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Capillary Electrochromatography: a Versatile Tool for Biochemical Analysis

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Capillary Electrochromatography: a Versatile Tool for Biochemical Analysis Int. J. Pharm. Sci. Rev. Res., 63(2), July - August 2020; Article No. 31, Pages: 195-201 ISSN 0976 – 044X Review Article Capillary Electrochromatography: A Versatile Tool for Biochemical Analysis Chandani Chandarana*1, Abhishek Joshi2, Shubham Lehene3 1. Department of Quality Assurance, Assistant Professor, SSR College of Pharmacy, Silvassa, India. ORCID ID:0000-0003-3049-0664. 2. Student, SSR College of Pharmacy, Silvassa, India. *Corresponding author’s E-mail: [email protected] Received: 12-05-2020; Revised: 24-07-2020; Accepted: 30-07-2020. ABSTRACT Capillary Electro chromatography (CEC) is a small scale separation technique which involves principle of both chromatography and capillary electrophoresis. Separation of analyte is done on the basis of differences in ratio of partition between stationary phase and mobile phase or due to differences in their electrophoretic mobility. Capillary Electro chromatography (CEC) combines the principles used in HPLC and CE. The mobile phases are driven across the chromatographic bed using electro osmosis instead of pressure (as in HPLC). This review describes the general introduction to CEC, the various advantages of CEC over HPLC and Electophoresis, instrumentation, history, various stationary phase and mobile phases, column technology and vast applications of CEC in a variety of fields. Keywords: Capillary Electro chromatography, Instrumentation, Biochemical analysis. INTRODUCTION Electrophoresis can be considered as a method of separation based on the differential rate of migration of he Capillary electro chromatography is a modern charged species in an applied electric field. An hybrid analytical tool that combines the Principle of electrophoretic separation can be done by injection of a both high performance liquid chromatography T small band of sample into aqueous buffer solution, a high (HPLC) and capillary electrophoresis (CE) Hence it is voltage is then applied along the length of buffer by means important to first know what actually is HPLC and CE. of electrodes. Two types of Electrophoresis are High Performance Liquid Chromatography is a type of characterized i.e slab electrophoresis and capillary column chromatography which uses columns in which electrophoresis. Capillary electrophoresis is carried out packing material (stationary phase), pump which moves with the help of phenomenon known as electro-osmotic mobile phase through the column, along with the detector flow. Electro-osmotic flow is generated when a high which shows the retention time of the analyte are present. voltage is applied across a fused silica capillary tube HPLC is generally used in the separation, identification and containing buffer solution, and the bulk liquid migrates quantification of active compounds. 1 It is very famous as towards the cathode.7 Thus in capillary electrophoresis the well as popular analytical tool used to separate each separation is performed in capillary column using electro- constituents of mixture. The pressurized fluid and sample osmotic flow (EOF) as the driving force.8 Capillary gel mixture both are passed with the help of pumps, thus both electrophoresis is miniature version of typical gel the mobile phase and sample enter into the column which electrophoresis. Basic system of capillary gel is filled with solid adsorbent material5. Different modes of electrophoresis consists of two buffer reservoirs, a operation or types of HPLC are normal phase capillary, a high voltage power supply, sample introduction chromatography, reversed phase chromatography, size device, detector. Electrophoresis is used to separate a exclusion chromatography, ion exchange chromatography, wide range of biological molecules including amino acids, affinity chromatography1. HPLC uses simple peptides, proteins, nucleic acids. Capillary electrophoresis chromatographic mechanisms which are as follows: has found its application in pharmaceutical analysis of adsorption, partition, size exclusion and ion exchange. basic drugs and related substances.9 A capillary gel electrophoresis method has also been developed for fast Partition chromatography is said to occur when liquid and selective characterization and quantification of viral stationary phase is not miscible with the eluent and that is proteins. 10 coated on an inert support)2. When an ionically charged surface that is different from charge of sample is used it is When electrophoretic and chromatographic adsorption termed as ion exchanged chromatography. When samples combines it gives a more versatile tool for analysis that is are separated by virtue of their molecular size it is termed capillary electro chromatography. Capillary electro as size exclusion chromatography. 6 chromatography can be defined as a separation technique wherein the flow of mobile phase is driven through a chromatographic column with the help of an electric field rather than pressure. Due to this the technique is a International Journal of Pharmaceutical Sciences Review and Research International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net 195 Available online at www.globalresearchonline.net ©Copyright protected. Unauthorised republication, reproduction, distribution, dissemination and copying of this document in whole or in part is strictly prohibited. Int. J. Pharm. Sci. Rev. Res., 63(2), July - August 2020; Article No. 31, Pages: 195-201 ISSN 0976 – 044X combination of separation and selectivity potential of Strain. EOF is important in electrically driven HPLC and high efficiency capillary electrophoresis.11 This chromatography was confirmed by the early work done on modern liquid chromatography uses electro-drive to separation of large biological entities which utilized electro improve chromatographic performance. Here the EOF drive to aid movement of analytes through separative drives the liquid through the column, which is movement medium. It is possible to analyze basic, neutral, acid of bulk of liquid except surface layers. This is unlike molecule using electro migration; this was reviewed and capillary electrophoresis wherein separation is mainly demonstrated by Strain and Sullvian. Separation of based upon the differential migration of charged species polysaccharides using EOF through colloidal membrane under an applied electric field.12 Based on the interactions was reported by Mould and Synge. Theoretical foundation between solutes and the stationary, the uncharged and of CEC was laid in 1980s as well as significant progress charged substances can be separated. There are different began. CEC is possible to be performed using open tubular separation modes of CEC which can be classified as: ligand columns was reported by Stud and co-workers. 12 affinity, chiral recognition, ion exchange, hydrophilic 1. THEORY AND PRINCIPLE partitioning and reversed phase. The main component of CEC are the columns. There are three types of columns In CEC instead of pressure, electro osmosis is used to move characterized namely: packed, monolithic and open the mobile phase across chromatographic bed 16. Electro tubular. As compared to packed columns, monolithic and osmosis is the main principle or phenomenon behind CEC. open tubular are mostly used due to its inherent Electro osmosis can be defined as a process which occurs advantages of unambiguous preparation and frit less when a liquid containing electrolyte moves to a stationary design. Open tubular columns are very much suitable for charged surface by the virtue of an applied electric field. introduction of innovative material as stationary phase. The surface charge is usually due to ionization. 15 The Monolithic columns offer higher efficiencies and generation of EOF in CEC is similar to that in CE. It occurs resolution.13 There are many advantages of CEC, these as follows: the stationary phase and the surface of capillary include: analysis of both neutral and charged solutes is wall both possess charge, when a voltage is applied beyond possible, sample requirements are quite low, the amount the column due to accumulation of counter ions an electric of organic solvents used is also quite low, the separation double layer is formed, thus bulk fluid flow results, and 8 efficiencies are also higher, shorter analysis time . The hence formation of EOF. The value of EOF generated µeo is band broadening in column chromatography is affected by directly proportional to the zeta potential ζ which is due to a number of processes such as eddy diffusion, molecular the double layer. This is explained mathematically by diffusion, resistance to mass transfer, system effects. CEC following equation: 8 is not much affected by eddy diffusion and resistance to 8 μeo= Ko K ζ / η mass transfer.12 Other advantages of CEC include separation of neutral compound occurs by Where, Ko = Permittivity of vacuum chromatographic mechanism(partition) and charged K= Dielectric constant compounds get separated by both chromatographic as well as electrophoretic mobility, the plug like flow in EOF η= viscosity of solvent yields in reduced band dispersion, back pressure There are various factors that affect electro osmotic flow generation in the column is absent in CEC this results in 15. These are: extremely high efficiency.14 To increase column efficiency and resolving power, smaller particles and longer column a. Column Packing- EOF generation is mainly dependent are utilized in CEC because velocity of EOF is independent upon the packing of column. EOF velocity gets of particle size in packed
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