By Jeyashree Sundaram, MBA
Developments in spectroscopic analysis and routine availability of X-ray crystallography have simplified structural elucidation of natural compounds. However, isolating bioactive components of interest from crude extracts of either plants or animals has always been challenging for two reasons: these extracts contain hundreds of compounds with a range of polarities, and the bioactivity of the compound of interest should be maintained throughout the purification process. This is where countercurrent chromatography (CCC) is helpful.
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Principles of countercurrent chromatography
CCC is a liquid–liquid chromatography technique in which one of a pair of immiscible liquids is placed in a tubular structure and moved in a planetary motion. This results in a combination of hydrodynamic and hydrostatic forces that hold the liquid in place (the stationary phase) while the other liquid (the mobile phase) is pumped countercurrently (allowing a general mixing and settling, often using magnetic stirring or ultrasound) through the liquid column.
There is a sample feed line in the middle of the column, which helps to inject the sample that has a mix of polar and nonpolar substances. Countercurrent distribution of the solutes happens in such a way that components with decreasing order of polarity are eluted in one liquid (say within a tubular system) and compounds with increasing order of polarity are eluted in the other liquid (mobile phase).
Advantages of using countercurrent chromatography in the life sciences
CCC has taken about 60 years to mature as a valid science, on par with other chromatographic techniques. The long time required for separation as well as the tedium of maintaining the countercurrent distribution have been overcome by the development of a number of highly efficient systems, with the result of CCC gaining popularity as a technique for the fractionation of natural products.
- CCC provides a method of achieving the best possible countercurrent distribution.
- In typical solid phase/liquid phase separations, complications arise on account of adsorbance of solutes to the column, in turn leading to degradation, deactivation or contamination—all of which are eliminated by using CCC.
- In CCC, separations are based only on the polarity of the solutes; classes of compounds with similar molarities can be separated without sample loss. In some cases, sequential CCC with different solvents or another HPLC step may be used for further separation.
- As CCC is basically a low-pressure technique (100-500 psi), equipment prices are lower than those for HPLC.
- CCC also helps in saving substantial costs associated with the large volumes of organic solvents used with HPLC.
- CCC can be scaled up from trace amounts to kilogram quantities (with potential for tonnes of production per annum).
- Normal and reversed-phased modes are possible by switching the mobile and stationary phases, and they produce mirror results.
Applications of CCC in the life sciences
The broad areas of application of CCC in the field of life sciences include isolation of target molecules, fractionation, separation of macromolecules, enantiomer separation, besides others.
Isolation: A chunk of bioactive compounds have been extracted from many medicinal plants listed in Traditional Chinese Medicine or Japanese Pharmacopoeia. Compounds isolated include alkaloids, flavonoids, carotenoids, and saponins. Alkane/ethyl acetate/methanol/water solvents are most commonly used for these compounds. Although the amount purified varies, depending on the sample, purities over 95% are often acheived.
Fractionation: In the field of drug discovery, receptor-binding assays or enzyme assays used for screening can be hampered by the non-specific binding of other active compounds. This suggests a fractionation step before the screening step. CCC with multichannel fractionation has proved to be a simple and fast method for the fractionation of extracts to a broad range of compounds.
Heparins (polyketide antibiotics), grape skins (anthocyanins), Sea Buckthorn juice concentrates (containing polyphenols), and ethyl acetate extracts of plants have all be purified using simple CCC procedures.
Separation of macromolecules: Polyethylene glycol and potassium phosphate are often used as aqueous two-phase systems in CCC for isolation and purification of macromolecules and proteins. It is known that hydrodynamic forces tend to cause emulsification of these two aqueous phases but that hydrostatic forces provide a clean separation of proteins. DNA and tRNA (for RNA sequencing) have also been purified using CCC.
Enantiomer separation: In CCC, a chiral separator is added to one of the phases in the biphasic liquid system appropriate to the target molecules. The chiral selector is soluble in the solvent mixture and facilitates separation of the enantiomers.
Polysaccharide derivatives, bovine serum albumin, vancomycin, and l-proline derivatives have been successfully used as chiral separators. Chlorpheniramine, gemifloxacin, and warfin enantiomers are common examples of the many enantiomer separations achieved using CCC. Although HPLC is often used for analytical enantiomer separations, CCC provides the advantage of scaling up to the preparative scale.
Others: CCC has also been successfully applied in the preparation of herbicides, pesticides, and some synthetic pharmaceuticals.