1. CHAPTER 21 SUPERCRITICAL-FLUID CHROMATOGRAPHY, CAPILLARY ELECTROPHORESIS, AND CAPILLARY ELECTROCHROMATOGRAPHY Introduction to Analytical Chemistry

2. Copyright © 2011 Cengage Learning 21-2 21A-1 Important Properties of Supercritical Fluids A supercritical fluid is formed whenever a substance is heated above its critical temperature. For example, carbon dioxide becomes a supercritical fluid at temperatures above 31°C.

3. Copyright © 2011 Cengage Learning 21-3 Table 21-1

4. Copyright © 2011 Cengage Learning 21-4 21A-1 Important Properties of Supercritical Fluids An important property of supercritical fluids related to their high densities is their ability to dissolve large nonvolatile molecules.

5. Copyright © 2011 Cengage Learning 21-5 21A-2 Instrumentation and Operating Variables Instruments for supercritical-fluid chromatography are similar in design to high-performance liquid chromatographs except that provision is made in the former for controlling and measuring the column pressure.

6. Copyright © 2011 Cengage Learning 21-6 21A-2 Instrumentation and Operating Variables The Effect of Pressure The density of a supercritical fluid increases rapidly and nonlinearly with pressure increases. Density increases also alter retention factors (k) and thus elution times.

7. Copyright © 2011 Cengage Learning 21-7 21A-2 Instrumentation and Operating Variables Columns Both packed columns and open tubular columns are used in supercritical fluid chromatography. Mobile Phases The most widely used mobile phase for supercritical- fluid chromatography is carbon dioxide. It is an excellent solvent for a variety of nonpolar organic molecules.

8. Copyright © 2011 Cengage Learning 21-8 21A-2 Instrumentation and Operating Variables Detectors A major advantage of supercritical-fluid chromatography is that the sensitive and universal detectors of gas-liquid chromatography are applicable to this technique as well.

9. Copyright © 2011 Cengage Learning 21-9 21A-3 Supercritical-Fluid Chromatography versus Other Column Methods Thus, like gas chromatography, supercritical-fluid chromatography is inherently faster than liquid chromatography. The intermediate diffusivities and viscosities of supercritical fluids result in faster separations than are achieved with liquid chromatography accompanied by less zone spreading than is encountered in gas chromatography.

10. Copyright © 2011 Cengage Learning 21-10 21B Capillary Electrophoresis Electrophoresis is a separation method based on the differential rates of migration of charged species in an applied dc electric field. A particular strength of electrophoresis is its unique ability to separate charged macromolecules of interest to biochemists, biologists, and clinical chemists.

11. Copyright © 2011 Cengage Learning 21-11 21B-1 Instrumentation for Capillary Electrophoresis A buffer-filled fused-silica capillary, extends between two buffer reservoirs. A potential difference of 5- to 30-kV dc is applied. Sample introduction is often accomplished by pressure injection. Alternatively, a vacuum is applied at the detector end of the tubing. Introduction may also be carried out by electroosmosis.

12. Copyright © 2011 Cengage Learning 21-12 Figure 21-4 Figure 21-4 Schematic of a capillary zone electrophoresis system.

13. Copyright © 2011 Cengage Learning 21-13 21B-2 Electroosmotic Flow The cause of electroosmotic flow is the electric double layer that develops at the silica/ solution interface. At pH values higher than 3, the inside wall of a silica capillary is negatively charged due to ionization of the surface silanol groups (Si － OH). 21-13

14. Copyright © 2011 Cengage Learning 21-14 Figure 21-5 Figure 21-5 Charge distribution at a silica /capillary interface and resulting electroosmotic flow. (From A. G. Ewing, R. A. Wallingford, and T. M. Olefirowicz, Anal. Chem., 1989, 61, 294A. With permission.)

15. Copyright © 2011 Cengage Learning 21-15 21B-2 Electroosmotic Flow The cations in the diffuse outer layer of the double layer are attracted toward the cathode. Since these cations are solvated, they drag the bulk solvent along with them. As shown in Figure 21-6, electroosmosis leads to bulk solution flow that has a flat profile across the tube because flow originates at the walls. 21-15

16. Copyright © 2011 Cengage Learning 21-16 21B-2 Electroosmotic Flow Because the profile is essentially flat, electroosmotic flow does not contribute significantly to band broadening. The rate of electroosmotic flow is generally greater than the electrophoretic migration velocities of the individual ions and effectively becomes the mobilephase pump of capillary zone electrophoresis.

17. Copyright © 2011 Cengage Learning 21-17 21B-2 Electroosmotic Flow The electroosmotic flow rate is usually sufficient to sweep all positive, neutral, and even negative species toward the same end of the capillary. 21-17

18. Copyright © 2011 Cengage Learning 21-18 Figure 21-6 Figure 21-6 Flow profiles for liquids under (a) electroosmotic flow and (b) pressure- induced flow.

19. Copyright © 2011 Cengage Learning 21-19 Figure 21-7 21-19 Figure 21-7 Velocities in the presence of electroosmotic flow. The length of the arrow next to an ion indicates the magnitude of its velocity; the direction of the arrow indicates the direction of motion. The negative electrode would be to the right and the positive electrode to the left of this section of solution.

20. Copyright © 2011 Cengage Learning 21-20 21B-3 The Basis for Electrophoretic Separations E is the electric field strength in volts per centimeter, V is the applied voltage, L is the length of the tube between electrodes, and μ e is the electrophoretic mobility. (21-1)

21. Copyright © 2011 Cengage Learning 21-21 21B-3 The Basis for Electrophoretic Separations The plate count N of a capillary electrophoresis column is given by where D is the diffusion coefficient of the solute (cm² s¯¹). Note that for electrophoresis, contrary to the situation in chromatography, the plate count does not increase with the column length. (21-2)

22. Copyright © 2011 Cengage Learning 21-22 21B-4 Applications of Capillary Electrophoresis Capillary electrophoretic separations are performed in several ways called modes. These include isoelectric focusing, isotachophoresis, and capillary zone electrophoresis (CZE). Figure 21-8 illustrates the unsurpassed quickness and resolution of electrophoretic separations of small anions.

23. Copyright © 2011 Cengage Learning 21-23 Figure 21-8 Figure 21-8 Electropherogram showing the separation of 30 anions. Capillary internal diameter: 50 μm (fused silica). Detection: indirect UV, 254 nm. Peaks: 1 = thiosulfate (4 ppm), 2 = bromide (4 ppm), 3 = chloride (2 ppm), 4 = sulfate (4 ppm), 5 = nitrite (4 ppm), 6 = nitrate (4 ppm), 7 = molybdate (10 ppm), 8 = azide (4 ppm), 9 = tungstate (10 ppm), 10 = monofluorophosphate (4 ppm), 11 = chlorate (4 ppm), 12 = citrate (2 ppm), 13 = fluoride (1 ppm), 14 = formate (2 ppm), 15 = phosphate (4 ppm), 16 = phosphite (4 ppm), 17 = chlorite (4 ppm), 18 = galactarate (5 ppm), 19 = carbonate (4 ppm), 20 = acetate (4 ppm), 21 = ethanesulfonate (4 ppm), 22 = propionate (5 ppm), 23 = propanesulfonate (4 ppm), 24 = butyrate (5 ppm), 25 = butanesulfonate (4 ppm), 26 = valerate (5 ppm), 27 = benzoate (4 ppm), 28 = l- glutamate (5 ppm), 29 = pentanesulfonate (4 ppm), 30 = d-gluconate (5 ppm). (From W. A. Jones and P. Jandik, J. Chromatogr., 1991, 546, 445. With permission.)

24. Copyright © 2011 Cengage Learning 21-24 Figure 21-11 21-24 Figure 21-11 CZE separation of a model protein mixture. Conditions: pH 2.7 buffer; absorbance detection at 214 nm; 22 kV, 10 A. Peaks are identified in the following table.

25. Copyright © 2011 Cengage Learning 21-25 21C Capillary Electrochromatography In CEC, a mobile phase is transported across a stationary phase by electroosmotic flow.

26. Copyright © 2011 Cengage Learning 21-26 21C-1 Packed Column Electrochromatography Electrochromatography based on packed columns. A capillary that is packed with a reversedphase HPLC packing.

27. Copyright © 2011 Cengage Learning 21-27 21-C2 Micellar Electrokinetic Capillary Chromatography A modification of the method that permits the separation of low-molecular-weight aromatic phenols and nitro compounds. In this technique, surfactants are added to the operating buffer in amounts that exceed the critical micelle concentration.

28. Copyright © 2011 Cengage Learning 21-28 21-C2 Micellar Electrokinetic Capillary Chromatography The surface of an ionic micelle of this type has a large negative charge, which gives it a large electrophoretic mobility. Most buffers, however, exhibit such a high electroosmotic flow rate toward the negative electrode that the anionic micelles are carried toward that electrode also, but at a much reduced rate.

29. Copyright © 2011 Cengage Learning 21-29 21-C2 Micellar Electrokinetic Capillary Chromatography When a sample is introduced into this system, the components distribute themselves between the aqueous phase and the hydrocarbon phase in the interior of the micelles. The positions of the resulting equilibria depend on the polarity of the solutes.

30. Copyright © 2011 Cengage Learning 21-30 THE END