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Counter-current chromatography (CCC) is a separation technique applying liquid-liquid phase. It is carried out without the aid of a solid supporting matrix to retain the stationary liquid in the chromatographic column (Conway, 1991). Separation of analytes in the sample is based on the affinities of mobile and stationary phases of the chromatography mode. CCC is carrying out based on three different stages. First are mixing, then settling; and lastly separation of analytes in the sample (Wikipedia, 2010).
CCC was developed since last centuries. It can be traced back the history of CCC where Cornish et al. (1934) had described a locular CCC column for purification of oil-soluble vitamin. In the early stage, the work on CCC had been done by of Ito et al. (1966), where they had first constructed an apparatus for differentiation of particles in suspension and solutes in a solvent system by applying centrifugal acceleration. Later, Foucault and Chevolot (1998) developed more advance technique based on a wide variety of CCC apparatuses using a variable-gravity field produced by a two-axis gyration mechanism and a rotary seal-free arrangement for the column.
This study reported the principles and practical applications of a few different CCC methods. The principles and advantages of various CCCs were explained. The practical applications of each CCC technique were described, and examples of each CCC techniques were provided, which linked to separation and isolation of analytes or bioactive compounds in plants.
TYPES OF COUNTER-CURRENT CHROMATOGRAPHY
CCC is generally divided into five forms: droplet CCC, elution extrusion CCC, centrifugal partition chromatography (CPC), simulated moving bed (SMB) chromatography and high-speed CCC.
2.1. Droplet counter-current chromatography (DCCC)
DCCC is known to be the oldest form of CCC. It uses the force of gravity to move the mobile phase through the stationary phase. It relies on the passage of droplets of a mobile phase through an immiscible stationary liquid phase for the continuous partition of a solute between the two phases (Tanimura et al., 1970). A schematic diagram of droplet counter-current chromatography is shown in Figure 1.
One phase is allowed to drip through the other in either ascending or descending mode. In last two decade, Hostettmann (1980) had developed all-liquid separation technique, which is based on the partitioning of solutes between a steady stream of droplets of mobile phase and a column of surrounding stationary phase. This technique is somewhat time-consuming and inefficient due to its low flow rates and poor mixing of solvent systems (Vogel, 1975). Tanimura et al. (1970) were the earliest researchers who used DCCC to effectively separate a mixture of dinitrophenylamino acids.
Figure 1: Droplet counter-current chromatography (Dynamic Extractions, 2010).
2.2. Elution extrusion counter-current chromatography (EECCC)
In 2003, Berthod et al. proposed the use of EECCC to extend the hydrophobicity window of CCC technique which extrudes the most retained solutes out of the column. It is thus, proposed that EECCC will be widely applied in fast separation of complex natural samples (Berthod et al., 2005).
2.3. Centrifugal partition chromatography (CPC)
The technique was established by Murayama et al. in 1982 whereby series of channels are linked in cascade by ducts and aligned in cartridges in a circle around a rotor, which is capable of creating constant centrifugal force. CPC was first developed by Japanese and later extensively developed in France by applying centrifugal force to speed separation and achieves higher flow rates than DCCC (Wikipedia, 2010). CPC is a typical method widely used as a routine preparative technique in research and industry, especially for separation of crude extracts and semi-pure fractions (Marston and Hostettmann, 1994). In CPC, reverse flow of direction and reversed-phased operation is possible to achieve (Himbert et al., 2004).
2.4. Simulated moving bed (SMB) chromatography
SMB chromatography was initially developed in 1961 by Broughton and Gerhold for petrochemicals separation. In the early 1990s, SMB was used for the first time in enantiomers separation and this has made it an important tool in pharmaceutical industries for racemic drugs separation (Rajendran et al., 2009). A schematic diagram of a four-section simulated moving-bed (SMB) unit is shown in Figure 2.
Figure 2: A four-section simulated moving-bed (SMB) unit (Max-Planck-Gesellschaft, 2010).
2.5. High speed counter-current chromatography (HSCCC)
HSCCC was developed by Ito (1981) and was at that time, "high-speed" in comparison to DCCC. Its mode of application is a kind of liquid-liquid partition chromatography without any solid matrix. This technique is developed to eliminate irreversible adsorption of samples on solid support in the conventional chromatographic column (Gu et al., 2004). It has been successfully applied in analysis and separation of various plants (Cao et al., 1999; Tian et al., 2000). Today, advance HSCCC has shown advance separation and purification performance for antibiotics and valid monomer of herbal compounds (Tauto Biotech, 2010) and it has been improved to deal with scaling-up problems in industries (Ito, 1986).
3.0 ADVANTAGES OF CCC AND ITS COMPARISON
Application of CCC in separation of organic and inorganic substances from a complicated mixture has many advantages due to its specificÂ characteristics. The types of CCC are demonstrated in Table 1. CCC is performing with no solid support, where it is free from adsorption of solutes to the solid column. Hence, 100% of the recovery of samples and reagents without contamination or decomposition is possible. An additional benefit is that it should be possible to use the same column repeatedly for separations with different stationary phases. Furthermore, the range of substances which can be extracted and pre-concentrated by CCC from aqueous sample solutions is widely. CCC can be used for the recovery of particulate matter, soluble polymers (e.g., humic substances), and other components of crude samples that may clog the HPLC columns and cause difficulties in capillary electrophoresis studies. The advantages of CCC had been compared with HPLC (Table 2). Besides, CCC is a multistage technique that enables incomparably better separations than conventional multiple extractions in separation funnels. As result, CCC is much more convenient for laboratory use.
Table 1: Comparison of four different types of CCC
Droplet Counter-Current Chromatography (DCCC)
Centrifugal Partition Chromatography (CPC)
High Speed Counter- Current Chromatography (HSCCC)
High Performance Counter-Current Chromatography (HPCCC)
Scale of sample capacity per run
Âµg to mg
mg to g
Âµg to mg
mg to g
Scale of run time
Hours to Days
Minutes to hours
Table 2: Comparison of counter-current chromatography (CCC) and high performance liquid chromatography (HPLC)
More cost effective
Less cost effective; high cost of columns&high solvent consumption
<100%; loss of sample byirreversible adsorptiononto the solid support
<99%; contamination or deactivation of sample
4.0 TECHNIQUES IN CCC
In order to fully optimize this technique, a few parameters need to be taken into consideration before applying CCC. Simple gradient system is applied with a known polarity range of the mixture sample, which is shown in Figure 3.
4.1. Solvent system and its ratio
The decision to use which solvent as mobile/stationary phase relies on partition coefficient (K) value. High K value (1.0 â‰¤ K â‰¤ 2.0) favor upper phase as mobile phase and a low K value (0.5 â‰¤ K â‰¤ 1.0) favor the lower phase as mobile phase. The types of mobile phases for polar, semi polar and polar compounds are shown in Table 3.
Figure 3: Gradient system for sample polarity. A: Very polar, B: Semi polar, C: Low polar (Leitao et al., 2001).
Table 3: Commonly used solvent system for polar, semi polar and low polar compound
Semi Polar Compound
Low Polar Compound
4.3. Flow rate of mobile phase
Better separation can be obtained in slow flow rate however it is time and mobile phase consuming with broader peaks.
Temperature has significant effect on K value, retention and separation capability and both phase solvency.
4.5. Revolution speed
Rotary speeds influence the retention of the stationary phase where high speed increases retention.
4.6. Liquid Motion in CCC
Head to Tail: Or known as descending mode where the heavier phase (ie aqueous) is pumped through as the mobile phase and lighter phase act as stationary phase.
Tail to Head: Reverse from descending mode where the less dense phase is used as the mobile phase.
Dual-Mode: This back flushing measurement uses both descending and ascending mode where mobile and stationary phases are reversed part way through the run. This is to ensure that compounds which are strongly retained during ascending mode will be forced out during descending mode (vice versa).
Gradient Mode: The concentration of one or more components in the mobile phase is varied throughout the run in order to achieve optimal resolution across a wider range of polarities.
Elution Extrusion Mode (EECCC): The mobile phase is extruded after a certain point by switching the phase being pumped into the system. EECCC raises the partition coefficient value (K) to more than 2 for better separation.
pH Zone Refining: Acidic and basic solvents are used to elute analytes based on their pKa.
CCC is mainly used in plant or natural product research due to its ability to separate very polar compound and high sample recovery. It is also due to its versatility in using solvent combinations and the ease in switching between normal phase and reverse phase. Main application of CCC is for isolation of compound of interest. It has been successfully applied to the separation, isolation of a numerous constituents of different classes of polar compounds from medicinal plants including, triterpene, alkaloids, falvonids, carotenoids, glycosides and peptides. Currently this technique is coupled with HPLC, NMR and also MS for compound identification and purity determination
i. Plant hormone and growth substances:
Cocktail of indole auxins, gibberellins and cytokinins were used separately in this study to determine the best solvent system and its respective ratio for separation of each respective compound-of-interest. Aqueous phase was used as mobile phase for separation of indole auxins and organic phase as mobile phase for separation of gibberellins (Mandava and Ito, 1982) (Table 4).
Table 4: Chromatographic conditions for analysis of indole auxins, gibberellins and cytokinins
Diethyl Ether:Methanol:Phosphate Buffer (pH7)
Ethyl Acetate:Methanol:Phosphate Buffer (pH7)
Peng et al. (2005).have reported that 2 lignans (schizandrin and gomisin A) were isolated and separated from crude extract of Schisandra chinensis with purity as high as 99.5% and 99.1%, respectively (Table 5).
Table 5: Chromatographic conditions for analysis of Schisandra chinensis
--Fraction I (schizandrin)
--Fraction II (gomisin A)
Separation and isolation of both chlorophyll a and b was successfully carried out using the same isolation technique from crude extract from spinach. Two different stationary phases were used for chlorophyll a (heptanes) and chlorophyll b (ethanol) and structure and purity for both chlorophylls were determined using MS and NMR (Jubert and Bailey, 2007) (Table 6).
Table 6: Chromatographic conditions for analysis of Spinach
iv. Organic synthesis purification:
Compounds from low and medium polarities were separated using HSCCC system. Oxazole and imidazole were separated in reverse phase condition with aqueous solvent system meanwhile Quinoxaline was isolated in normal phase with tail to head direction (Silva et al., 2007) (Table 7).
Table 7: Chromatographic conditions for analysis of oxazole, imidazole and Quinoxaline
Hexane : Methanol : Water
Hexane : Ethyl Acetate : Methanol : Water
N oxide from Quinoxaline and Phenazine
Hexane : Acetonitrile : Methanol
v. Polyphenolic compounds for tea characteristics:
Thearubigins were separated from black tea infusion and identified using TLC. These compounds were previously separated using cellulose column chromatography, ion exchange chromatography, paper electrophoresis, RP HPLC and gel filtration (Wedzhica and Donovan, 1990) (Table 8).
Table 8: Chromatographic conditions for analysis of black tea
Butanol : Ethyl Acetate : Water
vi. Glycoside and alkaloid:
In this study DCC technique is being applied to separate glucosides and alkaloid from crude extract (Ajuga pyramidalis). Due to the nature of the compounds, longer separation time is needed with the usage of less polar layer as mobile phase and vice versa (Hostettmann et al., 1979) (Table 9).
Table 9: Chromatographic conditions for analysis of Ajuga pyramidalis
Chloroform : Methanol : n-propanol : Water
9 : 12 : 1 : 8
5.2. Food analysis
CCC has the potential to play an important role in food analysis because it permits the analysis of crude and complex samples. One of the main advantages of CCC is that it can separate substances from large volumes of such samples, which is important for food analysis. Combining CCC separation with other analytical identification and detection methods, such as mass spectrometry, capillary electrophoresis, or high performance liquid chromatography (HPLC), is potentially useful for toxin analysis in food (Winterhalter, 2006).
One of the most frequent diseases is gastroenteritis resulting from food contaminated with Staphylococcal enterotoxin A (SEA) produced by the bacterium Staphylococcus auras. CCC was evaluated for its ability to separate SEA separation from milk. Although many foods can be analyzed for SEA directly by Western blot analysis, but milk samples generally require some purification because the high concentration of milk proteins distorts sodium dodecyl sulfate polyacrylamide gel electrophoresis' (SDS-PAGE) mobility. Hence, milk samples containing SEA were separated by toroidal coil CCC and the fractions were analyzed by Western immune-blotting (Rasooly and Ito, 1999).
CCC has been used for the separation of both native and heat-denatured SEA contained in crude samples of mushrooms in a column. This simple method does not require any sample preparation beyond homogenization. The method is approximately 10 times more sensitive than immunological methods such as western blotting because very large samples can be applied in CCC (Rasooly and Ito, 1998).
CCC was applied to the analysis of phosphocholine-containing glycoglycerolipids (GGPL-I and GGPL-III) of Mycoplasma fermentans, which is thought to be one of the causative microorganisms of rheumatoid arthritis (RA) (Matsuda et al., 1997). The CCC method is a powerful tool for the separation of lipids of microorganisms and, more importantly, it may become a useful tool for the analysis of a host-pathogen interaction or, in other words, a lipid-protein interaction at lipid micro-domains.
Hydrodynamic CCC columns were used to separate monomers, dimers, trimers, and oligomers of catechin or epicatechin from apple procyanidins or callsed as condensed tannins. Highly polymerized procyanidins have attracted attention in the fields of pharmacology and food chemistry because of their physiologic activities, such as hair-growth promotion, anti-allergic, antibiotic, and inhibitory activities against enzymes and receptors. The pharmacological properties of procyanidins depend on the degree of their polymerization, which makes it necessary to establish a reliable separation method. However, the application of liquid chromatography to the separation of these 'procyanidin oligomers' is difficult because the oligomers above hexamers tend to cause irreversible adsorption onto the column packing materials. Nevertheless, CCC is a liquid-liquid partition technique that eliminates various complications arising from the use of solid supports, and is particularly useful for the separation of hydrophilic, highly polymerized procyanidins (Shibusawa and Ito, 2005).
5.3. Environmental analysis
Organic pollutants present at trace amounts in water source can be identified and quantified by using gas chromatography/mass spectroscopy (GC/MS). However, these substances need to be concentrated in order to enhance the sensitivity of GC/MS. Yrieix et al. (1996) applied continuous CCC for such purpose and it was found that methylenechloride appeared to be the most efficient extractant for both non-polar and polar substances.
Besides, organic contaminants, especially polycyclic aromatic hydrocarbons (PAHs) are widely distributed in soils and sewage sludge. Fedotov et al. (2004) recovered PAH from soils directly by using rotating coiled columns (RCCs), which has been formerly used in CCC. RCC is based on the retention of a solid sample (stationary phase) in the rotating column under centrifugal force while mobile phase (acetone-cyclohexane) is continuously pumped through. Extract obtained in RCC required neither filtration nor clean-up steps.
Ito et al. (2006) utilized dual CCC to isolate N-methylcarbamate pesticides from vegetable oils and citrus fruits with a biphasic solvent system (n-hexane-acetonitrile). Dual CCC was chosen over gel permeation chromatography (GPC) because interfering substances, which are mostly aliphatic compounds, were retained in GPC. This increases the risk of sample contamination and is thus considered less efficient in comparison to dual CCC.
Biological pest control agent, Destruxin A, B, C, D, E and E-diol had been isolated from Metarhizium anisopliae Seger et al. (2006), where a three-step purification method was applied; starting with liquid-liquid extraction to remove hydrophilic fungal metabolites, followed by column chromatography with beaded cross-linked dextran gel (Sephadex) and finally purified with HSCCC. This method resulted in higher yield of dtx in contrast to separation by silica gel or preparative scale reversed phase chromatography.
CCC is an all-liquid separation technique which can be used to either prepare laboratory-scale or industrial-scale quantities. This technique is flexible and can be operated in a variety of modes. This flexibility of operation and the absence of a solid-phase support give CCC advantages over other adsorption methods. The most important point is the high recovery of sample from CCC with more than 99% purity in the isolated compound. For this reason, CCC is often applied by researchers in pharmaceutical and chemical industries for investigation of natural products.