|Year : 2009 | Volume
| Issue : 1 | Page : 8-15
High speed counter current chromatography: A support-free LC technique
Neha Sethi1, Ankit Anand1, Aarti Sharma1, Kaushal K Chandrul1, Garima Jain2, Kona S Srinivasa2
1 School of Pharmaceutical Sciences, Jaipur National University, Jaipur, India
2 Ranbaxy Research Laboratories, Gurgaon, India
|Date of Submission||14-Nov-2009|
|Date of Decision||25-Nov-2009|
|Date of Acceptance||04-Dec-2009|
|Date of Web Publication||23-Apr-2010|
School of Pharmaceutical Sciences, Jaipur National University, Jaipur
Source of Support: None, Conflict of Interest: None
| Abstract|| |
As separation of components is the major requirement of an analytical chemist, there is always a need for a convenient high throughput technique with minimum sample loss, high efficiency, high resolution, and ease of sample recovery, without contamination. This leads to the development of High Speed Counter Current Chromatography (HSCCC), in which the stationary phase is liquid instead of solid, and that provides a lot of advantages over other chromatographic techniques. In addition, advanced centrifugal partition technology is used to hold the liquid stationary phase in the column, while the liquid mobile phase is pushed through it, which provides high yield and purity. This review highlights the major applications of HSCCC that include extraction of medicinal drugs from plants and purification and isolation of active material, plant analysis, separation of rare earth elements, preparative-scale separations of chiral compounds, analysis of inorganic compounds and elements, drug discovery, and drug development. Separation of dipeptides and proteins, flavonoids, alkaloids, DNP amino acids, indole auxins, and so on, proves the versatile and dynamic nature of the technique.
Keywords: Centripetal accelerations, high speed counter current chromatography, solvent system
|How to cite this article:|
Sethi N, Anand A, Sharma A, Chandrul KK, Jain G, Srinivasa KS. High speed counter current chromatography: A support-free LC technique. J Pharm Bioall Sci 2009;1:8-15
|How to cite this URL:|
Sethi N, Anand A, Sharma A, Chandrul KK, Jain G, Srinivasa KS. High speed counter current chromatography: A support-free LC technique. J Pharm Bioall Sci [serial online] 2009 [cited 2019 Sep 22];1:8-15. Available from: http://www.jpbsonline.org/text.asp?2009/1/1/8/62680
With the development of science and technology, new advancements in chromatography are required for separation of complex compounds. A new dynamic all liquid separation technique, in the chromatographic era, has been developed with a new principle and characteristics of a support-free liquid stationary phase. It is called High Speed Counter Current Chromatography (HSCCC) and was first developed in the late 1970s, when it overshadowed other methods of chromatography with its superior capacity to achieve rapid and efficient separation.
High Speed Counter Current Chromatography (HSCCC) was developed in the early 1980s, by Yoichiro Ito and collaborators at the National Institutes of Health (Bethesda, MD). This technique is based on the effects of gravitational and centrifugal force on the solvent flow behavior in a helical-shaped tubing. When a coil containing two immiscible solvents is rotated within a certain 'critical' speed range (based on the helical measurements), an asymmetrical force distribution is created around the width of the coil. This causes preferential bilateral distribution, in which the heavier phase occupies the space closer to the 'head' of the coil (defined by direction of rotation), and, subsequently, the lighter phase occupies the 'tail' end. Therefore, for example, if a mobile phase is pumped through the tail end into a rotating coil filled with a less dense stationary phase, a better-than-expected stationary phase can be maintained because of its predilection for the column inlet. With the addition of a centrifugal field, the forces directed on the column become more complicated, but a right setup can greatly strengthen the propensity toward bilateral distribution. These centrifugal fields include speed regulators and rotating parts like rotors, gears, spools, rotating seals, motors, and so on. It is a liquid-liquid partition chromatography.  In general, countercurrent chromatography (CCC) involves the elution of a mobile solvent phase through a column filled with a 'stationary', immiscible liquid phase. To make this process an effective chromatographic technique, in terms of good resolution and reasonable retention times, several parameters are crucial. A rapid mobile phase must be used, and at the same time there must be significant stationary phase retention within the column. Stationary phase retention is critical because it directly controls the level of resolution that can be attained. In addition, effective mixing between solvents (as can be accomplished in a standard separatory funnel) must be achieved, to afford effective analyte partitioning. As there is no solid support, there is no chance of sample loss. Thus, separations are based solely on the partition coefficients of the analytes, with respect to the two solvent phases, and no absorption or degradation effects are possible.  The main aim of HSCCC is to obtain a stable support-free liquid stationary phase.
| Principle|| |
High Speed Counter Current Chromatography (HSCCC) is based on the principle of the partition of the solutes between the two immiscible liquid phases. It involves the use of a support-free liquid stationary phase, a liquid mobile phase, and a centrifugal force field. The centrifugal force field is used to retain the liquid stationary phase, while the liquid mobile phase is pushed through it.  This type of liquid partition chromatography is based on the differences between the solubilities of the components between the stationary and mobile phases. Each compound has a different relative solubility in each of the phases. The compound is effectively distributed between the two phases. The distribution can be quantified by taking the concentration of the compound in the upper phase and dividing it by the concentration of the same compound in the lower phase, that is, the ratio of the solute concentration in the stationary phase divided by the solute concentration in the mobile phase. This is known as distribution ratio, D.
D = C s /C m
Cs is the concentration of the sample component in the stationary phase (SP) and Cm is the concentration of the component in the mobile phase (MP). As a result of the partition, zones of mixing are formed and thus it leads to separation between different solutes. The amount of liquid stationary phase present in the column can be changed. If the amount of stationary phase retained in the column decreases, the chromatographic peak resolution decreases. For optimum separation, it has been found that there must be a good retention of the stationary phase and the solutes must have partition ratio between 0.5 and 2. 
| Mechanism of HSCCC|| |
Selection of the two-phase solvent system
Conventional liquid chromatography uses a single phase to elute the analytes released from the adsorptive or liquid phase coated on the solid support. In contrast, the CCC technique uses a two-phase solvent system made up of a pair of mutually immiscible solvents, one used as the stationary phase and the other as the mobile phase.  For effective HSCCC separations and to achieve the best peak resolution and retention of the stationary phase, the best pair of mobile and stationary liquid phases , must be selected. It is a very important step, as a minor change in composition of the mobile phase can affect the composition of the stationary phase. First of all, it should form two immiscible phases. Non-reactive and innocuous solvents must be used. Two similar compounds of almost identical polarity can have surprisingly different partition coefficients in a specific two-phase system, resulting in baseline separation by countercurrent chromatography.  The following factors should be satisfied while selecting a solvent system; (1) Solubility of the sample in the solvent system, (2) Stability of the sample in the solvent system, (3) Settling time of the solvent system, (4) Partition coefficient of the sample, (5) Satisfactory retention of the stationary phase in the column.
The partition coefficient (K) is the ratio of the solute distributed between the two mutually equilibrated solvent phases. Usually it is expressed by the amount of solute in the stationary phase divided by that of the mobile phase, as in conventional liquid chromatography. The higher the partition coefficient of the stationary phase better will be the resolution of peaks. When the lower phase is used as the mobile phase, the retention of the stationary phase is more, but it should be pumped into the column in the head-to-tail elution mode, however, when the upper phase is used as the mobile phase, it should be pumped into the column in the tail-to-head elution mode. A high retention of the stationary phase can be achieved by the low flow rate of the mobile phase, which results in better peak resolution. The sample may be dissolved in either phase or in a mixture of the two phases, and the injection volume must be less than 5% of the total column capacity.  Proper consideration must be given to the settling time of the solvent, to determine the proper elution mode, because it gives high correlation with the retention of the stationary phase.  If the settling time is less than 25 seconds, the upper phase is distributed toward the head of the coil. Thus the lower phase must be introduced from the head of the coil and the upper phase from the tail of the coil but if the settling time exceeds 25 seconds, this effect is reversed and the lower phase must be introduced from the tail and the upper phase from the head of the coil. The retention of the liquid stationary phase is also affected by the liquid-phase density difference, the mobile-phase viscosity, and interfacial tension. 
High Speed Counter Current Chromatography uses a multilayer coil separation column, which is prepared by winding a long piece of Polytetrafluoroethylene (PTFE) tubing directly around the spool-shaped column holder, making multiple layers. As mentioned earlier, when this coiled column is subjected to a planetary motion, it produces a familiar effect called the 'Archimedean screw,' which drives all objects of different density, either lighter or heavier than the suspended medium, toward one end of the coil called the 'head'; the other end is called the 'tail'. The separation column (multilayer coil) of the commercial HSCCC instrument is usually made up of the following three sizes of PTFE tubing: 2.6 mm i.d. (preparative), 1.6 mm i.d. (semi-preparative), and 0.85 - 1.0 mm i.d. (analytical). The analytical column is useful for optimization of the solvent system, as the same solvent system can be used to obtain the same elution profile in a large scale.
Various solvent systems
For polar compounds, the solvent system consists of 1-butanol / acetic acid / water, for moderately hydrophobic compounds, hexane / ethyl acetate / methanol / 0.1 M HCl is used, and for hydrophobic compounds, hexane / ethanol / water or hexane / acetonitrile can be used.  Carbon Tetrachloride and Chloroform were previously used as components of the solvent system because of their properties, such as, low viscosity and high density, but discovery of their carcinogenic activity stopped their use. Furthermore, Dichloromethane and Diethyl ether can also be used, but it leads to a vapor-lock that forces the stationary phase from the system and aborts the chromatography. Thus these solvents can be used when the instrument is equipped with a temperature controller. For the four component system, hexane, ethyl acetate, methanol, and water are commonly used. The lower phase consists of methanol and water and the upper phase consists of hexane and ethyl acetate. Due to the presence of low polarity organic solvents such as ethyl acetate, there is a chance of emulsification in the column. Thus, to prevent the solvent system from leading to the formation of an emulsion, a T-split with a small split ratio is used, through which only 1 / 20 of the flow stream can be introduced into the HPLC system.  When the solvent system has to be chosen for ionizable compounds, it must be seen that they are maintained in same ionization states throughout the chromatographic separation. A solvent system can be affected by addition of a small amount of acid or base or a low concentration of buffer as an aqueous component.  For the purification of proteins, two-phase solvent systems containing organic solvents are not suitable because of the property of denaturation of proteins in organic solvents. Thus, aqueous polymer two-phase systems are used as they provide non-denaturing media to the enzymes, sub-cellular particles, and cells.  A different solvent system is used for organic and inorganic separations. The PEO 6000/Dextran 500 systems provide a physiological environment, suitable for the separation of mammalian cells by optimizing osmolarity and pH with electrolytes.  For organic separations, heptane, ethyl acetate, methanol, and water are considered and for separation of inorganic species, a stationary phase should consist of extracting reagents, such as, cation-exchange, anion-exchange, and neutral in an organic solvent. ,,,,,,, The solvent system should contain specific complexing agents, which can bind elements under separation.
Flow rate of the mobile phase
The flow rate of the mobile phase determines the separation time, the amount of stationary phase retained in the column, and therefore the peak resolution. A lower flow rate usually gives higher retention level of the stationary phase  improving the peak resolution although it requires a longer separation time. The typical flow rates for the commercial multilayer coil are as follows: 5 - 6 ml/min for a preparative column, with 2.6 mm i.d. PTFE tubing (600 - 800 rpm) (up to 1 g sample load); 2 - 3 ml/min for a semi-preparative column with 1.6 mm i.d. PTFE tubing (800 - 1000 rpm) (up to 500 mg sample load); and 1 ml/min for an analytical column with 0.85 - 1.0mm i.d. PTFE tubing (1000 - 1200 rpm) (up to 50 mg sample load). The above-mentioned range of flow rates should be modified according to the settling time of the two-phase solvent system as well as other factors. When the settling time is around 20 seconds and the K value of the analyte is small, the use of a lower flow rate is recommended.
The optimum revolution speed (revolution and planetary rotation speeds are always the same) for the commercial HSCCC instrument, for preparative separation, ranges between 600 and 1200 rpm (40 - 160 Χ g) with a 10 cm revolution radius according to the i.d. of the separation coil, as described earlier. Use of a lower speed will reduce the volume of the stationary phase retained in the column leading to lower peak resolution. On the other hand, higher speeds may produce excessive sample band broadening by violent pulsation of the Column, because of elevated pressure.
The High Speed Counter Current Chromatography instrument consists of a column in which the liquid stationary phase is held by a centrifugal force field, while the immiscible mobile phase flows through it, a mobile phase reservoir, pump, injection valve, a detector, a fraction collector, and a recorder or data processor. [Figure 1] shows the schematic diagram of HSCCC.
The column consists of inert tubing that is helically coiled and made of teflon or stainless steel that rotates on its own axis. The planetary motion is produced by a gear assembly, which is arranged such that the helical coils revolve around a central axis. Separation coils are more than one and can be connected either in a series for one inlet and one outlet, or parallel for more inlets and outlets. When the helical coil rotates about its own axis and revolves around a central axis, an oscillating force field is produced at every given point along the length of the tubing.  As a result of this planetary motion, the solvent in the column is subjected to various velocities and centripetal accelerations and this helps the stationary phase to remain in column and the mobile phase is pumped with the sample components. These velocities and centripetal accelerations form alternating zones of mixing and the partition of the sample material occurs between the mobile and stationary phases, resulting in the separation of different components of the sample. Each planetary axis has a bobbin mounted on it that contains the coils of continuously wound Teflon tubing. In rotating bobbins with coiled tubes, the helix of the tube produces a force known as the Archimedean screw force that pushes the contained liquids toward one end of the tube called the head, that is, the higher-pressure end and the other end of the tube is called the tail, that is, the lower-pressure end. The intensity and direction of the Archimedean screw force depend on the rotational speed and the direction and helical pitch of the coiled tube. 
When the lower phase is chosen as the mobile phase in HSCCC, elution occurs in 'head-to-tail' mode and the opposite case is called 'tail-to-head' mode elution. Ethanol is mixed to the effluent at a volume ratio of 1:5 at the inlet of the detector in order to improve the tracing. To determine the volume of the stationary phase retained in the column, the column contents are collected in a graduated cylinder by pressured air. The retention of the stationary phase in the column can be calculated by dividing the volume of the retained stationary phase with the total column volume.  The HSCCC columns operate much more rapidly than the Craig, DCCC, and gravity-based columns. Furthermore, for the improvement of the stationary phase retention, the spiral disk assembly, which consists of multiple layers of plastic disks with spiral channels, can be used, which works on the principle of utilizing a radially acting centrifugal force, to retain a sufficient amount of stationary phase of highly polar solvent systems such as polymer phase systems. ,,, However, here a special concern should be given toward leakage of the solvents from each spiral disk. The main function of spiral disks is to improve the column efficiency and to reduce the dead space in the transfer tubes. The spiral tube assembly is embedded in polyethylene glycol (MW 3350), which protects the tubing damage caused by vibration under the fluctuating centrifugal force field. This improves the separation of proteins with PEG (polyethylene glycol).This apparatus is widely used for the separation of dipeptides and proteins. ,,
| Detectors for HSCCC|| |
The separated components obtained by HSCCC can be detected by high-performance liquid chromatography-diode array detection (HSCCC-HPLC-DAD). The effluent from the outlet of HSCCC is split into two parts from which one is collected and the other is introduced directly into an HPLC-DAD system, for purity analysis, through a switch valve. The purities of the obtained fractions from HSCCC are monitored, and fractions with high purities are collected.  A UV detector with Hg lamp 254 - 280 nm is widely used in the HSCCC separation technique. Other detectors such as conductivity detector, pH detector, evaporative light scattering detectors (ELSD), and mass spectrometric detectors can also be used. For example, ELSD is employed for systematic separation and purification of non-chromophoric chemical components from Chinese medicinal herb Adenophora tetraphlla. The 2996 photodiode array detector is used for the isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography.
Advantages of liquid stationary phases
Disadvantage of liquid stationary phase
- As there is no solid support matrix in the column, it eliminates irreversible adsorptive loss, denaturation, and contamination of samples from the solid support matrix used in the conventional chromatographic column. 
- Either of the two-phase solvent systems can be used as the mobile phase and the phase role can be changed during a run, to elute all injected species from the column.
- A wide variety of aqueous and non-aqueous solvent systems can be used.
- The sample can be easily dissolved in the whole stationary-phase volume.
It is difficult to maintain the liquid stationary phase in a steady state while the mobile phase is pushed through it. A variety of aqueous and non-aqueous solvent systems can be used, but at the same time it is time consuming for the right choice. It depends on the diffusion process between liquids and the efficiency of phase mixing. Any change in composition of one liquid may induce a change in the composition of another liquid.  Therefore, a lot of care must be taken while selecting the stationary phase and the mobile phase.
Advantages of HSCCC
The liquid stationary phase is used for chromatography, extraction, and purification of applications across a broad range of molecules. As the liquid stationary phase is used, the solutes have access to the whole volume of the stationary phase, leading to a high efficiency of extraction. There is no sample loss in this chromatography as both phases are liquid, and the sample can be totally recovered. In this technique, there is low solvent usage, high resolution, and separation speed. There is no need for column regeneration,  therefore, it gives high separation efficiency. In high speed counter current chromatography, an advanced Centrifugal Partition technology is used that enables quantitative recovery of the sample load.  The temperature of the experiment can be controlled accurately, and hence, enhances the repeatability and versatility of the experiment. The other advantage is that while the experiment is running, the noise is much less and the stability is effective. The low cost of the instruments, common usage, and chemicals make HSCCC more cost-effective than HPLC.  The scale up from milligrams to grams in drug discovery and then to kilograms in product development is important, and is easily possible with HSCCC.  It is used for enhancing the throughput performance and increasing the selectivity of liquid chromatography capability. It gives excellent target compound purity, a high yield, and an easy scale-up.
Disadvantages of HSCCC
It performs separations over a period of hours, rather than the tens of minutes typical for HPLC. It is unreliable, and therefore, there is a risk of losing valuable compounds. The range of equipment available is poor and typically only available at the preparative scale, requiring gram-size sample injections. This is a problem for small-molecule research and development. 
High Speed Counter Current Chromatography (HSCCC) provides a wide range of applications. [Table 1] includes the list of various HSCCC instruments available in the market.
Extraction of medicinal drugs from plants and purification and isolation of active material
HSCCC is a versatile technique used for a wide range of compounds including synthetics, natural products, and peptides.  It is the best method to obtain major kavalactones kavain and demethoxyyangonin in pure form.  It is used for isolation of the natural products from the raw material as well as from antibiotics and isomers. , For example, Flavonoids can be separated from a crude ethanol extract of sea buckthorn (Hippophae rhamnoides), obtained with a two-phase solvent system composed of chloroform-methanol-water (4:3:2, v/v/v). Tetracycline derivatives can be separated by using a salt-free solvent system such as ethyl acetate-n-butanol-water (3:2:5). Indole auxins can be separated and obtained by using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:1:1:1, v/v/v/v). DNP amino acid can be separated with the help of a two-phase solvent system composed of chloroform-acetic acid-0.1 M hydrochloric acid (2:2:1, v/v/v). Sulforaphane can be isolated from from Broccoli Seed Meal using a two-phase solvent system composed of n-hexane / ethyl acetate / methanol / water (1:5:1:5, v/v/v/v). 
Alkaloids palmatine, berberine, epiberberine, and coptisine can be isolated from Coptis chinensis Franch by using a system of chloroform-methanol-HCl solution (0.3 - 0.1 mol/L) at different volume ratios  Bioactive lignans schisanhenol and its acetate can be separated in a multilayer coil column using an n-hexane-ethanol-water (6:5:5) system. 
Purification of dyes and compounds
It can be used for the purification of dyes. It helps in quick purification of target compounds from crude samples or semi-pure fractions or complex mixtures  such as triterpenoic acids. It can be used for the separation of compounds with a wide range of polarities in natural product research. , Sample quantities ranging from submilligram to 100 mg are successfully separated within a few hours. It provides partition efficiencies of up to several thousand theoretical plates. As the main constituents are present in a minor quantity in extremely complex mixtures, for example, plant extracts and microbial fermentation products, it becomes difficult to isolate them. However, HSCCC with its liquid stationary phase proves to be beneficial in isolation of bioactive compounds from natural sources like plant extracts, microbial fermentation products, and animal tissues. , For example, the isolation of the components of bacitracin, which consists of a group of peptides with a bacteriocidic activity. It contains the major active component, BC-A, its oxidation product, BC-F, and over 20 other minor components, as shown by a reverse-phase HPLC analysis. It is also used in the analysis of various groups of wine constituents. Various labile wine aroma precursors, antioxidants, and pigments are studied. The fractionation of polymeric wine constituents helps in analytical research.  Quercetin-3-O-sambubioside can be separated and purified from the leaves of Nelumbo nucifera by using HSCCC.  It is also used in the purification of recombinant proteins directly from a crude E. coli lysate. Direct extraction and separation of some organic substances (e.g., xenobiotics) from a sewage sludge medium is a problem that is resolved by HSCCC. 
High Speed Counter Current Chromatography is used in the separation of small synthetic molecules from large biomolecules. Liquid-liquid partitioning helps in solute retention, which preserves biological activity. It provides an effective and fast separation of compounds with difficult separations or separations having problematic solubility in the existing reverse phase liquid chromatography purification or where low concentration components can be used; it has proved to be effective in the analysis of natural pigments such as anthocyanins, carotenoids, chlorophyll-related pigments, and betalains. Anthocyanins can be isolated from Purple Heart (Tradescantia pallida Rose), purple corn (Zea mays L.), elderberries (Sambucus nigra L.), red wine, and blackberries (Rubus fruticosus L. agg.).  Various polar and non-polar natural pigments, such as, tayberry, blood orange juice, black carrot juice, red pigmented potatoes, red wine, and so on, can be analyzed successfully by using HSCCC.  Coumarin, esculin, 2- and 3- or 4-hydroxycinnamic acids can be separated easily with a solvent system composed of ethyl acetate-10 mM potassium phosphate (1:1, v/v) at pH 6.5.  It helps in the identification of bioactive lignans from plant natural products.  When this technique is coupled with a mass spectrometer, it is used in the study of plant alkaloids. It is also useful in the determination of an unknown impurity and in validating the presence of a specific compound in a mixture and in the analysis of nonvolatile or thermally unstable molecules. The fractionation of complex mixtures becomes easy and cells and cell fragments can be easily separated.  Flavor analysis is also one of the important applications for the separation of labile substances like aroma compounds or their respective precursors (polyols and glycoconjugates), from complex natural mixtures. For example, the analysis of reactive flavor precursors from Rosa damascena flowers has been reported. 
Separation of rare earth elements
Rare earth elements are important for chlorophyll formation, nutrient uptake, plant root development, biomass, quality, and resistance against stress. HSCCC is used not only for the detection of rare earth elements and compounds in mixtures, but also for their separation, such as, yttrium,  thorium,  and lanthanides.  It is also used for the separation of lighter rare earth metal ions.  Lanthanides like La +3 , Pr +3 , and Nd +3 are successfully separated by using HSCCC. It is also used in the purification of xanthanolide.  Aqueous PEG-Na2SO4 biphasic system containing acetylacetone is used to separate La(III), Ce(III), Nd(III), and Yb(III) by HSCCC.  The multilayer coil system is used for metal-ion enrichment. It is superior to other analytical techniques because it can concentrate metal ions in very small quantities. It is widely used for the detection of metal ions at trace levels. The trace quantity of zinc in natural mineral water can be determined by the enrichment separation through an HSCCC column. 
Preparative-scale separations of chiral compounds
A multilayer coil planet centrifuge is used for the preparative-scale separations of chiral compounds.  The (+) and (-) enantiomers of a racemic compound mixture can be separated from each other using HSCCC. It can be done by adding a chiral selector to a first liquid phase of two pre-equilibrated immiscible liquid phases and then charging a counter current chromatographic centrifuge column with that liquid phase. As a result, a countercurrent chromatographic centrifuge column charged with a chiral selector is produced, and subsequently the racemic compound mixture is introduced into the centrifuge column, after which a second liquid phase is passed through the centrifuge column, thus charging it with a mixture, chiral selector, and first liquid phase, to elute the (+) enantiomer and (-) enantiomer from the countercurrent chromatographic centrifuge column.
Analysis of inorganic compounds and elements
As inorganic substances contain complexing agents, their separation is different from those of organic substances. This is a non-equilibrium process and thus the separation efficiency depends on the type of complexation process, its rate, and the mass transfer rate of the process. , Nickel, Copper, Iron, Magnesium, Cobalt, and other inorganic elements are separated successfully by HSCCC. Nd and Sm isotope concentrations are also determined in rock samples.
Drug discovery and drug development
Drug discovery and product development is a major application of HSCCC, where ease of scale-up from milligrams to grams and then to kilograms is possible. As 100% recovery of the sample is obtained by HSCCC, it is possible to develop methods for a drug.  Moreover, it is a high resolution separating method that can isolate various stable and unstable compounds, which helps in discovery of drugs. Fractionation of natural products by this technique also leads to drug discovery. Two compounds with almost similar polarity can be separated. Natural metabolites can be analyzed effectively because of the liquid stationary phase used in this technique. It can be used for separation of complex natural extracts. The amount of stationary phase retained in the column is responsible for the performance of the HSCCC column. A multiple-layer coiled column is found to be good for the separation of organic compounds. Planetary motion is also used to establish a hydrodynamic equilibrium between two immiscible solvent phases, which enables short separation times. Thus, it helps in the discovery of new drugs and development in the separation methods of the existing drugs.
| 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.
| References|| |
|1.||Ito Y, Bowman RL. Countercurrent chromatography: Liquid-liquid partition chromatography without solid support. Science 1970;167:281-3. [PUBMED] [FULLTEXT] |
|2.||Available from: http://www.pubs.acs.org/subscribe/journals/tcaw/10/i07/html/07inst.html. [retrieved on 2009 Oct 5]. |
|3.||Lee YW. Cross-axis countercurrent chromatography: A versatile technique for biotech purification. Vol. 82, Countercurrent chromatography. In: Menet JM, Thiebaut D, editors. New York: Marcel Dekker; 1999. p. 150. |
|4.||Freisen JB, Pauli GF. Rational development of solvent system families in counter-current chromatography, J Liq Chromatogr Relat Technol 2005;1151:51-9. |
|5.||Ito Y. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J Chromatogr A 2005;1065:145-68. [PUBMED] |
|6.||Colegate SM, Molyneux RJ. Application of high speed countercurrent chromatography to the isolation of bioactive natural products. Vol. 2. Bioactive natural products: Detection, isolation, and structural determination. NW: CRC Press; 2007. p. 269. |
|7.||Gu M, Ouyang F, Su Z. Comparison of high-speed counter-current chromatography and high-performance liquid chromatography on fingerprinting of Chinese traditional medicine. J Chromatogr A 2004;1022:142. |
|8.||Berthod A. Countercurrent chromatography: The support-free liquid stationary phase. Comprehensive analytical chemistry. In: Wilson, Wilson, editors. Elsevier: Amsterdam; 2002. p. 38. |
|9.||Ito Y, Conway WD. Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography: III: Effects of physical properties of the solvent systems and operating temperature on the distribution of two-phase solvent systems. J Chromatogr 1984;301:405-14. [PUBMED] |
|10.||Conway WD. Countercurrent chromatography apparatus. J Liq Chromatogr 1990;13:2409. |
|11.||Ito Y, Clary R, Powell J, Knight M, Finn TM. Spiral tube assembly for high-speed countercurrent chromatography: Choice of elution modes for four typical two-phase solvent systems. J Liq Chromatogr Relat Technol 2009;32:2013-29. |
|12.||Zhou T, Chen B, Fan G, Chai Y, Wu Y. Application of high-speed counter-current chromatography coupled with high-performance liquid chromatography-diode array detection for the preparative isolation and purification of hyperoside from Hypericum perforatum with online purity monitoring. J Chromatogr A 2006;1116:99-100. |
|13.||McAlpine JB, Morris P. Separation by high-speed countercurrent chromatography. Natural products isolation. In: Sarker DS, Latif Z, Gray AI, editors. New Jersey: Humana Press; 2006. p. 191-4. |
|14.||Mandava NB, Ito Y. Countercurrent chromatography. Chromatographic science series. New York: Marcel Dekker; 1988. p. 44. |
|15.||Zolotov YA, Spivakov BY, Maryutina TA, Bashlov VL, Fresenius VP. Partition countercurrent chromatography in inorganic analysis. J Anal Chem 1989;335:938. |
|16.||Muralidharan S, Freiser H. Fundamental aspects of metal-ions separations by centrifugal partition chromatography. In metal-ion separation and preconcentration. Progress and opportunities. In: Bond AH, Dietz ML, Rogers RD, editors. Vol. 716. ACS symposium series. US: 1999. p. 347. |
|17.||Abe H, Usuda S, Takeishi H, Tachimori S. Countercurrent chromatography: The support-free liquid stationary phase. J Liq Chromatogr A 1993;16:2661-72. |
|18.||Kitazume E, Bhatnagar M, Ito Y. Separation of rare earth elements by high-speed counter-current chromatograph. J Chromatogr A 1991;538:133. |
|19.||Maryutina TA, Fedotov PS, Spivakov BY. Countercurrent chromatography. Vol. 82. Chromatographic science series. In: Menet JM, Thiebaut D, editors. New York: Marcel Dekker; 1999. p. 171. |
|20.||Berthod A. High Speed countercurrent chromatography, Analusis 1990;18:352. |
|21.||Berthod A, Ruiz-Angel MJ, Carda-Broch S. Elution-extrusion countercurrent chromatography: Use of the liquid nature of the stationary phase to extend the hydrophobicity window. Anal Chem 2003;75:5886. [PUBMED] [FULLTEXT] |
|22.||Hamad EZ, Ijaz W, Ali SA, Hastaoglu MA. Influence of polymer structure on protein partitioning in two-phase aqueous systems. Biotechnol Prog 2008;12:173-7. |
|23.||Ito Y, Lee YW. Analytical high-speed counter-current chromatography with a coil planet centrifuge. J Chromatogr 1987;391:290-5. [PUBMED] |
|24.||Wood PL, Hawes D, Janaway L, Sutherland IA. Stationary phase retention in CCC: Modeling the J-type centrifuge as a constant pressure drop pump. J Liq Chromatogr Relat Technol 2003;26:1373-96. |
|25.||Ito Y, Yang FQ, Fitze PE, Sullivan JV. Spiral tube assembly for high-speed countercurrent chromatography, choice of elution modes for four typical two-phase solvent systems. J Liq Chromatogr Relat Technol 2003;26:1355-72. |
|26.||Ito Y, Yang F, Fitze P, Powell J, Ide D. Improved spiral disk assembly for high-speed counter-current chromatography. J Chromatogr A 2003;1017:71-81. [PUBMED] |
|27.||Ito Y, Qi L, Powell J, Sharpnack F, Metger H, Yost J, et al. Mixer-settler counter-current chromatography with a barricaded spiral disk assembly with glass beads. J Chromatogr A 2007;1151:108-14. [PUBMED] [FULLTEXT] |
|28.||Ito Y, Clary R, Sharpnak F, Metger H, Powell J. Mixer-settler counter-current chromatography with multiple spiral disk assembly. J Chromatogr A 2007;1172:151-9. |
|29.||Ito Y, Clary R, Powell J, Knight M, Finn TM. Spiral tube assembly for high-speed countercurrent chromatography choice of elution modes for four typical two-phase solvent systems. J Liq Chromatogr Relat Technol 2008;31:1346-57. [PUBMED] [FULLTEXT] |
|30.||Ito Y, Clary R, Powell J, Knight M, Finn TM. Improved spiral tube assembly for high-speed counter-current chromatography. J Chromatogr A 2009;1216:4193-200. [PUBMED] |
|31.||Zhou T, Chen B, Fan G, Chai Y, Wu Y. Application of high-speed counter-current chromatography coupled with high-performance liquid chromatography-diode array detection for the preparative isolation and purification of hyperoside from Hypericum perforatum with online purity monitoring. J Chromatogr A 2006;1116:97-101. [PUBMED] [FULLTEXT] |
|32.||Deng S, Deng Z, Fan Y, Li J, Liu R, Xiong D. Application of high-speed counter-current chromatography coupled with high performance liquid chromatography for the separation and purification of Quercetin-3-O-sambubioside from the leaves of Nelumbo nucifera. Frontiers Chem Engg China 2009;3:375-82. |
|33.||Berthod A, Maryutina T, Spivakov B, Shpigun O, Sutherland IA. Countercurrent chromatography in analytical chemistry. Pure Appl Chem 2009;81:355-87. |
|34.||Maryutina TA, Fedotov PS, Spivakov BY. Application of countercurrent chromatography in inorganic analysis. Vol. 82, Countercurrent chromatography. In: Menet JM, Thiebaut D, editors. New York: Marcel Dekker; 1999. p. 171-2. |
|35.||McAlpine JB, Morris P. Separation by high-speed countercurrent chromatography. Natural products isolation. In: Sarker SD, Latif Z, Gray AI, editors. New Jersey: Humana Press; 2006. p. 185. |
|36.||Marston A, Hostettmann K. Developments in the application of counter-current chromatography to plant analysis. J Chromatogr A 2006;1112:181-94. [PUBMED] [FULLTEXT] |
|37.||Keay D, Wood P. Reintroducing countercurrent chromatography to the chemist. Available from: http://www.iscnewsletter.com/newsletter/2008/nov/lin-2008/art4.pdf [cited in 2008]. |
|38.||Putterman GJ, Spear MB, Perini F. Synergistic use of countercurrent chromatography and high performance liquid chromatography for the purification of synthetic peptides. J Liq Chromatogr Relat Technol 1984;7:341-50. |
|39.||Katrien S, Peter W. Application of high speed countercurrent chromatography (HSCCC) to the isolation of kavalactones. J Liq Chromatogr Relat Technol 2005;28:1703-16. |
|40.||Nakazawa H, Riggs CE Jr, Egorin MJ, Redwood SM, Bachur NR, Bhatnagar R, et al. Continuous extraction of urinary anthracycline antitumor antibiotics with the horizontal flow-through coil planet centrifuge. J Chromatogr 1984;307:323-33. [PUBMED] |
|41.||Ito Y. High-speed countercurrent chromatography. CRC Crit Rev Anal Chem 1986;17:65-143. |
|42.||Liang H, Li C, Yuan Q, Vriesekoop F. Application of high-speed countercurrent chromatography for the isolation of sulforaphane from broccoli seed meal. J Agric Food Chem 2008;56:7746-9. [PUBMED] [FULLTEXT] |
|43.||Peng J, Han X, Xu Y, Qi Y, Xu L, Xu Q. New approach for application of high speed countercurrent chromatography coupled with direct injection of the powders of a raw material without any preparation, for isolation and separation of four alkaloids with high recoveries from coptis chinensis Franch. J Liq Chromatogr Relat Technol 2007;30:2929-40. |
|44.||Oka H. High-speed countercurrent chromatography. In: Ito Y, Conway WD, editors. Chichester: John Wiley; 1996. p. 73. |
|45.||Zhao CX, He CH. Sample capacity in preparative high-speed counter-current chromatography. J Chromatogr A 2007;1146:186-92. [PUBMED] [FULLTEXT] |
|46.||Owen RO, McCreath GE, Chase HA. A new approach to continuous counter-current protein chromatography: Direct purification of malate dehydrogenase from S. cervisiae homogenate as a model system. Biotechnol Bioeng 1997;53:427-41. [PUBMED] [FULLTEXT] |
|47.||Sutherland IA, Fisher D. Role of counter-current chromatography in the modernisation of Chinese herbal medicines. J Chromatogr A 2009;1216:740-53. [PUBMED] |
|48.||Winterhalter P. Application of countercurrent chromatography for wine research and wine analysis. Am J Enol Vitic 2009;60:123-9. |
|49.||Fedotov PS, Bauer C, Popp P, Wennrich R. Dynamic extraction in rotating coiled columns: A new approach to direct recovery of polycyclic aromatic hydrocarbons from soils. J Chromatogr A 2004;1023:305-9. [PUBMED] |
|50.||Schwarz M, Hillebrand S, Habben S, Degenhardt A, Winterhalter P. Application of high-speed countercurrent chromatography to the large-scale isolation of anthocyanins. Biotechnol Bioeng 2003;14:179-89. |
|51.||Winterhalter P. Application of countercurrent chromatography (CCC) to the analysis of natural pigments. Trends Food Sci Technol 2007;18:507-13. |
|52.||Shibusawa Y, Hagiwara Y, Chao Z, Ma Y, Ito Y. Application of high-speed counter-current chromatography to the separation of coumarin and related compounds. J Chromatogr A 1997;759:47-53. |
|53.||Lee YW, Voyksner RD, Pack TW, Cook CE, Fang QC, Ito Y. Application of countercurrent chromatography/thermospray mass spectrometry for the identification of bioactive lignans from plant natural products. Anal Chem 1990;62:244-8. [PUBMED] |
|54.||Al-Shammary FJ, Aziz Mian NA, Mian SM. The role of countercurrent chromatography in the fractionation of complex mixtures. J Sep Sci 2005;14:230-4. |
|55.||Winterhalter P, Knapp H, Straubinger M, Fornari S, Watanabe N. Challenges in isolation and characterization of flavor compounds. In: Mussinan CJ, Morello MJ, editors. Application of countercurrent chromatography. Washington, DC: American Chemical Society; 1998. p. 181. |
|56.||Nakamura S, Hashimoto H, Akiba K. Enrichment separation of rare earth elements by high-speed countercurrent chromatography in a multilayer coiled column. J Chromatogr A 1997;789:381-7. |
|57.||Barkley DJ, Blanchette M, Cassidy RM, Elchuk S. Dynamic chromatographic systems for the determination of rare earths and thorium in samples from uranium ore refining processes. Analy Chem 1986;58:2222-6. |
|58.||Akiba K, Sawai S, Nakamura S, Murayama W. Mutual separation of lanthanoid elements by centrifugal chromatography. J Liq Chromatogr 1988;11:2517-36. |
|59.||Araki T, Okazawa T, Kubo Y, Ando H, Asai H. Separation of lighter rare earth metal ions by centrifugal counter-current type chromatograpby with Di-(2-Ethylhexyl) phosphoric acid. J Liq Chromatogr 1988;11:267-81. |
|60.||Pinel B, Audo G, Mallet S, Lavault M, De La Poype F, Sιraphin D, et al. Multi-grams scale purification of xanthanolides from Xanthium macrocarpum: Centrifugal partition chromatography versus silica gel chromatography. J Chromatogr A 2007;1151:14-9. |
|61.||Hiroaki K, Kazuyuki I, Kazunori S, Hiroaki M, Masami S. Separation of rare earth elements by high-speed countercurrent chromatography with aqueous PEG-Na2SO4 biphasic systems. Nippon Kagakkai Koen Yokoshu 2005;85:30. |
|62.||Hosoda A, Tsuyoshi A, Akiba K. Enrichment and determination of zinc by high-speed countercurrent chromatography. Anal Sci 2002;18:897-901. [PUBMED] [FULLTEXT] |
|63.||Ma Y, Ito Y, Berthod A. Chiral separation by high-speed countercurrent chromatography. Vol. 82, Countercurrent chromatography. In: Menet JM, Thiebaut D, editors. New York: Marcel Dekker; 1999. p. 224. |
|64.||Eiichi K. Topics of analytical techniques: High-speed countercurrent chromatography. Chem Eng (Tokyo) 1999;44:853-64. |
|65.||Berthod A, Billardello B. Countercurrent chromatography: Fundamentally a preparative tool. Advances in chromatography. Vol. 40. Advances in chromatography. In: Brown P, Grushka E, editors. New York: Marcel Dekker; 2000. p. 503-538. |
|66.||Available from: http://www.tigger.uic.edu/~gfp/countercurrent/content/manufacturers.htm. [retrieved on 2006 Oct 5]. |
|This article has been cited by|
||Separation and purification of bioactive botrallin and TMC-264 by a combination of HSCCC and semi-preparative HPLC from endophytic fungus Hyalodendriella sp. Ponipodef12
| ||Ziling Mao,Ruiya Luo,Haiyu Luo,Jin Tian,Hongwei Liu,Yang Yue,Mingan Wang,Youliang Peng,Ligang Zhou |
| ||World Journal of Microbiology and Biotechnology. 2014; 30(9): 2533 |
|[Pubmed] | [DOI]|
||Analysis and Antioxidant Capacity of Anthocyanin Pigments. Part III: An Introduction to Sample Preparation and Extraction
| ||María José Navas,Ana María Jiménez-Moreno,Julia Martín Bueno,Purificación Sáez-Plaza,Agustin G. Asuero |
| ||Critical Reviews in Analytical Chemistry. 2012; 42(4): 284 |
|[Pubmed] | [DOI]|
||Analysis and Antioxidant Capacity of Anthocyanin Pigments. Part IV: Extraction of Anthocyanins
| ||María José Navas,Ana María Jiménez-Moreno,Julia Martín Bueno,Purificación Sáez-Plaza,Agustin G. Asuero |
| ||Critical Reviews in Analytical Chemistry. 2012; 42(4): 313 |
|[Pubmed] | [DOI]|