What is Capillary Electrophoresis?
Capillary electrophoresis (CE) is an analytical technique that utilizes the electrophoretic mobility of charged molecules in a buffer-filled capillary to effectuate their separation. This powerful technique boasts a high degree of resolution, sensitivity, and speed, making it an increasingly popular alternative to conventional separation methods like high performance liquid chromatography (HPLC).
The fundamental principle of CE is the separation of charged molecules by an electric field applied across a capillary filled with a buffer solution. Molecules migrate through the capillary at different rates based on their size, shape, and charge, and the separation is detected using a detector, such as a UV-Vis spectrophotometer, that tracks the migration of molecules through the capillary.
CE is capable of separating an extensive range of molecules, including peptides, proteins, nucleic acids, carbohydrates, and small organic molecules. This versatility makes CE well-suited for a variety of applications, including drug discovery, clinical diagnostics, environmental monitoring, and food analysis. Additionally, CE's high resolution and sensitivity enable it to effectively analyze complex mixtures such as biological fluids, providing valuable information that would be difficult to obtain otherwise.
Compared to traditional separation techniques, CE has several noteworthy advantages. For instance, CE requires only small sample volumes and exhibits high separation efficiency, allowing it to separate molecules that are otherwise difficult to isolate using other methods. Furthermore, CE is easily automated, making it a viable option for high-throughput analysis.
Materials:
- Capillary electrophoresis instrument
- Capillaries (fused silica, 50 or 100 μm i.d.)
- Electrolyte solution (e.g. 1 M sodium borate)
- Sample buffer (e.g. 0.1 M Tris-HCl pH 8.0)
- Standard marker solution
- Sample solution
Procedure:
1. Preparation of the electrolyte solution
- Weigh out the appropriate amount of sodium borate and dissolve it in distilled water.
- Adjust the pH to the desired value using a dilute solution of HCl or NaOH.
- Filter the solution through a 0.45 μm membrane filter and degas it under vacuum for 10-15 minutes.
2. Preparation of the capillary
- Cut a 50 or 100 μm i.d. fused silica capillary to the desired length.
- Flush the capillary with 0.1 M NaOH for 30 minutes to remove any contaminants.
- Rinse the capillary with distilled water for 10 minutes, followed by 0.1 M HCl for 10 minutes, and then distilled water again for 10 minutes.
- Fill the capillary with the electrolyte solution and place it in the instrument.
3. Calibration of the instrument
- Inject the standard marker solution into the capillary according to the manufacturer's instructions.
- Run the instrument and adjust the voltage and temperature until the marker peaks are well-separated and resolved.
- Record the migration time and peak area for each standard marker.
4. Sample preparation
- Mix the sample with an appropriate amount of sample buffer.
- Heat the sample at 90-100°C for 5-10 minutes to denature the proteins and break down any aggregates.
- Centrifuge the sample at 10,000 g for 5 minutes to remove any insoluble material.
- Inject the sample into the capillary using the instrument.
5. Data analysis
- Analyze the electropherogram to determine the migration time and peak area for each sample component.
- Compare the migration times and peak areas to those of the standard markers to identify the sample components.
- Calculate the concentration of each sample component using the standard curve generated from the standard marker peaks.
Note: This is a general protocol and may need to be modified based on the specific instrument and sample being used. Always consult the instrument and sample manuals for detailed instructions.
