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Capillary electrophoresis
- 1. By: Kasturi Banerjee M. Pharm-Pharmaceutics (1st Year) KLE College of Pharmacy, Bengaluru Capillary Electrophoresis
- 2. Introduction Terminologies Electroosmosis Instrumentation Flow Dynamics, Efficiencyand Resolution Capillary Diameter and Joule Heating Effects Of Voltage and Temperature Modes of C.E. Capillary Zone Electrophoresis Contents 2
- 3. Isoelectric Focusing Capillary GelElectorphoresis Isotachophoresis Micellar Electrokinetic Capillary Chromatography Advantages of C.E. Limitations of C.E. Common Applications of C.E. References 3
- 4. When an electricfield is applied across a tube containing a conductive solution, an electrolyte, which contains charged solutes, the solutes migrate through the solution. The rates and direction of their migration depend on the signs and magnitudes of their charges as well as their sizes. This phenomenon is called electrophoresis. Under the influence of an electrical field, electrophoresis flow (EOF) causes movement of the electrolyte through the tube. Capillary electrophoresis (CE) is a family of related techniques that employ narrow-bore capillaries (20-200 µm i.d.) to perform high efficiency separations of both large and small molecules. Introduction 4
- 5. The charged solutes,the ions, migrate through the tube, with the highly charged ions migrating the fastest and the lesser charged ions, the slowest. Neutral molecules are not affected by the electric field and move through the tube under the influence of just the EOF and are not separated from each other. The larger the diameter of the tube, the more joule heat is generated, which causes spreading of the zones giving poor separations. Molecules in the centre of the tube migrate faster than those near the wall because the viscosity of the electrolyte is lower in the centre. 5
- 6. These separations arefacilitated by the use of high voltages, which may generate electroosmotic and electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary. The properties of the separation and the ensuing electropherogram have characteristics resembling a cross between traditional polyacrylamide gel electrophoresis (PAGE) and modern high performance liquid chromatography (HPLC). 6
- 7. There are afew significant differences between the nomenclature of chromatography and capillary electrophoresis. For example, a fundamental term in chromatography is the retention time. In electrophoresis, under ideal conditions, nothing is retained, so the analogous term becomes migration time. The migration time (tm) is the time it takes a solute to move from the beginning of the capillary to the detector window. Other fundamental terms include the electrophoretic mobility, µep (cm2/Vs), the electrophoretic velocity, νep (cm/s), and the electric field strength, E (V/cm). Electrophoresis Terminologies 7
- 8. The relationships betweenthese factors are shown in equation 1. µep= ν 𝑒𝑝 𝐸 = 𝐿 𝑑 /𝑡𝑚 𝑉/𝐿𝑡 (1) Several important features can be seen from this equation: Velocities are measured terms. They are calculated by dividing the migration time by the length of the capillary to the detector, Ld. Mobilities are determined by dividing the velocity by the field strength. The mobility is independent of voltage and capillary length but is highly dependent on the buffer type and pH as well as temperature. 8
- 9. Two capillary lengthsare important: the length to the detector, Ld, and the total length, Lt. While the measurable separation occurs in the capillary segment, Ld, the field strength is calculated by dividing the voltage by the length of the entire capillary, Lt. The excess capillary length, Lt - Ld, is required to make the connection to the buffer reservoir. For the P/ACE system, this length is 7cm. By reversing the configuration of the system, this 7cm length of capillary can be used to perform very rapid separations. 9
- 10. One of thefundamental processes that drive CE is electroosmosis. This phenomenon is a consequence of the surface charge on the wall of the capillary. The fused silica capillaries that are typically used for separations have ionizablesilanol groups in contact with the buffer contained within the capillary. The pI of fused silica is about 1.5. The degree of ionization is controlled mainly by the pH of the buffer. The electroosmotic flow (EOF) is defined by νeo= ϵζ 4ηπ E Electroosmosis 10
- 11. where, • ϵ isthe dielectric constant, • η is the viscosity of the buffer, and • ζ is the zeta potential measured at the plane of shear close to the liquid-solid interface. The negatively-charged wall attracts positively-charged ions from the buffer, creating an electrical double layer. When a voltage is applied across the capillary, cations in the diffuse portion of the double layer migrate in the direction of the cathode, carrying water with them. The result is a net flow of buffer solution in the direction of the negative electrode. 11
- 12. This electroosmotic flowcan be quite robust, with a linear velocity around 2 mm/s at pH 9 in 20 mM borate. For a 50 µm i.d. capillary, this translates into a volume flow of about 4 nL/s. At pH 3 the EOF is much lower, about 0.5 nL/s. The zeta potential is related to the inverse of the charge per unit surface area, the number of valence electrons, and the square root concentration of the electrolyte. Since this is an inverse relationship, increasing the concentration of the electrolyte decreases the EOF. 12
- 13. EOF makes possiblethe simultaneous analysis of cations, anions, and neutral species in a single analysis. At neutral to alkaline pH, the EOF is sufficiently stronger than the electrophoretic migration such that all species are swept towards the negative electrode. The order of migration is cations, neutrals, and anions. Fig: Effect of pH on EOF13
- 14. The effect ofpH on EOF is illustrated in the figure. Imagine that a zwitterion such as a peptide is being separated under each of the two conditions described in the figure. At high pH, EOF is large and the peptide is negatively charged. Despite the peptide’s electrophoretic migration towards the positive electrode (anode), the EOF is overwhelming, and the peptide migrates towards the negative electrode (cathode). At low pH, the peptide is positively charged and EOF is very small. Thus, peptide electrophoretic migration and EOF are towards the negative electrode. 14
- 15. In untreated fusedsilica capillaries most solutes migrate towards the negative electrode regardless of charge when the buffer pH is above 7.0. At acidic buffer pH, most zwitterions and cations will also migrate towards the negative electrode. To ensure that a system is properly controlled, it is often necessary to measure the EOF. This is accomplished by injecting a neutral solute and measuring the time it takes to reach the detector. Solutes such as methanol, acetone, and mesityl oxide are frequently employed. 15
- 16. Table: Buffers forCapillary Electrophoresis 16
- 17. A capillary electrophoresissystem is represented in the following figure: Instrumentation 17
- 18. The basic instrumentalconfiguration for CE is relatively simple. All that is required is a fused-silica capillary with an optical viewing window, a controllable high voltage power supply, two electrode assemblies, two buffer reservoirs, and an ultraviolet (UV) detector. The ends of the capillary are placed in the buffer reservoirs and the optical viewing window is aligned with the detector. Electrophoresis is performed by filling the reservoirs and capillary with a buffer solution, the electrolyte. After filling the capillary with buffer, the sample can be introduced by dipping the end of the capillary into the sample solution and elevating the immersed capillary a foot or so above the detector-side buffer reservoir. Then the capillary inlet is placed back into the inlet reservoir and an electric field is applied. 18
- 19. The electric fieldcauses the solutes to migrate through the capillary and they are detected by the detector and its output is usually displayed at an integrator or computer. This output is a plot of detector response versus time and is called an electropherogram. Each peak in the electropherogram repesents one of the components as it migrates through the detector. Because the compounds migrate through the detector at different times, an electropherogram is produced in which the separated compounds appear as peaks with different migration times. The starting time, a time of zero, is the time when the electric field is turned on. Migration times are measured at the apices of the peaks, usually in the units of minutes. 19
- 20. When employing apressure-driven system such as a liquid chromatograph, the frictional forces at the liquid-solid interfaces, such as the packing and the walls of the tubing, result in substantial pressure drops. Even in an open tube, the frictional forces are severe enough at low flow rates to result in laminar or parabolic flow profiles. As a consequence of parabolic flow, a cross-sectional flow gradient, shown in figure, occurs in the tube, resulting in a flow velocity that is highest in the middle of the tube and approaches zero at the tubing wall. Flow Dynamics, Efficiency and Resolution 20
- 21. Fig: Capillary FlowProfiles21
- 22. This velocity gradientresults in substantial band broadening. In electrically driven systems, the driving force of the EOF is uniformly distributed along the entire length of the capillary. As a result, there is no pressure drop and the flow velocity is uniform across the entire tubing diameter except very close to the wall where the velocity again approaches zero. The efficiency of a system can be derived from fundamental principles. 22
- 23. The migration velocity,vep, is simply vep =μepE=μep 𝑉 𝐿 The migration time, t, is defined as t= 𝐿 vep = 𝐿2 v μep During migration through the capillary, molecular diffusion occurs leading to peak dispersion, s2, calculated as s2= 2Dmt = 2DmL2 v μep • where, Dm= the solute’s diffusion coefficient cm2/s. The number of theoretical plates is given as N= 𝐿2 s2 23
- 24. Substituting the dispersionequation into the plate count equation yields N= v μep 2Dm The dispersion, s2, in this simple system is assumed to be time- related diffusion only. The equation indicates that macromolecules such as proteins and DNA, which have small diffusion coefficients, D, will generate the highest number of theoretical plates. In addition, the use of high voltages will also provide for the greatest efficiency by decreasing the separation time. The practical voltage limit with today’s technology is about 30 kV. The practical limit of field strength (one could use very short capillaries to generate high field strength) is Joule heating. 24
- 25. Joule heating isa consequence of the resistance of the buffer to the flow of current. The resolution, Rs, between two species is given by the expression Rs= 1 4 ∆μep μep 𝑁 where, • Dmepis the difference in electrophoretic mobility between the two species, • μepis the average electrophoretic mobility of the two species and • N is the number of theoretical plates. 25
- 26. The production ofheat in CE is the inevitable result of the application of high field strengths. Two major problems arise from heat production: temperature gradients across the capillary and temperature changes with time due to ineffective heat dissipation. The rate of heat generation in a capillary can be approximated as follows 𝑑𝐻 𝑑𝑡 = 𝑖𝑉 𝐿𝐴 where L is the capillary length and A, the cross-sectional area. Capillary Diameter and Joule Heating 26
- 27. Since i =V/R and R = L/kA where k is the conductivity, then 𝑑𝐻 𝑑𝑡 = 𝑘𝑉2 𝐿2 The amount of heat generated is proportional to the square of the field strength. Either decreasing the voltage or increasing the length of the capillary has a dramatic effect on the heat generation. The use of low conductivity buffers is also helpful in this regard although sample loading is adversely affected. Temperature gradients across the capillary are a consequence of heat dissipation. Since heat is dissipated by diffusion, it follows that the temperature at the centre of the capillary should be greater than at the capillary walls. 27
- 28. Since viscosity islower at higher temperatures, it follows that both the EOF and electrophoretic mobility (EPM) will increase as well. Mobility for most ions increases by 2% per degree kelvin. The result is a flow profile that resembles hydrodynamic flow, and band broadening occurs. Operating with narrow-diameter capillaries improves the situation for two reasons: • the current passed through the capillary is reduced by the square of the capillary radius, and • the heat is more readily dissipated across the narrower radial path. 28
- 29. The temperature difference,∆t, between the centre of the tube and its wall is given by • where, W is the heat in watts m-3, • k is the thermal conductivity in cal sec-1 cm-1⁰C-1, and • r is the tube radius. The second problem is ineffective heat dissipation. If heat is not removed at a rate equal to its production, a gradual but progressive temperature rise will occur until equilibrium is reached. Depending on the specific experimental conditions, imprecision in migration time will result due to variance in both EOF and electrophoretic velocity.29
- 30. Narrow-diameter capillaries helpheat dissipation, but effective cooling systems are required to ensure heat removal. Liquid cooling is the most effective means of heat removal and capillary temperature control. Capillary inner diameters range from 20-200 mm. From the standpoint of resolution, the smaller the capillary i.d., the better the separation. However, smaller-bore capillaries yield poorer limits of detection due to reduced detector path length and sample loadability. Narrow capillaries are also more prone to clogging. As long as buffers are filtered through <0.5mm filters, clogging is seldom a problem in the above mentioned size range. 30
- 31. Both the electroosmoticand electrophoretic velocities are directly proportional to the field strength, so the use of the highest voltages possible will result in the shortest times for the separation. Theory predicts that short separation times will give the highest efficiencies since diffusion is the most important feature contributing to band broadening. The limiting factor here is Joule heating. Experimentally, the optimal voltage is determined by performing runs at increasing voltages until deterioration in resolution is noted. Effects of Voltage and Temperature 31
- 32. Viscosity is afunction of temperature; therefore, precise temperature control is important. As the temperature increases, the viscosity decreases; thus, the electrophoretic mobility increases as well. Some buffers such as Tris are known to be pH-sensitive with temperature. For complex separations such as peptide maps, even small pH shifts can alter the selectivity. Most separations are performed at 25C (i.e., near room temperature). With liquid cooling of the capillary it is possible to maintain excellent temperature control, even with high-concentration buffers and large-bore capillaries. 32
- 33. Whenever temperature controlstarts to become a problem, the usual strategy is to use a smaller-bore capillary (less current reduces the heat produced) or a longer capillary (more surface area dissipates the heat generated). An alternative is to reduce the buffer concentration, but this also reduces peak efficiency by decreasing the focusing effect. Inadequate temperature control is the main reason for using low-concentration (e.g., 20 mM) buffers or operating at elevated temperatures. 33
- 34. Capillary electrophoresis comprisesa family of techniques that have dramatically different operative and separative characteristics. The techniques are: • Capillary zone electrophoresis • Isoelectric focusing • Capillary gel electrophoresis • Isotachophoresis • Micellar electrokinetic capillary chromatography Modes of Capillary Electrophoresis 34
- 35. Capillary zone electrophoresis(CZE), also known as free solution capillary electrophoresis, is the simplest form of CE. The separation mechanism is based on differences in the charge- to-mass ratio. Fundamental to CZE are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. Following injection and application of voltage, the components of a sample mixture separate into discrete zones as shown in the figure. Capillary Zone Electrophoresis 35
- 36. Fig: Capillary ZoneElectrophoresis36
- 37. The fundamental parameter,electrophoretic mobility, mep, can be approximated from Debeye-Huckel-Henry theory µep= 𝑞 6𝜋𝜂𝑅 where, q is the net charge, R is the Stokes radius, and h is the viscosity. The net charge is usually pH dependent. For example, within the pH range of 4-10, the net charge on sodium is constant as is its mobility. Other species such as acetate or glutamate are negatively charged within that pH range and thus have negative mobilities (they migrate towards the positive electrode). 37
- 38. At alkaline pH,their net migration will still be towards the negative electrode because of the EOF. Zwitterions such as amino acids, proteins, and peptides exhibit charge reversal at their pI’s and, likewise, shifts in the direction of electrophoretic mobility. Advantages: Separations of both large and small molecules can be accomplished by CZE. Even small molecules, where the charge-to-mass ratio differences may not be great, may still be separable. 38
- 39. Capillaries: Capillaries with aninternal diameter of 25-75mm are usually employed. Fused silica is the material of choice due to its UV transparency, durability (when polyimide coated), and zeta potential. A new capillary must be conditioned before it can be used. Pretreat the capillary for 10 min with 0.1 M sodium hydroxide, 5 min with water, and 10 min with run buffer. This conditioning procedure is important to ensure that the surface of the capillary is fully and uniformly charged. Not all attempts to store a fused silica capillary are successful. 39
- 40. The smaller thecapillary i.d., the more prone it is to clogging. While this is not too serious for bare silica, capillary damage can be costly when using chemically modified capillaries. The following procedure will maximize the chances of successfully storing a capillary: • Rinse the capillary with 0.1 M NaOH for a few minutes.(Do not do this with chemically-modified capillaries.) • Rinse for 5 min with distilled water. • Place an empty, uncapped vial at the outlet end and blow N2 through the capillary for 5 min. • Remove the capillary cartridge from the instrument. 40
- 41. Applications of CZE: CZEis very useful for the separation of proteins and peptides since complete resolution can often be obtained for analytes differing by only one amino acid substituent. This is particularly important in tryptic mapping where mutations and post-translational modifications must be detected. Other applications where CZE may be useful include inorganic anions and cations such as those typically separated by ion chromatography. Small molecules such as pharmaceuticals can often be separated provided they are charged. In most cases, the technique of micellar electrokinetic capillary chromatography gives superior results for charged as well as neutral small molecules. 41
- 42. The fundamental premiseof isoelectric focusing (IEF) is that a molecule will migrate so long as it is charged. Should it become neutral, it will stop migrating in the electric field. IEF is run in a pH gradient where the pH is low at the anode and high at the cathode. The pH gradient is generated with a series of zwitter ionic chemicals known as carrier ampholytes. When a voltage is applied, the ampholyte mixture separates in the capillary. Ampholytes that are positively charged will migrate towards the cathode while those negatively charged migrate towards the anode. Isoelectric Focusing 42
- 43. The pH thenwill decrease at the anodic section and increase at the cathodic section. Finally, the ampholyte migration will cease when each ampholyte reaches its isoelectric point and is no longer charged. Initially, a solute with a net negative charge will migrate towards the anode where it encounters buffer of decreasing pH. Finally, the solute encounters a pH where its net charge becomes zero, the isoelectric point (pI), and migration halts. The greater the number of ampholytes in solution, the smoother the pH gradient. The pH of the anodic buffer must be lower than the pI of the most acidic ampholyte to prevent migration into the analyte. Likewise, the catholyte must have a higher pH than the most basic ampholyte. 43
- 44.
- 45. Resolving power: The resolvingpower, DpI, of IEF is described by the equation DpI=3 D(dpH/dx)/E(dμ/dpH) where, D is the diffusion coefficient, E is the electric field strength, and m is the electrophoretic mobility of the protein. A resolving power of 0.02 pH units has been calculated. Loading: The sample is mixed with the appropriate ampholytes to a final concentration of 1-2% ampholytes. The mixture is loaded into the capillary either by pressure or vacuum aspiration. 45
- 46. Focusing: The buffer reservoirsare filled with sodium hydroxide (cathode) and phosphoric acid (anode). Field strengths on the order of 500-700 V/cm are employed. As the focusing proceeds, the current drops to less than 1mA. Overfocusing can result in precipitation due to protein aggregation at high localized concentrations. Mobilization: Mobilization can be accomplished in either the cathodic or anodic direction. For cathodic mobilization, the cathode reservoir is filled with sodium hydroxide/sodium chloride solution. In anodic mobilization, the sodium chloride is added to the anode reservoir. The addition of salt alters the pH in the capillary when the voltage is applied since the anions/ cations compete with hydroxyl/hydronium ion migration. 46
- 47. Applications: In addition toperforming high resolution separations, IEF is useful for determining the pI of a protein. IEF is particularly useful for separating immunoglobulins, haemoglobin variants and post- translational modifications of recombinant proteins. 47
- 48. Traditional gel electrophoresisis conducted in an anticonvective medium such as polyacrylamide or agarose. The composition of the media can also serve as a molecular sieve to perform size separations. Furthermore, the gel suppresses the EOF. Because of the long history of this technique, the adaptation to CE is very desirable. This is particularly valuable for DNA separations since no other technique to date has provided such dramatic separations. Commercial capillary gel columns are now beginning to be introduced to the marketplace from numerous sources. Polyacrylamide gel-filled capillaries are usually employed, although new polymer formulations with greater stability to the applied electric field are likely to be introduced shortly. Capillary Gel Electrophoresis 48
- 49. Fig: Capillary GelElectrophoresis 49
- 50. Agarose gels areunable to withstand the heating produced by the high voltages used in capillary gel electrophoresis (CGE). There are two fundamental classes of gels that can be employed in CGE. The physical gel obtains its porous structure by entanglement of polymers and is quite rugged to changes in the environment. Hydroxy propyl methylcellulose and similar polymers can be used to form physical gels. Chemical gels use covalent attachment to form the porous structure. These gels are less rugged, and it is difficult to change the running buffers once the gels are formed. CGE is typically performed in 50- to 100-mm capillaries in lengths of about 10 cm to 1 m. 50
- 51. Fig: Capillary GelElectrophoresis 51
- 52. Prior to 1981,isotachophoresis (ITP) was the most widely used instrumental capillary electrophoretic technique, although the capillaries were quite wide (250-500 mm) by today’s standards. A commercial instrument, the LKB Tachophor, was introduced in the mid-1970s. Like IEF, ITP relies on zero electroosmotic flow, and the buffer system is heterogeneous. The capillary is filled with a leading electrolyte that has a higher mobility than any of the sample components to be determined. Then the sample is injected. A terminating electrolyte occupies the opposite reservoir, and the ionic mobility of that electrolyte is lower than any of the sample components. Isotachophoresis 52
- 53. Fig: Capillary AnionicIsotachophoresis 53
- 54. Separation will occurin the gap between the leading and terminating electrolytes based on the individual mobilities of the analytes. In the early instrumentation, detection was by conductivity or differential conductivity. Conductivity detection gave a stair-step pattern as each individual ion passed the electrodes. Differential conductivity could restore the isotachopherogram to a series of conventional peaks. Direct UV detection also gives a more familiar looking electropherogram in the presence of spacers. A spacer is a nonabsorbing solute with a mobility value that falls in between the mobilities of two peaks that need to be resolved. 54
- 55. The disadvantage ofITP is that unless spacers are employed, adjacent bands are in contact with each other. A second problem is that, compared to CZE, the selection and optimization of the buffer are less straight forward. For example, to determine cations, the leading cathodic electrolyte might contain highly mobile acid (H+) while the terminating anodic electrolyte might contain a weaker acid such as propionic acid. Isotachophoresis has two characteristics, the combination of which is unique to electrophoretic methods: all bands move at the same velocity, and the bands are focused. For example, highly mobile bands have high conductivity, and as a result, have a lower voltage drop across the band. 55
- 56. Perhaps the mostintriguing mode of CE for the determination of small molecules is MECC. The use of micelle-forming surfactant solutions can give rise to separations that resemble reverse-phase LC with the benefits of CE. Unlike IEF or ITP, MECC relies on a robust and controllable EOF. Micelles: Micelles are amphiphilic aggregates of molecules known as surfactants. They are long chain molecules (10-50 carbon units) and are characterized as possessing a long hydrophobic tail and a hydrophilic head group. Micellar Electrokinetic Capillary Chromatography 56
- 57.
- 58. Micelles form asa consequence of the hydrophobic effect, that is, they form to reduce the free energy of the system. The hydrophobic tail of the surfactant cannot be solvated in aqueous solution. Above a surfactant concentration known as the critical micelle concentration (CMC), the aggregate is fully formed. Physical changes such as surface tension, viscosity, and the ability to scatter light accompany micelle formation. Reverse micelles, which form in organic solvents, have not been studied in MECC. There are four major classes of surfactants: anionic, cationic, zwitter ionic, and nonionic, examples of which are given in the table. Of these four, the first two are most useful in MECC. 58
- 59. Table: Surfactant classesand properties 59
- 60. Separation mechanism: At neutralto alkaline pH, a strong EOF moves in the direction of the cathode. If SDS is employed as the surfactant, the electrophoretic migration of the anionic micelle is in the direction of the anode. As a result, the overall micellar migration velocity is slowed compared to the bulk flow of solvent. Since analytes can partition into and out of the micelle, the requirements for a separation process are at hand. When an analyte is associated with a micelle, its overall migration velocity is slowed. When an uncharged analyte resides in the bulk phase, its migration velocity is that of the EOF. Therefore, analytes that have greater affinity for the micelle have slower migration velocities compared to analytes that spend most of their time in the bulk phase. 60
- 61. Migration order: With SDSmicelles, the general migration order will be anions, neutrals, and cations. Anions spend more of their time in the bulk phase due to electrostatic repulsions from the micelle. The greater the anionic charge, the more rapid the elution. Neutral molecules are separated exclusively based on hydrophobicity. Cations elute last due to strong electrostatic attraction (e.g., ion- pairing with the micelle). While this is a useful generalization, strong hydrophobic interaction can overcome electrostatic repulsions and attractions. Likewise, the electrophoretic migration of the analytes can also affect the elution order. 61
- 62. Chiral Recognition: Chiral recognitionis dependent on the formation of diastereomers either through covalent or electrostatic interactions. There are several approaches for performing chiral separations by CE. When an analyte is complexed with the micellar or cyclodextrin additive, its migration velocity is slowed relative to the bulk phase. The enantiomer that forms the more stable complex will always show a longer migration time because of this effect. The main disadvantage of this approach towards chiral recognition is that it is difficult to predict which analytes will optically resolve with a particular additive. 62
- 63.
- 64. Applications: Food Analysis • Determinationof procyanidins and other phenolic compounds in lentil samples. • Determination of amoxicillin, ampicillin, sulfammethoxazole, sulfacetamide in animal feed. Advantages: Ability to separate chiral compounds. Low solvent consumption (environment friendly). Efficient separation. Relatively quicker compared to HPLC and GC. Cheaper. 64
- 65. Table: Selecting themode of CE 65
- 66. CE offers anovel format for liquid chromatography and electrophoresis that: employs capillary tubing within which the electrophoretic separation occurs; utilizes very high electric field strengths, often higher than 500 V/cm; uses modern detector technology such that the electropherogram often resembles a chromatogram; has efficiencies on the order of capillary gas chromatography or even greater; requires minute amounts of sample; Advantages of CE 66
- 67. is easily automatedfor precise quantitative analysis and ease of use; consumes limited quantities of reagents; is applicable to a wider selection of analytes compared to other analytical separation techniques. 67
- 68. General: Not well-suited fordetermination of nonpolar, low molecular weight, volatile compounds which are best determined by GC. Not well suited for determination of nonionic, high molecular weight polymers. Not as sensitive as HPLC. Accuracy Precision ranges of 1 to 2 relative standard deviation(%). Sensitivity and Detection Limits Sensitivities of mg/L (parts per million) to µg/L (parts per billion). Limitations of CE 68
- 69. Separation and identificationof polar and non polar compounds and some elements, including nonionic and ionic organic compounds, inorganic anion and cations, macromolecules (such as proteins and oligonucleotides) and chiral compounds. Quantitative and qualitative determination of compounds and some elements in mixtures. Determination of molecular weights of large biomolecules and isoelectric points of proteins. Depending on the type of detector used, can be non destructive or destructive. Common Applications of CE 69
- 70. Can be automatedfor analysis of liquid samples or solid samples dissolved in a liquid. Applicable to the separation and determination of many of the same types of samples as HPLC, ion chromatography and slab gel electrophoresis. Used in biochemical, clinical, environmental, food, forensic, and pharmaceutical applications. 70
- 71. 1. Introduction toCapillary Electrophoresis by Beckman and Coulter . 2. Principles of Instrumental Analysis, Douglas A. Skoog, F.James Holler, Stanley R. Crouch pg no – 865-878. 3. Pharmaceutical Analysis ,Volume II by Ashutosh Kar. References 71
- 72.