The Advantages and Disadvantages of Capillary Electrophoresis - Research Paper Example

Capillary Electrophoresis (CE) in Comparison with other High Resolution Separation Techniques for Different Molecules of Biological Significance Capillary electrophoresis (CE) is one of the methods for separation of molecules for scientific research purposes. It uses the principles of charge and frictional forces of ions to test and use them in different purposes. Compared to other types of electrophoresis, the said technique uses a small capillary to be able to segregate the components of an analyte into species of different charge ratio (Camilleri, 1998). The main applications of the said technology include the field of analytical chemistry, biochemistry, biotechnology, forensic science, food science, clinical science, neuro-science, medical research and production, pharmaceutical science and other fields of disciplines (MicroSolv Technology, 2005). Due to the importance of the CE technique in different fields of application, the study is undertaken to present a comparative analysis of the said technique with other high resolution separation techniques especially for application in different molecules of biological significance. The main focus of the study includes prior research related to molecules such as proteins, DNA, steroids or non-polar molecules and chiral molecules. Background Information on CE The development of the technique of separation referred to as capillary electrophoresis (CE) occurred years ago but the advent of its use in a multitude of fields occurred in the last decade. During the early years of introduction of CE, the main role of the said technique is related to educational and exploratory objectives. Through the course of its history, the importance of the said method in different analytical purposes paved the way to the present role in different fields for analysis of complex molecules (Camilleri, 1998). This is important since the main focus of the research undertaken involves complex biologically significant molecules. The role of CE in the present era had been established on the basis of the capabilities of the said method. Included in such capabilities are adaptability in different modes of operation due to simplicity of the method and high level of recovery and reproducibility. This can be attributed to the sharp separation of the components of analytes due to lower zone broadening. The advantages of the CE can be considered on the basis of the capability to allow high voltages for higher rate of heat dissipation (Camilleri, 1998). Due to the promising and significant results associated with the use of capillary electrophoresis, the study is targeted to present the positive and negative point in using the method for the analysis and separation of the different components of proteins, DNA, steroids or non-polar molecules and chiral molecules as compared to conventional procedures that are used to analyze these biologically significant molecules. Protein Protein is one of the most important molecules of biological significance. For that matter the importance of the different separation techniques such as mass spectrometry, various types of chromatography and electrophoresis. In proteins, mass spectrometry can be considered the main method for analysis. On the other hand the gel electrophoresis is the main technique use for separation of the components of different types of proteins. These are considered as the main methods that can be compared to capillary electrophoresis. The main mechanism in the capillary electrophoresis is the separation of the components of a biomolecule through the use of ionic charges which is undertaken through the use of capillary tube. As the positively charged ions are attracted to the negative electrode the negatively charged ions are attracted to the positive electrode (Schmitt-Kopplin, 2008). There are different aspects that are considered. One of the factors in the determination of the efficiency of the CE method is the rate of migration. In the CE, the migration of the different types of ions occurs in varying rates. For that matter, the separation of the components can be considered as more efficient. This phenomenon can be attributed to the charge of the molecule based on mass ratio which can be considered beneficial in cases of molecules considered to be structured and cohesive. Other parameters that are given importance in the CE method are ionic mobility, electro-osmotic flow (EOF) and plug flow in CE (MicroSolv Technology, 2005). The advantages of the CE can also be related to the different methods incorporated that can strengthen the efficiency of the method. These techniques include preconcentration and derivatization of the sample protein and reduction of protein adsorption to the capillary wall, aside from the combination of methods to separate the components of the biomolecule (Huck and Bonn, 2008). In the application of CE in the study of heparin disaccharides and nuclear magnetic resonance the method had been crucial in the achievement of the objectives specifically related to the electrophoretic mobility (Eldridge, Higgins, Dickey and Larive, 2009). This can be considered as one of the number of studies that apply the CE innovation. It is important to focus on the fact that the advantages of CE in the separation and analysis of proteins is the improvement on the basis of the application of electrodes. For that matter, the main strength is the increase in the rate of separation as compared to other separation techniques. On the other hand, the main disadvantage is the fact that needs other techniques in different stages of the process to be able to achieve an optimized result. This can specifically be related to the analytical applications of the CE in different types of proteins such as human proteins, food and agricultural product proteins, pharmaceutical proteins and other related types (Huck and Bonn, 2008). DNA DNA holds the genetic information that is important in the analysis of every biologically significant molecule. For that matter, the analysis of the importance and limitations of the CE and other high resolution separation methods can be based on the different phases. Through the application of CE, the positive attributes of the techniques are maximized which includes ‘high resolving capability, speed, low consumption of the sample and automation of sample loading. The process included in the separation of DNA can be considered related to the natural process of the said molecule specifically DNA sequencing, restriction mapping and genotyping (McCord, Hartzell-Bagueley and King, 2009). One of the main limitations of CE that had been the target of significant research activities the past few years is related to the query on the failure of the separation of DNA fragments based solely on size or length of the chain. Due to the said issue, the need for a sieving matrix had been realized. The said process became the main focus in the improvement of the CE for the application on the separation of DNA components for significant biomolecule research and analysis (McCord, Hartzell-Bagueley and King, 2009). In the research conducted to assess and analyze the DNA methylation as an application, the separation procedures and methods is one of the fundamental requirements. The quantification of the global methylation of the DNA had been undertaken through the utilization of the high-performance capillary electrophoresis (HPCE) and high-pressure liquid chromatography (HPLC), both with mass spectrometry. In the study, each of the methods had the individual advantages and disadvantages (Berdasco, Fraga and Esteller, 2009). One of the limitations of the two methods in the study is the inefficient digestion of RNA compound which affects the quantification of DNA methylation. Another is the susceptibility to changes in temperature that can lead to changes in the system. In terms of HPCE, one of the limitations is the susceptibility to changes in pH and molarity of the Tris and sulphate buffers that commonly result to unspecific DNA hydrolysis. For the case of the HPLC, it is affected by the pressure of the system as well as increase of organic acids (Berdasco, Fraga and Esteller, 2009). Each method has limitations and positive points, for that matter, there are different applications for the methods although both require mass spectrometry for analysis. In the DNA methylation analysis, the quantification though HPCE had been favoured. Steroids and Non-Polar Molecules Steroids and non-polar molecules can be considered as another biomolecule that require separation procedures to be able to be purified and analyzed. The steroids and related molecules can be classified in the group of molecules referred to as lipids on the basis of chemical structure of the component. In the study targeted to present the different potentials and limitations of capillary electrophoresis in the analysis of lipids, the application and utilization of the different types and varieties of CE had been recognized. Enumerated in the study are the different kinds of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary electrochromatography (CEC), electrokinetic chromatography (EKC) and microemulsion electrokinetic chromatography (MEEKC) (Otieno and Mwongela, 2008). In general, based on the study, CE can be considered to have the important potentials of GC and LC, which can be attributed to the speed of the technique and the efficiency in lipid determination due to the high resolution. The main factor that limits the optimization of the advantages is the low solubility of lipids in the buffer that is required in the systems that can hinder the separation of the components of the lipids. In the case of sterols, the most effective type of CE is the capillary electrochromatography (Otieno and Mwongela, 2008). In one of the specific applications of CE, stacking process had been improved on the basis of speed of recovery of natural and synthetic steroids had been accomplished. This is through the utilization carboxymethyl-β-CD enabling the separation of multiple types of steroids in a particular biological sample such as fish plasma. In the study undertaken, a simple biological sample was used. As the complexity of the sample increases, the set-up is compounded to be able to achieve the advantage of the speed of recovery associated with the method of CE (Otieno and Mwongela, 2008). Another application of CE in steroids and other non-polar molecules had been presented in the study related to the profiling of metabolites in steroid hormone metabolism. To be able to achieve the aim of the study to profile components of the mammalian steroid metabolism specific type of CE had been used. Through the use of partial filling micellar electrokinetic chromatography (PF-MEKC) with UV absorption, the required products had been achieved (Sirén, Seppänen-Laakso, and Oresic, 2008). One of the main advantages of the method as compared to other techniques is the capability for the achievement of fast results. In minutes the steroid hormone can be synthesized after the separation had been finished. This can be considered as an important basis for application in a huge variety of fields and in future researches that can be conducted related to the study of steroids (Sirén, Seppänen-Laakso, and Oresic, 2008). There is a wide variety of applications of CE and its specific types in the separation and analysis of steroids as well as in the synthesis of steroid products. The studies presented are the latest addition the said field. Chiral Molecules For chiral molecules and the unique chemical structure of the said biomolecules, the efficiency of CE is still the main reason for application. In the latest application of CE, the main focus is the analysis of chiral pharmaceutical molecules. The study specifically focused on the monolithic stationary phases due to the discovered importance in the onset of improvement in the separation methods (Tanret, Mangelings and Vander, 2009). In the said study, which is a representative of many studies related to chiral biomolecules, there are different reasons for the application of the CE method as well as pCEC. One of the main benefits of the CE technique is the ease in preparation that can be considered important in the replication of the empirical trials. In addition, the versatility of the method which can be defined as the capability of the set-up to be customized to suit the need of the experimentation is another important aspect. The rate of the process is considered as one of the main positive attributed of CE, thus, it is chosen for a significant number of experimentations not only in chiral molecules but in different types of biological experimentations. For the drawbacks of the CE types, the chemical compositions such as buffers and the susceptibility to different factors are considered to limit the efficiency of the technique (Tanret, Mangelings and Vander, 2009). Conclusion In the study undertaken, the focus had been given to the different types of biologically related molecules such as protein, DNA, steroids and non-polar molecules and chiral molecules. By accessing different researches conducted on the application of capillary electrophoresis and its specific types to the said molecules, the advantages and disadvantages of the method had been analyzed. Based on the data gathered, the most significant advantage of CE is the speed of recovery of the components of the biomolecules thus increasing the efficiency of the separation process. This is beneficial in replication and productivity of the experimentation. For the disadvantages of the CE, the susceptibility to chemical and physical factors can be a hindrance and a limiting factor to the achievement of the target results. References Berdasco, M., Fraga, M.F. and Esteller M. (2009). ‘Quantification of global DNA methylation by capillary electrophoresis and mass spectrometry’. Methods Mol Biol. 507:23-34. Bykova, L. and Holland, L.A. (2008). ‘Stacking enhanced determination of steroids by CE’. Electrophoresis, 29(18): 3794-800. Camilleri, P. (1998) Capillary Electrophoresis: Theory and Practice, New Directions in Organic and Biological Chemistry. USA: CRC Press. Eldridge, S.L., Higgins, L.A., Dickey, B.J. and Larive, C.K. (2009) ‘Insights into the capillary electrophoresis separation of heparin disaccharides from nuclear magnetic resonance, pKa, and electrophoretic mobility measurements’. Anal Chem, 81(17): 7406-15. Huck, C.W. and Bonn, G.K. (2008) ‘Analysis of Proteins by Capillary Electrophoresis’, in P. Schmitt-Kopplin (ed). Capillary electrophoresis: Methods and protocols. NJ: Humana Press. Larbi, N.B. and Jefferies, C. (2009) ‘2D-DIGE: comparative proteomics of cellular signalling pathways’. Methods Mol Biol., 517: 105-32. McCord, B., Hartzell-Bagueley, B. and King, S. (2009) ‘Separation of DNA by Capillary Electrophoresis’. Capillary electrophoresis: Methods and protocols. NJ: Humana Press. MicroSolv Technology (2005) ‘Basic Theory and Introduction on Separation Science’ [Online] Available at: http://www.microsolvtech.com/cebasic.asp (Accessed 29 September 2009). Moon, J.Y., Jung, H.J., Moon, M.H., Chung, B.C. and Choi, M.H. (2008). ‘Inclusion Complex-Based Solid-Phase Extraction of Steroidal Compunds with Entrapped β-cyclodextin Polymer’. Steroids, 78: 1090-97. Otieno, A.C. and Mwongela, S.M. (2008). ‘Capillary electrophoresis-based methods for the determination of lipids--a review’. Anal Chim Acta., 624(2):163-74. Sirén, H., Seppänen-Laakso, T. and Oresic, M. (2008). ‘Capillary electrophoresis with UV detection and mass spectrometry in method development for profiling metabolites of steroid hormone metabolism’. J Chromatogr B Analyt Technol Biomed Life Sci., 871(2): 375-82. Tanret, I., Mangelings, D. and Vander Heyden, Y. (2009). ‘Monolithic stationary phases in classic and chiral pharmaceutical analysis with CEC and pCEC’. J Chromatogr Sci., 47(6): 407-17. Read More