Capillary Electrophoresis (CE)
Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Capillary electrophoresis (CE) is a separation technique that separates molecules in…
1. CE Instrumentation Setup
- Turn on the CE instrument and computer. Using the computer software, turn on the light source for UV analysis to allow it to warm up. Some software has an indicator when the lamp is ready for use (lamp icon turns color).
- Make a methods file. Set the important parameters for running the CE. In this analysis the temperatures of the cartridge and sample storage are 35 °C. The wavelengthfor UV detection is 214 nm.
- Write a time program. The program generally consists of rinse steps (to clean the capillary before analysis), injection steps, and then an electrophoresis step. For the rinse step, perform 2 rinses for 1 min using 20 psi of pressure. The first rinse is with NaOH, which helps make sure the silanol groups on the capillary wall are deprotonated. The second rinse is with run buffer (0.025 M borate buffer here) to make sure the capillary is left equilibrated with buffer.
- For the injection, pressure injection is used at 0.5 psi for 5 s.
- For the electrophoresis step, the conditions are separation voltage: 20 kV, time: 5 min, normal polarity. For each step, also indicate which vial is the inlet and which vial is the outlet. Save the methods file after all the parameters are entered.
2. Preparation of the Standards and Soda Samples
- Make 500-ppm stock solutions of aspartame, caffeine, and benzoic acid in water. Make 50 mL of each, using a volumetric flask.
- Make a standard solution of 150 ppm aspartame, 150 ppm caffeine, and 100 ppm benzoic acid in a 10-mL volumetric flask.
- Make standard caffeine solutions of 50 ppm, 100 ppm, 150 ppm, and 200 ppm in a 10-mL volumetric flasks.
3. Run the Samples on the CE
- Place the vials containing either standards or soda samples into the sample vial holder. Make sure to write down which sample is in which slot. Two slots have the borate run buffer and the 0.1 M NaOH rinse solution.
- In the method file, input which slot the first sample vial is in.
- Acquire a single run, making sure to input all the data information the program requests.
- Continue to acquire data, changing the input vial for each sample. Run the combination standard, the 3 concentrations of caffeine, and a Pepsi and diet Pepsi sample.
- Insert the target into the instrument and choose the appropriate instrument.
- Analyze the data on the computer. Calculate the peak areas and overlay standards and real samples to help identify peaks. Make a calibration curve for the caffeine data.
Capillary electrophoresis, or CE, is a technique used in chemical analysis to separate molecules in an electric field according to size and charge.
Capillary Electrophoresis is performed in a sub-millimeter diameter tube, called a capillary, which contains a flowing electrolyte solution. The sample is injected into the capillary, and an electric field is applied. The molecules are then separated based on the difference in their velocity, which is influenced by charge, size, and the solvent's viscosity. CE is ideal for the separation of charged molecules and has a greater resolution than high-performance liquid chromatography, making it more efficient and sensitive.
This video will introduce the basics of capillary electrophoresis and demonstrate its use by determining the composition of a soft drink.
In CE, an electric field is applied to a capillary filled with an electrolyte. The electric field induces a positive charge at the capillary inlet, and a negative charge at the outlet.
The electrolyte flows within the capillary, induced by the electric field. This flow, called electro-osmotic flow, is caused by the movement of a discrete layer of positively-charged salt ions along the negatively-charged capillary walls.
As the electric current runs through the capillary, the cations along the wall move toward the negative end. This ion flow pulls the solution in the center through the tube.
The sample molecules are then separated based on their velocity within the capillary. This velocity, called electrophoretic mobility, depends on the molecules' charge and size, and how much it is attracted or repelled by an electric field.
Positively-charged molecules flow faster through the capillary, as they are more attracted to the potential at the outlet. Negatively-charged molecules flow much slower, as they are more attracted to the potential at the inlet. Neutral molecules are carried along with the bulk flow. Thus, the order of molecules exiting the capillary is positively-charged, neutral, and then negatively-charged. Additionally, the electrolyte flow pulls smaller molecules faster than larger molecules due to frictional forces.
Molecules are recorded by a detector, such as UV-Vis, as they exit the column, and are visualized in a plot of detector signal intensity versus time, called an electropherogram.
Electropherograms can yield a range of information; such as how many different compounds are present in a sample and the amount of each substance.
Now that you've seen a brief synopsis of capillary electrophoresis, let's take a look at how it is performed in the laboratory.
First, turn on the capillary electrophoresis instrument and computer. Then switch on the UV detector to allow it to warm up.
Set the parameters for running the experiment. First, set the temperature for the cartridge and sample storage to 35 ?C and the wavelength for UV detection to 214 nm.
Next, set the two rinse steps. The first rinse is with sodium hydroxide to ensure that the silanol groups on the capillary wall are protonated. The second rinse uses running buffer to equilibrate the capillary. Then, set the sample to inject at 0.5 psi for 5 s.
Set the electrophoresis step, by selecting the separation voltage. In this case, use 20 kV for 5 min using normal polarity, meaning that the electric field is positive at the inlet and negative at the outlet.
First, prepare 50 mL stock solutions of the soda components aspartame, caffeine, and benzoic acid at 500 parts-per-million in water.
From the stock solutions, make a standard solution of 150 ppm aspartame, 150 ppm caffeine, and 100 ppm benzoic acid.
Then, make 4 standard solutions of caffeine at 50, 100, 150, and 200 ppm. These samples will be used to make a calibration curve. Fore more information, see this collection's video on calibration curves.
Finally, prepare the soda samples by degassing them with nitrogen. The soda samples will be analyzed with no dilution.
Place the vials containing either standard or soda samples into the vial holder. Place the vials containing the run buffer and the sodium hydroxide rinse solution into the sample holder as well. Make sure to record the location of each.
In the CE instrument software, indicate which slots contain the two rinse solutions and the first sample vial. Now, run the first sample.
Then, run the combination standard, the 4 concentrations of caffeine, and a regular and diet soda sample by changing the input vial.
When all of the solutions have been separated, analyze the data.
First, use the standards to identify the peaks in the soda samples. A comparison of the three peaks observed in the diet soda sample to the standards shows that caffeine, aspartame, and benzoic acid are present in the diet soda. In the regular soda sample, only the caffeine peak is present, but not the aspartame and benzoic acid peaks.
Then, calculate the peak area for each caffeine standard solution, and make a calibration curve. The calibration curve for caffeine can be used to calculate the concentration of caffeine in each sample.
Capillary electrophoresis is used for many specialty separations in academic and industrial settings.
CE is often used as one component of pharmaceutical industry quality control testing. Drugs, either as small molecules or biologics, can be run through capillary electrophoresis to see if any side products are present. It can also be used to determine whether proteins are properly folded, as folding can affect protein charge.
CE can also be used to separate DNA. Using a microwell plate and multiple arrays of capillaries, researchers can increase the throughput of a single experiment, as shown here. DNA fragments were separated based on size, with a resolution down to 1 base pair. This makes sequencing fragments of DNA possible, along with determining other parameters like copy number variants, which is used to diagnose potential genetic diseases.
A protein can be modified by various functional groups that are chemically attached to different locations. Different copies of the same protein can vary with different modifications, which will change the charge and size of each protein. Running the purified proteins through a CE that is attached to a mass spectrometer can separate proteins based on which modifications are present, and also identify the type and location of the modification.
You've just watched JoVE's introduction to capillary electrophoresis. You should now understand how CE separates molecules based on charge and mass, and how to run a sample on the CE in the lab.
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Q1: How does capillary electrophoresis separate molecules?
Capillary electrophoresis separates molecules based on their electrophoretic mobility, which depends on charge, size, and solvent viscosity. An electric field applied across a capillary tube causes positively-charged molecules to move faster toward the negative outlet, while negatively-charged molecules move slower toward the positive inlet. Neutral molecules are carried along with the bulk flow, resulting in separation by charge and size.
Q2: What is electro-osmotic flow in capillary electrophoresis?
Electro-osmotic flow is the bulk movement of solution through the capillary caused by the electric field. Positively-charged salt ions along the negatively-charged capillary walls move toward the negative end, pulling the solution in the center through the tube. This flow carries all molecules, including neutral ones, and contributes to the overall separation efficiency in the capillary.
Q3: Why is capillary electrophoresis more efficient than high-performance liquid chromatography?
Capillary electrophoresis has a large surface area-to-volume ratio that dissipates heat efficiently, allowing voltages of 10,000–20,000 V to be applied without damaging the separation. This high voltage enables faster, more efficient separations with greater peak capacity than high-performance liquid chromatography. CE separations are particularly effective for charged molecules and provide superior resolution.
Q4: How are molecules detected and recorded in capillary electrophoresis?
Molecules are detected by a detector such as UV-Vis as they exit the capillary. The detector signal intensity is plotted versus time to create an electropherogram, which shows peaks corresponding to different compounds. The electropherogram reveals how many compounds are present in a sample and the amount of each substance based on peak position and area.
Q5: What sample preparation steps are necessary before running capillary electrophoresis?
Before running CE, prepare stock solutions of analytes at known concentrations, then create standard solutions and calibration standards at varying concentrations. For soda samples, degassing with nitrogen removes dissolved gases that could interfere with separation. Samples are placed in vials and loaded into the instrument's sample holder, with careful recording of each vial's location for accurate analysis.
Q6: How is a calibration curve used to determine analyte concentration in capillary electrophoresis?
A calibration curve is created by running standard solutions of known concentrations and measuring their peak areas. The relationship between concentration and peak area is plotted to establish a linear standard curve. Unknown sample peak areas are then compared to this curve to calculate the analyte concentration, enabling quantitative analysis of compounds like caffeine in soft drink samples.
Q7: What are the applications of capillary electrophoresis in pharmaceutical and research settings?
Capillary electrophoresis is used in pharmaceutical quality control to detect side products in drugs and verify proper protein folding. It separates DNA fragments with single base-pair resolution for sequencing and genetic analysis. When coupled with a mass spectrometer, CE can identify protein modifications and their locations, making it valuable for characterizing complex biological molecules in academic and industrial laboratories.