of the bicarbonate/carbonate ions can bevaried to optimize the retention time and selectivity. The elution strength of the mobile phase and, tosome extent, its selectivity, are affected by the type of ions of the eluent. A change of salts willnormally result in a change of the eluent pHcolumns of suitable packing.Ionic strength 17
    • The eluting ability of a mobile phase will increase as the ionic strength of the mobile phase isincreased. However, the change in the selectivity among equally charged ions is very small, whereasthe selectivity between ions of different charges (mono- or polyvalent) is far more sensitive tochanges in ionic strength.PHThe charge of the sample ions of weak acids or bases are controlled by the eluent pH and thus theretention times of such species will be affected by changes in PhTemperatureThe ion exchange rate between the stationary and the mobile phase increases with increasingtemperature. The viscosity of the eluent, and thereby the column backpressure, decreases and cangive a better separation efficiency. The temperature can also affect the column selectivity.Isocratic and Gradient ElutionThe most common type of elution in IC is isocratic where the mobile phase has a constantcomposition during the entire run. Gradient elution where the eluent concentration is changedduring the run can also be used. Gradient elution is effective when sample contains ions with widelydifferent retention times. Hydroxide eluents are usually used for gradient elution in anionchromatography. By gradually increasing the concentration of hydroxide ions, the eluting power ofthe mobile phase increases. As a result, ions with high retention stay at the top of the column They 18
    • then elute in sequence as sharp peaks. After each gradient run the column must be regenerated bypumping initial mobile phase for say 10-20 min (reproducibly getting the same baseline.COUNTER CURRENT CHROMATOGRAPHY COLUMS:From numerous column designs used to retain a liquid stationary phase [5–9], only two have had thepotential for sustained commercial development. They are called the hydrostatic and thehydrodynamic configurations.Hydrostatic CCC columns:The very first hydrostatic CCC columns used gravity to maintain the liquid stationary phase; theywere called droplet CCC (DCCC) columns. They needed very long elution times .The columns areno longer in use today. Modern hydrostatic CCC columns are known and marketed under the nameof centrifugal partition chromatographs Their two main characteristics are: they have a single axisof rotation generating a constant centrifugal field and they enclose geometrical volumes, tubes,channels, or locules that repeat themselves through connecting tubes forming a pattern .It can beseen that there is quite a significant volume of connecting ducts which only contain the mobilephase. 19
    • The liquid motion in CCC columns. A – Hydrostatic olumns or CPCs. There are a single axis ofrotation producing constant centrifugal field and no phase exchanges in the connecting ducts. B –Hydrodynamic columns. There are a variable and cyclic centrifugal field produced by the planetaryrotation of the bobbin around its own axis and the central rotor axis. There is contact between thetwo liquid phases throughout the tubing. The mobile phase is pictured in black, the stationary phaseis white. This design reduces the contact time for solute exchange with the stationary phase. Italso builds a small hydrostatic pressure that explains the significant pressure drop needed to operatehydrostatic centrifuges. All hydrostatic centrifuges contain two rotary seals; one at the top and theother one at the bottom. They are quiet to operate.In the toroidal coil CCC (helix CCC) system operated under a centrifugal force, the dimensions ofthe coil are reduced to that which is convenient for analytical separations. The coil is mountedaround the periphery of the centrifugal bowl so that the stable radially acting centrifugal force fieldretains the stationary phase in one side of the coil as in the basic hydrostatic system describedabove.Hydrodynamic columns:Hydrodynamic Counter current Chromatography columnsHydrodynamic centrifuges used in the CCC columns have two rotational axes, a main axis and aplanetar one which generates a variable centrifugal force field. There can be any number ofplanetary axes but the most common are single, double, and triple axes. Each planetary axis has abobbin or spool mounted on it that contains the coils of continuously wound Teflon tubing Inhydrodynamic columns, it is important to know the ratio of the spool radius, r, over the rotor radius,R. This ratio was traditionally termed is defined in LC as the phase ratio. 20
    • The tubing can be connected from the outside of the centrifuge, wound round the bobbins, andpassed back to the outside again without any rotary seals—hence, from the chromatography point ofview it is equivalent to one long thin continuous column. The variable force field produces mixingand settling zones throughout the whole length of the coiled column as indicated in Fig. 2B. There iscontinuous contact between the two liquid phases throughout the column with no significantpressure buildup. Hydrodynamic centrifuges work with low mobile-phase pressure but can generatenoise from the gear assembly, which can be reduced in well-designed centrifuges. These columnsare often called highspeed CCC (HSCCC) columns since they can operate much more rapidly thanthe Craig, DCCC, and gravity-based columns. Table 1 compares the features of the two kinds ofCCC columns. It is not possible to say that one kind is clearly superior to the other. The bestsituation is to have both kinds of CCC column to cover all possible cases. Studies are going on todevelop large-scale CCC centrifuges based on both types being able to produce significant mass ofpurified material (preparative CCC) [13–15]. Such centrifuges can be used to produce standardreference materials for analytical purposes and to purify analytical reagent INSTRUMENTATION:force of 1g (the earth’s gravity) which was not very effective at stabilising the SP. Furthermore, thescope was limited since only the more dense phase could be used as the SP. Apart from these issuesruns could take many days even with the later, mechanized and automated versions. 21
    • The time required to perform experiments could run into days but the instruments were capable ofresolving very complex mixtures. The technique was recognised to be gentle and offered highresolution and enjoyed favour as the purification method of choice for high value products such asChinese medicines and other natural products with many groups of chromatographers. Themethodology became, and until recent years remained, a niche application and was a technique veryrarely employed by medicinal chemists.The other significant 1g instrument was the Droplet Countercurrent Chromatograph, DCCC, whichwas invented by Ito (see below) and his group in 1970. These instruments also relied on the earth’sgravity alone to stabilise the stationary phase whilst MP was pumped through the instrument butlike the Craig machines, SP retention (stabilisation) was poor even at very low flow rates andexperiments could take days to complete. However, these instruments were able to use either phaseas SP although a significant disadvantage of these instruments was that phase mixing, an essentialpre-requisite of successful LLC, was poor.DESCRIPTION OF CRAIG’S TECHNIQUEA schematic representation of five stagcs in the Craig s technique of counter cirrent extraction. Theprocess of separation is carried cut by extracting a solution ofthc sample under test with animmiscible solvent. According to Craig, thi step is called ‘transfer’(n 0) and is shown i.nthe top row of the figure. The vessel in which the initial extraction is allowed to take place, has beenlabelled ‘tube zero’ (r= 0). The figure.also indicates four other additional vessels labelled tubes. 12,3 22
    • and 4. These vessels are a part of a very large numbe of identical containers. All of these vesselscontainorganic solvent a mobilc one and assuming for convenience that the weight Of solute is 1000 mg.and its partition ratio between the solvents is 1.00. It is also assumed that the volume of the organicsolvent is identical to that of aqueous phase. Thus when equilibrium is achieved-in tube 0 aftertransfer, 0.500 mg. of solute will be found in each phase.• The next step in the process (transfer no.1.) involves transfer of the organic solvent from tube 0 totube 1. This is also followed by an identical amount of fresh organic solvent into tube 0. When bothvessels are shaken, the solute will be distributed among the four solutions as indicated inline n=1 ofthe figure 3. This process of transfer and equilibrium is epeated with an identical vôlume of freshorganic solvent being added to tube 0 at each transfer. The distribution of solute after four transfers(n 4) has been depictcd in line 4, where it will be seen that solute has become dist’ributed among alltubes. Figure 4 indicates the movementof the so,lute for a much large number of transfers, when thefraction of total solute contained in the two layers of each tube (i.e., organic portion + aqueousportion) is plotted agailist the tube number. It has- been found thatthe distribution curves becomebroader’ and lower, with an increased number of transfers. It should also be noted that for a solutewith partition ratio larger than unity, the movement will be enhanced. 23
    • DERIVATION OF DISTRIBUTION CURRENTThe partition ratio data may be made use of in deriving the distribution curves, such as those slownin fig. 4. Suppose fn.r be the fraction of the initial solute A present in tube r after n transfers havebeen performed. Thus the sum of th total fractions must be equal to unity. That is,f.o+ fn + f2+ f3t .. 1We know that the partition ratio in all tubes is given by,C-where - Cm= Concenratinof solute in the mobile phase.C5 = Concentration of solute in stationary phase.If now x is designated as the fraction of’öute found in the mobile organic phase of any tube and y asthe fraction found in thetationary aqueoupiase, then x+ y= 1In any particular tube, the amount of solute found in eaëh phase is given by, weight in mobileorganic phase Cm . Vm.weight in stationary aqueous phase = C. V5 ‘ -V ri’ iho volumes of the two phases in millilitres ahdC, and C are expressed in grams per znillilitres. Thus, 24
    • andSubstitution of equation (i) and rearrangement gives,It may be made Dlear by taking the following examples. I. For n = 0. At this stage the solute isfound in tube 0 only. Thus,fo,o X+ y 1.00since x and y are dependent only on K’ when the volumes are fixed.POSITION WHEN LESS DENSELIQUID IS RECEIVED FROMPRECEDING TUBE ANDDURING EQUILIBRATION 25
    • (a) For r= 1. The solute in tube I comes.from the tranfer of the organic phase in tube 0, transfer 0,which contaiiied the fraction of the original solute. Hence.Now the transferred sDlute undergoes partition in tube 1. .A fraction x of the solute will remain inthe organic layer and a fraction y will go into the aqueous layer. Since the values of x and y aresame throughout we have(b) For r = 0. The solute present in tube 0 comes from the aqueous layer that ontains the fraction yof the original. Thus, 26
    • (3) For n =2. Here, the distribution of solut occurs among tubes 0,1 and 2.(a) For r =2. The solute in tube 2 comes from the organic layer of tube 1, transfer I containing thefraction x2 of the original sample.• This fraction will be again distributed to give x2 of the organic layer and x2y in the aqueous layerof tube 2. • 27
    • Solution— From(8) and (9), we havePutting n= 5, and 3 -r in equation (iv) 28
    • APPLICATIONS OF CRAIG’S TECHNIQUECraig’s technique has widely been dsed particularly in the field of biochemical’s. Many closelyrelated compounds have been separated by making use of this technique. Craig himself had resolveda mixture of 10 amino acids, despite the fact that the partition ratio for some of these compoundsdiffered by less than ?. 1. Complex mixtures of fatty acids, polypeptides nucleotides, aromaticamines, antibiotics and many other organic substances have been fully separated by. using countercurrent extraction.TECHNIQUES:TechniquesCCC can be thought of as occurring in three stages: mixing, settling, and separation (although theyoften occur continuously). Mixing of the phases is necessary so that the interface between them hasa large area, and the analyte can move between the phases according to its partition coefficient.A partition coefficient is a ratio of the amount of analyte found in each of the solvents at equilibriumand is related to the analytes affinity for one over the other. The mobile phase is mixing with thensettling from the stationary phase throughout the column. The degree of stationary phase retention(inversely proportional to the amount of stationary phase loss or "bleed" in the course of aseparation) is a crucial parameter. Higher quality instruments have greater stationary phaseretention. The settling time is a property of the solvent system and the sample matrix, both of whichgreatly influence stationary phase retention. 29
    • Droplet Countercurrent Chromatography (DCCC)Droplet CCC is the oldest form of CCC. It uses only gravity to move the mobile phase through thestationary phase. In descending mode, droplets of the denser mobile phase and sample are allowedto fall through a column of the lighter stationary phase using only gravity.If a less dense mobile phase is used it will rise through the stationary phase; this is called ascendingmode. The eluent from one column is transferred to another; the more columns that are used, themore theoretical plates can be achieved. The disadvantage of DCCC is that flow rates are low, andpoor mixing is achieved for most binary solvent systems, which makes this technique both time-consuming and inefficient.High-Performance Countercurrent Chromatography (HPCCC)The operating principle of CCC equipment requires a column consisting of a tube coiled around abobbin. The bobbin is rotated in a double-axis gyratory motion (a cardioid), which causes a variablegravity (G) field to act on the column during each rotation. This motion causes the column to see 30
    • one partitioning step per revolution and components of the sample separate in the column due totheir partitioning coefficient between the two immiscible liquid phases used.HPCCC works in much the same way as HSCCC but with one vital difference. A seven-year R&Dprocess that has produced HPCCC instruments that generated 240 g, compared to the 80 g of theHSCCC machines. This increase in g-level and larger bore of the column has enabled a tenfoldincrease in through put, due to improved mobile phase flow rates and a much higher stationaryphase retention.Countercurrent chromatography is a preparative liquid chromatography technique, however with theadvent of the higher g HPCCC instruments it is now prossible to operate instruments with sampleloadings as low as a few milligrams, whereas in the past 100s of milligrams have been necessary.Major application areas for this technique include natural products purification and also drugdevelopment.High-Speed Countercurrent Chromatography (HSCCC)The modern era of CCC began with the development by Dr. Yoichiro Ito of the planetary centrifugeand the many possible column geometries it can support. These devices make use of a little-knownmeans of making non-rotating connections between the stator and the rotor of a centrifuge. (It isbeyond the scope of this discussion to describe the method of accomplishing this. Any of the severalbooks available on CCC .Functionally, the high-speed CCC consists of a helical coil of inert tubing which rotates on itsplanetary axis and simultaneously rotates eccentrically about another solar axis. (These axes can bemade to coincide, but the most common or type J CCC is discussed here.) The effect is to createzones of mixing and zones of settling which progress along the helical coil at dizzying speed. Thisproduces a highly favorable environment for chromatography. 31
    • There are numerous potential variants upon this instrument design. The most significant of these isthe toroidal CCC. This instrument does not employ planetary motion. In some respects it is very likeCPC, but retains the advantage of not needing rotary seals. It also employs a capillary tube insteadof the larger-diameter tubes employed in the helices of the other CCC models. This capillarypassage makes the mixing of two phases very thorough, despite the lack of shaking or other mixingforces. This instrument provides rapid analytical-scale separations, which can nonetheless be scaledup to either of the larger-scale CCC instruments.Centrifugal Partition Chromatography (CPC)Centrifugal Partition Chromatography (CPC) was invented in the eighties by the Japanese companySanki Engineering Ltd, whose president was the late KanichiNunogaki. CPC has been extensivelydeveloped in France starting from the late nineties. CPC uses centrifugal force to speed separationand achieves higher flow rates than DCCC (which relies on gravity).The centrifugal partition chromatograph is constituted with a unique rotor (=column). This rotorrotates on its central axis (while HSCCC column rotates on its planetary axis and simultaneouslyrotates eccentrically about another solar axis). With less vibrations and noise, the CPC offers awider rotation speed range (from 500 to 2000 rpm) than HSCCC. That allows a better decantationand retention for unstable biphasic system (e.g., aqueous aqueous systems or Butanol/watersystems).Basics of CPC: The CPC rotor is constituted by the superposition of disks engraved with small cellsconnected by head / tail ducts. These cells, where the chromatographic separation takes place, canbe compared to lined-up separate funnels. The rotor is filled with the stationary phase, which staysinside the rotor thanks to the rotation speed, while the mobile phase is pumped through. CPC can beoperated in either descending or ascending mode, where the direction is relative to the forcegenerated by the rotor rather than gravity. According to the fast and permanent evolution of the cellsdesign, the efficiency and flow rate with low back pressure are improved. The CPC offers now thedirect scale up from the analytical apparatuses (few milliliters) to industrial apparatuses (someliters) for fast batch production. 32
    • MODES OPERATE:Modes of Operation • Reverse phase - Aqueous phase mobile - The denser phase is pumped through as the mobile phase. • Normal phase - Organic phase mobile - The less dense phase is used as the mobile phase. • Dual-Mode: The mobile and stationary phases are reversed part way through the run. • Gradient Mode: The concentration of one or more components in the mobile phase is varied throughout the run to achieve optimal resolution across a wider range of polarities. For example, a methanol-water gradient may be employed using pure heptane as the stationary phase. This is not possible with all binary systems, due to excessive loss of stationary phase. • Elution Extrusion Mode (EECCC): The mobile phase is extruded after a certain point by switching the phase being pumped into the system. For example, during the Elution portion of a separation using an EtOAcwater system running head to tail, the aqueous mobile phase is being pumped into the system, . In order to switch to extrusion mode, organic phase is pumped into the system. This can be accomplished either with a valve on the inlet of single pump, or ideally with an integrated system of two or three pumps, each dedicated either to a single phase of a binary mixture, or to an intermediate wash solvent. This also allows for good resolution of compounds with high mobile-phase affinities. It requires only one column volume of solvent and leaves the column full of fresh stationary phase. 33
    • • pH Zone Refining: Acidic and basic solvents are used to elute analytes . Achieving Resolution in Counter Current ChromomatographyUnderstanding how resolution is achieved in CCC is assisted by the use of the simple model of aseries of separating funnels numbered, each containing, let’s say 10ml of ethyl acetate which hasbeen pre-saturated with water and which will form the SP for our experiments. For our firstexperiment, 10ml of water, pre-saturated with ethyl acetate and 50 parts of a solute with a D valueof , when partitioned between ethyl acetate and water, are added to the first funnel. The funnel isstoppered, shaken and the phases allowed to settle. At this point each of the phases will contain 25parts of the solute. The lower aqueous phase is transferred to the second funnel and 10ml freshpre-saturated water are added to. Both funnels are stoppered, shaken and allowed to settle. At thispoint, the phases in will each contain parts of the solute as will those in The lower aqueous phase ofis transferred to, the lower phase of is transferred to and another portion of water is added to. Allthree are stoppered, shaken and allowed to settle. Each phase in will now contain parts will stillcontain 25 parts in each phase and the phases in will each contain part The process of transfer lowerphases to next funnels, add fresh portion of pre-saturated water to, shake and settle are repeated. Thesolute distribution profile after the fifth iteration is shown at the bottom of the Study of the figureshows that the solute is effectively moving through the series of funnels with a Gaussian distributionand as the solute band progresses, the maximum, single funnel concentration decreases. Aftersixteen or so such iterations, the outermost funnels, are essentially devoid of solute and the solute isdistributed between funnels with the highest concentration of solute around.The process of transfer lower phases to next funnels, add fresh portion of pre-saturated water to,shake and settle are repeated. The solute distribution profile after the fifth iteration is shown at the 34
    • bottom. If a total of 100 iterations are completed and the concentrations plotted as in the solute isdistributed over about 30 funnels with the maximum concentration found in funnel 50 or so as mightbe experted.Chromatographic ResolutionChromatographic resolution, i.e. complete separation of the components of a mixture, is the ultimategoal of a separation process. Such a process, performed on naturally occurring materials or those ofsynthetic origin, may involve many steps including techniques such as extraction, crystallization,distillation etc and may also include one or more chromatographic steps. It is these latter with whichwe are concerned i.e. the complete separation of the components of a mixture by means of achromatographic process.In both research and production, the most widely used, high performance (resolution)chromatographic method is RP-HPLC. HPCCC is a high performance (high resolution) techniqueand some comparison with RP-HPLC is justified.The Snyder resolution equation, Equation 4, tells us that resolution is the product of an efficiencyterm, a selectivity term and a retention term, respectively.This means that in order to maintain a given resolution, if the value of any single term is reduced,then the value of one or both of the others must increase to compensate for the reduction.Retention is a critical factor in any chromatographic process and ultimately determines columncapacity.shows a representation of the relative, characteristic SP and MP volumes in a HPCCCcolumn compared with those in a bonded RP-HPLC column. It should be noted that although theactive MP volume in both is much the same, in an HPCCC system the SP volume is much greater 35
    • and furthermore that the ratio of SP/MP is inverted. This factor means that the dynamic masscapacity of the HPCCC column is much grater than that of a rp- hplc columnHPLC chromatographers have only limited scope for improving selectivity, via Path B in the sinceC18 bonded phases differ little one from another and little more from, for example, a C8 bondedphase and the range of usable solvents is extremely limited so the usual solution to the problem foranalytical chromatographers is via Path A in the figure, i.e. moving to a higher efficiency, smallerparticle, packing. However, this is not a practicable proposition for preparative scalechromatographers since (large volume preparative columns filled with small particle media wouldbe prohibitively expensive) and in any case there are currently no commercially availablepreparative scale pumps capable of pumping mobile phase through a large bed of sub-media at apractically useful flow rate. Under such circumstances the preparative scale solid-liquidchromatographer is stymied. An alternative solution is to find a methodology which rather thanimproving efficiency can exploit controllable selectivity, i.e. a CCC methodology. Even therelatively high, compared with other CCC methods, efficiency of HPCCC is still measured in onlyhundreds of plates compared with the thousands or tens of thousands of plates found in anequivalent sized SLC column but by exploiting the optimization of selectivity the technique iscapable of high resolution. 36
    • APPLICATION OF COUNTER CURRENT CHROMATROGRAPHYCCC is playing an increasingly important role in separation science. All components in the samplesolution injected into the column can be recovered and irreversible adsorption and contamination ofsamples can be virtually eliminated. A crude sample can be injected directly into the column, whichsimplifies sample preparation. Now CCC is successfully used for the separation of organic andinorganic substances from a complicated mixture.ORGANIC SUBSTANCES; CCC has become a method of choice in natural products chemistry and has made possible theseparation of a number of biologically interesting natural products that are difficult or impossible toseparate by other techniques Crude extracts of plants or other organisms are often too complex forthe direct analysis by HPLC. Certain materials may irreversibly bind to the packing material or mayplug the column inlet filters, and hence reduce the column life. Those restrictions do not apply toanalytical CCC, which represents an interesting method for enrichment and separation of variousanalytes. The technique is also used for the separation of bio chemicals and pharmaceuticals. CCCis especially suitable for the separation of alkaloids from medical herbs using simple solventsystems, for the total hormonal analysis of natural samples and for the screening of new bioactivecompounds in crude extracts and other complex samples CCC has been also suggested as analternative to the shake flask method to measure liquid–liquid partition coefficients as a way tocharacterize the lipophilic–hydrophilic nature of a compound ,below some interesting methods arebriefly described which have been successfully used in the analysis of various samples or which canbe applied to analytical purposes without serious modification of the procedures and apparatus. 37
    • Analysis of plant and different natural products Different types of hydrodynamic (HSCCC, cross-axis coil) and hydrostatic (toroidal coil) centrifuges can be used for separation and concentration ofvarious compounds from plant and different natural products. The quantity of separated compoundsmay range from trace to gram amounts. Toroidal coil CentrifugesToroidal coil centrifuges have been successfully applied to the separation and purification of planthormones,namely, indole auxins, gibberellins, cytokinins, and abscisic acid. Indole auxins wereseparatedby either hexane-ethyl acetate–methanol–water (volume ratio 0.6:1.4:1.0:1.0) orchloroform–acetic- acid–water (2:2:1) in a column with a total capacity of 18 mL. The latter solventsystem was especially useful for the separation of abscisic acid from indole-3-acetic acid.Gibberellins (GA3, GA4, and GA7)were separated from each other in ether-methanol-phosphatebuffer (pH 7) (3:1:2). The CCC method was suitable for the separation of four cytokinins in ethylacetate-methanol-phosphate buffer (pH 7)(3:1:3) [2,4,5].A toroidal coil planet centrifuge foranalytical-scale separations was used for the purification of abscisic acid (ABA) obtained from crudeplant extracts and its determination in several plant tissues using HPLC and GC-MS [34].The results of theisolation of 3-oxo-5-steroid isomerase (KSI) from crude E. coli lysate were published [35]. A separation wasperformed on ca. 3 mg of 15N-labeled KSI using a polymer-containing system based on PEO 3350. Thepresent method eliminates sample loss and denaturation caused by the solid support and yields pure proteinsin both preparative and analytical separations . 38
    • High-speed hydrodynamic centrifuges (HSCCC)Numerous applications in the analysis and preparation of natural products by CCC have beenreported. Separation of poly phenolic natural products such as flavonoids are difficult because thesecompounds tend to show “peak tailing” in RP-HPLC, as well as irreversible adsorption on silica gel.Those difficulties do not exist in CCC and are the reason why CCC has been recognized as a mostvaluable technique for the isolation of polyphenols . The flavonoids and hydroxyanthraquinones canbe easily separated by CCC with a high selectivity Zhang has published the results of the separationof alkaloids (from Stephania tetrandra S.Moore) using n-hexane–ethyl acetate–methanol–watersystems at different volume ratios, hydroxyanthraquinones (from the rhizome of Rheum palmatumL.) using a system of hexane–ethyl acetate–methanol–water (9:1:5:5) and flavonoids (from seabuckthorn Hippophae rhamnoides) using a system of chloroform–methanol–water (4:3:2) by CCC;the total capacity of the column was 43 mL, the maximum revolution speed of centrifuge was 2000rpm By increasing the flow-rate of the mobile phase in these analytical separations, the separationtime for a crude sample mixture was shortened to within 15 min, which is quite comparable withthat of analytical HPLC. Milligram and even gram amounts of substances can be isolated by theCCC technique.This makes it possible to produce standard reference materials for any analytical study. For the moreefficient separation of compounds having a wide range of polarity, lower and upper phases of thesolvent system were used as the mobile phase in succession. This method achieved a completeseparation of five components present in a 1-mg sample mixture. The peak fraction of eachcompound was subjected to mass spectrometric analysis for compound structure confirmationshows the countercurrent chromatogram of five major compounds in the crude extract of rhizome ofRheum palmatum L. 39
    • Three peaks were eluted with the upper phase in NP mode followed by two peaks which wereeluted with the lower phase in RP mode. The results indicated that peaks were corresponding tochrysophanol, emodin, physcion, aloe-emodin, and rhein, respectively. CCC was used for thesystematic selection and optimization of a two-phase solvent system to separate alkaloids fromCoptis chinensis Franch using a system of chloroform–methanol–HCl solution. at different volumeratios .One separation run yielded four pure alkaloids, including palmatine, berberine, epiberberine,and coptisine from a crude alkaloid extract. Analytical application of CCC was successfullydemonstrated for the separation of microgram quantities of flavonoids from a crude ethanol extractof sea buckthorn in a multilayer coil with a total capacity of 8 mL using a two-phase solvent systemcomposed of chloroform–methanol–water (4:3:2) [43]. Five peaks, including isorhamnetin andquercetin, were well resolved and eluted within 8 min. An artificial mixture of three common plantcoumarins (herniarin, scopoletin, and umbelliferone) and one flavanone (hisperetin) was separated 40
    • with a hydrodynamic CCC column connected to a photodiode array detector. The lower phase of achloroform–methanol–water.CONCLUSION: 41
    • High Speed Counter Current Chromatography is a very good preparative separation technique. Theuse of support-free liquid stationary phase and no sample loss, with high separation efficiency andresolution by using the centrifugal field, are the characteristics that make it superior to all otherseparation techniques. As there is no solid support, it is free from adsorption of solutes to thecolumn and the recovery of samples and reagents is without contamination or decomposition.Another advantage is that it is possible to use the same column repeatedly for separations, withdifferent stationary phases. The use of HSCCC in drug discovery and product development wherethere is ease of scale-up from milligrams to grams and then to kilograms, makes it an excellenttechnique for the separation of natural pigments and other bioactive constituents, which are presentin minute quantities.HPCCC is an orthogonal and complementary chromatography technique toHPLC in the armamentarium available to separation scientists and worthy of a place in thechromatographer’s toolbox. Neither HPCCC, nor any other modern LLC methodology, is a ‘magicbullet’ solution but with controllable, tunable selectivity it is able to very effectively tackleproblems that are difficult or totally intractable when HPLC is used. In many instances the use ofselectivity instead of efficiency can prove to be the optimal way of separating components,especially from whenever preparative quantities require purification. More and morechromatographers are realizing the real benefits that accrue from the use of LLC. The list ofapplications of LLC grows day by dayREFERENCES 42
    • 1. Dr.S. Ravi shankar2. B.K.sharma3. Sikdar, Cole, et al. Aqueous Two-Phase Extractions in Bioseparations: An Assessment. Biotechnology 9:254. 19914. Szlag, Giuliano. A Low-Cost Aqueous Two Phase System for Enzyme Extraction. Biotechnology Techniques 2:4:277. 19885. Dreyer, Kragl. Ionic Liquids for Aqueous Two-Phase Extraction and Stabilization of Enzymes. Biotechnology and Bioengineering. 99:6:1416. 20086. Boland. Aqueous Two-Phase Systems: Methods and Protocols. Pg 259-2697. 11. A. Berthod, B. Billardello. Advances in Chromatography, Vol. 40, P. Brown, E. Grushka (Eds.),8. p. 8, Marcel Dekker, New York (2000).9. 12. A. Berthod. In Centrifugal Partition Chromatography, A. P. Foucault (Ed.), Chromatographic10. Science Series, Vol. 68, p. 167, Marcel Dekker, New York (1995).11. I. A. Sutherland, J. de Folter, P. L. Wood. J. Liq. Chromatogr. Rel. Technol. 26, 1449 (2003).12. P. L. Wood, D. Hawes, L. Janaway, I. A. Sutherland. J. Liq. Chromatogr. Rel. Technol. 26, 137313. (2003).14. A. Marston, K. Hostettmann. J. Chromatogr., A 658, 315 (1994).15. Y. Ito. J. Chromatogr., A 106516. 11. A. Berthod, B. Billardello. Advances in Chromatography, Vol. 40, P. Brown, E. Grushka (Eds.),17. p. 8, Marcel Dekker, New York (2000). 43
    • 18. 12. A. Berthod. In Centrifugal Partition Chromatography, A. P. Foucault (Ed.), Chromatographic19. Science Series, Vol. 68, p. 167, Marcel Dekker, New York (1995).20. 13. I. A. Sutherland, J. de Folter, P. L. Wood. J. Liq. Chromatogr. Rel. Technol. 26, 1449 (2003).21. 14. P. L. Wood, D. Hawes, L. Janaway, I. A. Sutherland. J. Liq. Chromatogr. Rel. Technol. 26, 1373(2003).22. 15. A. Marston, K. Hostettmann. J. Chromatogr., A 658, 315 (1994).23. 16. Y. Ito. J. Chromatogr., A 1065,,24. . Oka. In High-Speed Countercurrent Chromatography, Y. Ito, W. D. Conway (Eds.), p. 73, John25. Wiley, Chichester (1996)26. Kanichi Nunogaki was invented centrifugal partition chromatography japans company engineering limited whose president27. Coptis chinensis was invented counter current chromatography .for the development of applications 44
    • 18. 12. A. Berthod. In Centrifugal Partition Chromatography, A. P. Foucault (Ed.), Chromatographic19. Science Series, Vol. 68, p. 167, Marcel Dekker, New York (1995).20. 13. I. A. Sutherland, J. de Folter, P. L. Wood. J. Liq. Chromatogr. Rel. Technol. 26, 1449 (2003).21. 14. P. L. Wood, D. Hawes, L. Janaway, I. A. Sutherland. J. Liq. Chromatogr. Rel. Technol. 26, 1373(2003).22. 15. A. Marston, K. Hostettmann. J. Chromatogr., A 658, 315 (1994).23. 16. Y. Ito. J. Chromatogr., A 1065,,24. . Oka. In High-Speed Countercurrent Chromatography, Y. Ito, W. D. Conway (Eds.), p. 73, John25. Wiley, Chichester (1996)26. Kanichi Nunogaki was invented centrifugal partition chromatography japans company engineering limited whose president27. Coptis chinensis was invented counter current chromatography .for the development of applications 44