ABSTRACT

Countercurrent chromatography (CCC) is a generic term covering all forms of liquid–liquid

chromatography that use a support-free liquid stationary phase held in place by a simple

centrifugal or complex centrifugal force field. Biphasic liquid systems are used with one

liquid phase being the stationary phase and the other being the mobile phase. Although

initiated almost 30 years ago, CCC lacked reliable columns. This is changing now, and the

newly designed centrifuges appearing on the market make excellent CCC columns. This

review focuses on the advantages of a liquid stationary phase and addresses the

chromatographic 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 to

obtain a liquid stationary phase using centrifugal forces, the hydrostatic way and the

hydrodynamic way. These two kinds of CCC columns are described and compared. The

reported applications of CCC in analytical chemistry and comparison with other separation

and enrichment methods show that the technique can be successfully used in the analysis of

plants 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 the

studies of the last two decades, recommendations are also given for the application of CCC

in trace inorganic analysis and in radio analytical chemistry.

1

INTRODUCTION

Counter current chromatography is a method of multiple liquid liquid extraction technique where

separation of components’ having variable solubility in two immisible liquid is achieved

In a conventional liquid liquid extraction, 2 components example a and b are distributed between 2

immisible liquids; according to their partition coefficient still pure a and b are not present in these 2

liquids even after reaching equilibrium

In the counter current chromatography, two immisible solvents flow in an opposite direction in

multiple 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 individual

components through equilibrium distribution between two phases. Essentially, the technique of

chromatography is based on the differences in the rate at which the components of a mixture move

through a porous medium called stationary phase under the influence of some solvent or gas called

mobile phase

2

PRINCIPLE

In counter current chromatography when 2 components’ a and b having varying affinity or partion

co-efficient, is distributed between 2 immisible solvents eg. X and Y which are allowed to flow in

opposite direction separation of pure a and b takes places in multiple stages

In the first stage when equilibrium is achieved in container 1, solvent X lighter or upper phase and

solvent Y heavier or lower phase will have both components a and b based on their distribution

coefficient ,let us say a is present more in X and b is present more in Y. the upper phase solvent x is

transferred to next container 2, with similar composition of solvents. Fresh solvents X is added to

container 1.

After achievement of equilibrium in container 2, now the upper phase will contains less of b, due to

its low solubility in X and more of a. this upper phase is then transferred to container 3 with similar

composition of solvents now, the upper layer of container 1 is then transferred to container 2 and

fresh solvent is added to container 1. The above steps are repeated till the upper layer contains pure

a in the n container, where n is the last container the lower phase solvent Y of container 1 contains

the 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 distribution

coefficient when the difference between is more few steps are required. But when the difference in

distribution coefficient between a and b is less then more steps are required.

3

4

ADVANTAGES OF COUNTER CURRENT CHROMATOGRAPHY

Modern CCC technology has many advantages over traditional preparative techniques:

Fast

CCC provides high throughput preparative separations.

Inexpensive

After 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 preparative

chromatography techniques, such as HPLC.

Gentle Technique

CCC is a very gentle technique in which the sample is only in contact with solvents and teflon (or

other inert material). Therefore CCC provides the least chance for sample

degradation/decomposition.

Versatile Selectivity

Separation of virtually every compound class has been demonstrated with CCC. Selectivity over a

full range of polarities is achieved through the use of appropriate CCC Solvent Systems.

Scaleable

CCC is able to range from milligrams to tens of grams on the same instrument. Furthermore, many

Manufacturers 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 with

surface-to-volume ratios and flow dynamics; this is avoided when both phases are liquid.

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100% Sample Recovery

Since CCC does not use a solid support, permanent adsorption of analyte onto the column is

avoided, and a 100% recovery of the analyte can be achieved in practice

TYPES OF COUNTER CURRENT CHROMATOGRAPHY

INTRODUCTION

LIQUID LIQUID CHROMATOGRAPHY

SOLID LIQUID CHROMATOGRAPHY

Countercurrent chromatography (CCC) is a liquid chromatography (LC) technique that uses two

immiscible liquid phases without any solid support. As an LC technique, CCC uses many terms

already defined for chromatography [1]. This article will give the fundamentals of the CCC

technique and briefly describe the special chromatographic columns capable of maintaining a static

liquid phase using centrifugal fields. A rapid approach to selecting solvent systems that can be used

in CCC

Liquid–liquid extraction, also known as solvent extraction and partitioning, is a method to

separate compounds based on their relative solubilities in two different immiscible liquids, usually

water and an organic solvent. It is an extraction of a substance from one liquid phase into another

liquid phase. Liquid–liquid extraction is a basic technique in chemical laboratories, where it is

performed using a separatory funnel. This type of process is commonly performed after a chemical

reaction as part .

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The term partitioning is commonly used to refer to the underlying chemical and physical processes

involved in liquid–liquid extraction but may be fully synonymous. The term solvent extraction can

also refer to the separation of a substance from a mixture by preferentially dissolving that substance

in a suitable solvent. In that case, a soluble compound is separated from an insoluble compound or a

complex matrix.

Solvent extraction is used in nuclear reprocessing, ore processing, the production of fine organic

compounds, the processing of perfumes, the production of vegetable oils and biodiesel, and other

industries.

Liquid–liquid extraction is possible in non-aqueous systems: In a system consisting of a molten

metal in contact with molten salts, metals can be extracted from one phase to the other. This is

related to a mercury electrode where a metal can be reduced, the metal will often then dissolve in

the mercury to form an amalgam that modifies its electrochemistry greatly. For example, it is

possible for sodium cations to be reduced at a mercury cathode to form sodium amalgam, while at

an inert electrode (such as platinum) the sodium cations are not reduced. Instead, water is reduced to

hydrogen. A detergent or fine solid can be used to stabilize an emulsion, or third phase

Techniques

Batchwise single stage extractions

This 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 using

ether, 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 organic

extract with sodium bicarbonate. The acetic acid reacts with the sodium bicarbonate to form sodium

acetate, carbon dioxide, and water.

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Multistage countercurrent continuous processes

These are commonly used in industry for the processing of metals such as the lanthanides; because

the separation factors between the lanthanides are so small many extraction stages are needed. In the

multistage processes, the aqueous raffinate from one extraction unit is fed to the next unit as the

aqueous feed, while the organic phase is moved in the opposite direction. Hence, in this way, even if

the separation between two metals in each stage is small, the overall system can have a higher

decontamination factor.

Multistage countercurrent arrays have been used for the separation of lanthanides. For the design of

a good process, the distribution ratio should be not too high (>100) or too low (<0.1) in the

extraction portion of the process. It is often the case that the process will have a section for

scrubbing unwanted metals from the organic phase, and finally a stripping section to obtain the

metal back from the organic phase.

Multistage Podbielniak contactor centrifuges produce three to five stages of theoretical extraction in

a single countercurrent pass, and are used in fermentation-based pharmaceutical and food additive

production facilities.

Centrifugal extractors mix and separate in one unit. Two liquids will be intensively mixed between

the spinning rotor and the stationary housing at speeds up to 6000 RPM. This develops great

surfaces for an ideal mass transfer from the aqueous phase into the organic phase. At 200 – 2000 g

both 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 current

and cross current extractions are easily established.

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Extraction without chemical change

Some solutes such as noble gases can be extracted from one phase to another without the need for a

chemical reaction . This is the simplest type of solvent extraction. When a solvent is extracted, two

immiscible liquids are shaken together. The more polar solutes dissolve preferentially in the more

polar solvent, and the less polar solutes in the less polar solvent. Some solutes that do not at first

sight appear to undergo a reaction during the extraction process do not have distribution ratio that is

independent of concentration. A classic example is the extraction of carboxylic acids into non polar

media such as benzene. Here, it is often the case that the carboxylic acid will form a dimer in the

organic layer so the distribution ratio will change as a function of the acid concentration (measured

in either phase).

Solvation Mechanism

Using 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 PUREX

process that is commonly used in nuclear reprocessing uses a mixture of tri-n-butyl phosphate and

an 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 contact

with a dilute nitric acid solution; the equilibrium is shifted away from the organic soluble uranium

complex and towards the free TBP and uranyl nitrate in dilute nitric acid. The plutonium(IV) forms

a 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.

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This oxidation state does not form a stable complex with TBP and nitrate unless the nitrate

concentration is very high (circa 10 mol/L nitrate is required in the aqueous phase). Another method

is to simply use dilute nitric acid as a stripping agent for the plutonium. This PUREX chemistry is a

classic example of a solvation extraction

General Procedure for Experiments on a Centrifugal LLC Instrument

Performing an LLC experiment on a centrifugal instrument usually follows most of the steps

outlined below:

1. Find a suitable solvent system The HEMWat system is well tried and tested and proves adequate

for most separations

2. Perform partition studies with different steps in the HEMWat series, with and without, acidic or

basic modifiers if there are ionisable species present in the sample to be purified. Determine

partition coefficient/distribution ratio (D and 1/D) for phases, examine the D and 1/D values to

ascertain whether resolution is feasible. D ≥ 0.5 usually produces a satisfactory separation

3. The results of step 2 show whether the experiment should be performed in NP or RP mode. D ≈ 1

i.e. in the range of approximately 0.5 – 2.5 (the sweetspot), for a component of interest is desirable

NP mode: the more polar phase is designated as SP. For the HEMWat series this is always the lower

phase

4. Prepare the solvent mixture chosen for the separation and separate the phases

5. Perform scouting experiments and refine the experimental conditions or preparative runs as

described in steps 6 - 9

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6. Set up the instrument with respect to the choice for SP and fill the column with the chosen SP at

the highest usable flow rate

7. Equilibrate the column with MP at the chosen elution flow rate. This step will displace some

quantity of SP and from the displaced volume and the system and column volumes the initial SP

retention can be calculated. These data allow prediction of elution volumes and times for the

components of interest

8. Dissolve the appropriate quantity in a volume, equal to or less than 5 – 10% of the column

volume, of either phase or preferably a mixture of the phases

9. Inject the sample, perform the elution, collect fractions and analyze then work up the required

fractions

Control of CCC selectivity is effected when using a stepped polarity solvent combination series

such 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 how

retention 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

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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

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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 flavors

Liquid-Solid Chromatography (LSC)

This type of chromatographic technique is also called adsorption chromatography since the

mechanism of separation depends on adsorption of solutes on the stationary phase. The stationary

phase is a solid which is usually silica or alumina with the former being most widely used. The

retention times of some compound categories are as follows:Carboxylic acids > amides > amines ~

alcohols > ketones ~ aldehydes ~ esters >nitrocompounds > halides > hydrocarbons.

Mobile Phase Selection

The only factor that is used to optimize α and k’ is the mobile phase composition since the

stationary phase is a solid. Great variations in α and k’ can be obtained by variations in nature and

composition of the mobile phase. The polarity index of solvents can be used as a guide for

estimating the polarity index of the mobile phase. However, a better scale is optimally dependent

upon the adsorption energy per unit area of the solvent which is called the eluent strength, εo. The

values 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 the

other with low eluent strength. k’ is then optimized by variation of the volume ratio of the two

solvents. A small increase in εo value will significantly change k’. Therefore, large variations in k’

are possible by variation in mobile phase

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Composition.

In case of getting overlapping peaks but acceptable k’, the type of mobile phase constituents must

be changed in order to change α. It is usually possible to carry a thin layer chromatographic

separation to optimize the mobile phase composition with regards to both eluent strength and

composition

Applications of Adsorption Chromatography

LSC 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 aqueous

solvents. It should be remembered that the mobile phase in LSC should be non polar modified with

a polar solvent. However, the solvent polarity must not be very large since irreversible adsorption

on the stationary phase can occur precluding the use of LSC. Therefore, water is usually excluded

from mobile phases to be used in LSC. Separations of difficult to separate isomers were possible

with LSC.

Ion-Exchange Chromatography (IEC or IC

Separation of ionic species is efficiently done using ion-exchange chromatography (IEC) or simply

ion chromatography (IC). Anions can be separated on an anionic exchange resin while cations can

be separated on a cationic exchange resin.

Separation of ionic species is efficiently done using ion-exchange chromatography (IEC) or simply

ion chromatography (IC). Anions can be separated on an anionic exchange resin while cat ions can

be separated on a cationic exchange resin.

Ion-Exchange Resins and Equilibria

Cationic exchange resins are mainly of two types:

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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 cationic

exchange 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

Ion-Exchange Resins and Equilibria

Cationic exchange resins are mainly of two types:

a. Sulfonic acid group like ~SO3H

b. Carboxylic acid group, ~COO-H+

Anionic exchange resins are mainly of the tertiary amine type, ~(CH3)3N+ OH Thecationic

exchange 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+]aq

Ion-Exchange Resins and Equilibria

Cationic exchange resins are mainly of two types:

a. Sulfonic acid group like ~SO3 H+

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b. Carboxylic acid group, ~COOH+

Anionic exchange resins are mainly of the tertiary amine type, ~(CH3)3N+ OH The cationic

exchange 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

kex = [RSO3- B+]s[H+]aq/[ RSO3- H+]s[B+]aq

K = Cs/CM = [RSO3- B+]s/[B+]aq

Where, 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 depend

on 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 Columns

Unfortunately, detectors available for use with IC are rather limited. Conductivity detectors are most

common where simply ions will increase the conductivity. A major drawback to using conductivity

detectors is the high salt concentration in the mobile phase which may make it very difficult to

determine differences in conductivity, especially at low solute concentrations. The problem of high

salt content of the mobile phase was solved by the use of what is called suppressor columns. The

suppressor column is packed with a second ion exchange resin that converts the ions in the

mobile 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 anion

exchange packing. The following equilibrium takes place in the suppressor column:

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H+aq + Claq+ Resin+ OH- (solid) = Resin+ Cl- (solid) + H2O For separations of anions, the

suppressor column contains a hydrogen ion cationic exchange resin and the eluent is a

carbonate/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 electrolyte

which will contribute very little to conductivity. Therefore, in both cases the conductivity of the

mobile phase was eliminated using suppressor .Parameter Effect on retention in anion IC

The Mobile Phase

The mobile phase transports the sample through the system and affects both retention and selectivity

of 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 is

the 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 mobile

phases for anionic separations are based on hydroxide or carbonate as eluting anion. In carbonate

based 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 be

varied to optimize the retention time and selectivity. The elution strength of the mobile phase and, to

some extent, its selectivity, are affected by the type of ions of the eluent. A change of salts will

normally result in a change of the eluent pHcolumns of suitable packing.

Ionic strength

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The eluting ability of a mobile phase will increase as the ionic strength of the mobile phase is

increased. However, the change in the selectivity among equally charged ions is very small, whereas

the selectivity between ions of different charges (mono- or polyvalent) is far more sensitive to

changes in ionic strength.

PH

The charge of the sample ions of weak acids or bases are controlled by the eluent pH and thus the

retention times of such species will be affected by changes in Ph

Temperature

The ion exchange rate between the stationary and the mobile phase increases with increasing

temperature. The viscosity of the eluent, and thereby the column backpressure, decreases and can

give a better separation efficiency. The temperature can also affect the column selectivity.

Isocratic and Gradient Elution

The most common type of elution in IC is isocratic where the mobile phase has a constant

composition during the entire run. Gradient elution where the eluent concentration is changed

during the run can also be used. Gradient elution is effective when sample contains ions with widely

different retention times. Hydroxide eluents are usually used for gradient elution in anion

chromatography. By gradually increasing the concentration of hydroxide ions, the eluting power of

the mobile phase increases. As a result, ions with high retention stay at the top of the column They

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then elute in sequence as sharp peaks. After each gradient run the column must be regenerated by

pumping 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 the

potential for sustained commercial development. They are called the hydrostatic and the

hydrodynamic configurations.

Hydrostatic CCC columns:

The very first hydrostatic CCC columns used gravity to maintain the liquid stationary phase; they

were called droplet CCC (DCCC) columns. They needed very long elution times .The columns are

no longer in use today. Modern hydrostatic CCC columns are known and marketed under the name

of centrifugal partition chromatographs Their two main characteristics are: they have a single axis

of 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 be

seen that there is quite a significant volume of connecting ducts which only contain the mobile

phase.

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The liquid motion in CCC columns. A – Hydrostatic olumns or CPCs. There are a single axis of

rotation 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 planetary

rotation of the bobbin around its own axis and the central rotor axis. There is contact between the

two liquid phases throughout the tubing. The mobile phase is pictured in black, the stationary phase

is white.

This design reduces the contact time for solute exchange with the stationary phase. It

also builds a small hydrostatic pressure that explains the significant pressure drop needed to operate

hydrostatic centrifuges. All hydrostatic centrifuges contain two rotary seals; one at the top and the

other one at the bottom. They are quiet to operate.

In the toroidal coil CCC (helix CCC) system operated under a centrifugal force, the dimensions of

the coil are reduced to that which is convenient for analytical separations. The coil is mounted

around the periphery of the centrifugal bowl so that the stable radially acting centrifugal force field

retains the stationary phase in one side of the coil as in the basic hydrostatic system described

above.

Hydrodynamic columns:

Hydrodynamic Counter current Chromatography columns

Hydrodynamic centrifuges used in the CCC columns have two rotational axes, a

main axis and a planetar one which generates a variable centrifugal force field.

There can be any number of planetary axes but the most common are single,

double, and triple axes. Each planetary axis has a bobbin or spool mounted on

it that contains the coils of continuously wound Teflon tubing In hydrodynamic

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.

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The tubing can be connected from the outside of the centrifuge, wound round

the bobbins, and passed back to the outside again without any rotary seals—

hence, from the chromatography point of view it is equivalent to one long thin

continuous column. The variable force field produces mixing and settling zones

throughout the whole length of the coiled column as indicated in Fig. 2B. There

is continuous contact between the two liquid phases throughout the column

with no significant pressure buildup. Hydrodynamic centrifuges work with low

mobile-phase pressure but can generate noise from the gear assembly, which

can be reduced in well-designed centrifuges. These columns are often called

highspeed CCC (HSCCC) columns since they can operate much more rapidly

than the Craig, DCCC, and gravity-based columns. Table 1 compares the

features of the two kinds of CCC columns. It is not possible to say that one kind

is clearly superior to the other. The best situation is to have both kinds of CCC

column to cover all possible cases. Studies are going on to develop large-scale

CCC centrifuges based on both types being able to produce significant mass of

purified material (preparative CCC) [13–15]. Such centrifuges can be used to

produce standard reference materials for analytical purposes and to purify

analytical reagent

INSTRUMENTATION:

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force of 1g (the earth’s gravity) which was not very effective at stabilising the SP. Furthermore, the

scope was limited since only the more dense phase could be used as the SP. Apart from these issues

runs could take many days even with the later, mechanized and automated versions.

The time required to perform experiments could run into days but the instruments were capable of

resolving very complex mixtures. The technique was recognised to be gentle and offered high

resolution and enjoyed favour as the purification method of choice for high value products such as

Chinese medicines and other natural products with many groups of chromatographers. The

methodology became, and until recent years remained, a niche application and was a technique very

rarely employed by medicinal chemists.

The other significant 1g instrument was the Droplet Countercurrent Chromatograph, DCCC, which

was invented by Ito (see below) and his group in 1970. These instruments also relied on the earth’s

gravity alone to stabilise the stationary phase whilst MP was pumped through the instrument but

like the Craig machines, SP retention (stabilisation) was poor even at very low flow rates and

experiments could take days to complete. However, these instruments were able to use either phase

as SP although a significant disadvantage of these instruments was that phase mixing, an essential

pre-requisite of successful LLC, was poor.

DESCRIPTION OF CRAIG’S TECHNIQUE

22

A schematic representation of five stagcs in the Craig s technique of counter cirrent extraction. The

process of separation is carried cut by extracting a solution ofthc sample under test with an

immiscible solvent. According to Craig, thi step is called ‘transfer’(n 0) and is shown i.n

the top row of the figure. The vessel in which the initial extraction is allowed to take place, has been

labelled ‘tube zero’ (r= 0). The figure.also indicates four other additional vessels labelled tubes. 12,3

and 4. These vessels are a part of a very large numbe of identical containers. All of these vessels

contain

organic 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 organic

solvent is identical to that of aqueous phase. Thus when equilibrium is achieved-in tube 0 after

transfer, 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 to

tube 1. This is also followed by an identical amount of fresh organic solvent into tube 0. When both

vessels are shaken, the solute will be distributed among the four solutions as indicated inline n=1 of

the figure 3. This process of transfer and equilibrium is epeated with an identical vôlume of fresh

organic 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 all

tubes. Figure 4 indicates the movementof the so,lute for a much large number of transfers, when the

fraction of total solute contained in the two layers of each tube (i.e., organic portion + aqueous

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portion) is plotted agailist the tube number. It has- been found thatthe distribution curves become

broader’ and lower, with an increased number of transfers. It should also be noted that for a solute

with partition ratio larger than unity, the movement will be enhanced.

DERIVATION OF DISTRIBUTION CURRENT

The partition ratio data may be made use of in deriving the distribution curves, such as those slown

in fig. 4. Suppose fn.r be the fraction of the initial solute A present in tube r after n transfers have

been performed. Thus the sum of th total fractions must be equal to unity. That is,

f.o+ fn + f2+ f3t .. 1

We 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 as

the fraction found in thetationary aqueoupiase, then x+ y= 1

In any particular tube, the amount of solute found in eaëh phase is given by, weight in mobile

organic phase Cm . Vm.

24

weight in stationary aqueous phase = C. V5 ‘ -V ri’ iho volumes of the two phases in millilitres ahd

C, and C are expressed in grams per znillilitres. Thus,

and

Substitution 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 is

found in tube 0 only. Thus,

fo,o X+ y 1.00

since x and y are dependent only on K’ when the volumes are fixed.

POSITION WHEN LESS DENSE

LIQUID IS RECEIVED FROM

PRECEDING TUBE AND

DURING 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 in

the organic layer and a fraction y will go into the aqueous layer. Since the values of x and y are

same throughout we have

(b) For r = 0. The solute present in tube 0 comes from the aqueous layer that ontains the fraction y

of 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 the

fraction x2 of the original sample.

• This fraction will be again distributed to give x2 of the organic layer and x2y in the aqueous layer

of tube 2. •

27

Solution— From(8) and (9), we have

Putting n= 5, and 3 -

r in equation (iv)

28

APPLICATIONS OF CRAIG’S TECHNIQUE

Craig’s technique has widely been dsed particularly in the field of biochemical’s. Many closely

related compounds have been separated by making use of this technique. Craig himself had resolved

a mixture of 10 amino acids, despite the fact that the partition ratio for some of these compounds

differed by less than ?. 1. Complex mixtures of fatty acids, polypeptides nucleotides, aromatic

amines, antibiotics and many other organic substances have been fully separated by. using counter

current extraction.

TECHNIQUES:

Techniques

CCC can be thought of as occurring in three stages: mixing, settling, and separation (although they

often occur continuously). Mixing of the phases is necessary so that the interface between them has

a 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 equilibrium

and is related to the analyte's affinity for one over the other. The mobile phase is mixing with then

settling 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 a

separation) is a crucial parameter. Higher quality instruments have greater stationary phase

retention. The settling time is a property of the solvent system and the sample matrix, both of which

greatly 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 the

stationary phase. In descending mode, droplets of the denser mobile phase and sample are allowed

to 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 ascending

mode. The eluent from one column is transferred to another; the more columns that are used, the

more theoretical plates can be achieved. The disadvantage of DCCC is that flow rates are low, and

poor 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 a

bobbin. The bobbin is rotated in a double-axis gyratory motion (a cardioid), which causes a variable

gravity (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 to

their 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&D

process that has produced HPCCC instruments that generated 240 g, compared to the 80 g of the

HSCCC machines. This increase in g-level and larger bore of the column has enabled a tenfold

increase in through put, due to improved mobile phase flow rates and a much higher stationary

phase retention.

Countercurrent chromatography is a preparative liquid chromatography technique, however with the

advent of the higher g HPCCC instruments it is now prossible to operate instruments with sample

loadings 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 drug

development.

High-Speed Countercurrent Chromatography (HSCCC)

The modern era of CCC began with the development by Dr. Yoichiro Ito of the planetary centrifuge

and the many possible column geometries it can support. These devices make use of a little-known

means of making non-rotating connections between the stator and the rotor of a centrifuge. (It is

beyond the scope of this discussion to describe the method of accomplishing this. Any of the several

books available on CCC .

Functionally, the high-speed CCC consists of a helical coil of inert tubing which rotates on its

planetary axis and simultaneously rotates eccentrically about another solar axis. (These axes can be

made to coincide, but the most common or type J CCC is discussed here.) The effect is to create

zones of mixing and zones of settling which progress along the helical coil at dizzying speed. This

produces a highly favorable environment for chromatography.

31

There are numerous potential variants upon this instrument design. The most significant of these is

the toroidal CCC. This instrument does not employ planetary motion. In some respects it is very like

CPC, but retains the advantage of not needing rotary seals. It also employs a capillary tube instead

of the larger-diameter tubes employed in the helices of the other CCC models. This capillary

passage makes the mixing of two phases very thorough, despite the lack of shaking or other mixing

forces. This instrument provides rapid analytical-scale separations, which can nonetheless be scaled

up to either of the larger-scale CCC instruments.

Centrifugal Partition Chromatography (CPC)

Centrifugal Partition Chromatography (CPC) was invented in the eighties by the Japanese company

Sanki Engineering Ltd, whose president was the late KanichiNunogaki. CPC has been extensively

developed in France starting from the late nineties. CPC uses centrifugal force to speed separation

and achieves higher flow rates than DCCC (which relies on gravity).

The centrifugal partition chromatograph is constituted with a unique rotor (=column). This rotor

rotates on its central axis (while HSCCC column rotates on its planetary axis and simultaneously

rotates eccentrically about another solar axis). With less vibrations and noise, the CPC offers a

wider rotation speed range (from 500 to 2000 rpm) than HSCCC. That allows a better decantation

and retention for unstable biphasic system (e.g., aqueous aqueous systems or Butanol/water

systems).

Basics of CPC: The CPC rotor is constituted by the superposition of disks engraved with small cells

connected by head / tail ducts. These cells, where the chromatographic separation takes place, can

be compared to lined-up separate funnels. The rotor is filled with the stationary phase, which stays

inside the rotor thanks to the rotation speed, while the mobile phase is pumped through. CPC can be

operated in either descending or ascending mode, where the direction is relative to the force

generated by the rotor rather than gravity. According to the fast and permanent evolution of the cells

design, the efficiency and flow rate with low back pressure are improved. The CPC offers now the

direct scale up from the analytical apparatuses (few milliliters) to industrial apparatuses (some

liters) 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 Chromomatography

Understanding how resolution is achieved in CCC is assisted by the use of the simple model of a

series of separating funnels numbered, each containing, let’s say 10ml of ethyl acetate which has

been pre-saturated with water and which will form the SP for our experiments. For our first

experiment, 10ml of water, pre-saturated with ethyl acetate and 50 parts of a solute with a D value

of , when partitioned between ethyl acetate and water, are added to the first funnel. The funnel is

stoppered, shaken and the phases allowed to settle. At this point each of the phases will contain 25

parts of the solute. The lower aqueous phase is transferred to the second funnel and 10ml fresh

pre-saturated water are added to. Both funnels are stoppered, shaken and allowed to settle. At this

point, the phases in will each contain parts of the solute as will those in The lower aqueous phase of

is transferred to, the lower phase of is transferred to and another portion of water is added to. All

three are stoppered, shaken and allowed to settle. Each phase in will now contain parts will still

contain 25 parts in each phase and the phases in will each contain part 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 bottom of the Study of the figure

shows that the solute is effectively moving through the series of funnels with a Gaussian distribution

and as the solute band progresses, the maximum, single funnel concentration decreases. After

sixteen or so such iterations, the outermost funnels, are essentially devoid of solute and the solute is

distributed 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 is

distributed over about 30 funnels with the maximum concentration found in funnel 50 or so as might

be experted.

Chromatographic Resolution

Chromatographic resolution, i.e. complete separation of the components of a mixture, is the ultimate

goal of a separation process. Such a process, performed on naturally occurring materials or those of

synthetic 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 which

we are concerned i.e. the complete separation of the components of a mixture by means of a

chromatographic 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) technique

and some comparison with RP-HPLC is justified.

The Snyder resolution equation, Equation 4, tells us that resolution is the product of an efficiency

term, 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 column

capacity.shows a representation of the relative, characteristic SP and MP volumes in a HPCCC

column compared with those in a bonded RP-HPLC column. It should be noted that although the

active 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 mass

capacity of the HPCCC column is much grater than that of a rp- hplc column

HPLC chromatographers have only limited scope for improving selectivity, via Path B in the since

C18 bonded phases differ little one from another and little more from, for example, a C8 bonded

phase and the range of usable solvents is extremely limited so the usual solution to the problem for

analytical chromatographers is via Path A in the figure, i.e. moving to a higher efficiency, smaller

particle, packing. However, this is not a practicable proposition for preparative scale

chromatographers since (large volume preparative columns filled with small particle media would

be prohibitively expensive) and in any case there are currently no commercially available

preparative scale pumps capable of pumping mobile phase through a large bed of sub-media at a

practically useful flow rate. Under such circumstances the preparative scale solid-liquid

chromatographer is stymied. An alternative solution is to find a methodology which rather than

improving efficiency can exploit controllable selectivity, i.e. a CCC methodology. Even the

relatively high, compared with other CCC methods, efficiency of HPCCC is still measured in only

hundreds of plates compared with the thousands or tens of thousands of plates found in an

equivalent sized SLC column but by exploiting the optimization of selectivity the technique is

capable of high resolution.

36

APPLICATION OF COUNTER CURRENT CHROMATROGRAPHY

CCC is playing an increasingly important role in separation science. All components in the sample

solution injected into the column can be recovered and irreversible adsorption and contamination of

samples can be virtually eliminated. A crude sample can be injected directly into the column, which

simplifies sample preparation. Now CCC is successfully used for the separation of organic and

inorganic substances from a complicated mixture.

ORGANIC SUBSTANCES;

CCC has become a method of choice in natural products chemistry and has made possible the

separation of a number of biologically interesting natural products that are difficult or impossible to

separate by other techniques Crude extracts of plants or other organisms are often too complex for

the direct analysis by HPLC. Certain materials may irreversibly bind to the packing material or may

plug the column inlet filters, and hence reduce the column life. Those restrictions do not apply to

analytical CCC, which represents an interesting method for enrichment and separation of various

analytes. The technique is also used for the separation of bio chemicals and pharmaceuticals. CCC

is especially suitable for the separation of alkaloids from medical herbs using simple solvent

systems, for the total hormonal analysis of natural samples and for the screening of new bioactive

compounds in crude extracts and other complex samples CCC has been also suggested as an

alternative to the shake flask method to measure liquid–liquid partition coefficients as a way to

characterize the lipophilic–hydrophilic nature of a compound ,below some interesting methods are

briefly described which have been successfully used in the analysis of various samples or which can

be 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 of

various compounds from plant and different natural products. The quantity of separated compounds

may range from trace to gram amounts.

Toroidal coil Centrifuges

Toroidal coil centrifuges have been successfully applied to the separation and

purification of plant hormones,namely, indole auxins, gibberellins, cytokinins,

and abscisic acid. Indole auxins were separatedby either hexane-ethyl acetate–

methanol–water (volume ratio 0.6:1.4:1.0:1.0) or chloroform–acetic- acid–water

(2:2:1) in a column with a total capacity of 18 mL. The latter solvent system

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-

phosphate buffer (pH 7) (3:1:2). The CCC method was suitable for the separation of four cytokinins

in ethyl acetate-methanol-phosphate buffer (pH 7)(3:1:3) [2,4,5].A toroidal coil planet centrifuge for

analytical-scale separations was used for the purification of abscisic acid (ABA) obtained from crude

plant extracts and its determination in several plant tissues using HPLC and GC-MS [34].The results of the

isolation of 3-oxo-5-steroid isomerase (KSI) from crude E. coli lysate were published [35]. A separation was

performed on ca. 3 mg of 15N-labeled KSI using a polymer-containing system based on PEO 3350. The

present method eliminates sample loss and denaturation caused by the solid support and yields pure proteins

in both preparative and analytical separations.

38

High-speed hydrodynamic centrifuges (HSCCC)

Numerous applications in the analysis and preparation of natural products by CCC have been

reported. Separation of poly phenolic natural products such as flavonoids are difficult because these

compounds 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 most

valuable technique for the isolation of polyphenols . The flavonoids and hydroxyanthraquinones can

be easily separated by CCC with a high selectivity Zhang has published the results of the separation

of alkaloids (from Stephania tetrandra S.Moore) using n-hexane–ethyl acetate–methanol–water

systems at different volume ratios, hydroxyanthraquinones (from the rhizome of Rheum palmatum

L.) using a system of hexane–ethyl acetate–methanol–water (9:1:5:5) and flavonoids (from sea

buckthorn 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 2000

rpm By increasing the flow-rate of the mobile phase in these analytical separations, the separation

time for a crude sample mixture was shortened to within 15 min, which is quite comparable with

that of analytical HPLC. Milligram and even gram amounts of substances can be isolated by the

CCC technique.

This makes it possible to produce standard reference materials for any analytical study. For the more

efficient separation of compounds having a wide range of polarity, lower and upper phases of the

solvent system were used as the mobile phase in succession. This method achieved a complete

separation of five components present in a 1-mg sample mixture. The peak fraction of each

compound was subjected to mass spectrometric analysis for compound structure confirmation

39

shows the countercurrent chromatogram of five major compounds in the crude extract of rhizome of

Rheum palmatum L.

Three peaks were eluted with the upper phase in NP mode followed by two peaks which were

eluted with the lower phase in RP mode. The results indicated that peaks were corresponding to

chrysophanol, emodin, physcion, aloe-emodin, and rhein, respectively. CCC was used for the

systematic selection and optimization of a two-phase solvent system to separate alkaloids from

Coptis chinensis Franch using a system of chloroform–methanol–HCl solution. at different volume

ratios .One separation run yielded four pure alkaloids, including palmatine, berberine, epiberberine,

and coptisine from a crude alkaloid extract. Analytical application of CCC was successfully

demonstrated for the separation of microgram quantities of flavonoids from a crude ethanol extract

of sea buckthorn in a multilayer coil with a total capacity of 8 mL using a two-phase solvent system

composed of chloroform–methanol–water (4:3:2) [43]. Five peaks, including isorhamnetin and

quercetin, were well resolved and eluted within 8 min. An artificial mixture of three

common plant coumarins (herniarin, scopoletin, and umbelliferone) and one

flavanone (hisperetin) was separated with a hydrodynamic CCC column

40

connectedto a photodiode array detector. The lower phase of a chloroform–methanol–

water.

CONCLUSION:

High Speed Counter Current Chromatography is a very good preparative separation technique.  The

use of support-free liquid stationary phase and no sample loss, with high separation efficiency and

resolution by using the centrifugal field, are the characteristics that make it superior to all other

separation techniques. As there is no solid support, it is free from adsorption of solutes to the

column 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, with

different stationary phases. The use of HSCCC in drug discovery and product development where

there is ease of scale-up from milligrams to grams and then to kilograms, makes it an excellent

technique for the separation of natural pigments and other bioactive constituents, which are present

in minute quantities.HPCCC is an orthogonal and complementary chromatography technique to

HPLC in the armamentarium available to separation scientists and worthy of a place in the

chromatographer’s toolbox. Neither HPCCC, nor any other modern LLC methodology, is a ‘magic

bullet’ solution but with controllable, tunable selectivity it is able to very effectively tackle

problems that are difficult or totally intractable when HPLC is used. In many instances the use of

41

selectivity instead of efficiency can prove to be the optimal way of separating components,

especially from whenever preparative quantities require purification. More and more

chromatographers are realizing the real benefits that accrue from the use of LLC. The list of

applications of LLC grows day by day

REFERENCES

1. Dr.S. Ravi shankar

2. B.K.sharma

3. Sikdar, Cole, et al. Aqueous Two-Phase Extractions in Bioseparations: An Assessment.

Biotechnology 9:254. 1991

4. Szlag, Giuliano. A Low-Cost Aqueous Two Phase System for Enzyme Extraction.

Biotechnology Techniques 2:4:277. 1988

5. Dreyer, Kragl. Ionic Liquids for Aqueous Two-Phase Extraction and Stabilization of

Enzymes. Biotechnology and Bioengineering. 99:6:1416. 2008

6. Boland. Aqueous Two-Phase Systems: Methods and Protocols. Pg 259-269

7. 11. A. Berthod, B. Billardello. Advances in Chromatography, Vol. 40, P. Brown, E. Grushka

(Eds.),

42

8. p. 8, Marcel Dekker, New York (2000).

9. 12. A. Berthod. In Centrifugal Partition Chromatography, A. P. Foucault (Ed.),

Chromatographic

10. 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,

1373

13. (2003).

14. A. Marston, K. Hostettmann. J. Chromatogr., A 658, 315 (1994).

15. Y. Ito. J. Chromatogr., A 1065

16. 11. A. Berthod, B. Billardello. Advances in Chromatography, Vol. 40, P. Brown, E. Grushka

(Eds.),

17. p. 8, Marcel Dekker, New York (2000).

18. 12. A. Berthod. In Centrifugal Partition Chromatography, A. P. Foucault (Ed.),

Chromatographic

19. 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,

John

25. Wiley, Chichester (1996)

43

26. Kanichi Nunogaki was invented centrifugal partition chromatography japans company

engineering limited whose president

27. Coptis chinensis was invented counter current chromatography .for the development of

applications

44