Capillary electrophoresis

Methods:

• buffer: 25 mM NH₄CH₃COOH, pH=9.0
• capillary: l_eff= 33 cm (or l_eff= 8 cm), internal diameter= 50 μm
• injection: 100 mbar∙s (-100 mbar∙s)
• T: 25°C
• Separation voltage: +25 kV (-25 kV)
• Detection: λ=200 nm

Samples:

1. Benzoic acid, Salicylic acid and Acetylsalicylic acid solutions (1 mg/mL)

2. 4 solutions containing acetylsalicylic acid and benzoic acid in known concentration

3. Unknown sample (acetylsalicylic or and benzoic acid)

Tasks:

Students have to prepare a three-component sample solution and 4 solutions for the calibration.

Students have to analyse the prepared solutions with the CE instrument.

Related tasks:

1. Determination of migration times, effective electrophoretic mobilities and resolutions.

2. Plotting a calibration curve by measuring the 4 solutions of exactly known concentration, giving the equation of the line.

3. Qualitative and quantitative determination of the unknown sample based on the results obtained.

Possible questions in the short test:

1. What is the electroosmotic flow (EOF)? In which conditions does it develop? How can it be controlled? What is its significance?
2. Describe the two most important sample injection methods used in capillary electrophoresis.
3. Describe schematically the structure of the capillary electrophoresis instrument, explain in detail the capillaries, how to condition them.
4. What are the problems of UV-VIS detection in the case of capillary electrophoresis?
5. Describe the followings: electrophoresis, electroosmotic flow, migration time, theoretical plate number, dispersion, resolution.
6. 3-3 components are separated by CE in an untreated quartz capillary, using positive voltage. What is the order of the migration time of the following components?
7. Li ⁺, Na ⁺, K ⁺ ions?
8. for methylammonium, ethylammonium, propylammonium ions?
9. Write the following parameters for the capillary zone electrophoresis technique:
10. applied voltage, field strength
11. length and inner diameter of capillary
12. volume of injected sample, parameters of hydrodynamic injection
13. theoretical plate number
14. Our two components to be separated (compounds A and B) are anions under the separation conditions. We use a quartz capillary in which the mobility of the EOF has direction towards the cathode.
15. In which polarity (voltage) can the two components be determined?

Suppose that two components are cations under the separation conditions.

• Which polarity is used to obtain the fastest separation? Why?
• In which polarity cannot the separation be performed? Why?

Literature:

H.Engelhardt, W.Beck, T.Schmitt: Capillary electrophoresis, Friedr.Vieweg & Sohn Verlagsgesellschaft mbH, Braunschweig/Wiesbaden, 1996 (ISBN 3-528-06668-7)

R.Kuhn, S.Hoffstetter-Kuhn: Capillary electrophoresis, Springer-Verlag, New York, 1993 (ISBN 0-387-56434-9)

D.N.Heiger: High Performance Capillary Electrophoresis, Hewlett-Packard GmbH, Waldbronn, 1992 (ISBN 12-5091-6199E)