Separation of nitrophenol isomers by capillary zone electrophoresis (CZE) experiment

Summary

This experiment is from the official website of Wuhan University School of Pharmacy

Operation method

Separation of nitrophenol isomers by capillary zone electrophoresis (CZE) experiment

Principle

Capillary electrophoresis (CE): A chromatographic method in which a high voltage electric field is used as the driving force, electrolyte is used as the electrophoresis medium, and the capillary tube is used as the separation channel, and the separation of sample components is realized according to the difference of flow rate and distribution behavior. Due to the small inner diameter of the capillary, the ratio of surface area to volume is large, and it is easy to dissipate heat, so capillary electrophoresis can reduce the generation of Joule heat, which is the fundamental difference between CE and traditional electrophoresis technology. It has various separation modes and can be used in various detection methods in liquid chromatography, CE can separate charged solutes and also analyze neutral solutes by capillary micellar electrokinetic chromatography and other separation modes, CE's high separation efficiency, high detection sensitivity, and minimal sample dosage make it show outstanding superiority in the analysis of biomedical samples, etc. Quartz capillary columns used in CE can be used for the analysis of biomedical samples under the condition of pH>3.0, and its inner surface area and volume ratio are large. 3.0, its inner surface is negatively charged, and a double electric layer is formed when it comes into contact with the solution. Under the action of high voltage, the directional movement of charged ions in a certain medium under the action of DC electric field is called electrophoresis. The electrophoretic speed per unit electric field is called electrophoretic mobility or electrophoretic mobility. The magnitude of electrophoretic velocity is related to the strength of the electric field, the properties of the medium, the effective charge of the particles and their size and shape. The hydrated cations in the bilayer cause the fluid to move toward the negative pole as a whole, and the phenomenon is called electroosmosis, and the electroosmosis speed per unit electric field is called electroosmosis flow rate. The electroosmotic velocity is related to the dielectric constant and viscosity of the electrolyte solution in the capillary, the ζ-potential of the bilayer, and the strength of the applied DC field. Particle migration velocity in the capillary electrolyte is equal to the vector sum of electrophoresis and electroosmotic flow (EOF) two speeds, the direction of motion of positive ions and electroosmotic flow consistent, so the first outflow; neutral particles of the electrophoretic flow rate of "zero", so its migration speed is equivalent to the speed of the electroosmotic flow; the direction of motion of the negative ions and the direction of the opposite direction of electroosmosis, but because of the general direction of electroosmosis, the electroosmotic velocity is the same as the electroosmotic velocity. But because the electro-osmotic flow speed is generally greater than the electrophoretic flow speed, so it will flow out after the neutral particles, so as to realize the separation of various particles migrate at different speeds. Electro-osmosis is the driving force in CE to push the fluid forward, it makes the whole fluid like a plug to move forward at a uniform speed, so that the whole flow pattern is similar to the flat type of "plug flow". It makes the solute zone in the capillary tube will not expand in principle. However, in HPLC, the pressure-driven mode makes the fluid in the column parabolic, and the velocity at the center is twice the average velocity, which leads to the expansion of the solute zone itself, and causes a decrease in the column efficiency, so that the separation efficiency is not as good as that of CE.Nitrophenol is a weakly acidic substance, and its neighboring, inter-, and para-isomers are differently ionized in the buffer solution at a certain pH due to their different pKa values. Therefore, they showed different migration speeds during capillary electrophoretic separation to achieve separation.

Move

1. Turn on the capillary electrophoresis instrument and preheat until the detector output signal is stable.

2. Measure the length of the capillary accurately. About 15cm from one end of the capillary tube, remove about 2mm of the capillary polymer protective layer as the detection window, and measure the length from the inlet end of the capillary tube to the detection window.

3. Align the detection window of the capillary with the optical path of the detector and install the capillary.

4. Sequentially rinse the capillary with sodium hydroxide solution (1.0 mol-L-1), secondary distilled water, hydrochloric acid solution (0.1 mol-L-1), and secondary distilled water for 5 min each, and finally inject a buffer solution into the capillary and insert each end of the capillary into the bottle of buffer solution located at the electrode. The DC voltage was adjusted to 20kV.

5. After the baseline of the recorder is stabilized, turn off the high-voltage power supply, feed the sample by pressure injection and set the injection time, record the migration time after the sample peak appears, operate the mixed sample according to the same steps and record the spectrum.

6. Change the applied voltage (e.g. 15kV or 25kV) and repeat steps 4 and 5.

7. After the experiment is completed, turn off the power of the instrument and rinse the capillary with secondary distilled water.

Common Problems

1. Calculate the electroosmotic velocity, the electroosmotic mobility, the electrophoretic mobility of each component, and the theoretical number of plates of m-nitrophenol according to the obtained experimental data. Calculate the separation between the components according to the separation diagram.

2. Plot the relationship between applied voltage and electroosmotic velocity and give an explanation.

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