1. Lecture 9 Introduction to Capillary Electrophoresis Lecture 9- PHCM662-SS2016 Dr. Rasha Hanafi 1© Dr. Rasha Hanafi, GUC

2. Lecture 9- PHCM662-SS2016 By the end of this session, the student should be able to: 1.Define general theory of capillary electrophoresis (CE). 2.Compare between CE and chromatography. 3.Define electrophoretic/electroosmotic mobilities, apparent mobilities, migration velocity. 4.Investigate the pumping mechanism in CE. 5.Compare between electroosmotic and hydrodynamic flow profiles. 6.Investigate the effects of electrophoresis and electroosmosis on CE. 2© Dr. Rasha Hanafi, GUC LEARNING OUTCOMES

3. Electrophoresis is a separation method based on the differential rate of migration of ions by attraction or repulsion in a buffer soln. across which has been applied a dc electric field. at steady speed: Accelerating force = frictional force qE = fv ep v ep = (q/f)E=μ ep E μ ep = Electrophoretic mobility (α charge and inversely α hydrodynamic radius of ion) © Dr. Rasha Hanafi, GUCLecture 9- PHCM662-SS20163 What is Electrophoresis? ------ ------ ++++++ ++++++ + + q.Ef.v ep q = charge of an ion. E = field strength (V cm -1 ). f= friction coefficient. v ep = migration velocity of charged particle in the potential field (cm sec -1 ).  ep = electrophoretic mobility (cm 2 V -1 sec -1 ). V = applied voltage (V) (E=V/L). L = length of capillary (cm).

4. The ion in an electric field When an ion of charge q (coulombs) is placed in an electric field E (Voltage V/ length of capillary m), the force on the ion is qE (newtons). In solution, the retarding frictional force is fv ep, where v ep is the velocity of the ion and f is the friction coefficient. The ion quickly reaches a steady speed when the accelerating force equals frictional force. Electrophoretic mobility  ep is the constant of proportionality between the speed of the ion and the strength of the electric field.  ep is proportional to the charge of the ion and inversely proportional to the friction coefficient. For molecules of similar size, the magnitude of mobility increases with charge. The greater the radius the lower the mobility, i.e., as the radius of the hydrodynamic volume of the ion increases, “f” increases. © Dr. Rasha Hanafi, GUCLecture 9- PHCM662-SS20164

5. Set up of capillary electrophoresis instrument © Dr. Rasha Hanafi, GUCLecture 9- PHCM662-SS20165 All ions move to the cathode. All???even the anions???? How???? The basic instrument is made of 1.fused silica capillary. 2.a controllable high voltage power supply. 3.2 electrode assemblies. 4.2 buffer reservoirs. 5.detector (ex: UV). 6.data acquisition system. The ends of the capillary are placed in the buffer. After filling the capillary with buffer, the sample can be introduced by dipping the end of the capillary in the sample solution. Pressure, suction or application of an electric field drives the sample in the capillary. In CE nothing is retained so the analogous term to “retention time” is migration time.

6. How small is the capillary tube used in CE? In typical CE instruments, the inner diameter of the capillary is 25 to 150 μm, the length is usually 50 cm. It also could be small enough to insert into a single, large living cell to analyze its contents (sensitivity of enzyme analysis can reach zeptomol, 10 -21 mol). Smaller cells can be taken one at a time into the capillaries, burst open and analyzed. © Dr. Rasha Hanafi, GUCLecture 9- PHCM662-SS20166

7.  The inside wall of the capillary is covered by silanol groups (SiOH) that are deprotonated at pH > 3  SiO -.  SiO - attracts cations of the buffer solution which become tightly adsorbed and hence immobile (THE RIGID LAYER).  The following layer is rich in cations but still mobile (THE DIFFUSE LAYER).  These 2 layers represent an “electrical double layer”. © Dr. Rasha Hanafi, GUC7Lecture 9- PHCM662-SS2016 What happens in the capillary of CE?

8.  Predominance of cations in the diffuse part produces ELECTROOSMOTIC FLOW (EOF) towards the cathode, exceeding the opposite flow towards the anode.  i.e. Net flow occurs as solvated cations drag along the solution. The net flow becomes quite large for high pH situations – a 50 mM pH 8 buffer flows through a 50-cm capillary at 5 cm/min with 25 kV applied potential. © Dr. Rasha Hanafi, GUC8Lecture 9- PHCM662-SS2016 Electroosmosis, the PUMP of CE…

9. EOF contributes significantly to high resolution unlike pressure-driven flow in LC and related techniques, explain. © Dr. Rasha Hanafi, GUC9Lecture 9- PHCM662-SS2016 Electroosmotic versus hydrodynamic Flow Profiles CathodeAnode Electroosmotic flow profile Hydrodynamic flow profile High Pressure Low Pressure 1.Driven by charge along capillary wall. 2.no pressure drop is encountered. 3.flow velocity is uniform across the capillary. 1.Driven by pressure difference. 2.Frictional forces at the column walls cause differences in flow velocity across the capillary.

10. v eo = μ eo E μ eo = Electroosmotic mobility is 1.proportional to the surface charge density on silica. 2.inversely proportional to the square root of the ionic strength of buffer. i.e. electroosmosis decreases at low pH and high ionic strength. Apparent Mobility μ app = μ ep + μ eo © Dr. Rasha Hanafi, GUCLecture 9- PHCM662-SS201610 Electroosmosis, the pump of CE… Electrophoretic mobility Total apparent mobility ++ + - - ++ + - - Electroosmotic mobility CATHODECATHODE ANODEANODE

11.  Combining the two effects for migration velocity of an ion (also applies to neutrals, but with  ep = 0:  At pH > 2, cations flow to cathode because of positive contributions from both  ep and  eo.  At pH > 2, anions flow to cathode in spite of the negative contribution from  ep, due to the significant positive contribution from  eo (if EOF is strong enough). At low pH,  eo is weak and anions may never reach the detector, where  ep predominates.  At pH > 2, neutrals flow to the cathode because of  eo only. © Dr. Rasha Hanafi, GUC11Lecture 9- PHCM662-SS2016 Electrophoresis and Electroosmosis

12.  Net EOF becomes large at higher pH: –A 50 mM pH 8 buffer flows through a 50-cm capillary at 5 cm/min with 25 kV applied potential  EOF can be quenched (stopped) by protection of silanols or low pH.  Uniform EOF contributes to the high resolution of CE, by minimizing peak width. © Dr. Rasha Hanafi, GUC12Lecture 9- PHCM662-SS2016 Silanols fully ionized above pH = 9 Electroosmotic Flow Profile

13. Unprecedented resolution in CE! N= 50,000-500,000 routinely!! WHY????? Van Deemter Equation: relates the plate height H to the velocity of the carrier gas or liquid mobile phase. Where A, B, C are constants, and a lower value of H corresponds to a higher separation efficiency. © Dr. Rasha Hanafi, GUC13Lecture 9- PHCM662-SS2016 CE versus chromatography (HPLC)

14.  In CE, a very narrow open-tubular capillary is used –No A term (multipath) because there is NO stationary phase (fused silica is not a stationary phase, i.e. There is no retention). –No C term (mass transfer) because there is NO stationary phase. –Only the B term (longitudinal diffusion) remains:  Cross-section of a capillary: © Dr. Rasha Hanafi, GUC14Lecture 9- PHCM662-SS2016 CE versus chromatography (HPLC)

15.  The flow of ions in the capillary generates heat. “Joule heating” is a consequence of the resistance of the solution to the flow of current. Typically, the centerline of the capillary channel is 0.02 to 0.3 K hotter than the edge of the channel.  If heat is not sufficiently dissipated from the system the resulting temperature and density gradients can reduce separation efficiency (effect on peak shape, how?) : viscosity is lower in the warmer region disturbing the flat electroosmotic profile of the fluid.  Heat dissipation is key to CE operation, for smaller capillaries (d= 50μm) heat is already dissipated due to the large surface area to volume ratio, yet it is a serious problem if d>1 mm.  The ability to dissipate heat results in extremely fast separations with excellent resolution are possible by application of high potentials (30kV) © Dr. Rasha Hanafi, GUC15Lecture 9- PHCM662-SS2016 Joule Heating

16.  Detectors are placed at the cathode since under common conditions, all species are driven in this direction by EOF.  Detectors similar to those used in LC, typically UV absorption, fluorescence, and Mass spectrometry. © Dr. Rasha Hanafi, GUC16Lecture 9- PHCM662-SS2016 Detection and Quantitative Analysis  For quantitative analysis, normalized peak areas are used: analytes with different apparent mobilities pass through the detector at different rates. The higher the apparent mobility, the shorter the migration time, and the less time the analyte spends in the detector which may erroneously decrease the detector’s response to it. Hence, normalization involves dividing each peak area by its migration time. Remember? In chromatography, each analyte passes through the detector at the same rate under the effect of pressure, so peak area is proportional to the quantity of analyte. For maximum precision, mobilities are measured relative to an internal standard. Absolute variations from run to run should not affect relative mobilities.

17. Proteins (enzymes at zeptomol level 10 -21 !), Peptides, Amino acids. Nucleic acids (RNA and DNA) also analyzed by slab gel electrophoresis. Inorganic ions. Organic bases, acids. Whole cells (if large cells, the capillary is introduced into them!). © Dr. Rasha Hanafi, GUC17Lecture 9- PHCM662-SS2016 Types of Molecules that can be Separated by CE

18. 1.CE is based on the principles of Electrophoresis and Electroosmosis. 2.The speed of movement or migration of solutes in CE is determined by their electrophoretic mobility which depends on ions‘ charge and size. Small highly charged solutes will migrate more quickly then large less charged solutes. Also the pH and ionic strength of the buffer change their electroosmotic mobilities. 3.Bulk movement of solutes is caused by EOF. 4.The speed of EOF can be adjusted by changing the buffer pH. 5.The flow profile of EOF is flat, yielding high separation efficiencies. 6.http://bio-animations.blogspot.com/2008/04/capillary- electrophoresis.htmlhttp://bio-animations.blogspot.com/2008/04/capillary- electrophoresis.html © Dr. Rasha Hanafi, GUC18Lecture 9- PHCM662-SS2016 Conclusion

19. QUIZ 2 Quiz 2 will take place on 14-04-2016 at 4:00 PM and will include lectures 6, 7 and 8 + tutorials 6,7. No make up is offered for the quiz Please respect locations of your groups. © Dr. Rasha Hanafi, GUCLecture 9- PHCM662-SS201619

20. HallDate / TimeGroups H3Thursday, 14-4-2016, 4:00-4:30 1-4 H5 6-9 H12 10-13 H13 14-17 H14 18-20 Location and time of Quiz 2 © Dr. Rasha Hanafi, GUCLecture 9- PHCM662-SS201620

21. 1.Textbook: Principles of instrumental analysis, Skoog et al., chapter 30. 2.Textbook: Quantitative Chemical Analysis, Harris, chapter 26. 3.Capillary Electrokinetic Separations, lecture in University of Villanova. 4.Extra resources on intranet folder: V:\Faculties\Pharmacy & Biotechnology\Pharmaceutical Chemistry\Instrumental Analysis II_ PHCM662_ SS2014\Extra resources © Dr. Rasha Hanafi, GUCLecture 9- PHCM662-SS201621 References