Introduction

Countercurrent chromatography or CCC is also known as liquid-liquid chromatography. Here, no solid matrix is used, and the liquid phases separate the samples. Here the liquids use the columns to interact with each other. The gravitational or centrifugal force holds the stationary liquid phase while the mobile phase moves in the columns. The first made Countercurrent chromatography equipment took a long time to separate. Still, with new technologies, the system has become quick and efficient and has gained popularity for the fractionation of natural products. Countercurrent chromatography shows a variety of instrumentations and differs from the conventional liquid chromatography method.

History and advancement of Countercurrent chromatography

Countercurrent chromatography is a type of liquid partition chromatography. The idea of counter-current chromatography was first pitched by Post and Craig in the late 1940s. The proposed machine consisted of an interconnecting glass separating funnels held with 1g gravitational force. The force was not efficient to stabilize the stationary phase. As a result, only a denser stationary phase could be used in this counter-current chromatography equipment. Even though the machine took days to complete a single experiment, it could resolve any highly complex mixture. The technique was simple and high resolution and made high-valued products like natural products and Chinese medicines. 

All the Countercurrent chromatography systems can be broadly divided into two major classes:

  • Hydrodynamic equilibrium system: It has an Archimedian screw effect to facilitate constant mixing of the two phases while keeping one of the phases as a stationary phase. Countercurrent chromatography (CCC) systems belonging to this class are high-speed CCC,  slow rotary CCC and nonsynchronous CCC.

 

  • Hydrostatic equilibrium system: It uses stable field force to retain the stationary phase while the mobile phase flows through the column. Countercurrent chromatography systems belonging to this class are helix Countercurrent chromatography, droplet Countercurrent chromatography, locular Countercurrent chromatography, and centrifugation partition chromatography. 

The droplet counter-current chromatography equipment was developed in the 1970s. It used only one phase as a stationary phase. After advancement and further research, it can use a two-phase stationary phase. The high-performance counter-current chromatography or HPCCC was invented in the early 2000s. This technology uses a centrifugal force of about 240g and approximately 10 minutes. 

 

Principle of Countercurrent chromatography

The principle of counter-current chromatography is somewhat similar to liquid chromatography. Countercurrent chromatography instrumentation uses two solvent phases, where two immiscible liquids are mixed and then separated several times consecutively. This provides us with a partition coefficient (K) which a test tube experiment can easily determine before separation. The formula for partition coefficient is :

K= amount of solute in stationary phase/ amount of solute in the mobile phase

The partition coefficient determines the relative proportion of solute passing through the two-phased solution. 

The procedure of Countercurrent chromatography

The whole CCC is a chromatographic column. The procedure is given in points below:

  • The sample solution is filtered
  • The centrifugal phase helps mix the phases so that the analyte can move between them.
  • The mobile phase mixes with the analyte settling the stationary phase in the column.
  • Maximum retention of stationary phase is obtained using less viscous phase as mobile phase.

 

The solvent system used in the experiment can be of three types;

  • The biphasic liquid system, constituting of water and octanol
  • The tertiary liquid system with chloroform, methanol and water
  • The quaternary liquid system with hexane, ethyl-acetate, acetone and water.

 

Some counter-current chromatography applications are:

  • Droplet counter-current chromatography is used to separate plant constituents and natural products.
  • Some new applications of counter-current chromatography are steroids, plant hormones, essential oils, etc.
  • Different levels of centrifugation help in separating efficient end products helping in fractionation.
  • Preparation of herbicides and pesticides
  • Analysis of food
  • Separation of macromolecules
  • DNA and tRNA purification

Advantages and Disadvantages of counter-current chromatography

Some advantages of counter-current chromatography are:

  • It is inexpensive as only the cost of solvent is the major rest of the instrument is a one-time investment. 
  • The process is quick.
  • The result is versatile; the product will not decompose quickly.
  • The phase can be switched from reversed-phase to normal.
  • There is no irreversible adsorption to solid support giving us a 100% sample after extraction.
  • Uses low-pressure techniques.

One major disadvantage of counter-current chromatography is:

  • Cannot experiment with small sample volumes. 

Conclusion

Countercurrent chromatography is one of the oldest chromatography methods, which has evolved with time to give us great results and applications. The use of Countercurrent chromatography in the extraction of biological products like hormones, enzymes, essential oils and plant-based extraction. Countercurrent chromatography has evolved to give us a better and more efficient result. The extractions are pure, and the output equals the input. This results in no wastage of the crude material and is cost-efficient. Different types of Countercurrent chromatography can be used for different sample experiments depending on the requirements.