1. Droplet Counter-Current Chromatography prof. aza Department of Pharmacy, Andalas University STIFI Perintis, Padang STIFAR, Pekan Baru STIFI Bhakti Pertiwi, Palembang

2. Introduction DCCC is a liquid-liquid separation technique relying on the partition of the solute between two immiscible solvents, the relative proportions of solute passing into each of the 2 phases being determined by the respective partition coefficient. The method, developed by Tanimura et al (1970), exploits the observation that a light liquid phase with low wall surface affinity forms discrete droplets that rise through a heavy phase with visible evidence of very active interfacial motion. Under ideal condition, each droplet could become a plate if kept more or less discrete through the system. By partitioning a solute between the stationary phase and the droplets a separation is achieved.

3. Schematic illustration of the principle of DCCC Apparatus

4. Apparatus, continued In the typical commercially available DCCC apparatus, consisting of 200 to 600 vertical column (20-60cm in length) of narrow-bore silanized glass tubing, interconnected in series by capillary teflon tubes, droplets of mobile phase are passed through the columns filled with stationary phase. Collection of mobile phase is effected at the end of the battery of columns. The sample, dissolved in the lighter phase or the heavier phase or in a mixture of both phase, is injected into the sample chamber. Mobile phase is subsequently pumped via the sample chamber into the first of the columns, displacing the sample and forming a stream of droplets in the immiscible stationary phase. Depending on the choice of solvents for the mobile phase and stationary phases, these droplets are caused either ascend or descend.

5. Apparatus, continued As the mobile phase moves through the column in the form of droplets, turbulence within the droplets promotes efficient partitioning of the solute between the two phases. Separation occurs according to the difference in the partition coefficient of the components of the sample. The size and mobility of the droplets are functions of the column bore, the flow rate of the mobile phase, the diameter of the inlet tip, the difference in specific gravities of the two liquid phases, the viscosity of the solvents and surface tension. Columns with an internal diameter of 2 mm are most often used for separations but internal diameters of 2.7, 3.0. 3.4 mm are now commercially available

6. Fig.6.2 shows that increasing the flow rate from 18 ml/h to 50 ml/h actually leads to an increase in resolution from 120 to 240 theoretical plate.

7. Choice of Solvents Binary solvent systems are impractical for the formation of suitable droplets because of the large difference in polarity between the two components. Ternary or quaternary systems are required for the preparation of the two phases, such that the addition of a third or fourth component, miscible with the other components, diminishes the difference in polarity between the two phases. The selectivity of the systems is thus increased, the interfacial tension and the viscosity of the system should diminished by addition of the third component.

8. Substance classBasic binary systemAuxiliary solvent Very polar Polar Lipophilic n-Butanol-water Chloroform-water n-Heptane-methanol n-Heptane-acetonitrile Methanol, ethanol, acetone, acetic acid (pyridine) Methanol, n- propanol, i-propanol Chlorinated hydrocarbons, acetone Solvent systems for droplet counter-current chromatography

9. Choice a solvent system A tlc on silica gel is run, with water-saturated organic layer as eluen. If the R f values of the compounds to be separated are higher than about 0.50 (less polar solutes), given that there is a suitable separation of the components of the mixture, the less polar layer is suitable for use as the mobile phase. For more polar solutes (Rf < 0.50), the more polar layer should be used as the mobile phase. When the R f values lie in the range 0.40 – 0.60, the separation can be achieved by using either the more polar layer or the less polar layer as the mobile phase. If the R f values of the compounds to be separated are either too low (< 0.20-0.30) or too high 0.70-0.80 no elution will take place in reasonable time.

10. Choice a solvent system When non-aqueous DCCC systems containing n- heptane are being investigated, chromatography on RP-8 HPTLC plates with the lower layer is employed for analysis of samples. An alternative method of selecting suitable solvent is to monitor the distribution of 5 to 10 mg sample between 5 to10 ml of each of the two phases constituting the solvent system. A system in which 15-25% of the samples the distributed in one of the phases is chosen. The stationary phase is chosen as the phase that contain the most sample.

11. Choice of DCCC modes by TLC, Support Silica gel, Eluent: less polar layer of two-phase solvent system

12. DCCC of a crude extract of Hedera helix berries (1,2 g) with CHCl3-MeOH-H2O) 7:13:18, mobile phase lower layer.

13. Hedera helix L. (Araliaceae)

14. Choice a solvent system A tlc on silica gel is run, with water- saturated organic layer as eluent. If the R f values of the compounds to be separated are higher than about 0.50 (less polar solutes), given that there is a suitable separation of the components of the mixture, the less polar layer is suitable for use as the mobile phase.

15. DCCC elution chromatogram of fractionated croton oil (510 mg), using hexane-diethyl ether-n-propanol-95% ethanol-water (4:8:3:5:4) in descending mode, I =solvent front, II = 4 a -phorbol, III = phorbol

16. DCCC of a crude extract of Ajuga pyramidalis (1.4g) with CHCl3- MeOH-H2O = 43:37:20 by ascending method (mobile phase: upper layer).

17. DCCC separation of xanthone O-glycosides with CHCl3-MeOH-PrOH –H2O=9:12:1:8), a)ascending mode, b) descending mode

18. Rotation Locular Counter-current Chromatography prof. aza

19. Description of the Method This technique, like DCCC, relies on liquid- liquid partition, and thus on a two-phase systems, for separation of a mixture (Signar et al, 1956). The RLCC apparatus consists of 16 glass columns (50cmx1.1 cm.id) arranged around a rotational axis and connected in series with teflon tubing (1 mm.id). Both the speed of rotation of the columns and their angle of inclination can be varied.

20. Description of the Method, continued Each column is divided into 37 compartments (loculi) by teflon disc and as each disc is perforated by a small hole (1 mm diameter) in the centre, solvent can flow between the compartments. The columns are moved to the vertical position and stationary phase is pumped into each column in turn from the bottom, using a constant flow pump connected to the inlet tube. At the same time, the set of 16 columns is rotated about the central axis and care must be taken to remove all air bubbles from the loculi. The columns are then usually tilted to an angle of 20- 40 0 with the horizontal and system phase is pumped into the

21. Description of the Method, continued While the mobile phase enters the columns at a rate of 15-50 ml/h, the columns rotate about the central axis at 60-80 rpm. The liquid entering the first loculus displaces stationary phase until it reaches the level of the hole leading to the next loculus. The lighter, mobile phase then passes into the second loculus and so the process continuous until the mobile phase emerges from the uppermost loculus. It is then directed to the bottom of the next column and the displacement of stationary phase is repeated until all the columns are charged with mobile phase.

23. Solvent Selection Contrary to DCCC, the formation of droplets in a two-phase solvent system is not a necessary condition of RLCC. Consequently, a wide range of solvent system is available. In addition, ethyl acetate-water system, which are often incompatible with DCCC, present no problem in RLCC. Binary systems ma be employed but, for reasons of selectivity, ternary or quaternary systems are preferred.

24. Solvent Selection, continued The selection of appropriate biphasic solvent systems can be performed by TLC, in which each of the two phases is used in turn. If the sample has R f values greater than 0.8 in one phases and between 0.2 and 0.4 in the other phase, the solvent system is suitable for separation. A small amount e.g. 5 mg of sample is subsequently mixed with 5-10 ml of each of the two phases and a solvent system is chosen such that 15-25% of the sample remains in one phase. The phase containing the majority of sample is chosen as the stationary phase.