1. Rafal Gajos, Adam Chomicki, Beata Polak and Tadeusz H. Dzido Medical University of Lublin, Poland International Conference and Exhibition on Advances in HPLC & Chromatography Techniques – its application to micro-preparative separation The Orthogonal Pressurized Planar Electrochromatography (OPPEC)

2. Outline OPLC in combination with PPEC for simultaneous 2D separation = OPPEC OPPEC as a example of 2D separation technique OPPEC - device and its principle of action Examples of micropreparative OPPEC separation The concern of two-dimensionality in preparative mode of the OPPEC separation Conclusion

3. PPEC – Pressurized Planar Electrochromatography OPLC – Overpressured Layer Chromatography OPPEC – Orthogonal Pressurized Planar Electrochromatography Overpressure Layer Chromatography (OPLC) in combination with Pressurized Planar Electrochromatography (PPEC)

4. 2D simultaneous separation in the OPPEC technique Successful of 2D separation is based on different selectivity in each separation system involved Two dimensional process seems to be especially attractive for continuous separation liquid chromatography electrophoresis

5. Orthogonal pressurized planar electrochromatography (OPPEC) – device

6. 1 – OPPEC chamber, 2 – microinjection valve, 3 – sample syringe pump, 4 – mobile phase pomp, 5 – hydraulic pump, 6 – high voltage power supply, 7 – thermostat, 8 – fraction collector, 9 – security cabinet Conceptual view of the device for OPPEC The OPPEC equipment comprises of:

7. The picture of the complete device for OPPEC

8. The OPPEC chamber

9. Special sealing margin sealing margin chromatographic plate The chromatographic plate is equipped with special sealing margin on the whole periphery of its adsorbent layer

10. OPPEC - its principle of action

11. Principle of action of the OPPEC chamber The schematic view of the OPPEC device for application to micro-preparative separation 1 - continuous feeding the sample solution for the separation system, 2 - sample inlet capillary, 3 - continuous collecting the separated fraction at the outlet of the separation system, 4 - solute bands, 5 - direction of mobile phase flows, 6 – outlet capillary, 7 – plate’s areas in which the eluate is carrying from the separation system, 8 - reservoirs of a fraction collector, 9 - metal plate with circulating cooling medium inside, 10 - chromatographic plate, 11 – electrodes.

12. Examples of micropreparative (continuous) OPPEC separation

13. Separation of mixture dyes The picture of the chromatographic plate after continuous an OPPEC separation of the test mixture of three components (PAR, AZ and PB), HPTLC RP 18W plates from Merck and 45% methanol in buffer pH 3.2 as the mobile phase, U = 1.5 kV, t = 150 min. the numbers of reservoirs in which collected colored fractions are consistent with the plate’s areas in which the eluate was carried from the separation system The digital photograph of collected colored fractions plate’s areas in which the eluate was carried from the separation system – +

14. The purity test of the collected fractions The picture of the chromatographic plate after the TLC separation of eight fractions of the mobile phase (labeled from 1 to 8), and standard solution (labeled as W), A - direction of the chromatogram development the collected fractions proved to be pure the deviation angles of the solute bands were constant and individual components, which left the separation system, did not mix with each other

15. Separation of amino acids – + The picture of the chromatographic plate after continuous an OPPEC separation of the test mixture of two components, HPTLC RP 18W plates from Merck and 10% acetonitrile in buffer pH 3.2 as the mobile phase, U = 2.0 kV, t = 60 min. 1 – phenylalanine (pI = 5.48) 2 – aspartic acid (pI = 2.77 )

16. The concern of two-dimensionality in preparative mode of the OPPEC separation

17. Two-dimensionality in the preparative mode of the OPPEC separation The picture of the chromatographic plate after OPPEC separation of the test mixture of three components (PAR, GS and PB), HPTLC RP 18W plates from Merck and 45% methanol in buffer pH 3.2 as the mobile phase, U = 1.5 kV, t = 18 min.

18. Two-dimensionality in the preparative mode of the OPPEC separation The picture of the chromatographic plate after continuous an OPPEC separation of the test mixture of three components (PAR, GS and PB), HPTLC RP 18W plates from Merck and 45% methanol in buffer pH 3.2 as the mobile phase, U = 1.5 kV, t = 60 min. PAR PB + GS

19. Two-dimensionality in the preparative mode of the OPPEC separation The picture of the chromatographic plate after the TLC separation, A - direction of the chromatogram development cutting line PAR PB + GS

20. cutting line Two-dimensionality in the preparative mode of the OPPEC separation 2 3 A PB + GS PAR T 1 = 20 min T 2 = 15 min T 3 = 10 min Despite that these substances formed a single band, they are partially separated when the preparative mode of the OPPEC separation is applied. This means that these substances are separated as a result of chromatografic effect. 1, 2 and 3 - migration distance for a for a different time of the experiments, A - direction of the chromatogram development 1 A A

21. the obtained results proves the two-dimensionality in the preparative mode of OPPEC separation, the substances which have the same electrophoretic mobility can be separated, because their solutes usually demonstrate different retention and then these undergo separation based on principle of frontal chromatography, this approuch can be used for enantiomer separation. Two-dimensionality in the preparative mode of the OPPEC separation

22. Conclusion Combination of chromatography and electrophoresis techniques into a single separation technique in which both processes proceed simultaneously and orthogonally seems to be a attractive mode of 2-D separation. The OPPEC can be used for enantiomer separation, based on principle of frontal chromatography. The results presented demonstrate the high potential of the OPPEC technique for preparative separation.

23. Thank you for your attention !!!