Capillary Zone Electrophoresis

CHAPTER

2

Capillary Zone Electrophoresis Basic Concepts

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14

Electrical Conduction in Fluid Solution The Language of Electrophoresis Electroendoosmosis Efficiency Resolution Joule Heating Optimizing the Voltage and Temperature Capillary Diameter and Buffer Ionic Strength Optimizing the Capillary Length Buffers Temperature Effects Buffer Additives Capillaries Sources of Band Broadening References

2.1 ELECTRICAL CONDUCTION IN FLUID SOLUTION Several simple concepts are important for understanding the physical processes that occur u p o n passage of an electrical current through an ionic solution. ^ These processes are far more complex than the passage of current through a metal. In metals, uniform and v^eightless electrons carry all the current. In fluid ^See any basic text on physical chemistry for a thorough description of electrical conduction in fluid solution. 25

26

Chapter 2 Capillary Zone Electrophoresis

solution, the current is carried by cations and anions. The molecular weight of these charge bearing ions ranges from a simple proton to tens of thousands for large complex ions such as proteins and polynucleotides. Conduction in fluid solution is still described by Ohm's law, E = IR,

(2.1)

where E is the voltage or applied field, I is the current that passes through the solution, and R is the resistance of the fluid medium. The reciprocal of resistance is conductivity. Kohlrausch found that the conductivity of a solution resulted from the independent migration of ions. As illustrated in Figure 2.1, when a current passes through an ionic solution, anions migrate toward the anode (positive electrode) while cations migrate toward the cathode (negative electrode) in equal quantities. Despite the passage of current, electroneutrality of the solution is always maintained because of electrolysis at each electrode. This is important because electrolysis produces protons at the anode and hydroxide at the cathode (Figure 2.2). The resultant pH changes are due to the process known as buffer depletion (1-3). Since pH is the single most important experimental parameter in capillary electrophoresis, this effect must be minimized by 1. Using the appropriate buffers 2. Having sufficiently large buffer reservoirs 3. Replacing buffers frequently The introduction of a sample into the capillary changes the situation dramatically (Figure 2.3). The Ohm's law equation changes as well to that for a series circuit: E = IR^ + IRj .

(2.2)

This process and the equation have important implications in HPCE. When lowconductivity samples (relative to the BGE) are injected, a process known as stacking (Section 8.6) occurs. This permits the use of large-volume injections

CATHODE

ANODE

FIGURE 2.1

The independent migration of ions.

27

2.1 Electrical Conduction in Fluid Solution

POWER SUPPLY

CAPILLARY

DETECTOR OH GENERATED AT CATHODE

CATHOLYTE

H GENERATED AT ANODE

ELECTRODES

ANOLYTE (INLET)

(OUTLET) FIGURE 2.2

Buffer depletion.

to be employed without excessive band broadening because zone compression occurs. On the other hand, if high-conductivity samples are injected relative to the BGE, antistacking or zone broadening will occur. The conductivity of a solution is determined by two factors: 1. The concentration of the ionic species. 2. The speed of movement or mobility of the ionic species in an electric field. In other words, highly mobile species are also highly conductive, and vice versa.

IR

IR, ©,

FIGURE 2.3

Impact of the sample injection on the IR drops in a capillary.

28

Chapter 2 Capillary Zone Electrophoresis

The mobility of ions in fluid solution is governed by their charge to size ratio. The size of the molecule is based on the molecular weight, the three-dimensional structure, and the degree of solvation (usually hydration). Data given in Table 2.1 (4) for alkali metals illustrate several of these important points: 1. The orders for the mobilities of the metal ions are the reverse of what is expected based on the metal or crystal radii data. These smaller ions are more hydra ted than their larger counterparts. 2. The current generated by 100 mM solutions of various acetate salts is proportional to the ionic mobility of the cation. This feature becomes important when selecting the appropriate counterion for preparing buffer solutions. The forces governing this behavior are expressed by Stoke's law, / = 67rrirv ,

(2.3)

where 7] = viscosity, r = ionic radius, and v = ionic velocity. The competing forces of mobility (velocity) and viscosity are illustrated in Figure 2.4 for an ion of radius r. Ionic size modifies mobility because of a solute's exposure to frictional drag as it migrates through the supporting electrolyte. The frictional drag is directly proportional to viscosity, size, and electrophoretic velocity. An expression for mobility that contains these terms is

M—)=-5^^^^^ = ^ - , Vs

E(V/cm)

(2.4)

6nrir

where q = the net charge and E = the electric field strength. Thus, mobility is considered a charge-to-size ratio. Since the units for velocity are centimeters per second and the field strength is expressed as volts per centimeter, the units of mobility are cm^A^s.

2.2

THE LANGUAGE OF ELECTROPHORESIS

There are several distinguishing differences between the terminology of chromatography and that of capillary electrophoresis. For example, a fundamental parameter in chromatography is the retention time. In electrophoresis nothing should ever be retained (except for CEC), so a more descriptive term is migration time: the time it takes a solute to travel from the beginning of the capillary to the detector window.

2.2

t

T1

0 u


fi o

, ^

2 »OS

o <

3

"rn

Tl rr!

Sf2

U Pi

II

< X;>>^

OH

P^nd

B T!UJ X 0

—1

d

00

rn

O

The Language of Electrophoresis

< I—I

2 o

1^

hs. ^

.-I

ON

in

(N

^ O

s

in rH

ON ON

u

u

=^0 ^ r ^ ON

5 o ^j

<-.

29

30

Chapter 2 Capillary Zone Electrophoresis

MOBILITY FRICTIONAL FORCES

FIGURE 2.4

The competing forces of electrophoretic mobility and viscous drag.

The use of a detection window in HPCE (on-capillary detection) as opposed to postcolumn detection must also be considered. In HPLC, the length of the chromatographic column must be included in all methods. Figure 2.5 is a drawing of a capillary. Both the total length of the capillary (L^ or L) and the length to the detector (L^ or I) must be described. The segment of capillary that occurs after the detector window is necessary to make electrical contact with the outlet or detector-side electrolyte reservoir. Ideally, L^ - L^ should be as short as practical. Otherwise, some system voltage (V) is wasted on maintaining field strength (E) over part of the capillary that lies beyond the detector window and hence does not participate in the separation.

Lt

DETECTION WINDOW FIGURE 2.5 tor (Ld).

Illustration of a capillary defining the total length (Lj) and the length to the detec-

2.3

Electroendoosmosis

31

Expressions for some other fundamental terms are given in the following equations: (2.5)

"ep

Mep

E

(2.6)

.jjtrn The preceding include the electrophoretic mobility (^ep^ cm^A^s), the electrophoretic velocity (Vgp, cm/s), and the field strength (E, V/cm). These equations define some fundamental features of HPCE: 1. Velocities are measured experimentally (Eq. 2.5). They are determined by dividing the length of capillary, from the injection side to the detector window (Ld), by the migration time t^. 2. MobiUties are calculated by dividing the electrophoretic velocity v^p by the field strength (Eq. 2.6). The field strength is simply the voltage divided by the total capillary length (L^). The field strength is the important parameter governing electrophoretic migration. Field strength is changed when either the voltage or the capillary length is altered. Mobility is the fundamental parameter of capillary electrophoresis. This term is independent of voltage and capillary length. Equations (2.6) and (2.7) define only the relative mobility. To calculate the true mobility, a correction for a phenomenon known as electroendoosmotic flow (Section 2.3) must first be made.

2.3 ELECTROENDOOSMOSIS A.

THE CAPILLARY SURFACE

One of the fundamental processes that accompanies electrophoresis is electroosmosis. One of the "pumping" mechanisms of HPCE, electroosmosis occurs because of the surface charge, known as the zeta potential, on the wall of the capillary. Fused silica is the most common material used to produce capillaries for HPCE. Technology developed for manufacturing capillary columns for GC readily transferred to HPCE. Fused silica is a highly crosslinked polymer of silicon dioxide with tremendous tensile strength (5), although it is quite brittle. With its polyimide coating, fused silica is quite durable, although some polyimide must

32

Chapter 2 Capillary Zone Electrophoresis

be removed to create a ultraviolet (UV) transparent optical window for detection. Other materials such as Teflon and quartz have been used (6), but performance and cost are less favorable. Before use, capillaries are usually conditioned with 1 N sodium hydroxide. The base ionizes free silanol groups and may cleave some silica epoxide linkages as well. An anionic charge on the capillary surface results in the formation of an electrical double layer. The resulting ionic distribution is shown in Figure 2.6 (7). Anions are repelled from the negatively charged wall region, whereas cations are attracted as counterions. Ions closest to the wall are tightly bound and immobile, even under the influence of an electric field. Further from the wall is a compact and mobile region with substantial cationic character. At a greater distance from the wall, the solution becomes electrically neutral as the zeta potential of the wall is no longer sensed. Expressions describing this phenomenon were derived by Gouy and Chapman in 1910 and 1913, respectively This diffuse outer region is known as the Gouy-Chapman layer. The rigid inner layer is called the Stem layer. When a voltage is applied, the mobile positive charges migrate in the direction of the cathode or negative electrode. Since ions are solvated by water, the fluid in the buffer is mobilized as well and dragged along by the migrating charge. Although the double layer is perhaps 100 A thick, the electroendoosmotic flow (EOF) is transmitted throughout the diameter of the capillary, presumably through hydrogen bonding of water molecules or van der Waals interactions between buffer constituents. The electroosmotic flow as defined by Smoluchowski in 1903 is given by Veo = ^ E ,

(2.8)

where £ is the dielectric constant, 77 is the viscosity of the buffer, and f is the zeta potential of the liquid-solid interface. The equation is only valid for capillaries sufficiently large that the double layers on opposite walls do not overlap each other (8). Practical use of this equation is not forthcoming, as the zeta potential is rarely measured and data for the dielectric constants of mixtures are not readily available. Like electrophoretic mobility, the EOF is inversely proportional to the viscosity of the BGE.

B.

MEASURING THE ELECTROOSMOTIC FLOW

Since the migration time of a solute is influenced by the EOF, calculation of the actual mobility requires measurement of the EOF:

Here jH^^ = the actual mobility, jU^pp = apparent (observed) mobility, and jLl^o = electroosmotic mobility. The use of mobility as the "migration parameter"

2.3

33

Electroendoosmosis

inttrfaisa

®,

' " ^ l A J Z*^^ /'""x x*i*'

c .^ g—: SI

I

•X-

adsorbed compact layer layer

diffuse layer

B interf8€0

1 « i

4N#

0 fit i

1 1

s

1 1

u <*• ^ l ^ \ r compact. dlffuM***" Llayer 1 ''yr

'

^

•

HMliiiiiiliilnpi

dislanai from lh# eolynin wail FIGURE 2.6 Representation of the electrical double layer versus distance from the capillary wall. Reprinted with permission from J. Chromatogr., 559, 69 (1991), copyright © 1991 Elsevier Science Publishers.

will frequently yield greater precision compared to the use of migration time, since the impact of the EOF is factored out of the calculation (Section 10.6). Routine measurement of the EOF is also necessary to ensure the integrity of the separation. If the EOF is not reproducible, it is likely that the capillary wall is being affected by some component in the sample or an experimental parameter is not being properly controlled (see Section 2.3F).

34

Chapter 2 Capillary Zone Electrophoresis

The simplest method for measuring the EOF is to inject a dilute solution containing a neutral solute and measure the time it takes to transit the detector (9-11). Since the capillary length is known, the velocity in centimeters per second is easily calculated. Dividing that value by the field strength yields the electroosmotic mobility in units of c m W s . Neutral solutes such as methanol, acetone, benzyl alcohol, and mesityl oxide are frequently employed. When MECC is the mode of separation (Chapter 4), a further requirement that the marker solute not partition into the micelle is imposed. When the EOF is slow, the migration time can be quite long. To reduce the experimental time, it is useful to use the short end of the capillary to make the measurement. The short end is the section of capillary normally found between the detector window and capillary outlet. The injection can be made at the outlet side and the system operated using reversed polarity Now the EOF is measured using a short capillary length of 6-10 cm depending on the brand of instrument. As will be shown later, the short end of the capillary can be very useful when performing screening runs during methods development. When the EOF is very slow, as in the case with certain coated capillaries, special techniques must be employed (12). It is seldom necessary to measure very weak EOF, since it does not notably affect mobility or experimental precision. C.

EFFECT OF BUFFER P H

The impact of pH on the EOF and the mobility is illustrated in Figure 2.7. At high pH the silanol groups are fully ionized, generating a strong zeta potential and dense electrical double layer. As a result, the EOF increases as the buffer pH is elevated (9, 13). A robust flow, typically around 2 mm/s at pH 9 in 20 mM borate buffer at 30 kV, 30°C is realized. For a 50 |Lim capillary, this translates to 235 nL/min. Since the total volume of a 50 cm x 50 jim i.d. capillary is only 980 nL, a neutral compound would reach the detector in 4.2 min. At pH 3, the EOF is much lower, about 30 nL/min. The EOF must be controlled or even suppressed to run certain modes of HPCE. On the other hand, the EOF makes possible the simultaneous separation of cations, anions, and neutral species in a single run. For example, a zwitterion like a peptide will be negatively charged at a pH above its pi. The solute will electromigrate toward the positive electrode. However, the EOF is sufficiently strong that the solute's net migration is toward the negative electrode (Figure 2.7, top). At low pH, the zwitterion has a positive charge and will migrate as well toward the negative electrode (Figure 2.7, bottom). In untreated fused-silica capillaries, most solutes migrate toward the negative electrode unless buffer additives or capillary treatments are used to reduce or reverse the EOF (Section 3.3). The EOF is exquisitely sensitive to pH (9,14,15). Hysteresis effects have been reported (15) wherein the direction of approach to a particular pH value produces a different pH (Figure 2.8). When approaching from the acid side, the measured

35

2.3 Electroendoosmosis

HIGH pH ++++•'•+•'•+++++++++++++++++++++++++"'•+•*'+''"

M,0 4 . 4 . ^ 4 . 4 , 4 »

/ * ep

4.4>

4.

4.4.4.

4 . 4 . 4 . 4 . 4 * 4 .

+ + + + + 4* + 4- + + +^+ + -f + -I" + -f +

FIGURE 2.7 Behavior of electroendoosmotic flow and electrophoretic migration of a zwitterion (pi = 7) at high and low pH.

EOF is always lower, and vice versa. This means there is a kinetic parameter with regard to the estabUshment of a stable charge on the capillary wall. Longer equilibration times would reduce hysteresis at the expense of increased total run time. Since the EOF will affect migration time precision, it is important to design experiments with these features in mind. The problems with EOF reproducibility are often most severe in the pH range 4-6 (15). D.

EFFECT OF BUFFER CONCENTRATION

The expression for the zeta potential is (16)

c=

47r5e

(2.10)

where £ = the buffer's dielectric constant, e = total excess charge in solution per unit area, and 5 is the double-layer thickness or Debye ionic radius. The Debye radius is 5 = (3 x 10'')(Z)(Ci/^), where Z = number of valence electrons and C = the buffer concentration. As the ionic strength increases, the zeta potential and, similarly, the EOF decreases in proportion to the square root of the buffer concentration. This was

36

Chapter 2 Capillary Zone Electrophoresis

10

9+

8+ E

o

% 6 X

u.

o 5

4+ 3+ H 2

3

1

1

1

1—I

4

5

6

7 8 PH

1 9

1 10

1 1 11

FIGURE 2.8 Effect of experimental design on the EOF. Key: • , high pH titrated to low pH; • , low pH titrated to high pH. Data from reference (15).

confirmed experimentally (17) for a series of buffers where the EOF was found hnear to the natural logarithm^ of the buffer concentration. It was reported that equivalent EOF is found for different buffer types as long as the ionic strength is kept constant (17). The effect of buffer concentration and field strength is shown in Figure 2.9 (18). The electroosmotic mobility is plotted against field strength for phosphate buffer at three different concentrations using a 50-|Lim-i.d. capillary. As expected, the higher buffer concentrations showed lower EOF at all field strengths. Since ^The linear relationship of EOF with the buffer concentration is a square root relationship as indicated by Eq. (2.10).

37

2.3 Electroendoosmosis

71 u. ^^ u

z

o

S liini

UJ

50

100

150 200 E (V/cm)

250

FIGURE 2.9 Effect of buffer concentration and field strength (E, V/cm) on the electroosmotic flow in a 50-|xm-i.d. capillary. Buffer: phosphate at a concentration of (a) 10 mM; (b) 20 mM; (c) 50 mM. Redrawn with permission from J. Chromatogr., 516, 223 (1990), copyright © 1990 Elsevier Science Publishers.

mobility was plotted, all three lines should be flat. Slight positive slopes were reported for all three concentrations, presumably due to heating effects (Section 2.6). The same data produced using a lOO-jiim-i.d. capillary will be examined in that section.

E. EFFECT OF ORGANIC SOLVENTS Organic solvents can modify the EOF because of their impact on buffer viscosity (17) and zeta potential (19). Linear alcohols such as methanol, ethanol, or isopropanol usually decrease the EOF because they increase the viscosity of the electrolyte. Acetonitrile either does not affect or may slightly increase the EOF (20). Organic solvents are often employed in HPCE to help solubilize the sample. Selectivity can be affected as well in both CZE (20) and MECC (21). Because of the sensitivity of organic solvent concentration on selectivity, evaporation must

38

Chapter 2 Capillary Zone Electrophoresis

be carefully controlled. In this regard, wholly aqueous separations are often advantageous.

F.

CONTROLLING THE ELECTROOSMOTIC FLOW

The EOF is a double-edged sword. It allows the separation of cations, anions, and neutral solutes in a single run. It is also the single most important contributor to migration time variability on a run-to-run, day-to-day, and capillary-tocapillary basis. The EOF is affected by many parameters, including Buffer pH Buffer concentration Temperature Viscosity Capillary surface Field strength Organic modifiers Cellulose polymers Surfactants In this list, the only factor not under direct experimental control is the capillary surface. This single factor is often implicated as the cause for migration time variation in HPCE. It is important to ensure that the capillary surface is properly reconditioned after each run to maintain a reproducible surface. Coated capillaries that suppress the EOF are useful here, as long as the coating is stable. Some new reagents^ that form a dynamic surface coating show great promise for stabilizing the capillary surface (Section 3.3). For some modes of HPCE, it is advantageous to suppress the FOE Capillary isoelectric focusing (CIEF) and capillary isotachophoresis (CITP) separations are usually performed under conditions of very low or carefully controlled EOF Additives such as 0.5% hydroxypropylmethyl cellulose are effective in suppressing the EOF, particularly in conjunction with a coated capillary (22). Cationic surfactants such as cetyltrimethylammonium bromide can actually reverse the direction of electroosmotic flow (14). This can be employed to prevent proteins from sticking to the capillary wall (23, 24). While complete suppression of the EOF is unnecessary for most applications, control is critical to obtain reproducible migration times and resolution. ^CElixir, Scientific Resources, Inc., Eatontown, NJ.

2.4

2.4

Efficiency

39

EFFICIENCY

The high efficiency of HPCE is a consequence of several unrelated factors: 1. A stationary phase is not required for HPCE. The primary cause of band broadening in LC is resistance to mass transfer between the stationary and mobile phases. This mass transfer problem is illustrated in Figure 2.10. When a solute is in the mobile phase, its linear velocity is determined by the linear velocity of the mobile phase. When attached to the stationary phase, the linear velocity becomes zero. The solute is not of a single velocity as it moves down the chromatographic tube. Whenever differing velocities occur during a separation, band broadening will occur. Minimizing the particle size of the packing improves but does not eliminate this problem. Thus, the parameter that results in separation also causes band broadening. The greater the retention, the greater the problem—as evidenced by broadened peaks as retention time increases. For most modes of HPCE (except CEC), this dispersion mechanism does not operate. Similarly, other HPLC dispersion mechanisms such as eddy diffusion and stagnant mobile phase are unimportant in HPCE. 2. In pressure-driven systems such as LC, the frictional forces of the mobile phase interacting at the walls of the tubing result in radial velocity gradients throughout the tube. As a result, the fluid velocity is greatest at the middle of the tube and approaches zero near the walls (Figure 2.11). This is known as laminar or parabolic flow. These frictional forces, together with the chromatographic packing, result in a substantial pressure drop across the column. In electrically driven systems, the EOF is generated uniformly down the entire length of the capillary. There is no pressure drop in HPCE, and the radial flow profile is uniform across the capillary except very close to the walls, where the flow rate approaches zero (Figure 2.11).

FIGURE 2.10

The mass transport problem in HPLC.

40

Chapter 2 Capillary Zone Electrophoresis

Jorgenson and Lukacs derived the efficiency of the electrophoretic system from basic principles (25-27) using the assumption that diffusion is the only source of band broadening. Other sources of dispersion—including Joule heating (Section 2.6), capillary wall binding (Section 3.5), injection (Section 9.1), detection (Section 9.5), and electromigration dispersion (Section 2.13)—lead to fewer theoretical plates than the simple theory predicts. The migration velocity for a solute is V = luE =

HV

(2.11)

where // = the mobility, E = field strength, V = voltage, and L = capillary length. The time t for a solute to migrate the length L of the capillary is (2.12) V

fiV

ELECTROOSMOTIC FLOW

HYDRODYNAMIC FLOW

FIGURE 2.11

Capillary flow profiles resulting from electroosmotic and hydrodynamic flow.

2.5

Resolution

41

Diffusion in liquids that leads to broadening of an initially sharp band is described by the Einstein equation (yl = 2Dt = =^^,

(2.13)

where D = the diffusion coefficient of the individual solute. The number of theoretical plates N is given by N=—.

(2.14)

Substituting Eq. (2.11) into Eq. (2.12) gives an expression for the number of theoretical plates: N = ^ . 2D

(2.15)

Some important generalizations can be made from this expression: 1. The use of high voltage gives the greatest number of theoretical plates, since the separation proceeds rapidly, minimizing the effect of diffusion. This holds true up to the point where heat dissipation is inadequate (Section 2.6). 2. Highly mobile solutes produce high plate counts, because their rapid velocity through the capillary minimizes the time for diffusion. 3. Solutes with low diffusion coefficients give high efficiency due to slow diffusional band broadening. Points 2 and 3 appear contradictory. This is clarified by Figure 2.12 and supplemented with some calculations in Table 2.2. Because of the indirect but inverse relationship between mobility and diffusion, high-efficiency separations occur across a wide range of molecular weights. HPCE can yield high-efficiency separations for both large and small molecules. The greatest number of theoretical plates is found in capillary gel electrophoresis (CGE). The use of an anticonvective gel matrix furthers the advantages of HPCE. The combination of HPCE in the gel or polymer network format (Chapter 6) can yield millions of theoretical plates.

2.5

RESOLUTION

While high efficiency is important, resolution is the key for all forms of separation. In a high-efficiency system, inadequate resolution may result in a single very sharp peak.

42

Chapter 2 Capillary Zone Electrophoresis

DIFFUSION

MOBILITY

SMALL MOLECULES

RAPID

HIGH

LARGE MOLECULES

SLOW

LOW

FIGURE 2.12

Diffusion and mobihty of small and large molecules.

The resolution (R) between two solutes is defined as 1 AAI^PVN

(2.16)

R. = 4 Mep + Meo

where A^ is the difference in mobility between two solutes, /i^^ is the average mobility of the two solutes, and N is the number of theoretical plates. Substituting the plate count equation (Eq. (2.15) and V = EL) yields (25) EL

R, = O.UlAfl^

(/^ep +

(2.17) I^J^n

This expression suggests that increasing the voltage is not very effective in improving resolution, since that parameter falls inside of the square root of the resolution equation. A doubling of voltage results in only a 41% improvement in resolution. The production of heat quickly limits this Table 2.2

Solute Horse heart myoglobin Quinine sulfate

Calculated Theoretical Plates for a Small and Large Molecule

MW

Mobility (10-^ cmW-s)

Diffusion Coefficient (10-^cmVs)

N

13,900

0.65

1

975,000

4

7

857,000

747

2.6 Joule Heating

43

approach. Another means of improving resolution as predicted by Eq. (2.17) is to adjust the EOF. Akhough this also falls under the square root sign of the resolution equation, this technique can be quite effective. There are three categories in this regard: 1. Both electrophoresis and electroosmosis are in the same direction. This normally occurs when cations are being separated. In this case, decreasing the EOF will enhance resolution at the expense of run time. Doubling the run time produces a 41% improvement in resolution. 2. Electrophoresis and electroosmosis are in opposite directions. This occurs on bare silica capillaries when anions are separated. Decreasing the EOF will enhance run time at the expense of resolution, and vice versa. 3. Electrophoresis and electroosmosis are equal but in opposite directions. Here the resolution is infinite, but so is the separation time. However, this concept was used to generate ultrahigh theoretical plate numbers (28). It is clear that improvements in resolution are best addressed by adjustments to AjUgp, the difference in mobility between the two most closely eluting solutes in a separation. Since A/i^p falls outside of the square root sign of the resolution equation, the improvement in resolution is directly proportional to the change in mobilities. This subject forms the basis for many of the chapters in this book.

2.6 JOULE HEATING The conduction of electric current through an electrolytic solution generates heat via frictional collisions between migrating ions and buffer molecules. Since high field strengths are employed in HPCE, ohmic or Joule heating can be substantial. There are two problems that can result from Joule heating: 1. Temperature changes due to ineffective heat dissipation 2. Development of thermal gradients across the capillary If heat is not dissipated at a rate equal to its production, the temperature inside the capillary will rise and eventually the buffer solution will outgas. Even a small bubble inside of the capillary disrupts the electrical circuit. At moderate field strengths, outgassing is not usually a problem, even for capillaries that are passively cooled. The rate of heat production inside the capillary can be estimated by ^ =-^, dT LA

(2.18)

where L = capillary length and A = the cross-sectional area. Rearranging this equation using J = V/R, where the resistance R = L/kA and k = the conductivity.

44

Chapter 2 Capillary Zone Electrophoresis

kV'

(2.19)

dT

The amount of heat that must be removed is proportional to the conductivity of the buffer, as well as the square of the field strength. Lacking catastrophic failure (bubble formation), the problem of thermal gradients across the capillary can result in substantial band broadening (29-31). This problem is illustrated in Figure 2.13. The second law of thermodynamics states heat flows from warmer to cooler bodies. In HPCE, the center of the capillary is hotter than the periphery. Since the viscosity of most fluids decreases with increasing temperature, Eq. (2.4) and (2.8) predict that both mobility and EOF increase as the temperature rises. This situation becomes similar to laminar flow where the electrophoretic or electroosmotic velocity at the center of the capillary is greater than the velocity near the walls of the capillary. The temperature differential of the buffer between the middle and the wall of the capillary can be estimated from AT = 0.24

(2.20)

4K

where W = power, r = capillary radius, and K = thermal conductivity of the buffer, capillary wall, and polyimide cladding. A 2-mm-i.d. capillary filled with 20 mM CAPS buffer draws 18 rtiA of current at 30 ky giving a AT of 75°C. A 50-|lm-i.d. capillary filled with the same buffer draws only 12 |LIA of current, yielding a AT of 50 m°C. Since the thermal gradient is proportional to the square of the capillary radius, the use of narrow capillaries facilitates high resolution. On the other hand, the use of dilute buffers or isoelectric buffers (32) permits the use of wider bore capillaries, but the loading capacity of the separation is reduced.

r ep

AT

FIGURE 2.13 Impact of the radial temperature gradient on electrophoretic and electroosmotic flow.

45

2.6 Joule Heating

The requirement for narrow-bore capillaries comes with a price due to the short optical path length. If a solution is injected equivalent to 1% of the capillary volume of a 50 cm x 50 |im i.d. capillary, the injection size is 9.8 nL. This small-volume injection coupled to a 50-|im optical path length provides for concentration limits of detection (CLOD) that are about 50 times poorer than by LC. Fortunately, through the use of stacking procedures (33) and extended path length capillaries (34), this gap has been narrowed considerably The compromise between sensitivity and resolution is illustrated in Figures 2.14 and 2.15. Note in particular the cluster of peaks centered at a migration time of 31 min (26 min in Figure 2.15) in Figure 2.14. With the 50-|im-i.d. capillary, none of these peaks are baseline-resolved, but there is virtually no noise in the electropherogram. Separation of the same sample in a 25-|im-i.d. capillary (Figure 2.15) presents a different picture. The peaks are nearly baselineresolved, but there is substantial noise in the output. This presents one of several compromises that must be made in HPCE. In this case, sensitivity and resolution are competing analytical goals.

0.300-!

0.262H

0.225H

0. I87H

0. 150H

0. uaH 0.075H

0.037H

\MJ

WAAAJ

L

0.00OH —J—

io

15

20

—~r-—1—25 30 MINUTES

" "1— 45

•'"••'"

3S

40

!

50

""•"

'" 1 55

FIGURE 2.14 Separation of heroin impurities by MECC on a 50-|lm-i.d. capillary. Buffer: 85 mM SDS, 8.5 mM borate, 8.5 mM phosphate, 15% acetonitrile, pH 8.5; capillary: 50 cm (length to detector) X 50 (Xm i.d.; voltage: 30 kV; temperature: 50°C; detection: UV, 210 nm. Reprinted with permission from Anal. Chem., 63, 823 (1991), copyright © 1991 Am. Chem. Soc.

46

Chapter 2 Capillary Zone Electrophoresis

Even these electropherograms must be carefully interpreted. In both cases, the injection time was kept constant at 1 s. This means that the amount of material injected in the 25-|Ltm-i.d. capillary was a factor of four lower relative to the 50-|Llm-i.d. tube (Section 8.1). This contributes to the decreased signal-to-noise ratio observed when using the 25-|lm-i.d. capillary. The problem of Joule heating depends on the capillary diameter, the field strength, and the buffer concentration. Recalling Figure 2.9 (50-|Lim-i.d. capillary), there was a slight increase in |Lieo as the field strength was increased. Figure 2.16 contains data from the same experiments, except a 100-|Lim-i.d. capillary is used. A marked departure from linearity is found at the higher buffer concentrations. Higher concentration buffers are more conductive, draw higher currents, and produce more heat than more dilute solutions. In the 100-|im-i.d. capillary, this heat is not properly dissipated. As a result, the internal temperature rises, reducing the viscosity of the buffer. Since Eq. (2.8), the basic expression for electroosmotic velocity, contains a viscosity parameter in the denominator, Vgo increases with decreasing buffer viscosity. Because the buffer viscosity depends on temperature, the capillary heat removal system plays an important role in deciding the maximum field strength, buffer concentration, and capillary diam0.080-1

0.07CH

0.060H

0. 050H

0. 040-H

0.030H

0. 020H

0. oiCH

0. GOOH

N'wWW ]

12

\^\I\KJMM I 16

1 1 20 24 MINUTES

1 28

1 32

1 36

1" 40

FIGURE 2.15 Separation of heroin impurities by MECC on a 25-fxm-i.d. Conditions as per Figure 2.14 except for capillary diameter. Reprinted with permission from Anal. Chem., 63, 823 (1991), copyright © 1991 Am. Chem. Soc.

2.7

47

Optimizing the Voltage and Temperature

11

59 u. ^ o

E7 to '

o

j>5

50

100

200 150 E (V/cm)

250

FIGURE 2.16 Effect of buffer concentration and field strength (E, V/cm) on the electroosmotic flow in a 100-|lm-i.d. capillary. Buffer: phosphate at a concentration of (a) 10 mM; (b) 20 mM; (c) 50 mM. Redrawn with permission from J. Chromatogr., 516, 223 (1990), copyright © 1990 Elsevier Science Publishers.

eter that can be successfully employed. Insufficient heat removal begins a vicious cycle leading to viscosity reduction, greater current draw, and higher temperature, further reducing the viscosity.

2.7 OPTIMIZING THE VOLTAGE AND TEMPERATURE A.

OHM'S LAW PLOTS

A means of optimizing the voltage and/or the temperature despite the buffer concentration and capillary cooling system is very desirable. An Ohm's law plot provides this tool with very little experimental work (35, 36). Simply fill the capillary with buffer, set the temperature, vary the voltage, record the current, and plot the results.

48

Chapter 2 Capillary Zone Electrophoresis

Some Ohm's law plots are shown in Figures 2.17 and 2.18 for an air-cooled and water-cooled temperature control system, respectively. Whenever the graph shows a positive deviation from linearity, the heat removal capacity of the system is being exceeded. Operating on the linear portion of the curve will generally yield the highest number of theoretical plates. As a rule of thumb, it is best to keep operating currents below 100 joA. Often, separations are run in the nonlinear section to optimize speed at the expense of plates, but it is not wise to push things too far. Lowering the temperature below ambient can be used to extend the linear range of the Ohm's law plot. This is useful when high-ionic-strength buffers are

A

^

100

•B

V

• •

80

•

" •

< V

UJ

*>'

60 '

XT

^

^ •

O

.c

•

V

40

•

^

X T .

^

D

0

•^ •=-•

0 D

^ ^

D

V ^- u"

20 ¥

1 l±lll

I

^

D

^^o°

^ ^t°

^¥ ^ 0 ° ¥^^D

1

H

10 20 VOLTAGE (kV)

_—

1-

30

FIGURE 2.17 Ohm's law plots for capillary temperature control by air circulation. (A) no control; (B) 25°C; (C) 10°C; (D) 4°C. Redrawn with permission from J. High Res. Chromatogr., 14, 200 (1991), copyright © 1991 Dr. Alfred Heuthig Publishers.

2.7

49

Optimizing the Voltage and Temperature

A

100

' "V

1

•

• B •

80

V

•

1 LU

•V

•

•

^ C

60 ^

•

^

•

OL

-^

A

•

O

•

A

A

0

0

^ 0 „ «

°

40 • •

20

•

X •

-6. a -<^ 0 ^ 0

^0

^0

,^^t° •^a

*i^a

«ELf

1

1

10 20 VOLTAGE (kV)

I—

30

FIGURE 2.18 Ohm's law plots for capillary temperature control by water circulation. (A) no control; (B) 25°C; (C) 10°C; (D) 4°C. Redrawn with permission from J. High Res. Chromatogr., 14, 200 (1991), copyright © 1991 Dr. Alfred Heuthig Publishers.

necessary. These concentrated buffers are particularly useful in micropreparative CE (Section 9.10), increasing the linear dynamic range (Section 10.4) and suppressing wall effects (Section 3.3). Increasing the temperature can also be employed to speed the separation, since both v, and Vep increase about 2%/K due to the decreased viscosity of the buffer medium For various instruments the Ohm's law plot is an effective means of evaluating the efficiency of their capillary cooling systems. Fluid-cooled systems are

50

Chapter 2 Capillary Zone Electrophoresis

generally more effective than air-cooled systems, since the heat capacity of most fluids exceeds that of air.

B. CONSTANT VOLTAGE OR CONSTANT CURRENT? Power can be applied to the system in one of two ways. The voltage can be fixed, allowing the current to float based on the resistance of the buffer. Alternatively the current can be fixed. Most published work in HPCE is in the constant-voltage mode. There has been one report that found constant current more reproducible than constant voltage (37). Until this is better understood, both modes should be studied during methods development for CZE, MECC, and CGE separations. CITP is typically performed in the constant-current mode, or else separation time becomes long. CIEF may also benefit from the constant-current mode as well, although there is no evidence published to that effect.

2.8 CAPILLARY DIAMETER AND BUFFER IONIC STRENGTH Some very subtle effects due to Joule heating can occur when comparing separations run on capillaries with different inner diameters, or even the same capillaries run on various instruments with different capillary cooling systems. Some of these issues are illustrated in Figures 2.19 and 2.20. The ionic strength of the buffer influences not only the EOF and jil^^, but indirectly the viscosity of the medium. More concentrated buffers have greater conductivity and generate more heat when the voltage is applied. The viscosity depends on the temperature, and so there is also a dependence on the capillary diameter. This is shown for a series of runs in 50- and 75-|Llm-i.d. capillaries (38). With the 50-|lm capillary, the migration times lengthen as the buffer concentration is increased. Ions in solution are always surrounded by a double layer of ions of the opposite charge. The migration of these counterions is in a direction opposite to that of the solute (Figure 2.21); hence, increasing the concentration of the buffer reduces the mobility of the solute, due to increased drag caused by countermigration of the more densely packed counterions. With the 75-|Lim capillary, the solute migration times first increase as expected, but then they decrease. This decrease is a consequence of the significant effects of Joule heating at higher buffer concentrations. Note as well the impact of buffer concentration on peak width. Sharper peak widths at the higher buffer concentrations are due to stacking (Section 8.6). The

2.8

51

Capillary Diameter and Buffer Ionic Strength

.32

50nm Capillary

0200M

^'ULlJlJLJIjLii^

liujLi

0.075M 0.050M O.O^M

8

10

12

14

16

TIME Crnin,) FIGURE 2.19 Effect of buffer ionic strength on peptide separations in a 50-|im-i.d. capillary. Buffers: 0.025-0.200 M phosphate, pH 2.44; voltage: 30 kV; capillary: 50 cm to detector x 50 |Lim i.d.; key: (1) bradykinin; (2) angiotensin 11; (3) TRH; (4) LRHR; (5) bombesin; (6) leucine enkephalin; (7) methionine enkephalin; (8) oxytocin; (9) dynorphin. Reprinted with permission from Techniques in Protein Chemistry 11, 1991, Academic Press, 3-19, copyright © 1991 Academic Press.

Stacking effect is more evident when the 75-|im-i.d. capillary is used. Injection time was held constant for this comparison; thus, a larger injection was made on the latter capillary. Stacking is not very noticeable when small injections are made.

52

Chapter 2 Capillary Zone Electrophoresis

.45

7S[im Capillary

2B 0.125M

I

11 I I

11^

.27 BS

UuulJJLIL

.18H

lOOM

I . II I 0.075U

JIU

ILJJIUL

"ililiiL!

050M

1 ? 3.4

6*

UMJC 6

8

10

12

02511 14

16

TIME (min.) FIGURE 2.20 Effect of buffer ionic strength on peptide separations in a 75-(Xm-i.d. capillary. Conditions as per Figure 2.19 except for capillary diameter. Reprinted with permission from Techniques in Protein Chemistry U, 1991, Academic Press, 3-19, copyright © 1991 Academic Press.

2.9

OPTIMIZING THE CAPILLARY LENGTH

The efficiency of the separation (theoretical plates) is directly proportional to the capillary length (38), provided the field strength is kept constant. The

2.9

Optimizing the Capillary Length

FIGURE 2.21

53

Countermigration of a solute against its ionic atmosphere.

limitation here is available voltage. Most instruments produce a maximum of 30 kV. Once the capillary length reaches a certain point, the field strength must be reduced and no further gains in efficiency are realized (6). Based on Eq. (2.16), the electrophoretic resolution depends on the square root of the number of theoretical plates and, thus, on the square root of the capillary length (38). Increasing the capillary length beyond the limits imposed by the voltage maximum lengthens the separation time without any substantial benefits. These effects are illustrated in Figure 2.22, where the number of theoretical plates is linear with the capillary length until 30 kV is reached. Note that the resolution increase is proportional to the square root of the number of theoretical plates. Most chemists overly rely on the length of the capillary to perform their separations. This results in lengthy separations. Since diffusion is time related, the sensitivity of the method declines as well. If more time is spent optimizing the separation chemistry, shorter capillaries can be employed, with obvious benefits.

54

Chapter 2 Capillary Zone Electrophoresis

Theoretical Plates (1OOO's)

Resolution

AAA

UUU

4-„™

^p^zzz . ..4^'^'^'"

x:—

ji

800

-

Jf

i *

"i 1.5

^ x'^

600

400 0.6 200

...

20

30

40

50

i

60

! 70

1

i

,1

80

90

100

Length to Detector (cm) FIGURE 2.22

2.10 A.

Impact of capillary length on the number of theoretical plates and resolution.

BUFFERS THE ROLE OF THE BUFFER

A wide variety of buffers (Table 2.3) can be employed in CZE. The buffer is frequently called the background or carrier electrolyte. These terms are used interchangeably throughout this book. Other terms frequently employed are co-ion and counterion. A co-ion is a buffer ion of like charge compared with the solute, a counterion a buffer ion of opposite charge. The purpose of the buffer is to provide precise pH control of the carrier electrolyte. This is important, since both mobility and electroendoosmosis are sensitive to pH changes. The buffer may also provide the ionic strength necessary for electrical continuity. Usual buffer concentrations range from 10 to 100 mM, though there are many exceptions. Dilute buffers provide the fastest separations, but the sample loading capacity is reduced. Buffer solutions should resist pH change upon dilution and addition of small amounts of acids and bases. Concentrated buffer solutions do this well, but they

2.10

55

Buffers

Table 2.3

Buffers for HPCE

BUFFER

pKa

MobiUty^

ZWITTERIONIC BUFFERS Aspartate j3-Alanine

3.55

+31.6 +36.7

)8-Alanine Histidine MES

10.24 9.34 6.13

-30.8 +29.6 -26.8

L99

ACES

6.75

-31.3

MOPSO BES MOPS

6.79 7.16 7.2

-23.8 -24.0

TES

7.45

-22.4

DIPSO

7.5

HEPES TAPSO

7.51 7.58

-21.8

HEPPSO EPPS

7.9 7.9

-22.0

POPSO DEB

7.9 7.91

Tricine

8.05 8.2

Glygly Bicine TAPS CHES CAPS

-24.4

-26.2

8.25 8.4 9.55 10.4

CONVENTIONAL (Nonzwitterionic) BUFFERS Citrate Formate Acetate Lactate Phosphate Borate Creatinine

3.12, 4.76, 6.40 3.75

-28.7 (low pKg value)

4.76

-42.4 -35.8 -35.1 (low pKg value) -40.0 (estimate)

3.85 2.14, 7.10, 13.3 9.14 4.89

-56.6

+33.1

Data from J. Chromatogr., 1991; 545:391. ^Effective mobility for fully ionized buffers at 25°C (10~^ c m W s ) .

are often too conductive for use in HPCE. The buffering capacity of a weak acid or weak base is limited to ±1 pH unit of its pK^. Operation outside of that range requires frequent buffer replacement to avoid pH changes (39). The buffer

56

Chapter 2 Capillary Zone Electrophoresis

should have a low temperature coefficient and not absorb significantly in the UV region, where detection occurs. Table 2.4 presents some of these data for a few common buffers. B.

BUFFER SELECTION

The selection of the appropriate buffer need not be difficult. For acids, start with borate buffer, pH 9.3, and for bases, phosphate buffer pH 2.5. These two buffer systems along with the appropriate additives will work well for most applications. If bases are not soluble in phosphate buffer, acetate buffer pH 4 may be more effective. Higher pH values may be required for basic proteins to avoid adherence to the capillary wall. Phosphate buffers are often used for low-pH protein separations (39-41). McCormick (41) proved that phosphate ions bind to the capillary wall, reducing the impact of protein binding to anionic silanol groups. Prewashing the capillary with pH 2.5 phosphate buffer was reported to reduce protein binding as well (42). Capillaries aggressively pretreated with phosphate buffer have also been useful in this regard (43). Borate buffers are useful for separating carbohydrates (44-49) and catecholamines (50, 51) because of specific complexation chemistry. Unless such specific interactions are identified, buffer selection based on the desired pH is usually satisfactory. Borate buffers are used to control pH in the range 8.3-10.3. Phosphate and borate buffers have adequate buffer capacity over a wide pH range and are useful as general purpose buffers. They may not be useful for cer-

Table 2.4

Characteristics of Some Buffer Solutions

pH at 25°C

Dilution Value^

Buffer Capacity^

Temperature Coefficient^^

50 mM KH2C6H5O7 (citrate)

3.776

+0.02

0.034

-0.0022

25 mM KH2PO4, Na2HP04

6.865

+0.080

0.029

-0.0028

8.7 mM KH2PO4+ 3.0 mM Na2HP04

7.413

+0.07

0.16

-0.0028

10 mM Na2B407 (borate)

9.180

+0.01

0.020

-0.0082

50 mM Tris-HCl; 16.7 mM Trls

7.382

na

Buffer

na

^pH change upon 50% dilution. ^pH change upon mixing 1 L of buffer with 1 gram equivalent of strong acid or base. ^Change in pH per °C. Data from pH Measurements, 1978, Academic Press.

-0.026

2.10

Buffers

57

tain protein separations, particularly if biological activity must be maintained. These buffers also lack buffer capacity around pH 4, where acetate is quite good. The selection of the buffer cation also plays a role in buffer conductivity (4). The correlation of the atomic radii and mobility was discussed in Section 2.1. In this regard, lithium and sodium salts are best used, since they contribute least to buffer conductivity. Dual buffering systems with lower mobility counterions (Tris-phosphate, Tris-borate, or Tris-Tricine) are used to reduce heating problems in the slab gel and can also be used in HPCE. Buffers used in the slab gel must be unreactive with Commassie or silver stain. This is not a guarantee that they will not absorb in the UV. The UV spectrum of a buffer should be checked prior to use to avoid detection problems. In capillary isoelectric focusing this problem is more acute, as ampholytes can absorb even at 280 nm. Aromatic buffer constituents such as phthalates should be avoided because of their strong UV absorption characteristics, unless indirect detection is planned (Section 3.6). Strongly absorbing components such as carrier ampholytes (Chapter 5) prevent the use of the low-UV region for detection. Zwitterionic buffers such as bicine, Tricine, CAPS, MES, and Tris are also used, particularly for protein and peptide separations. They are all amines, and some buffers such as Mes, Tricine, and glycylglycine bind calcium, manganese, copper, and magnesium ions. PIPES, HEPES, and Tris do not bind any of these metals, whereas BES binds only copper (52). Metal binding may be useful, or it may interfere with subsequent separations. The advantage of the zwitterionic buffer is low conductivity when the buffer is adjusted to its pi. There is little buffer capacity when pK^ and pi are separated by more than 2 pH units. When the pi and pK^ are close together, the buffer is known as an isoelectric buffer (32). The advantage here is low current draw and reduced Joule heating, allowing higher buffer concentrations to be used. However, isoelectric buffers are poor stacking buffers because of their extremely low conductivity. Table 2.3 provides data describing the mobility of the fully ionized buffer component. Mobility matching between the buffer and solute is used to improve the peak symmetry when the solute concentration is high relative to the buffer concentration (53). This problem, known as electromigration dispersion or simply electrodispersion (Section 2.14), is particularly troublesome when indirect detection is employed, because the buffer concentration must be low. A software program (Buffer Workshop, Scientific Resources Inc., Eastontown, NJ) is useful to help calculate buffer mobilities at any pH. C.

BUFFER PREPARATION

Titration of a buffer to the appropriate pH has some operational subtleties. In the trivial but ideal case, equimolar solutions of two different salts of the identical anion are blended to the appropriate pH. For example, to prepare a 50 mM,

58

Chapter 2 Capillary Zone Electrophoresis

pH 7 phosphate buffer, titrate a 50 mM disodium salt with 50 mM phosphoric acid. Under all possibilities, the final phosphate concentration must be 50 mM and the ionic strength must be consistent. For very critical separations, it is best to prepare buffers in large batches to reduce batch-to-batch variation. When this is done, pay attention to buffer stability and the potential for microbial growth. In other cases, the buffer is often titrated with acid or base to adjust the pH. Under these conditions, both pH and ionic strength are being adjusted (54). Unusual effects, such as the reduction of EOF with increasing pH, have been observed that are attributable to this problem. Selecting a buffer that requires no titration or only a minor titration will minimize these ionic strength effects. In any event, it is important to exactly specify the buffer preparation in methodology. All buffers should be filtered before use through 0.21-|Llm filters. Prepared buffers are available from many instrument manufacturers and suppliers. These solutions are manufactured in large batches, prefiltered, and put through quality control. Common recipes containing phosphate and borate along with specialty preparations for application-based methods are readily available. For small laboratories without water purification systems, water for HPCE can be purchased as well. Since reagent usage in capillary electrophoresis is minimal, the costs are low

2.11

TEMPERATURE EFFECTS

The impact of temperature on a series of peptides is illustrated in Figure 2.23. As the temperature is increased, the migration time always decreases because of the reduced viscosity of the BGE. The current increases as well due to this effect. The viscosity is, of course, inversely proportional to the temperature. There are no significant changes in selectivity as the temperature is increased for these peptide separations. This will not always hold true, particularly when secondary equilibrium is employed. When proteins are being separated, temperature can have profound effects on the separation if unfolding occurs. This is illustrated in Figure 2.24 for a-lactoglobulin (55). At 20°C, a single peak is found at 4.5 min. As the temperature is increased, the band broadens until, finally, a sharp peak is found when the temperature reaches 50°C. The peak at 20°C represents the native form of the protein. At the intermediate temperature, multiple forms of the protein are found as unfolding begins. At 50°C, a peak representing the unfolded protein is found. Note that the migration times always decrease as the temperature is raised due to the aforementioned viscosity effects. Elevated temperatures are frequently used during methods development to speed separations without having to cut the capillary. When secondary equilibrium is employed, particularly for chiral separations, subambient temperatures are often used.

59

15 25 35^ 45 55 Temperatiire (C)

-jL. 4

6

Jl—LJuL....li 8

I

10

Time ( Minutes )

n

14

" 15

25

35

45

55

Temperature (C)

FIGURE 2.23 Effect of temperature on time, current, and viscosity. Buffer: 50 mM phosphate, pH 2.5; voltage: 20 kV; capillary: 50 cm x 75 )im i.d. Reprinted with permission from Techniques in Protein Chemistry U, 1991, Academic Press, 3-19, copyright © 1991 Academic Press.

2.12

BUFFER ADDITIVES

Reagents are often added to the buffer solution for reasons other than controlhng pH. Known as buffer additives, these materials are used for several functions: 1. 2. 3. 4.

To modify mobility (secondary equilibrum) To modify electroosmotic flow To prevent solutes from adhering to the capillary wall To maintain solubility

Table 2.5 lists of some of the reagents used for these purposes. These will be discussed in further detail throughout this text.

60

Chapter 2 Capillary Zone Electrophoresis 1129

1L-

mt W

}l

m ^

m L m m

'

0

'

^•'•••^'f"*"'"'*"""'^''"'*'"^'"lB"''l''*'T"""*'^*^"*i'"T"t''T™"f¥'''^P'^*^

t

2

^

3

.

i^

,

1

*

5

«

Tmetmin) FIGURE 2.24 Influence of temperature on the electrophoretic behavior of cc-lactoglobulin. BGE: 100 mM borate, pH 8.3; voltage: 350 V/cm; capillary: 50 cm bare siUca. Reprinted with permission from Anal. Chan., 63, 1346 (1991), copyright © 1991 Am. Chem. Soc.

2.13 CAPILLARIES A.

FUSED SILICA

Fused-silica, polyimide-coated capillaries, similar to those used in capillary gas chromatography, are the material of choice for HPCE (Figure 2.25). Internal diameters ranging from 25 to 100 |lm are usually employed. Capillaries covering the range of 2 - to 700 |Lim i.d. are commercially available.^ Between 10- and 75-|im i.d., the i.d. tolerance is 1 |Lim. Fused silica is a good (though not ideal) material due to its UV transparency, durability (when polyimide coated), and zeta potential. The variation of the surface charge along with solute-capillary wall interaction are the most important limitations of the material. Functionalized capillaries and buffer additives are used to overcome this limitation. Gelfilled capillaries (Chapter 6) for CGE are also commercially available. Most instrument manufacturers employ a cartridge to contain the capillary This allows for more optimal integration with the capillary cooling system. The ^Polymicro Technologies, Phoenix, AZ 85017.

2.13

61

Capillaries

Table 2.5

Buffer Additives

Purpose

Reagent

To modify mobility

Transition metals

Complex formation

Cyclodextrins

Inclusion comple

Surfactants

Micelle interaction

Organic solvents

Solvation

To modify EOF

To reduce wall effects

To maintain solubility

Mechanism

Sulfonic acids

Ion-pair formation

Quaternary amines

Ion-pair formation

Borate

Complex with carbohydrates, diols

Chelating agents

Complex formation with metals

Crown ethers

Inclusion complex

Macrocyclic antibiotics

Inclusion complex

Calixarenes

Inclusion complex

Dendrimers

Inclusion complex

Cationic surfactant

Dynamic coating, EOF reversal

Organic solvents

Affects viscosity

Linear polymers

Dynamic coating

Zwitterionic surfactant

Dynamic coating

Cationic surfactant

Dynamic coating, EOF reversal

Polyamines

Covers silanols

Linear polymers

Dynamic coating

Zwitterionic surfactant

Dynamic coating

Organic solvents

Hydrophobicity

Urea

"Iceberg effect"

alignment between the capillary detection window and the detector optics is simple when a cartridge is used. Capillaries can be purchased from the instrument manufacturers for $35-60 per capillary. The detection window is in place and the outlet side may be premeasured for a particular instrument. Polyimide is removed at the inlet and outlet side to prevent shards of polyimide from clinging to the capillary opening. Alternatively, high-quality fused-silica capillaries can be purchased in bulk at a cost of $10-20 per meter depending on quantity. A new fused-silica capillary can be prepared and conditioned in half an hour with materials costs of under $5.00. At such a low cost, these home-cut capillaries are disposable items. Rather than attempt regeneration of a suspect capillary, it is wise to simply replace it. It is best to dedicate a capillary to a particular application.

62

Chapter 2 Capillary Zone Electrophoresis

SeOjttm

Fused Silica

Polyimide Coating

12/im FIGURE 2.25

B.

25-75/im

Cross-sectional view of a fused-silica capillary.

PREPARING A FUSED-SILICA CAPILLARY

The procedure for preparing a bare silica capillary is given below. Only a ruler, a cutter (silicon wafer or ceramic cutter), a butane lighter, methanol, and a tissue are required. The method for introducing the detection window should not be used with gel-filled or surface-treated capillaries. L Nick the polyimide coating near the edge of the capillary as squarely as possible. Pull the capillary directly apart, making sure not to pull at too much of an angle. Measure from the cut end to the desired total length of the capillary (L^), and cut again. Observe the ends of the capillary under magnification to ensure that they are cut squarely. 2. Measure the separation length of the capillary (L^) and flame a length of about 2-3 mm with the butane lighter.^ Clean the burnt polyimide with a tissue moistened with methanol. The capillary can now be inserted into the instrument, taking care not to bend the now fragile 5Heat burns off the coating without making the glass brittle. Alternatively, concentrated sulfuric acid at 130°C will remove the coating in a few seconds. This method is required when gel-filled or functionalized capillaries are used.

2.13

Capillaries

63

detection window.^ The ends of the capillary can be flamed as well to remove about 1 mm of coating. This prevents problems from shards of polyimide obstructing the inlet and outlet. Swelling of the polyimide due to interaction with BGE components is prevented as well. 3. Wash the capillary for 15 min each with 1 N sodium hydroxide, 0.1 N sodium hydroxide, and BGE. Change the detector-side reservoir to BGE. The system is ready to run. The base conditioning procedure is important to ensure the surface of the capillary is fully charged. For some methods, it is necessary to regenerate this surface with 0.1 N sodium hydroxide—in extreme cases, 1 N sodium hydroxide. This regeneration procedure is often necessary if migration times change on a regular basis. Regeneration may help if the zeta potential at the capillary wall is altered. Binding of solutes or sample matrix components may be the cause of this problem. When working at low pH, a wash with O.IN hydrochloric acid is useful to reduce the EOF C.

STORING A FUSED-SILICA CAPILLARY

For overnight shutdown, simply leave BGE in the capillary and be sure that the capillary ends are immersed in BGE. For prolonged shutdown or when removing the capillary from the system, all buffer materials must be removed from the capillary. Otherwise, the BGE will clog the capillary when evaporation occurs. 1. 2. 3. 4.

Rinse the capillary with water for several minutes. Change both buffer reservoirs to distilled water. Rinse for five minutes with distilled water. Empty the appropriate buffer reservoir, and draw air through the capillary for five minutes. 5. Remove the capillary from the instrument. 6. For capillaries not part of a cartridge assembly, slide some 0.5- to 1-mmi.d. Teflon tubing over the optical window and gently secure with tape or septa.

Cleaning a capillary can be done offline using a simple 1-mL syringe and some polyethylene tubing. This is particularly advantageous during methods development, since instrument time is not wasted. Sleeve some tubing over the syringe needle and capillary. It is possible to flush water and then air through ^For those not wishing to prepare a detection window, a fluorocarbon-coated capillary (CElectUVT) is available from Supelco, Bellefonte, PA. This capillary cannot be used in with liquid cooling systems containing fluorocarbons, since the coating will become brittle.

64

Chapter 2 Capillary Zone Electrophoresis

the capillary manually. A kit for conditioning CEC capillaries, available from Unimicro Technologies, can also be used on bare silica.

D.

COATED CAPILLARIES

Coated capillaries are often used to prevent proteins from adhering to the capillary wall. They are also used to reduce the EOF if the application or mode of electrophoresis requires such a reduction. Hundreds of papers describing various coatings and coating procedures have appeared in the literature. As traditional silane chemistry is used to prepare the coatings, most of these papers describe coatings that are not stable at alkaline pH. The advantage of the coated capillary for protein separations is that it is often possible to get a good separation without using additives to the background electrolyte. The disadvantages include cost and the aforementioned problems with capillary stability. Table 2.6 lists commercially available capillaries. The uses of some of these capillaries will be described in the appropriate sections of this book. When a capillary is purchased from a vendor, the polyamide at the point of detection is already removed. Should you need to remake a window, do not flame the capillary since that will destroy the inner coating. Instead, place a drop of concentrated sulfuric acid where the window is to be made. Then place the capillary a few inches above a butane lighter flame. The window will appear when the acid temperature reaches 130°C. Carefully rinse the acid off of the capillary and examine the window under magnification to determine clarity

2.14 SOURCES OF BAND BROADENING In Section 2.4, Jorgenson and Lukacs's model describing the efficiency of HPCE is described. This model assumes that diffusion is the only cause of band broadening. At low voltages, molecular diffusion is a leading cause of band broadening. At higher voltages, the Joule heating problem causes parabolic flow. Adsorption of solutes at the wall also leads to dispersion, as does electromigration dispersion (see below). Extracolumn processes such as injection and detection can also lead to dispersion (Sections 8.1 and 9.5). The peak variance or dispersion can be expressed by ^'

= ^diff
+ < a l l + ^det + ^heat + ^ e d >

(2-21)

where cTdiff, ofnj. ^ap. <^et> ^eat. ^^d (jJd are the respective variances due to diffusion, injection, capillary, detection. Joule heating, and electromigration

2.14

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66

Chapter 2 Capillary Zone Electrophoresis

dispersion. Because of the additivity of the square of the variances, the greatest contributor to variance becomes the hmiting factor. The problem can best be articulated as follows. Since (2.22)

N =

a 1,000,000-plate separation on a 50-cm capillary gives G^ = 0.25 mm^. For a 100,000-plate separation, G^ = 2.5. Thus, it is reasonably simple to maintain the efficiency for a 100,000-plate separation, but quite difficult when the plate count approaches 1,000,000. Some of the key sources of dispersion along with their importance and remedy are given in Table 2.7. On-capillary dispersion comprises contributions from injection overload, hydrostatic flow (siphoning), diffusion, adsorption (wall effects), and Joule heating. Injection overload, electrodispersion, antistacking (high-ionic-strength sample), and hydrostatic flow (57) due to fluid imbalances are easily avoided. Siphoning is unimportant for small-diameter capillaries unless the height differential becomes extreme. The contribution from diffusion, expressed by Eq. (2.13), is directly proportional to the separation time. The faster the separation, the sharper the peak. The variance contribution from Joule heating has been extensively studied by Hjerten (58), Foret et al (59), Grushka et al (30), Knox (31), and Jones and Grushka (29). Grushka et al (30) concluded that temperature effects are negligible in narrow-bore capillaries. Use of wide-bore capillaries is possible when Table 2.7

Sources of Band Broadening in HPCE

Process

Importance

Solution

Antistacking

++++

Reduce injection size Increase buffer concentration Reduce ionic strength of sample

Adsorption on wall

++++

Use coated capillary or buffer additive

Detection window

+

Reduce slit width if possible

Diffusion

+++

Reduce separation time

Electrodispersion

+++

Increase buffer concentration Reduce solute concentration Mobility match buffer to solute

Injection size

+++++

Reduce injection size Use stacking buffers

Joule heating

++

Reduce voltage

Poorly cut capillary

++

Ensure capillary is cut squarely

Siphoning

+

Balance fluid level of reservoirs

2.14

Sources of Band Broadening

67

low-conductivity buffers are used. The problem with low-conductivity buffers is decreased loading capacity and increased wall effects. Using Ohm's law plots (Section 2.7) helps ensure that Joule heating is not a significant cause of dispersion. Operation at the voltage prescribed by the Ohms's law plot minimizes diffusion-related dispersion since the field strength is maximized as well. The mathematics for some of these sources of peak dispersion is covered in the individual sections along with more experimental detail. For a thorough review of this often complicated theory, the paper written by Gas et al. (60) should be consulted. Another form of band broadening is known as electromigration dispersion or simply electrodispersion. The appearance of triangular or saw-toothed peaks when the solute concentration is high is indicative of this phenomenon. This process, which is intrinsic to the electrophoretic process, may be observed whenever two conditions simultaneously occur: 1. The solute concentration approaches the concentration of the BGE co-ion. 2. The solute's mobility differs from the mobility of the BGE co-ion. To totally eliminate electrodispersion, the solute concentration must be at least be 50-fold lower than the BGE concentration or the mobilities of both solute and co-ion must closely match. In most cases, it is not possible to completely eliminate the effect; however, some electrodispersion is easily tolerable. Quantitative analysis is not affected as long as sufficient resolution is designed into the separation, the integration parameters are properly set, and peak areas are employed. The dispersion problem only becomes excessive at the higher (relative to BGE) solute concentrations (Section 10.4). Electrodispersion is usually observed when employing indirect detection for small ions. The nature of indirect detection (Section 3.6) limits the concentration of the BGE. Furthermore, electrodispersion is diffusion mediated (see below), and small ions have very high diffusion coefficients. Other times, electrodispersion may appear at the higher end of a calibration curve (Section 10.4), during micropreparative separations, or on the major component of the separation when performing trace impurity determinations. If the major-component concentration is limited to 1 mg/mL and the buffer concentration is 50 mM, electrodispersion is seldom observed. Electrodispersion was originally reported by Mikkers et al. (61). The mechanism for electrodispersion is described in Figure 2.26 for three conditions using cations as the solutes: (1) /LL^ > IX^GE^ 0-) Mc = MBGE. ^^^ O) Mc < MBGE. where /a is the symbol for mobility. We analyze the first case in detail. If a solute has a high mobility relative to the BGE, the electrical conductivity of the solute zone is relatively high. It follows that the resistance of the solute zone is low; therefore, the field strength expressed over the zone is low relative to the field over the BGE. As diffusion occurs over the left boundary, the field strength that the solute experiences increases. The resultant acceleration in migration velocity

68

Chapter 2 Capillary Zone Electrophoresis

LOW FIELD

UNIFORM FIELD

HIGH FIELD DIFF.

®® ®®

®® C
UNSTABLE BOUNDARY

STABLE BOUNDARY

STABLE BOUNDARY UNSTABLE BOUNDARY

TIME FIGURE 2.26

Mechanism of electromigration dispersion.

causes the ions in that portion of the zone to move ahead of the other solute ions. At the right-handed boundary, the same effect occurs, except the cations recombine or restack since they are migrating toward the cathode. In case 2, no such effects can occur—the field strengths are held constant over both the solute and the BGE, since their mobilities are equal. Similar arguments can be made for case 3. If the solute concentration is sufficiently low relative to the BGE, the impact of the solute on the field strength is negligible and symmetric peaks are obtained. Electrodispersion can be minimized by 1. Diluting the sample 2. Increasing the BGE concentration 3. Matching the mobility of the BGE co-ion to the solute Sample dilution is often not possible because of the concomitant loss of sensitivity It is possible to use dilution to reduce electrodispersion in conjunction with an extended path length flowcell. If there is sufficient sensitivity, sample dilution is the simplest means of reducing electrodispersion. Increasing the BGE concentration is not possible when employing indirect detection because of the reduction in sensitivity When direct detection is

References

69

employed, this technique works well up to the point at which Joule heating becomes a problem. In some cases, the capillary diameter can be reduced, thereby permitting the use of a highly concentrated BGE. Mobility matching is very effective when indirect detection is employed; however, it is not possible to mobility match the BGE to all solutes when simultaneous separation of multiple ions is required. In this case, some of the peaks may be skewed while others are symmetric. Other advanced techniques have been proposed to simplify mobility matching (62, 63). Since electrodispersion is not usually a problem during the course of everyday HPCE, the reader should consult the references for more information.

REFERENCES l.Macka, M., Andersson, P., Haddad, P. R. Changes in Electrolyte pH Due to Electrolysis during Capillary Zone Electrophoresis. Anal Chem., 1998; 70:743. 2. Zhu, T., Sun, Y., Zhang, C , Ling, D., Sun, Z. Variation of the pH of the Background Electrolyte as a Result of Electrolysis in Capillary Electrophoresis. J. High Res. Chromatogr., 1994; 17:563. 3.Bello, M. S. Electrolytic Modification of a Buffer during a Capillary Electrophoresis Run. J. Chromatogr., A, 1996; 744:81. 4.Atamna, I. Z., Metral, C. J., Muschik, G. M., Issaq, H.J. Factors That Influence Mobility, Resolution and Selectivity in Capillary Zone Electrophoresis. II. The Role of the Buffer's Cation. J. Liq. Chromatogr, 1990; 13:2517. 5.Jennings, W, Analytical Gas Chromatography. 1987, Academic Press. 6.Lukacs, K. D., Jorgenson, J. W. Capillary Zone Electrophoresis: Effect of Physical Parameters on Separation Efficiency and Quantitation. HRC & CC, 1985; 8:407. 7.Saloman, K., Burgi, D. S., Helmer, J. C. Evaluation of Fundamental Properties of a Sihca Capillary Used for Capillary Electrophoresis. J. Chromatogr, 1991; 559:69. 8. van de Goor, T. A. A. M., Janssen, P S. L., van Nispen, J. W, van Zeeland, M.J. M., Everaerts, E M. Capillary Electrophoresis of Peptides. Analysis of Adrenocorticotropic Hormone-Related Fragments. J. Chromatogr, 1991; 545:379. 9.Tsuda, T., Nomura, K., Nakagawa, G. Separation of Organic and Metal Ions by High-Voltage Capillary Electrophoresis. J. Chromatogr, 1983; 264:385. lO.Lauer, H. H., McManigill, D. Capillary Zone Electrophoresis of Proteins in Untreated Fused Silica Tubing. Anal. Chem., 1986; 58:166. 11. Walbroehl, Y., Jorgenson, J. W. Capillary Zone Electrophoresis of Neutral Organic Molecules by Solvophobic Association with Tetraalkylammonium Ion. Anal. Chem., 1986; 58:479. 12.Ermakov, S. V, Capelli, L., Righetti, P. G. Method for Measuring Very Weak, Residual Electroosmotic Flow in Coated Capillaries. J. Chromatogr, A, 1996; 744:55. 13.Fujiwara, S., Honda, S. Determination of Cinnamic Acid and Its Analogues by Electrophoresis in a Fused Silica Capillary Tube. Anal. Chem., 1986; 58:1811. 14. Altria, K. D., Simpson, C. F. High Voltage Capillary Zone Electrophoresis: Operating Parameter Effects on Electroendosmotic Flows and Electrophoretic Mobilities. Chromatographia, 1987; 24:527. 15. Lambert, W. J., Middleton, D. L. pH Hystersis Effect with Silica Capillaries in Capillary Zone Electrophoresis. Anal. Chem., 1990; 62:1585. 16.Tsuda, T., Nomura, K., Nakagawa, G. Open-Tubular Microcapillary Liquid Chromatography with Electro-osmotic Flow Using a UV Detector. J. Chromatogr, 1982; 248:241. 17. VanOrman, B. B., Liversidge, G. G., Mclntire, G. L., Olefirowicz, T M., Ewing, A. G. Effects of Buffer Composition on Electroosmosis Flow in Capillary Electrophoresis. J. Microcolumn Sep., 1990; 2:176.

70

Chapter 2 Capillary Zone Electrophoresis

IS.Rasmussen, H. T., McNair, H. M. Influence of Buffer Concentration, Capillary Internal Diameter and Forced Convection on Resolution in Capillary Zone Electrophoresis. J. Chromatogr., 1990; 516:223. 19.Schwer, C , Kenndler, E. Electrophoresis in Fused-Silica Capillaries: The Influence of Organic Solvents on the Electroosmotic Velocity and the f Potential. Anal. Chem., 1991; 63:1801. 20.Fujiwara, S., Honda, S. Effect of Addition of Organic Solvent on the Separation of Positional Isomers in High-Voltage Capillary Zone Electrophoresis. Anal. Chem, 1987; 59:487. 21. Corse, J., Balchunas, A. T., Swaile, D. E, Sepaniak, M.J. Effects of Organic Mobile Phase Modifiers in Micellar Electrokinetic Capillary Chromatography. J. High Resolut. Chromatogr., 1988; 11:554. 22.Hjerten, S. High-Performance Electrophoresis: Elimination of Electroendosmosis and Solute Adsorption. J. Chromatogr, 1985; 347:191. 23.Emmer, A., Jansson, M., Roeraade, J. A New Approach to Dynamic Deactivation in Capillary Zone Electrophoresis. HRC & CC, 1991; 14:738. 24.Wiktorowicz, J. E., Colburn, J. C. Separation of Cationic Proteins via Charge Reversal in Capillary Electrophoresis. Electrophoresis, 1990; 11:769. 25.Jorgenson, J. W, Lukacs, K. D. Zone Electrophoresis in Open Tubular Glass Capillaries. Anal. Chem., 1981; 53:1298. 26.Jorgenson, J. W, Lukacs, K. D. Free-Zone Electrophoresis in Glass Capillaries. Clin. Chem., 1981; 27:1551. 27.Jorgenson, J. W, Lukacs, K. D. Zone Electrophoresis in Open-Tubular Glass Capillaries: Preliminary Data on Performance. HRC & CC, 1981; 4:230. 28. Culbertson, C. T., Jorgenson, J. W. Flow Counterbalanced Capillary Electrophoresis. Anal. Chem., 1994; 66:955. 29.Jones, A. E., Grushka, E. Nature of Temperature Gradients in Capillary Zone Electrophoresis. J. Chromatogr, 1989; 466:219. 30. Grushka, E., McCormick, R. M., Kirkland, J. J. Effect of Temperature Gradients on the Efficiency of Capillary Zone Electrophoresis Separations. Anal. Chem., 1989; 61:241. 31. Knox, J. H. Thermal Effects and Band Spreading in Capillary Electro-separations. Chromatographia, 1988; 26:329. 32. Stoyanov, A. V, Righetti, P G. Fundamental Properties of Isoelectric Buffers for Capillary Zone Electrophoresis. J. Chromatogr, A, 1997; 790:169. 33.Burgi, D., Chien, R.-L. Optimization in Sample Stacking for High-Performance Capillary Electrophoresis. Anal. Chem., 1991; 63:2042. 34.Moring, S., Reel, R. T., van Soest, R. E. J. Optical Improvements of a Z-Shaped Cell for HighSensitivity UV Absorption Detection in Capillary Electrophoresis. Anal. Chem., 1993; 65:3454. 35. Nelson, R. J., Paulus, A., Cohen, A. S., Guttman, A., Karger, B, L. Use of Peltier Thermoelectric Devices to Control Column Temperature in High-Performance Capillary Electrophoresis. J. Chromatogr, 1989; 480:111. 36.Kurosu, Y., Hibi, K., Sasaki, T, Saito, M. Influence of Temperature Control in Capillary Electrophoresis. HRC & CC, 1991; 14:200. 37.Kurosu, Y., Satou, Y, Shisa, Y, Iwata, T. Comparison of the Reproducibihty in Migration Times between a Constant-Current and a Constant-Voltage Mode of Operation in Capillary Zone Electrophoresis. J. Chromatogr, A, 1998; 802:391. 38. McLaughlin, G., Palmieri, R., Anderson, K., Benefits ojAutomation in the Separation ofBiomolecules by High Performance Capillary Electrophoresis, in Techniques in Protein Chemistry II, J. J. Villafranca, Ed. 1991, Academic Press: 3. 39.Tran, A. D., Park, S., Lisi, P J., Huynh, O. T, Ryall, R. R., Lane, P A. Separation of Carbohydrate-Mediated Microheterogeneity of Recombinant Human Erythropoietin by Free Solution Capillary Electrophoresis. Effects of pH, Buffer Type and Organic Modifiers. J. Chromatogr, 1991; 542:459. 40. Strickland, M., Strickland, N. Free-Solution Capillary Electrophoresis Using Phosphate Buffer and Acidic pH. American Lab, 1990; November:60.

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41.McCormick, R. M. Capillary Zone Electrophoretic Separation of Peptides and Proteins Using Low pH Buffers in Modified Silica Capillaries. Anal. Chem., 1988; 60:2322. 42. Zhu, M., Rodriguez, R., Hansen, D., Wehr, T. Capillary Electrophoresis of Proteins under Alkaline Conditions. J. Chromatogr., 1990; 516:123. 43.McNerney, T. M., Watson, S. K., Sim, J.-H., Bridenbaugh, R. L. Separation of Recombinant Human Growth Hormone from Escherichia coli Cell Pellet by Capillary Zone Electrophoresis. J. Chromatogr., A, 1996; 744:223. 44. Honda, S., Iwase, S., Makino, A., Fujiwara, S. Simultaneous Determination of Reducing Monosaccharides by Capillary Zone Electrophoresis as the Borate Complexes of N-2-Pyridylglycamines. Anal. Biochem., 1989; 176:72. 45.Hoffstetter-Kuhn, S., Paulus, A., Gassmann, E., Widmer, H. M. Influence of Borate Complexation on the Electrophoretic Behavior of Carbohydrates in Capillary Electrophoresis. Anal. Chem., 1991; 63:1541. 46.Honda, S., Suzuki, S., Nose, A., Yamamoto, K., Kakehi, K. Capillary Zone Electrophoresis of Reducing Mono- and Oligo-saccharides as the Borate Complexes of Their 3-Methyl-l-phenyl2-pyrazolin-5-one Derivatives. Carbohydrate Research, 1991; 215:193. 47.Klockow, A., Amado, R., Widman, H. M., Paulus, A. The Influence of Buffer Composition on Separation Efficiency and Resolution in Capillary Electrophoresis of 8-Aminonaphthalene-1,3,6trisulfonic Acid Labeled Monosaccharides and Complex Carbohydrates. Electrophoresis, 1996; 17:110. 48. Plocek, J., Chmelik, J. Separation of Disaccharides as Their Borate Complexes by Capillary Electrophoresis with Indirect Detection in the Visible Range. Electrophoresis, 1997; 18:1148. 49. Rydlund, A., Dahlman, O. Efficient Capillary Zone Electrophoretic Separation of Wood-Derived Neutral and Acidic Mono- and Oligosaccharides. J. Chromatogr, A, 1996; 738:129. 50.Tanaka, S., Kaneta, T., Yoshida, H. Separation of Catecholamines by Capillary Zone Electrophoresis Using Complexation with Boric Acid. Anal. Sciences, 1990; 6:467. 51. Kaneta, T., Tanaka, S., Yoshida, H. Improvement of Resolution in the Capillary Electrophoretic Separation of Catecholamines by Complex Formation with Boric Acid and Control of Electroosmosis with a Cationic Surfactant. J. Chromatogr, 1991; 538:385. 52.Westcott, C. C , pH Measurements. 1978, Academic Press. 53.Sustacek, V, Foret, F, Bocek, P. Selection of the Background Electrolyte Composition with Respect to Electromigration Dispersion and Detection of Weakly Absorbing Substances in Capillary Zone Electrophoresis. J. Chromatogr, 1991; 545:239. 54.Vindevogel, J., Sandra, P Simultaneous pH and Ionic Strength Effects and Buffer Selection in Capillary Electrophoretic Techniques. J. Chromatogr, 1991; 541:483. 55. Rush, R. S., Cohen, A. S., Karger, B. L. Influence of Column Temperature on the Electrophoretic Behavior of Myoglobin and a-Lactalbumin in High-Performance Capillary Electrophoresis. Anal. Chem., 1991; 63:1346. 56. Nashabeh, W, Rassi, Z. E. Capillary Zone Electrophoresis of Proteins with Hydrophilic FusedSilica Capillaries. J. Chromatogr, 1991; 559:367. 57. Grushka, E. Effect of Hydrostatic Flow on the Efficiency in Capillary Electrophoresis. J. Chromatogr, 1991; 559:81. 58.Hjerten, S. Free Zone Electrophoresis. Chromatogr Rev., 1967; 9:122. 59. Foret, F, Demi, M., Bocek, P Capillary Zone Electrophoresis: Quantitative Study of the Effects of Some Dispersive Processes on the Separation Efficiency. J. Chromatogr, 1988; 452:601. 60. Gas, B., Stedry, M., Kenndler, E. Peak Broadening in Capillary Zone Electrophoresis. Electrophoresis, 1997; 18:2123. 61.Mikkers, F E. P., Everaerts, F M., Verheggen, T. P E. M. Concentration Distributions in Free Zone Electrophoresis. J. Chromatogr, 1979; 169:1. 62. Williams, R. L., Vigh, G. N-(Polyethyleneglycol Monomethyl Ether)-N-MethylmorpholiniumBased Background Electrolytes in Capillary Electrophoresis. J. Chromatogr, A, 1997; 763:253. 63. Williams, R. L., Vigh, G. Polyethylene Glycol Monomethyl Ether Sulfate-Based Background Electrolytes in Capillary Electrophoresis. J. Chromatogr, A, 1996; 744:75.