Countercurrent partition chromatography

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Countercurrent distribution chromatography (English: countercurrent chromatography , abbreviated to CCC ) is an umbrella term for chromatographic techniques that are mostly used for the isolation of natural substances such as flavonoids (e.g. anthocyanins ) or as pre-separation for other analytical and preparative techniques. The separation is based on the distribution of the analytes between two immiscible liquid phases, similar to the separation in a separating funnel (see also partition chromatography ). The separation efficiency is, however, significantly improved by the special apparatus methods. One of the two phases flows through one or more spiral hose systems referred to as coils , while the other phase remains in the coil . Numerous mixing and segregation processes take place here.

The chromatogram is usually recorded using a UV detector with a flow cell or a diode array detector . Coupling techniques similar to LC / MS couplings are also occasionally in use. With the help of a time-controlled fraction collector and the information obtained from the chromatogram, several substances can be preparatively isolated simultaneously.

In addition to the very good separation performance, another advantage of these techniques is the almost complete recovery of the substances used in the event of malfunctions etc.

Disadvantages of the technology are the relatively long separation time and the rather high susceptibility to malfunctions caused by the increased wear of the moving Teflon hoses and the complex apparatus structure.

The countercurrent distribution chromatographic techniques are divided into hydrostatic and hydrodynamic techniques.

Hydrostatic systems

The oldest countercurrent distribution chromatography techniques are coil planet centrifuge ( CPC ) and droplet countercurrent chromatography (DCCC for short).

In DCCC and CPC , a rigid coil filled with one phase of the two-phase mixture represents the stationary phase. The second component, known as the mobile phase, runs through the stationary phase from top to bottom. This results in the distribution processes between the phases, which leads to the separation of the analyte.

The disadvantage is that the flow velocities are low. In comparison to more modern techniques, this results in few mixing processes in most binary solvent systems and a high expenditure of time.

Hydrodynamic systems

More modern variants of countercurrent distribution chromatography are, for example, high-speed countercurrent chromatography ( HSCCC for short ) and low-speed rotating countercurrent chromatography ( LSRCCC , sometimes just LSCCC ).

  • High-speed countercurrent chromatography (HSCCC)

The HSCCC consists of a so-called multi-layer coil . This is a Teflon tube with a volume of around 1000 mL wound around a spool several times. The coil is rotatably mounted around the central axis of a motor and rotatable in the same direction around its own axis. If the central axis is set in rotation, this leads to a planetary movement of the coil. Depending on the speed of rotation, two immiscible liquids in the coil form a special hydrodynamic distribution. The mobile phase is fed into the coil with an HPLC pump , which displaces the stationary phase to the other end. The permanently changing forces acting through the planetary motion lead to frequent mixing and separation processes of the mobile and stationary phases in the separation hose. In the turns near the axis of rotation, both phases are mixed, while they are separated in the turns further away. The permanent distribution of the analytes between the mobile and stationary phase results in approx. 50,000 distribution processes per hour at rotation speeds of 800 to 1000 revolutions per minute. With separation times in the range of several hours, this means that a very efficient separation takes place.

The elution of the separated components depends on the partition coefficient in the selected solvent system . Depending on the stationary phase used, a distinction is made between the head-to-tail and tail-to-head elution modes . If the phase of lower density is used as the stationary phase, it is referred to as head to tail , since the light phase moves towards the end of the head when it rotates. The phase of greater density serves as the mobile phase; the amount of light phase in the coil remains almost constant. Elution takes place at the tail end. If the heavy phase is used as the stationary phase, the elution mode is tail to head .

  • Low-speed rotating countercurrent chromatography (LSRCCC)

A variant of the HSCCC is the LSRCCC . Here, in a coil rotating with 60–80 revolutions around a central axis with a volume of around 5000 mL , the so-called convoluted tubes are mixed with an Archimedean screw - and segregation processes take place between the mobile and stationary phase. Due to the large volume of the coil , this technology enables the isolation of bioactive substances on a gram scale.

literature

  • Yoichiro Ito: Development of Countercurrent Chromatography and Other Centrifugal Separation Methods. In: Haleem J. Issaq (Ed.): A Century of Separation Science. Marcel Dekker, New York 2001, ISBN 0-8247-0576-9 , pp. 293-308.
  • Yoichiro Ito: Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. In: Journal of Chromatography A. Vol. 1065, No. 2, 2005, pp. 145-168, doi : 10.1016 / j.chroma.2004.12.044 .
  • Qizhen Du, Peter Winterhalter, Yoichiro Ito: Large Convoluted Tubing for Scale-Up of Slow Rotary Countercurrent Chromatograph. In: Journal of Liquid Chromatography & Related Technologies. Vol. 26, No. 12, 2003, pp. 1991-2002, doi : 10.1081 / JLC-120021766 .

Individual evidence

  1. ↑ Andreas Degenhardt, Saskia Habben, Peter Winterhalter: Isolation of Physiologically Active Compounds from Nutritional Beverages and Vegetables by Countercurrent Chromatography. In: Fereidoon Shahidi, Deepthi K. Weerasinghe (Ed.): Nutraceutical Beverages. Chemistry, Nutrition, and Health Effects (= ACS Symposium Series. Vol. 871). American Chemical Society, Washington DC 2004, ISBN 0-8412-3823-5 , pp. 443-456.
  2. ↑ Stéphane Vidal, Yoji Hayasaka, Emmanuelle Meudec, Véronique Cheynier, George Skouroumounis: Fractionation of Grape Anthocyanin Classes Using Multilayer Coil Countercurrent Chromatography with Step Gradient Elution. In: Journal of Agricultural and Food Chemistry. Vol. 32, No. 4, 2004, pp. 713-719, doi : 10.1021 / jf034906a .
  3. ^ Ito: Development of Countercurrent Chromatography and Other Centrifugal Separation Methods. In: Issaq (Ed.): A Century of Separation Science. 2001, pp. 293-308.
  4. ↑ Ito: Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. In: Journal of Chromatography A. Vol. 1065, No. 2, 2005, pp. 145-168.
  5. ↑ Du, Winterhalter, Ito: Large Convoluted Tubing for Scale-Up of Slow Rotary Countercurrent Chromatograph. In: Journal of Liquid Chromatography & Related Technologies . Vol. 26, No. 12, 2003, pp. 1991-2002.