Organic-High Ionic Strength Aqueous Two-Phase Solvent System Series
illustrates compositions of a set of 21 two-phase solvent systems which are arranged from the top to the bottom in an order of increasing polarity. Important physical properties of the solvent system such as a volume ratio between the two phases, settling time, and density difference between the two phases are also presented. All systems show acceptable volume ratios between the two phases. The settling times of all these systems are longer that 20 seconds suggesting that the conventional multilayer coil may not be efficiently used for separation. However, the spiral tube configuration used in the present studies can retain a satisfactory volume of the stationary phase at over 50% by utilizing the centrifugal force gradients acting along the spiral channel. A large difference in density between the two phases due to a high concentration of ammonium sulfate in the lower phase also enhances the retention of the stationary phase in the spiral column. A set of these polar solvent systems can be used to determine the suitable two-phase solvent systems for a single compound or a multiple sample mixture by only two steps of K measurements using a simple graphic analysis as described in the following sections. Although lower phase fraction contains a large amount of ammonium sulfate, it is largely removed by adding methanol followed by centrifugation.
Graphic Selection of Two-Phase Solvent System for a Single Compound
A suitable K value for separation of a single target compound is near unity. In order to achieve this goal, one may often spend an enormous amount of time to optimize the solvent system. However, the ideal two-phase solvent system can be easily found from the solvent series listed in by two-step K measurements followed by a simple graphic analysis as follows: For each sample prepare three two-phase solvent systems, Systems 0, 10 and 20 in . Then, measure the partition coefficient of the sample in System 10 to get K10. If the sample is a pure standard as in the present study, K value may be determined with a spectrophotometer at a suitable wavelength. If the sample is a crude mixture, the standard HPLC method should be used to get the K value of the target compound. Then if K10 is greater than 1, the second K measurement is performed in System 0 to get K0. If K10 is above 1, the second measurement is performed in System 20 to get K20. Then, these two Log K values are plotted in the graph as shown in . Then, these two points were connected with a straight line to get the crossing point to the Log K = 0 line which indicates the ideal solvent system for the separation of this compound. Several compounds including tartrazine, methyl green, tryptophan, and catecholamines have been tested, the results of which are shown in . It should be noted that all these compounds are not efficiently separated by the standard HSCCC method with existing two-phase solvent systems due to their low K values.
Graphic selection of two-phase solvent system for single polar compounds by two-step partition coefficient measurements. Optimum K values and solvent systems are listed in .
Optimized Log K at the crossing point by a graphic method
Graphic Selection of Two-Phase Solvent System for Multiple Target Compounds
The present method can also be applied to the selection of the two-phase solvent system for separation of multiple compounds as follows: The sample mixture is first distributed in System 10. If the averaged K values are greater than 1, the second measurement is performed in System 0. But if the averaged K value is smaller than 1, the second measurement is performed in System 20. In either case, a pair of log K values for each target compound is plotted on the graph and connected with a straight line. In , a set of three sulfonic acids, P3S, HP3S and P4S were each plotted according to this procedure. From this graph the separation of these three compounds is easily visualized by distribution of three lines at each solvent system. In this case System 8 or 9 will provide satisfactory peak resolution between three compounds. shows the separation of D&C Green No. 8 which contains three compounds at a flow rate of 1 ml/min at 800 rpm.
Figure 2 Graphic selection of two-phase solvent system for a set of sulfonic acids by two-step partition coefficient measurements. Sample: 1,3,6-pyrenetrisulfonic acid (P3S), 8-hydroxy-1,3,6-pyrenetrisulfonic acid (HP3S) and 1,3,6,8-pyrenetetrasulfonic acid (P4S) (more ...)
Figure 3 Separation of sulfonic dyes by spiral HSCCC with Solvent system 9. Experimental conditions: apparatus: type-J HSCCC coil planet centrifuge with 10 cm revolution radius; separation column: 1.6 mm ID, flat-twisted PTFE spiral column with 70 ml capacity; (more ...)