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CAPILLARY ELECTROPHORESIS

Prepared by:

Dr.Elsadig H.KH.Adam

Instrumental Analysis -Dr.Elsadig H.kh.Adam1

Electrophoresis It is another class of separation techniques in which

analytes are separated based on their ability to movethrough a conductive medium, usually an aqueous buffer,in response to an applied electric field.

In capillary electrophoresis the conducting buffer isretained within a capillary tube whose inner diameter istypically 25–75 mm.

Samples are injected into one end of the capillary tube.Under the effect of applied electric field, the samplemigrates through the capillary, its components separateand elute from the column at different times.

The resulting electropherogram looks similar to thechromatograms obtained in GC or HPLC and provides bothqualitative and quantitative information.


Theory of Capillary Electrophoresis In capillary electrophoresis the sample is injected into a

buffered solution retained within a capillary tube. When anelectric field is applied to the capillary tube, the sample’scomponents migrate as the result of two types of mobility:electrophoretic mobility and electroosmotic mobility.

Electrophoretic mobility is the solute’s response to theapplied electric field. Cations move toward the negativelycharged cathode, anions move toward the positivelycharged anode, and neutral species, which do not respondto the electric field, remain stationary.

The other contribution to a solute’s migration iselectroosmotic flow, which occurs when the buffer solutionmoves through the capillary in response to the appliedelectric field.

Under normal conditions the buffer solution moves towardthe cathode, sweeping most solutes, even anions, towardthe negatively charged cathode.


Electrophoretic Mobility The velocity with which a solute moves in response to the

applied electric field is called its electrophoretic velocity, it isdefined as

Electrophoretic velocity = µEwhere µ is the solute’s electrophoretic mobility, and E is themagnitude of the applied electric field. A solute’s electrophoretic mobility is defined as

µ = q/6ɳπrwhere q is the solute’s charge, ɳ is the buffer solvent’s viscosity, and ris the solute’s radius.

From the above equations, electrophoretic mobility, and,therefore, electrophoretic velocity, is largest for more highlycharged solutes and solutes of smaller size. Since q is positivefor cations and negative for anions, these species migrate inopposite directions. Neutral species, for which q is 0, have anelectrophoretic velocity of 0.


Electroosmotic Mobility

What is observed under normal conditions,however, is that the buffer solution movestoward the cathode. This phenomenon is calledthe electroosmotic flow.

Electroosmosis occurs because the walls of thecapillary tubing are electrically charged. Thesurface of a silica capillary contains large numbersof silanol groups (Si–OH). At pH levels greaterthan approximately 2 or 3, the silanol groupsionize to form negatively charged silanate ions(Si–O–). Cations from the buffer are attracted tothe silanate ions.


From the above figure, some of the cations bind tightly to thesilanate ions, forming an inner, or fixed, layer. Other cations aremore loosely bound, forming an outer, or mobile, layer. Togetherthese two layers are called the double layer.

Cations in the outer layer migrate toward the cathode. Becausethese cations are solvated, the solution is also pulled along,producing the electroosmotic flow.


Electroosmotic flow velocity is a function of themagnitude of the applied electric field and the buffersolution’s electroosmotic mobility (µeof)Electroosmotic flow velocity = µeofEElectroosmotic mobility is defined asµeof = ƐƇ/4ɳπwhere Ɛ is the buffer solution’s dielectric constant, Ƈ is thezeta potential, and ɳ is the buffer solution’s viscosity. From the above equations, zeta potential plays an

important role in determining the electroosmotic flowvelocity. The zeta potential is directly proportional to thecharge on the capillary walls, with a greater density ofsilanate ions corresponding to a larger zeta potential.

Below a pH of 2, for example, there are few silanate ions;thus, the zeta potential and electroosmotic flow velocityare 0. As the pH level is increased, both the zeta potentialand the electroosmotic flow velocity increase.


N.B.The electroosmotic flow profile is very different from that for aphase moving under forced pressure. The flow profile forelectroosmosis with that for hydrodynamic pressure. Theuniform, flat profile for electroosmosis helps to minimize bandbroadening in capillary electrophoresis, thus improvingseparation efficiency.


Total Mobility A solute’s net, or total velocity, is the sum of its electrophoretic

velocity and the electroosmotic flow velocity. Under normal conditions the following relationships hold:

(vtot)cations > veof (vtot)anions < veof (vtot)neutrals = veof

Thus, cations elute first in an order corresponding to theirelectrophoretic mobilities, with small, highly charged cationseluting before larger cations of lower charge. Neutral specieselute as a single band, with an elution rate corresponding tothe electroosmotic flow velocity. Finally, anions are the lastcomponents to elute, with smaller, highly charged anionshaving the longest elution time.


N.B. The solute migration time is governed by the following

relation:

𝑡 =𝑙𝐿

µ𝑒𝑝 + µ𝑒𝑜𝑓 𝑉Where l is the distance from the site of injection to the detector site, L is the length of the capillary tube, V is the applied potential. So we can decrease a solute’s migration time (and thus

the total analysis time) by applying a higher voltage orby using a shorter capillary tube. Increasing theelectroosmotic flow also shortens the analysis time,but, as we will see shortly, at the expense of resolution.


Efficiency

The efficiency of capillary electrophoresis is characterized by thenumber of theoretical plates, N, just as it is in GC or HPLC.

In capillary electrophoresis, the number of theoretic plates isdetermined by the following equation:

𝑁 =µ𝑒𝑝 + µ𝑒𝑜𝑓 𝑉

2𝐷where D is the solute’s diffusion coefficient.

From the above equation it is easy to see that the efficiency of acapillary electrophoretic separation increases with higher voltages.

Again, increasing the electroosmotic flow velocity improvesefficiency, but at the expense of resolution.

Efficiency in capillary electrophoresis is independent on thecapillary’s length (opposite to chromatography).

Typical theoretical plate counts are approximately 100,000–200,000 for capillary electrophoresis.


Selectivity In chromatography, selectivity is defined as the ratio of the capacity factors for two

solutes. In capillary electrophoresis, the analogous expression for selectivity is

α =µ𝑒𝑝1

µ𝑒𝑝2

where µ𝑒𝑝1 and µ𝑒𝑝2 are the electrophoretic mobilities for solutes 1 and 2,

respectively, chosen such that α > 1.

Selectivity often can be improved by adjusting the pH of the buffer solution.

For example, ammonium ion is a weak acid with a pKa of 9.24. At a pH of 9.24 the

concentrations of ammonium ion and ammonia neutral molecule are equal.

Decreasing the pH below 9.24 increases its electrophoretic mobility because a

greater fraction of the solute is present as the cation. On the other hand, raising

the pH above 9.24 increases the proportion of the neutral ammonia, decreasing its

electrophoretic mobility.Instrumental Analysis -Dr.Elsadig H.kh.Adam12

Resolution

The resolution between two solutes is

where µav is the average electrophoretic mobility for the two solutes.

From the above equation, increasing the applied voltage and decreasing

the electroosmotic flow velocity improves resolution.

The latter effect is particularly important because increasing

electroosmotic flow improves analysis time and efficiency while

decreasing resolution.

𝑅 =0.177 µ𝑒𝑝2 − µ𝑒𝑝1 √𝑉

(µ𝑎𝑣 + µ𝑒𝑜𝑓) 𝐷


INSTRUMENTATION

The basic instrumentation for capillary electrophoresis includes apower supply for applying the electric field, anode and cathodecompartments containing reservoirs of the buffer solution, asample vial containing the sample, the capillary tube, and adetector.


Capillary Tubes

Most capillary tubes are made from fused silica coated with a 20–35-µmlayer of polyimide to give it mechanical strength. The inner diameter istypically 25–75 µm, which is smaller than that for a capillary GC column,with an outer diameter of 200–375 µm.

The narrow bore of the capillary column and the relative thickness of thecapillary’s walls are important. When an electric field is applied to acapillary containing a conductive medium, such as a buffer solution,current flows through the capillary.


This current leads to Joule heating, the extent of which isproportional to the capillary’s radius and the magnitude of theelectric field.

Joule heating is a problem because it changes the buffersolution’s viscosity, with the solution at the center of thecapillary being less viscous than that near the capillary walls.Since the solute’s electrophoretic mobility depends on thebuffer’s viscosity and so solutes in the center of the capillarymigrate at a faster rate than solutes near the capillary walls. .

The result is additional band broadening that degrades theseparation.

Capillaries with smaller inner diameters generate less Jouleheating, and those with larger outer diameters are moreeffective at dissipating the heat.


Injecting the Sample

The mechanism by which samples are introduced in capillaryelectrophoresis is quite different from that used in GC orHPLC. Two types of injection are commonly used:hydrodynamic injection and electrokinetic injection.

In both cases the capillary tube is filled with buffer solution.One end of the capillary tube is placed in the destinationreservoir, and the other is placed in the sample vial.

Hydrodynamic injection

It uses pressure to force a small portion of the sample into thecapillary tubing.

To inject a sample hydrodynamically a difference in pressure isapplied across the capillary by either pressurizing the samplevial or by applying a vacuum to the destination reservoir.


Electrokinetic injection

It is made by placing both the capillary and theanode into the sample vial and briefly applying anelectric field so the introduction of the sample ismainly dependent on the electrophoreticmobility of the samples.

The solutes with the largest electrophoreticmobilities (smaller, more positively charged ions)are injected in greater numbers than those withthe smallest electrophoretic mobilities (smaller,more negatively charged ions).


StackingWhen a solute’s concentration in the sample is very

small, it may be possible to inject the solute in amanner that increases its concentration in the capillarytube. This method of injection is called stacking.

Stacking is accomplished by placing the sample in asolution whose ionic strength is significantly less thanthat of the buffering solution. Because the sample plughas a lower concentration of ions than the bufferingsolution, its resistance is greater. Since the electriccurrent passing through the capillary is fixed, so theelectric field in the sample plug is greater than that inthe buffering solution.

Ohm Law E = iR


Since electrophoretic velocity is directlyproportional to the electric field ,thus, ions in thesample plug migrate with a greater velocity.When the solutes reach the boundary betweenthe sample plug and the buffering solution, theelectric field decreases and their electrophoreticvelocity slows down, “stacking” together in asmaller sampling zone as shown in the followingfigure


Applying the Electric Field

Migration in electrophoresis occurs inresponse to the applied electric field.

Application of large electric field is importantbecause higher voltages lead to shorteranalysis times, more efficient separations, andbetter resolution.

Voltages up to 40 kV can be applied.


Detectors Most of the detectors used in HPLC also find use in capillary

electrophoresis. UV/Vis detectors are among the most popular. Because

absorbance is directly proportional to path length, the capillarytubing’s small diameter leads to signals that are smaller thanthose obtained in HPLC.

Several approaches have been used to increase the path length,including a Z-shaped sample cell and bubble cell.

Better detection limits are obtained using fluorescence,particularly when using a laser as an excitation source.


Capillary Electrophoresis Methods

1- Capillary Zone Electrophoresis (CZE). In CZE the capillary tube is filled with a buffer solution and,

after loading the sample, the ends of the capillary tube areplaced in reservoirs containing additional buffer solution.

Under normal conditions, the end of the capillarycontaining the sample is the anode, and solutes migratetoward the cathode at a velocity determined by theirelectrophoretic mobility and the electroosmotic flow.

Cations elute first, with smaller, more highly chargedcations eluting before larger cations with smaller charges.

Neutral species elute as a single band. Finally, anions arethe last species to elute, with smaller, more negativelycharged anions being the last to elute.


The direction of electroosmotic flow and, therefore, theorder of elution in CZE can be reversed. This isaccomplished by adding an alkylammonium salt to thebuffer solution. The positively charged end of thealkylammonium ion binds to the negatively chargedsilanate ions on the capillary’s walls.

The alkylammonium ion’s “tail” is hydrophobic andassociates with the tail of another alkylammonium ion.

The result is a layer of positive charges to which anionsin the buffer solution are attracted.

The migration of these solvated anions toward theanode reverses the electroosmotic flow’s direction. Theorder of elution in this case is exactly the opposite ofthat observed under normal conditions.


Capillary zone electrophoresis also can beaccomplished without an electroosmotic flow bycoating the capillary’s walls with a nonionic reagent.

In the absence of electroosmotic flow only cationsmigrate from the anode to the cathode.

Capillary zone electrophoresis provides an effectiveseparations of any charged species, including inorganicanions and cations, organic acids and amines, and largebiomolecules such as proteins. For example, CZE hasbeen used to separate a mixture of 36 inorganic andorganic ions in less than 3 minutes. Neutral species, ofcourse, cannot be separated.


2- Micellar Electrokinetic Capillary Chromatography

One limitation to CZE is its inability to separate neutralspecies. Micellar electrokinetic chromatography (MEKC)overcomes this limitation by adding a surfactant, such assodium dodecylsulfate to the buffer solution.

Sodium dodecylsulfate, (SDS) has a long-chain hydrophobic“tail” and an ionic functional group, providing a negativelycharged “head.” When the concentration of SDS issufficiently large, a micelle forms. A micelle consists of anagglomeration of 40–100 surfactant molecules in which thehydrocarbon tails point inward, and the negatively chargedheads point outward.

Because micelles are negatively charged, they migratetoward the cathode with a velocity less than theelectroosmotic flow velocity.


Neutral species partition themselves between the micellesand the buffer solution in much the same manner as theydo in HPLC. Because there is a partitioning between twophases, the term “chromatography” is used.

Note that in MEKC both phases are “mobile.” The elutionorder for neutral species in MEKC depends on the extent towhich they partition into the micelles.

Hydrophilic neutrals are insoluble in the micelle’shydrophobic inner environment and elute as a single bandas they would in CZE. Neutral solutes that are extremelyhydrophobic are completely soluble in the micelle, elutingwith the micelles as a single band. Those neutral speciesthat exist in a partition equilibrium between the buffersolution and the micelles elute between the completelyhydrophilic and completely hydrophobic neutrals.

Micellar electrokinetic chromatography has been used toseparate a wide variety of samples, including mixtures ofpharmaceutical compounds, vitamins, and explosives.


3- Capillary Gel Electrophoresis In capillary gel electrophoresis (CGE) the capillary tubing is filled with

a polymeric gel. Because the gel is porous, solutes migrate through thegel with a velocity determined both by their electrophoretic mobilityand their size.

The ability to effect a separation based on size is useful when thesolutes have similar electrophoretic mobilities. For example, fragmentsof DNA of varying length have similar charge-to-size ratios, makingtheir separation by CZE difficult. Since the DNA fragments are ofdifferent size, a CGE separation is possible.

The capillary used for CGE is usually treated to eliminateelectroosmotic flow, thus preventing the gel’s extrusion from thecapillary tubing.

Samples are injected electrokinetically because the gel provides toomuch resistance for hydrodynamic sampling.

The primary application of CGE is the separation of largebiomolecules, including DNA fragments, proteins, andoligonucleotides.


4- Capillary Electrochromatography Another approach to separating neutral species is

capillary electrochromatography (CEC). In this technique the capillary tubing is packed with

1.5–3-mm silica particles coated with a bonded,nonpolar stationary phase.

Neutral species separate based on their ability topartition between the stationary phase and the buffersolution (the mobile phase).

Separations are similar to the analogous HPLCseparation, but without the need for high-pressurepumps.

The efficiency in CEC is better than in HPLC, withshorter analysis times.


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