Capillary Electrophoresis - Andrew Maloney, Tim Towner
Background
The basic principle of electrophoresis is the movement of charged particles as the result of an electric gradient. Capillary electrophoresis frequently substitutes the gel that is typical of an electrophoresis medium, with a capillary. These tubes typically have dimensions of roughly 50 μm by 100cm in length and are operated from 20-30kV.[1] These high voltages enable a fast separation, and well-defined “zones” of the analyte. Each end of the capillary tube is dipped into a buffer solution and one end is put in the inlet solution with the anode and the other end runs through the detector to the cathode vial. The analytes will migrate through the capillary due to the electric gradient and will separate into bands from each other based on size. In zone electrophoresis, these zones are well defined with areas of buffer separating zones. As the zones pass the detector they are typically detected with UV absorption near the 200nm range. Because of the high voltage, and thus tighter bands, the resultant electropherogram will display narrow peaks that correspond to the respective analyte.[2] Unlike in capillary isotachophoresis, the capillary zone electrophoresis should separate into bands with the buffer between bands. These buffer bands mean that the electropherogram should return to 0 between analyte detections.
Governing Equations
Some of the governing equations for the flow regime in capillary electrophoresis include:[3]
The velocity of an analyte v:
v=μE = μV/L
Where μ is the electrophoretic mobility, V is the voltage, and L is the length of the tube.
Therefore the time for a zone of analyte to transverse the tube t is governed by:
t=L/v or L*L/μV
The molecular diffusion σ that contributes to the broadening of the zones can be modeled by:
σ=2Dt
Where D is the diameter of the capillary and combined with the previous equation, the spatial variance can be modeled by:
σ=2DL*L/μV
A relevant equation for analyzing electropherograms is the resolution which is the distance between peaks divided by the average peak width. [7]
Transition to the Microscale
In the case of Capillary Electrophoresis, there are many benefits of transitioning to the microscale including the ability to apply high voltages because of the heat transfer on the small diameter of the capillary. [6] This high voltage is what yields the strong zone definition and allows the process to be run relatively quickly. The small diameter allows for heat to be generated uniformly throughout the tube's cross-section and dissipated to the tube walls.[1]
Factors to consider at the microscale are viscosity, pH, and polarity of the analytes and all affect the flow within the capillary. Viscosity and pH can form gradients which can cause zone dispersion and vary the time that the analytes spend near the center or the wall of the capillary. The polarity of the anayltes can cause sample overloading, where a sufficiently concentrated substance can perturb the chemical and physical properties of the medium. As most analytes have acidic or basic properties, a high concentration may decrease the mobility of the substance or affect the conductive properties of the medium. Therefore, a low concentration of analytes to electrolyte buffer is used in Capillary Zone Electrophoresis to avoid sample overloading. The lower concentration and the smaller volume also place a higher priority on greater levels of detection sensitivity. [1]
Methods of Detection
Once the analytes can be separated from the electrophoresis, it is imperative to detect them. There are many different methods for detecting analytes in capillary electrophoresis including, mass spectrometry, optical spectroscopy, and others. [10] Mass spectroscopy is one of the most popular analytical techniques used in characterizing the differences in analytes. Mass spectrometry paired with capillary electrophoresis or CE-MS can create highly sensitive, chemical information about the analytes. CE-MS works once the analytes have been separated based on molecular weight in the capillary and the mass spectrometer will detect the difference.
Optical spectroscopy for detection methods primarily consists of fluorescence microscopy and UV absorption for capillary electrophoresis. Using fluorescence as a method of detecting information from capillary electrophoresis, analytes can be excited by a beam of light. Depending on the chemical structures of the different compounds in the analytes, they will fluoresce differently. [10] UV absorption of compounds as a detection method for capillary electrophoresis works similarly to fluorescence but in the opposite way. UV absorption with capillary electrophoresis works by shining light on the column and determining which compounds absorb the light. The difference in which the compounds absorb different wavelengths of light is directly related to the difference in chemical compounds located inside the analyte. Proteins are usually not good candidates for fluorescence detection because they have high variability and weak fluorescence. [1]
Applications
Capillary electrophoresis continues to be a very highly sensitive technique capable of analyzing a wide variety of compounds. As of recent, the primary applications for capillary electrophoresis consist of the analysis of proteomics and amino acids.[4] Combined with some of the detection techniques mentioned before, amino acids can easily be detected. Depending on the type of amino acids, the ones containing chromophores can easily absorb UV. If the analyte were to contain amino acids consisting of the aromatic ones and the nonaromatic compounds, there would be a noticeable absorption of UV for the compounds with aromaticity present in their structure. This relationship paired with high voltage applied to the system creates a strong separation technique by providing very narrow and well-defined peaks. Similarly, fluorescence microscopy can also be used to show the difference in amino acids based on their structures. Although these types of amino acids can be detected with these techniques, not all amino acids are UV-active. Capillary electrophoresis can also use the buffer as a way of separating amino acids based on their pH in a solution. [11] Rainelli et. al was able to differentiate a variety of amino acids, like threonine and methionine, based on their pKa in the buffer solution.
Capillary zone electrophoresis can also be applied to separate larger things like proteins. For a proper run of a capillary zone electrophoresis separation with proteins, the proper pH level needs to be maintained via the buffer to overcome the Coulombic repulsion of the protein and the silica walls. [5] This technique also offers a low-cost and user-friendly way to analyze the quality of antibiotics such as penicillin. Paul et al. describe a method for analyzing the quality of pre or post-commercialized antibiotics using capillary zone electrophoresis. [6]
References
1. James W. Jorgenson. Capillary Zone Electrophoresis New Directions in Electrophoretic Methods. March 18, 1987, 182-198. DOI: http://dx.doi.org/10.1021/bk-1987-0335.ch013
2. Clinical and Forensic Applications of Capillary Electrophoresis.[[1]]
3. Jorgenson, J. W.; Lukacs, K. DeArman. Zone Electrophoresis in Open-Tubular Glass Capillaries. Anal. Chem. 1981, 53 (8), 1298–1302. https://doi.org/10.1021/ac00231a037
4. Y.-F. Cheng and N. J. Dovichi, "Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence". Science 28 Oct 1988: Vol. 242, Issue 4878, pp. 562-564 DOI: http://dx.doi.org/10.1126/science.3140381
5. Lauer, . H.; McManlgHI, D. Anal. Chem. 1986, 58, 166-170 DOI: http://dx.doi.org/10.1021/ac00292a041
6. Capillary Zone Electrophoresis | Science. https://www.science.org/doi/10.1126/science.6623076?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed (accessed 2024-04-14).
7. A simple, low‐cost and robust capillary zone electrophoresis method with capacitively coupled contactless conductivity detection for the routine determination of four selected penicillins in money‐constrained laboratories - Paul - 2018 - ELECTROPHORESIS - Wiley Online Library. https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/full/10.1002/elps.201800033 (accessed 2024-04-14).
8. Perry, S. ChE590E Microfluidics and Analysis. Lecture 6: Separations. 2018. University of Massachusetts, Department of Chemical Engineering.
9. By Apblum [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons
10. Voeten, R. L. C.; Ventouri, I. K.; Haselberg, R.; Somsen, G. W. Capillary Electrophoresis: Trends and Recent Advances. Anal. Chem. 2018, 90 (3), 1464–1481. https://doi.org/10.1021/acs.analchem.8b00015.
11. Rainelli, A.; Hauser, P. C. Fast Electrophoresis in Conventional Capillaries by Employing a Rapid Injection Device and Contactless Conductivity Detection. Anal Bioanal Chem 2005, 382 (3), 789–794. https://doi.org/10.1007/s00216-005-3063-1.
