Electrophoresis refers to the migration of charged electrical species when dissolved or suspended in an electrolyte through which an electric current is passed.Cations migrate toward the negatively charged electrode (cathode),while anions are attracted toward the positively charged electrode (anode).Neutral particles are not attracted toward either electrode.

The use of capillaries as a migration channel in electrophoresis has enabled analysts to perform electrophoretic separations on an instrumental level comparable to that of high-performance liquid chromatography (HPLC),albeit with some distinct operational differences,advantages,and disadvantages relative to HPLC.This method of analysis is commonly known as capillary electrophoresis (CE).During typical CEoperation with an uncoated capillary filled with a buffer,referred to as the “operating buffer,”silanol groups present on the inner wall of the glass capillary release hydrogen ions to the buffer and the wall surface becomes negatively charged,even at a fairly low pH.Cations,or solutes having partial positive charges in the medium,are electrostatically attracted to the negatively charged wall,forming an electrical double layer.The initiation of electrophoresis by applying voltage across the length of the capillary causes the solution portion of the electrical double layer to move toward the cathode end of the capillary,drawing the bulk solution.This movement of the bulk solution under the force of the electrical field is called the electroosmotic flow (EOF).The degree of ionization of the inner-wall capillary silanol groups depends mainly on the pHof the operating buffer and on the modifiers that may have been added to the electrolyte.At low pH,the silanol groups generally have a low ionization and the EOFis low.At higher pH,silanol groups become more ionized and the EOFincreases.In some cases organic solvents,such as methanol or acetonitrile,are added to the aqueous buffer to increase the solubility of the solute and other additives or to affect the degree of ionization of the sample.The addition of such organic modifiers generally causes a decrease in the EOF.The detector is located toward the cathode end of the capillary.The EOFis usually greater than the electrophoretic mobility;thus,even anions are swept toward the cathode and the detector.When an uncoated capillary containing pH7.0phosphate buffer is used,the usual order of appearance of solutes in an electropherogram is cationic species,neutral solutes,and anionic species.

Currently,there are five major modes of operation of CE:capillary zone electrophoresis (CZE),also referred to as free solution or free flow capillary electrophoresis;micellar electrokinetic chromatography (MEKC);capillary gel electrophoresis (CGE);capillary isoelectric focusing (CIEF);and capillary isotachophoresis (CITP).

In CZE,separations are controlled by differences in the relative electrophoretic mobilities of the individual components in the sample or test solution.The mobility differences are functions of analyte charge and size under specific method conditions.They are optimized by appropriate control of the composition of the buffer,its pH,and its ionic strength.

In MEKC,ionic surfactants are added to the operating buffer at a concentration above their critical micelle concentration.The micelles provide a pseudostationary phase with which analytes can partition.This technique is useful for the separation of neutral and ionic species.

CGE,which is analogous to gel filtration,uses gel-filled capillaries to separate molecules on the basis of relative differences in their respective molecular weight or molecular size.It was first used for the separation of proteins,peptides,and oligomers.Gels may have the advantage of decreasing the EOFand also significantly reducing protein adsorption onto the inner wall of the capillary,which can significantly reduce analyte peak tailing effects.

In CIEF,substances are separated on the basis of their relative differences in isoelectric points.This is accomplished by achieving steady-state sample zones within a buffer pHgradient,where the pHis low at the anode and high at the cathode.The gradient is established by applying a voltage across a capillary filled with a mixture of carrier components consisting of amphoteric substances having different pIvalues.

CITPemploys two buffers that enclose the analyte zones between them.Either anions or cations can be analyzed in sharply separated zones.In addition,the analyte concentrations are the same in each zone;thus,the length of each zone is proportional to the amount of the particular analyte.

The most commonly utilized capillary electrophoresis techniques are CZEand MEKC.These are briefly discussed in the following sections.Pertinent general principles and theory,instrumental considerations,analysis,and operational considerations and parameters are discussed as well.

PRINCIPLES OF CAPILLARY ZONE ELECTROPHORESIS

µEP=q/(6phr),

nEP=µEP(V/L),

nEO=µEO(V/L),

t=l/E(µEP+µEO)=lL/V(µEP+µEO),

N=(µEP+µEO)V/2D,

PRINCIPLES OF MICELLAR ELECTROKINETIC CHROMATOGRAPHY

tR=(1+k¢)t0/[1+(t0/tMC)],

k¢=(tR/t0-1)/(1-tR/tMC),

k¢=tR/t0-1,

INSTRUMENTAL CONSIDERATIONS

ANALYTICAL CONSIDERATIONS

Capillary Dimensions— Variation of the capillary diameter and length can affect the electrophoretic resolution.Increasing the capillary length results in longer migration times,usually increasing resolution and generating a lower current.Increasing the capillary diameter will usually increase current and associated internal temperature gradients that decrease resolution.Conversely,a reduction in capillary diameter will result in lower heat and better resolution.However,larger capillary diameters have advantages of better mass loading and improved signal-to-noise ratio.

Voltage Effects— When higher voltages are applied,additional internal heating of the operating buffer occurs because of the current flow through the buffer.This heating effect,known as Joule heating,must be controlled because resistance,dielectric constant,and viscosity are temperature-dependent and alter the velocity of the EOFand solute mobilities.

Ionic Strength Effects— Control of ionic strength and its manipulation allow adjustment of resolution,efficiency,and sensitivity.Increasing ionic strength will generally improve resolution,peak efficiency,and peak shape.Sensitivity may be improved because better focusing is achieved.However,because the current generated is directly proportional to the buffer concentration,more heat is produced when ionic strength of the buffer is increased,hence limiting the ionic strengths that can be utilized.

pH Effects— Resolution,selectivity,and peak shape can be dramatically altered by changes in pHas this parameter affects the extent of solute ionization and the level of EOF.The EOFis high at high pHand low at low pHin uncoated fused-silica capillaries.

OPERATIONAL PARAMETERS

System Setup— An appropriate capillary of specific length,inner diameter,and coating is selected,with considerations made for separation and resolution,ionic strength of buffer,and pHeffects.Abuffer of appropriate composition,ionic strength,and pHis prepared,degassed,if necessary,and passed through an appropriate filter.All solvents,including water,are HPLCor CEgrade.

Capillary Rinsing Procedure— Improved consistency of migration times and resolution may generally be obtained if a defined rinsing procedure is followed.Capillary conditioning and rinsing procedures are very specific to the analyte,matrix,and method.Therefore,these procedures are developed as part of the method and are specified in the individual monograph.Rinsing may involve the use of solutions such as 0.1Mphosphoric acid,water,and 0.1Msodium hydroxide.Before beginning analysis of the test specimen,the capillary may be rinsed with five column volumes of the operating buffer that is to be used for the test.When changing buffer composition,it is advisable to rinse the capillary with five column volumes of each new buffer to allow the capillary to be cleansed of the previous buffer.Use of a new uncoated fused-silica capillary usually requires a regeneration procedure to activate the surface silanol groups.This procedure may include an extended rinse with a sodium hydroxide solution.Coated capillaries are rinsed according to the manufacturer's guidelines because inappropriate rinsing can remove or damage the coating.Columns may be dedicated to particular methods or buffer types to prevent cross-contamination.

Running a Sample— An appropriate capillary,electrolyte,and injection procedure are selected to achieve adequate resolution,sensitivity,and separation,with well-shaped and well-defined peaks.The required injection precision for a specific method may require use of an internal standard.The internal standard is selected with consideration of its ability to adequately separate from the analyte.The performance of the system may be improved by rinsing the capillary between injections and supplying fresh buffer to the source and destination vials used during voltage application,namely,vials 2and 4in Figure 1.Replicate injections from the same sample vial may be performed provided that no cross-contamination occurs.If cross-contamination occurs,the capillary tip may be rinsed by briefly inserting it into a vial containing the buffer prior to inserting the capillary into the electrolyte or sample vial.

System Suitability— Parameters measured may include injector reproducibility,system selectivity,system efficiency,and tailing.Resolution between the analytes and other compounds may be determined by using test mixture standards.

Sample Analysis— Once the suitability of the CEsystem has been established,aliquots of both the Standard preparation and the test preparation are injected.Standards are injected before or after the samples and intermittently throughout the run.

Data Handling— Time-normalized peak areas are often used in quantitative calculations.These are determined by dividing the observed integrated peak area by the migration time of the analyte.This compensates for the fact that in CE,unlike HPLC,each analyte travels through the detector at a different velocity.Unless this normalization is performed,slowly moving (later-migrating)analytes will have disproportionately large peak areas compared with those for early migrating components.

System Shutdown— After analysis,the capillary is rinsed according to the directions specified in each monograph or as recommended by the manufacturer.For example,the capillary might be rinsed with distilled water to remove buffer components and then filled with air or nitrogen by performing a rinse from an empty vial.Naturally,the destination and source vials,namely,vials 4and 2in Figure 1,are emptied of buffer and rinsed thoroughly with deionized water.