1. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Chapter 9Capillary Electrophoresis (CE) References: Dale R. Baker, Capillary Electrophoresis, John Wiley & Sons, 1995. M.G. Khaledi, Ed., High-Performance Capillary Electrophoresis, John Wiley & Sons, 1998.

2. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 1.An Overview of capillary electrophoresis

3. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/21/2008Chapter 9Capillary Electrophoresis Column offers:  Ease of quantitation  Automation  Fraction collection  On-line coupling to structure specific detectors, including MS, NMR  Jorgenson and Lukacs (Anal. Chem., 1981, 53, 1298) were the first to produce an operational capillary electrophoresis unit and demonstrate its high resolving power.

4. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis

5. 1.1Some concepts  Electrophoresis: is the movement of electrically charged particles or molecules in a conductive liquid medium, usually aqueous, under the influence of an electric field.

6. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis

7.  Electroosmotic flow: Under the influence of an electric field, the buffer and the neutral molecules also move through the tube, due to Electroosmotic flow (we’ll discuss the Electroosmotic flow in more details later).

8. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis

9.  Zone Spreading Theoretically, in electrophoresis, the compounds will travel through the conductive medium as zone that do not diffusion or spread out in the absence of any other influences except for the electric field.

10. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis  Zone electrophoresis refers to the migration of molecules as zones which do not undergo zone spreading due to diffusion. Longitudinal and radical diffusion Do not contribute much zone spreading Molecular diffusion rate in a liquid is relatively small compared to the rate at which they migrate through the liquid

11. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Thermal or convective diffusion Contributes significantly to zone spreading Electric current causes Joule heating Molecules in the warmer, center of a tube migrate faster than those near the cooler wall, leading to zone spreading Minimize the amount of heat generated and dissipate that heat. In addition to causing zone spreading, high T may also cause thermal degradation of some molecules

12. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Influence of tube diameter on convective diffusion In a round tube, the temperature difference between the center and the wall of the tube,  T, can be calculated from:  T = (0.239Q/4k)r 2 [1] where Q is the power density in watts/m 3, k is thermal conductivity of the solution, and r is tube radius.

13. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/21/2008Chapter 9Capillary Electrophoresis Minimize convective diffusion: decrease diameter of the capillary –Less current is generated for a given voltage, and less joule heat is produced. –Fast dissipation of the heat (increase in the inner surface area-to- volume ratio of the tube). Therefore: –Narrow capillary has been selected: 50 -75  M i.d. –400,000 theoretical plates with 80-100 cm long (20,000 by HPLC)

14. CE is referred to: Capillary electrophoresis is sometimes referred to as Capillary zone Electrophoresis (CZE), Free solution Capillary Electrophoresis (FSCE) High-performance Capillary Electrophoresis (HPCE) Capillary Electrophoresis (CE). Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/21/2008Chapter 9Capillary Electrophoresis

15. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 1.2CE system overview

16. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis

17. CE Electropherograms and HPLC chromatograms Assuming equal solute concentrations and detector responses

18. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis HPLC chromatograms: In isocratic HPLC, the longer the retention time, the broader and the shorter the peaks. This is because solutes are diluted more as they spend more time inside the column. Area of all the peaks are approximately the same since all solutes through the detector at the same rate.

19. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis CE Electropherograms Peak height remain constant as retention gets longer because the solutes move through the detector in zones of approximately the same length and, therefore, the same concentration. The peak get wider with time because the later eluting solutes move through the detector more slowly, and consequently, reside in the detector cell longer. For equal concentrations and detector responses, the peak areas increase with time.

20. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Sample injection: Hydrodynamic: by pressure or siphoning (gravity). Electrokinetic: an electric field is applied to the sample vial, causing the sample components to migrate into the capillary.

21. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/21/2008Chapter 9Capillary Electrophoresis Capillaries: Fused silica capillaries (30-100 cm long with inner diameters of 50-75  m and outer diameters of 375  m). Detectors: UV/Vis, Fluorescence, conductivity, MS, ICP/MS, NMR (new)...... Power supply: Voltages up to 30 kV, currents up to 300  A, and power up to 6 W.

22. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 1.3Comparison of CE to other separation techniques  Efficiency Efficiency, N, expressed as the number of the theoretical plates, is related to how narrow the peaks are in a chromatogram or electropherogram. N = 16(t/w) 2 The narrower the peak, the higher the efficiency, and the better the separation. CE has very high efficiency compared to HPLC and GC.

23. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis  Sample type HPLC and GC are complementary. HPLC and CE are more competitive with each other. CE can use buffers that covers a wide pH range, whereas most silica-based HPLC column cannot be used with mobile phases at pH above about eight.  Sample volume CE: the relatively small volume (few nl) of sample. A capillary that is 50 cm long and 50  m i.d. has a volume of only 0.98  l.

24. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis  Detection limit The Concentration detection limit of CE is not as good as HPLC and GC, roughly 100-1000 times higher. Confused about: Concentration detection limit Instrument detection limit Sensitivity  Reagent requirements Compared to HPLC, CE requires much less amount solvents, typically a few ml for a day of analysis.

25. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.Principles 2.1Electroosmotic flow (EOF) In CE, in addition to the solutes, the buffer solution usually also moves through the capillary under the influence of an electric field. This phenomenon is termed electroosmotic flow. In normal operation, the direction of EOF is toward the negatively charged cathode. 2.1.1Benefits of EOF Separate anions and cations in a single run. Neutral solutes would not move through the capillary tube. Reduces the analytical time

26. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis

27. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/23/2008Chapter 9Capillary Electrophoresis 2.1.2Formation of EOF Silanol groups have strong affinity for polar organic molecules. Can be deactivated by silanization with dimethylchlorosilane (DMCS).

28. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis solvated

29. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis solvated http://www.chemsoc.org/ExemplarChem/entries/2003/leeds_chromatograph y/chromatography/eof.htm The build up of ions at the capillary wall (image courtesy of Agilent Technologies)Agilent Technologies

30. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Silanol (Si-OH) groups are ionized to negatively charged silanoate (Si-O-) groups at pH above about three. This ionization can be enhanced first by passing a basic (KOH or NaOH) solution through the capillary followed by the buffer. The negatively charged silanoate groups then attract positively charged cations from the buffer, which form an inner layer of cations at the capillary wall. These cations are not of sufficient density to neutralize all the negative charges, so a second, outer layer of cations forms. The inner layer is tightly held by the Si-O- groups and is referred to as the fixed layer. The outer layer of cations is not tightly held because is it further away from the silanoate groups, and it is referred to as mobile layer. These two layers make up the diffuse double layer of cations. When an electric field is applied, the mobile, outer layer of cations is pulled toward the negatively charged cathode. Since these cations are solvated, they drag the bulk buffer solution with them, thus causing EOF.

31. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Zeta potential (  ): is an electrical imbalance created at the plane of shear, which is the potential difference across the layers.  = 4  e/  where  is the thickness of the diffuse double layer, e is the charge per unit surface area,  is the dielectric constant of the buffer. EOF is proportional to the zeta potential.

32. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.1.3EOF Velocity and Mobility The velocity, v EOF, v EOF =   E/4  where E is the applied electric field in volts/cm, and  is the viscosity of the buffer. The mobility,  EOF,  EOF =   /4  Note that  EOF is dependent solely on buffer characteristics, and independent of the applied electric field.

33. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Significant parameters that affect EOF: (1)Applied Voltage Migration time Currents and Joule heating Zone spreading.

34. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/23/2008Chapter 9Capillary Electrophoresis Excessive heat produced Resistance goes down Causing increase in current Question? Effects of capillary length and diameter on maximum voltage selection

35. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis (2) Buffer pH Changes zeta potential. As pH increases, EOF increases, primarily because at higher pH, there more dissociation of Si-OH to Si-O- on the inner surface of the capillary. Influence the degree of ionization of the solutes and hence their mobility. Must consider the effect on separation

37. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/23/2008Chapter 9Capillary Electrophoresis (3) Buffer concentration Effect on zeta potential The run buffer concentration should be 100 times that of sample, typically 10-100 mM. Question? Why cannot the buffer concentration be too low?

38. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/23/2008Chapter 9Capillary Electrophoresis (4) Temperature Effect on viscosity of the buffer. A temperature increase of 1  C, from 20 to 21  C, reduces the viscosity of water by 2.4%.  = 4  e/   EOF =   /4  Temperature has effect on , but cancelled (see above equations). Zone spreading Compound decomposition

39. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.1.4Measurement of EOF  Neutral marker: Requirement of neutral markers: Uncharged under the pH of the buffer Detectable by detector No interaction with the capillary and the buffer.

40. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis  Current monitoring: The capillary and destination vials are filled with buffer, and the source vial filled with the same buffer, but at a slightly different concentration. Current Time

41. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.1.5Reverse EOF “Normal” CE Reverse EOF

42. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Detector a c Eletrophoretic mobility of formate ion: 5.7 x 10 -4 cm 2 /V EOF: 4.2 x 10 -4 cm 2 /V

43. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Why and How

44. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.2Electrophoretic mobility E lectrophoretic velocity, ν EP, in cm/s ν EP =  EP E[1]  EP :eletrophoretic mobility E:applied electric field.

45. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Electrophoretic mobility  EP  EP = q/6  r[2] q:the charge of the ionized solute  :buffer viscosity r:solute radius ν EP is dependent on both mobility and electric field.  EP is dependent only on solute and buffer properties Neutral molecules:  EP = 0

46. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis A solute’s velocity is influenced both by its velocity of EOF, v EOF and ν EP. The observed electrophoretic velocity, v OBS v OBS = v EOF + v EP [3] In “normal” CE, that is the detector is on the negatively charged side, and EOF is from source to detector. Anions:v OBS < v EOF Cations: v OBS > v EOF Neutals: v OBS = v EOF

47. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis The observed electrophoretic mobility,  OBS  OBS =  EOF +  EP [4]

48. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.2.1Measure electrophoretic velocity and mobility v OBS = l/t m [5] t m : migration time l: the effective length of capillary, from inlet to detector. The electroosmotic velocity, v EOF, can be determined by measuring the migration time of a neutral marker, t nm. Then v EP = l/t m - l/t nm [6]

49. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Electrophoretic mobility: E=V/L V:is the voltage L:is the total length v EP =  EP E  EP = v EP L/V[7]  EP = (l/t m - l/t nm )(L/V)[8]

50. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.2.2Parameters influencing  EP  EP = q/6  r Solute charge Solute size Buffer viscosity

51. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.3Effects of electrophoretic parameters on separation Chromatographic Parameters

52. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis

53. 2.3.1Migration Time (t m ) t m = l/ v OBS [9] l:effective capillary length v OBS =  OBS E[10] t m = l/(  OBS E) [11] t m = lL/(  OBS V) [12] t m = lL/[(  EP +  EOF )V] [13]

54. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis  2.3.2Efficiency Definition of plate height in Chromatography

55. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis H =  2 /L [14] The area of triangle is ~96% of total area under the peak. (± 2  ) W = 4 

56. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Since W = 4  [16] [17] Because H =  2 /L, H = L/N, N = L 2 /  2 [18]

57. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis N = 16(t R /w) 2 [19] Or N = 5.54(t R /w 1/2 ) 2 [20] Same for CE: N = 16(t m /w) 2 [21] Or N = 5.54(t m /w 1/2 ) 2 [22] Efficiency, N, is expressed as the number of theoretical plates. w: peak width measured at the base of the peak w 1/2: peak width measured at the half the peak height

58. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis The amount that a zone spreads is given by its spatial variance,  2, which is how much an infinitely thin zone will diffuse over time, t:  2 = 2Dt[23] D: the solute’s diffusion coefficient in cm 2 /s.

59. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Assuming that all adsorption of sample, and no radial diffusion, Substituting Eq 13 for t in [23]: t m = lL/[(  EP +  EOF )V] [13]  2 = 2DlL/[(  EP +  EOF )V] [24] Because N = L 2 /  2 Substituting Eq 24 for  2 : N =(  EP +  EOF )V/2D[25]

60. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.3.3Selectivity (relative migration rates) In HPLC, selectivity, , is given by  = (t 2 -t o )/(t 1 -t o ) A similar expression as that used in Chromatography can be used for in CE.  = (t 2 -t nm )/(t 1 -t nm )[26] where t 2 and t 1 are migration times of adjacent peaks, and t nm is the migration of a neutral marker.

61. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis 2.3.4Resolution The most important separation parameter is resolution, that is, how well the components in a mixture are separated. Resolution, R, can be calculated from an electropherogram using R =  t/w AVE R = 2(t 2 -t 1 )/(w 1 +w 2 )[27] where w 1 and w 2 are peak widths of adjacent peaks.

62. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Recall in chromatography, we assume W A = W B = W R = [(t R ) B – (t R ) A ]/W N = 16(t R /W) 2

63. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis In CE? James W. Jorgenson et al. Anal. Chem. 1981, 53, 1298- 1302. James W. Jorgenson et al.,Science, 1983, 222,266-272. J.C. Giddings, Separation Sci. 1969, 4, 181.

64. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis