counter current chromatography

introduction of ccc,cpc and dccc,applications

1. Counter current chromatography Presented by S. Suma Under the guidance of Mrs. Srividya M .pharm Assistant professor

2. CONTENTS INTRODUCTION PRINCIPLE PROCEDURE INSTRUMENTATION COLUMNS OF CCC TYPES OF CCC DROPLET CCC CENTRIFUGAL PARTITION CHROMATOGRAPHY CHOICE OF SOLVENTS APLLICATIONS OF CCC CONCLUSION REFERENCES

3. I ntroduction Counter-current chromatography is a liquid chromatography that uses two immiscible liquid phases i.e.., one act as stationary liquid and mobile liquid phase and no solid support . The inventor Yochiro Ito(1978) ,named it after the counter current partition method . Definition Counter-current chromatography is a method of distribution of two (or) more substances by repeated distribution between two immiscible liquid phases that flow in opposite directions .

4. Principle Of CCC The principle of separation involves the partition of a solute between two immiscible solvents (mobile phase and stationary phase). The relative proportion of solute passing into each of the two solvents determined by partition coefficient. Here both phases are liquid. Partition of solute in two phases is based on differences in capacity factor, k and distribution coefficient , kd of the analyte .

5. PROCEDURE T he best results are obtained by filtering the sample solution before introduction into the column. The whole ccc is a chromatographic column centrifugal fields imply rotating parts, rotors, gears, spools, motors and speed regulators. Mixing of phases is done so that the analyte can move between the phases. Mobile phase is mixing with then settling from stationary phase throughout the column. Maximum retention of stationary phase is obtained by using less viscous phase as mobile phase.

6. Instrumentation Of CCC

7. Hydrostatic ccc column The very first hydrostatic CCC columns used gravity to maintain the liquid stationary phase; they were called droplet CCC (DCCC) columns. They needed very long elution times (days ).Their two main characteristics are : (1) they have single axis of rotation generating a constant centrifugal field and (2) they enclose geometrical volumes ,tubes , channels, or locules that repeat themselves through connecting tubes forming a pattern. It can be seen that there is quite a significant volume of connecting ducts which only contain the mobile phase .

8. Hydrodynamic ccc column Hydrodynamic centrifuges used in the CCC columns have two rotational axes, a main axis and a planetary one which generates a variable centrifugal force field. There can be any number of planetary axes but the most common are single, double, and triple axes. Each planetary axis has a bobbin or spool mounted on it that contains the coils of continuously wound Teflon tubing.

9. Types o f ccc 1. Droplet counter current chromatography. 2. Elution Extrusion Counter Current Chromatography 3. Centrifugal Partition Chromatography 4. High Speed Counter Current Chromatography.

10. Droplet counter current chromatography 1 . It uses only gravity to move the mobile phase through the stationary phase . 2. In descending mode, droplets of the denser mobile phase and sample are allowed to fall through a column of the lighter stationary phase using only gravity. 3. If a less dense mobile phase is used it will rise through the stationary phase, this is called ascending mode. 4. The eluent from one column is transferred to another; the more columns that are used, the more theoretical plates can be achieved.

11. Centrifugal Partition Chromatography 1 . CPC use CENTRIFUGAL FORCE to speed separation and achieves higher flow rates than DCCC. 2 . The centrifugal partition chromatograph is constituted with a unique rotor(column ). This rotor rotates on its central axis. 3 . With less vibrations and noise, the CPC offers a wider rotation speed range(from 500 to 2000 rpm) . 4. That allows a better decantation and retention for unstable biphasic system.

12. cpc rotor

13. Choice of solvents Critical points in selection are Sample solubility Partition coefficient Chloroform based system (or) Ternary phase diagram is used for the selecting the solvent system

14. - Foucault suggested three criteria to follow ternary phase diagrams -Select the best solvent in which sample can be completely dissolved, - Select two solvents (one is less polar another is more polar), best solvent will partition into the two other solvents, - The less polar fraction of the sample will preferentially go into the less polar phase and the more polar fraction will preferentially go into the more polar phase so that average partition coefficient stay around 1.

15. Solvent systems Biphasic liquid system -octanol and water Tertiary liquid system -chloroform-methanol-water Quaternary liquid system - hexane-ethyl acetate- acetone-water

16. Applications Droplet counter-current chromatography (DCCC) has found wide application for preparative separation of plant constituents and other natural products. It is particularly indicated for the isolation of polar compounds. New fields of applications include essential oils, triterpenoids and steroids, plant growth regulators (gibberellins), antibiotics . Different types of centrifuges are used for seperation of various compounds from plant and different natural products.

17. REFERENCE www.wikipedia.org/wiki/counter-cureent-chromatography http://www.authorstream.com/Presentation/aSGuest111335-1159749-cntrifugal-partion-chromatography / http :// www.tandfonline.com/doi/abs/10.1080/10826070701277067?journalCode=ljlc20 http:// pubs.acs.org/doi/abs/10.1021/jf00076a020 http:// www.slideshare.net/kalpanaraj/abstrct https:// en.wikipedia.org/wiki/Droplet_countercurrent_chromatography https://www.ncbi.nlm.nih.gov/pubmed/7031776