Strategies for Electromigration Separations of Biologically Relevant Compounds

Advanced Chromatographic and Electromigration Methods in Biosciences Z. Deyl, 1. MikSlk, F. Tagliaro and E. Tesafov4, editors 01998 Elsevier Science B.V.All rights reserved

CHAPTER 2

Strategies for Electromigration Separations of Biologically Relevant Compounds Hidetoshi ARAKAWA'. Masako MAEDA' and Toshihiko HANA12'*

'School of Pharmaceutical Science, Showa University, Shinagawa-ku, Tokyo 142-8555,Japan 'International Institute of TechnologicalAnalysis 4-944-13 Matsumi Matsumidai Haitsu 409, Kanagawaku, Yokohama 221-0005, Japan

CONTENTS 2.1 2.2 2.3

2.4

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Strategy for the application of electromigration techniques for biological samples . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . 54 Analysis of low molecular mass solutes by capillary electrophoresis and related techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 2.3.1 Measurement of electroosmotic flow-rate . . . . . . . . . . . . . . 57 2.3.2 Relationship between migration time and pKa . . . . . . . . . . . 6 1 2.3.3 Reproducibility of migration time . . . . . . . . . . . . . . . . . . 64 2.3.4 Selection of the background electrolyte . . . . . . . . . . . . . . . 68 2.3.5 Sample loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2.3.5.1 Selection of column size . . . . . . . . . . . . . . . . . 69 2.3.5.2 Multi-channel column . . . . . . . . . . . . . . . . . . 70 2.3.5.3 Selection of injection method . . . . . . . . . . . . . . . 73 Analysis of biological macromolecules . . . . . . . . . . . . . . . . . . . 73 2.4.1 Separation of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 75 2.4.1.1 Gels used for separation and nucleic acid sequencing . . 75 2.4.1.2 Size separation of single-strand DNA for DNA sequencing76 2.4.1.3 Analysis of single-strand DNA-conformation polymorphism (SSCP) . . . . . . . . . . . . . . . . . . . . , 77 2.4.1.4 Separation of double-strand DNA fragments . . . . . . . 80 2.4.2 Separation of proteins . . . . . . . . . . . . . . . . . . . . . . . . 82 2.4.2.1 Separation by capillary zone electrophoresis . . . . . . . 84

54

Chapter 2

2.5 2.6

Capillary isoelectric focusing . . . . . . . . . . . . . . . 85 Capillary sodium dodecyl sulfate-gel (SDS-gel) electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Binding of low-molecular-mass solutes to proteins . . . . . . . . . . . . . 87 Future of CE as a method for the separation of biological materials’ 89 constituents. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 2.4.2.2 2.4.2.3

2.7 2.8

2.1 INTRODUCTION

Electromigration methods, ie.,electrophoresis and isoelectric focusing, have been in use for years for the separation of biopolymers and their fragments. Gels (of polyacrylamide, agarose and - in the early days - starch) have been used widely as anticonvective media. Today, one-dimensional electrophoresis in gels is used mainly for molecular-mass estimation: for the analysis of complex protein mixtures, twodimensional separations are preferred. In the two-dimensional mode the sample is first separated by isoelectric focusing then in the perpendicular run the charge of different proteins is equilibrated by the addition of sodium dodecyl sulfate (SDS). The resulting maps can reveal up to 10,000 protein zones, and catalogues of such protein maps revealing the composition of, for example, different tissues under physiological and/or pathological conditions are available [ 1-41, As the technique of two-dimensional gel separations is generally well known (including the detection methodology) it is not described here, and the reader is referred to monographs on this subject. Some additional information can be found in Chapter 13.

2.2 STRATEGY FOR THE APPLICATION OF ELECTROMIGRATION TECHNIQUES FOR BIOLOGICAL SAMPLES When selecting an electromigration method from the numerous techniques available the first property of the solutes to consider is their molecular mass. For biopolymers and their fragments, gel separations (no matter whether in the one- or two-dimensional mode) are the methods of first choice. This does not mean that other techniques, such as capillary electrophoresis (see later), could not be applied. However, gel-based separations, particularly in their two-dimensional mode, offer much higher selectivity and can separate more protein zones, for example, than any of the other approaches. As a matter of fact, two-dimensional gel electrophoresis exploiting isoelectric focusing in the first dimension, followed by SDS-electrophoresis in the second dimension, offers the highest selectivity of all separation techniques used today.

Strategies for Electromigration Separations

55

For low molecular mass solutes discrimination is made on their solubility in water or aqueous buffers. If the solutes are water soluble, zone electrophoresis is recommended. If they are poorly- or practically insoluble in water, more sophisticated capillary techniques are recommended. These include micellar- or microemulsion electrokinetic chromatography, electrochromatography or electrophoresis in nonaqueous media. In the last category, methanol-acetonitrile mixtures are typical. The conductivity is obtained by adding an organic salt - sodium or ammonium acetate in about 20 mM concentration. 2.3 ANALYSIS OF LOW MOLECULAR MASS SOLUTES BY CAPILLARY ELECTROPHORESIS AND RELATED TECHNIQUES Since Mikkers and Everaerts first used a narrow PTFE tube for electrophoresis [5] electrically driven separation methods using capillary tubes became very popular, particularly because their high theoretical-plate-numbers cut down the overall analysis time, which is highly appreciated in routine analyses. The Golay equation reveals that a narrower internal diameter of the tube gives a higher theoretical-plate number. This theory has been successhlly put into practice with capillary gas chromatography. However, in capillary liquid chromatography the main obstacle is the high back-pressure resulting from the high viscosity of the mobile phase. The internal diameter of the tube is usually 200-500 pm for gas chromatography, but less than 5 pm for column liquid chromatography. Such a requirement limits the development of capillary liquid chromatography, owing to the difficulty of miniaturizing the injector, connector and detector for high-pressure operation. In contrast, the electrically-driven separation methods using a capillary tube can be performed at atmospheric pressure. The internal diameter of the tube is usually 25-100 pm, and only a relatively short tube is required (compared to capillary gas chromatography). Several modes of capillary electrophoretic techniques have been developed over the course of time. These techniques can be classified into five groups, according to the column type and background-electrolyte composition. 1) Capillary electrophoresis in a free solution (capillary zone electrophoresis) 2 ) Capillary electrophoresis using a micellar or microemulsion solution as background electrolyte, i e . , micellar electrokinetic capillary chromatography (micellar electrophoresis). 3) Capillary electrochromatography and micellar electrochromatography 4) Capillary electrophoresis in colloidal sols, (colloidal sol electrochromatography and gel electrochromatography) 5) Host-guest electrophoresis, complexation electrophoresis

References pp. 91-93

56

Chapter 2

Capillary electrophoresis and capillary liquid electrochromatography have similarities and differences based on the type of column and instrumentation. Both separation methods use capillary tubes which can be bare, coated or bonded with surface modifiers, or gel-filled or even gel-packed. However, the driving forces of the liquid and analytes are different. The analytes migrate by electrophoresis and electroosmotic flow in capillary electrophoresis, and the graphical presentation is called the electropherogram. In capillary electrochromatography the analytes move generally in a sorbent-packed capillary tube by electrophoresis and electro-osmotic flow. The main difference here is the presence of the column packing which can be of considerably differing nature, from reversed-phases (used most frequently), up to ion exchangers. Capillary electrochromatography now represents a fast-growing type of endo-osmoticflow-driven separations. Separations are made in a capillary tube which contains pressurized liquid or is operated with very diluted buffers in order to reduce the electrolysis and bubble formation. The graphical recording is called a chromatogram. The migration of the liquid phase in electrophoresis and electrochromatography differs from pressure-driven liquid separations (HPLC) in that the carrier front moves physically with a flat profile rather than the parabolic profile of a pressurized liquid (which is the case, for example, in HPLC). This, of course, improves the separation possibilities of capillary electrophoresis and electrochromatography [6]. The physical differences between capillary electrochromatography and capillary liquid chromatography are easily seen. The former requires a high-power electrical supply to maintain the electro-endo-osmotic flow and electrophoresis, while the latter requires a high-pressure pump to control the pressurized liquid flow. Capillary electrophoresis and capillary electrochromatography , in contrast, are essentially identical separation methods - at least as far as the equipment is concerned. However, the interaction mechanisms differ, interactions between the internal wall of the capillary tube, the surface of the packed particles and the separated solutes are involved. If the interaction between the internal wall of the tube and/or the surface of the packed particles is eliminated, the separation process turns into electrophoresis. If the internal wall of the capillary and/or the surface of the packed particles act as a stationary phase (sorbent), the separation process can be called electrochromatography. On the other hand, capillary electrophoresis in colloidal sols, and gel electrochromatography, are very close - if not the same, as far as the molecular interaction mechanism is concerned - and also the migration of analytes is ensured only by electrophoresis. The smaller-size molecules move faster than the larger molecules. This migration order is the opposite of that in pressure-driven size-exclusion liquid chromatography (see also reference book by Heftman).

Strategies for Electromigration Separations

57

2.3.1 Measurement of electro-osmotic flow-rate The reproducibility of the migration time of analytes measured in the electrically driven separations is smaller than that of the elution time of analytes measured in pressure-driven liquid chromatography. Although several factors have been found to influence the flow-rate [7-91, the results reported have been inconsistent [ 10-151. The migration time, mt, is given by the equation:

the electrowhere L is the length of the column, E the electric field strength, kOs osmotic flow-rate, and pep the electrophoretic flow-rate. The electro-osmotic flowrate is obtained from the migration time of neutral compounds or solvent using eq. (2.2):

where mto is the migration time of neutral compound. The relationship between the applied voltage and the current cannot be expressed in terms of the simple Ohm’s Law equation R=V/A where R, V and A are the resistance of the solution, the applied potential, and the current, respectively. As shown in Fig. 2.1, the migration times of dansylalanine, dansylisoleucine and fructose diminish as a fimction of applied voltage; however, the current is not linearly increased. The resistance is constant for a solid at a given temperature, but the electrophoretic effect and the zeta potential affect the current. An increase in either the buffer concentration or the voltage applied causes a reduction in the resistance of the background-electrolyte-filled capillary by the electrophoretic effect, or an increase in the zeta potential of the inside wall, or the static potential of the outer wall of the column (or a combination of all three). In the same way as the retention factor, k, is used for the theoretical treatment of pressure-driven liquid chromatography, so the relative migration time, Rmt, is used in the electrically-driven separations. Under normal conditions, Rmt is calculated from the following equations:

for cations, Rmt = mts/mto

References pp. 91-93

(2.3)

Chapter 2

58

200

20

4 100

10

E E L

3

0

0

10 Power

15

kV

Fig. 2.1, Migration time related to applied power. Conditions: column, 50 cm x 50 pm I.D.; separation length, 30 cm; carrier, 50 mM sodium phosphate buffer @H 7.0); injection, hydrostatic method; symbols: 0, dansylalanine (DnsAl); 0 , dansylisoleucine; B, fructose (Vo marker); 0, measured current for anions, Rmt = (mts - mto)/mto

where mts and mto are the migration times of an ionic solute and a neutral compound, respectively. The measurement of mto is important in the electrically-driven separations. The migration time of fructose, which can be used as a void-volume marker in pressure-driven reversed-phase liquid chromatography [ 161, is not linearly related to the applied voltage or current flow in 10-500 mM sodium phosphate buffers (pH 7.0). The migration time of fructose as a function of the current is plotted in Fig. 2.2. The vertical axis is the migration time obtained by the transformation of eq. (2.2),

where mt is the measured migration time and A the current. The slope, a, is related to the buffer concentration by eq. (2.6): a = 1,419e-4(M-') + 1.362e-4

59

Strategies for Electromigration Separations

0.8

0.4

0.0

0

250

Measured current

5 '0

PA

Fig. 2.2. Buffer concentration effect. Conditions: column, 50 cm x 50 pm I.D.; separation length, 30 cm; carrier, sodium phosphate buffer (pH 7.0); injection, hydrostatic method; sample, fructose; symbols: 0 , 0.025 M; 0 , 0 . 0 5 M; A, 0.5 M. (From [ 181 with permission.)

where M is the molar concentration of the buffer. The migration time of fructose increases as the concentration of sodium chloride is increased, as shown in Fig. 2.3: the dependence of the slope of eq. (2.5) on the sodium chloride concentration is expressed by eq. (2.7):

a = 1.607e-3 x log(M-')

- 4.228e'4

(2.7)

Increasing the buffer concentration slows the migration speed; however, the theoretical-plate numbers increase for a better separation, as shown in Fig. 2.4, which shows that the separation of dansylisoleucine from dansylalanine is improved by increasing the sodium phosphate concentration from 6.3 to 50 mM. The theoreticalplate number is linearly related to the current. The higher the current, the higher the theoretical-plate number. By increasing the applied current from 57 to 230 PA, the separation time is markedly reduced (Fig. 2.4). The effects of changing the concentrations of buffer and sodium chloride are different, and the electro-osmotic flow-rate is influenced by the nature of the anion or cation; for instance the phosphate ion has a greater influence on the electro- osmotic flow-rate than chloride. References p p 91-93

Chapter 2

60

1.0

.-c I

E

!i -.cI

0.5

c

.-0 CI

2 I

.-0 0.0 0

4

200

Measured current

I0

pA

Fig. 2.3. Sodium chloride concentration effect. Conditions, column, 50 cm x 50 pm I.D.; separation length, 30 cm; carrier, 50 mM sodium phosphate buffer @H 7.0); injection, hydrostatic method; sample, fructose; symbols: QO.01 M; 0 , O . O S M; A, 0.45 M. (From [18] with permission.)

conc.NaP

voltage current

0.05 14 230

0.0063 M 6 kV 22 pA

0.05 6 57

k

Dnslle 0.87 DnsAl 1.03

1

1

I

I

5

6

25

28

Migration time

I

I

8

10

min

Fig. 2.4. Relative migration time influenced by buffer concentration. Conditions, column, 50 cm x 50 pm I.D.; separation length, 30 cm; carrier, sodium phosphate buffer (pH 7.0); injection, hydrostatic method; Nap, sodium phosphate buffer; DnsIle, dansylisoleucine; DnsAl, dansylalanine.

61

Strategies for Electromigration Separations

.-S E

30

1 /

I

10

o

12

z

0

10

0

30

20

conc. Methanol

16 kV

%

Fig. 2.5. Methanol concentration effect. Conditions: column, 50 cm x 50 pm ID.; separation length, 30 cm; carrier, 50 mM sodium phosphate buffer (pH 7.0); injection, hydrostatic method; open symbols, dansy lalanine; fused symbols, dansylisoleucine.

The addition of methanol to the background electrolyte produces a significant improvement in the peak shape of nitrogen-containing compounds [ 171, as seen in Fig. 2.5, while the electro-osmotic flow-rate is affected only slightly by this addition. The addition of sodium dodecyl sulfate does not affect the electro-osmotic flow-rate, and that of tetrabutylammonium bromide increases the flow-rate [ 181.

2.3.2 Relationship between migration time and pKa The electro-osmotic flow usually carries even negatively charged solutes from the inlet (anode) of a capillary column to the outlet (cathode). It is controlled by the nature of the ionic species present, the salt concentration, the pH, other additives (modifiers) present in the background electrolyte, the column (capillary) dimensions, and the applied voltage, as explained in Section 2.3.1. The elution time in pressuredriven liquid chromatography depends upon the physical flow-rate of the eluent, and the physico-chemical molecular interactions of solutes between the stationary phase and eluent. Although both positively and negatively charged compounds move in the same direction in capillary electrophoresis (owing to the strong endo-osmotic flow which in most cases carries the contents of the capillary from anode to cathode), the migration time of ionized compounds is significantly affected by the electrophoretic process whereby positively charged solutes are attracted to the cathode and negatively References pp. 91-93

62

Chapter 2

charged solutes are attracted to the anode. This electrophoretic partition can be related to the interactions governing ion-exchange liquid chromatography, as will be explained later. The differences between the migration times of charged solutes are related to their dissociation constants. In reversed-phase liquid chromatography it is possible to predict the retention times of ionized compounds mathematically, by a combination of the hydrophobicity (log P values obtained from the octanol-water partition) and the dissociation constants of the solutes [19,20]. The log P values are calculated by Rekker’s method [21], and the dissociation constants, pKa, are usually calculated using Hammett’s equation [22]. The hydrophobicity can be further related to the van der Waals volume (VWV) and the selectivity of the solutes [23]. The retention factor (k) is then obtained from eqs. (2.8) and (2.9).

where kmax is the maximum retention factor of a solute which retains its molecular form, and kmin is the minimum retention factor of a solute which is completely ionized. The quantity [K] is the dissociation constant, related to a solute’s PKa value, and [H’] is given by the pH of the eluent. The van der Waals volume was, in this case, calculated by Bondi’s method [24] and the selectivity, R*, was obtained chromatographically [25]. In the above equations, the relative retention time is not influenced by the flow-rate and is controlled by the degree of ionization of the solutes. The degree of ionization of the solutes affects the hydrophobic interaction between the solutes and the stationary phase. The migration time (mt) in capillary electrophoresis, in contrast, is given by eq. (2.1). In the absence of an interaction between the solutes and the internal wall of the column, poS is measured from the migration time of neutral compounds, as explained in Section 2.3.1. For ionized solutes, the migration speed depends on lep: in other words, the degree of ionization of the solutes affects hydrophobicity in reversed-phase liquid chromatography and the electrophoretic mobility in capillary electrophoresis. The relationship between the retention factor and the pH of the eluent is given by eq. (2.8). A similar relationship between the relative migration time (Rmt) and pH is shown in Fig. 2.6, where experimental data are given for aniline, N,N-dimethylaniline, and benzylamine.

63

Strategies for Electromigration Separations

1.o

,--

N,N-Di&aniline

E

/

0.5

0 2

4

6

8

10

12

PH Fig. 2.6. Relative migration time (Rmt) of nitrogen containing compounds related to pH of carrier. Conditions: column. SO cm x SO pm I.D.; separation length, 30 cm; carrier, 50 mM sodium phosphate solution; injection, hydrostatic method; Vo marker, fructose.

Rmt = (mt of a solute)/(mt of water)

(2.10)

From eq. (2.8), it is apparent that the pH change between kmax and kmin is two pH units in reversed-phase liquid chromatography. In contrast, in capillary electrophoresis the number of pH units depends on the dissociation constants of the solutes, as seen in Fig. 2.6. The higher the dissociation constant, the higher the number of units. The mathematical fit was examined on the basis of experimental results for eight nitrogen-containing compounds, giving eq. 2.1 1.

Rmt = { 1.OO + 0.1SO([H+]KK)"}/{ 1.OO + ([H'IKK)"}

(2.1 1)

In this equation, KK is derived from the dissociation constant, pKa, influenced by the electrophoretic force [see eq. (2.12)]. This value for [H'] is obtained from the pH of the carrier, and w controls the shape of the curve in Fig. 2.6.

References p p 91-93

Chapter 2

64

KK =

0-(0.604 x pKa + 1.53 1)

(2.12)

w is also related to the dissociation constant, which leads to eq. (2.13):

w = 1/{0.0915 x

pKa)}

(2.13)

The constants of eqs. (2.12) and (2.13) can change according to the system used, and are especially dependent on the components of the background electrolyte. The migration time of nitrogen-containing compounds can be predicted if their dissociation constants are known. However, the molecular interaction between solutes and the surface of the fused silica tube is not negligible [26,27]: just as careful surface treatment can eliminate such molecular interactions in reversed-phase liquid chromatography [28,29], surface treatment will be necessary to improve the precision of theoretical approaches in capillary electrophoresis. Of course, all these considerations refer to sets of model compounds and their generalization requires additional experimental work.

2.3.3 Reproducibility of migration time Lack of reproducibility of migration time in capillary electrophoresis can be a problem even if the capillary is repeatedly rinsed with 0.1 M KOH, water, and the background electrolyte [30]. This is a critical problem for the applycation of this analytical technique to quantitative analysis, and it can be overcome only if the reproducibility is quantitatively analysed. Electrophoretic migration depends on two basic phenomena, one being the migration of the solute ions in the background electrolyte, and the second being the zeta potential of the inner surface of the capillary tube. Further, electrolysis of the electrolyte components and solutes cannot be neglected when operating at high voltage, particularly if the concentration of salts is rather high. The background-electrolyte components should therefore be selected carefully, and if necessary the inner wall of the capillary should also be chemically treated. The stability of the electro-osmotic flow-rate can be measured from the migration time of an uncharged solute (e.g., fructose, thiourea, or benzyl alcohol) and the pH change of the background electrolyte in both the anode- and cathode reservoirs. The endo-osmotic flow-rate has a linear relationship to the applied current [ 181. The flow-rate can be obtained from eq. 2.14 at any given pH in a solution with the same buffer components. Thus, for example, the migration time of fructose was measured in a pH 2-1 1.5, 0.05 M sodium phosphate buffer, at an applied voltage from 2 to

Strategies for Electromigration Separations

3 0 LL

0

2

4

6

8

65

1

1 0 12

PH Fig. 2.7. Change of the ratio of electroosmotic flow divided by the current. Conditions: column, 50 cm x 50 pm I.D.; separation length, 30 cm; carrier, 50 d v l sodium phosphate solution; injection, hydrostatic method; sample, fructose. 16 kV, which changed the current (A) from 12 to 115 PA. In Fig. 2.7 the ratio of the electro-osmotic flow divided by the current is plotted versus the pH of the buffer; the values on the y-axis (b) are therefore related to the endo-osmotic migration by a simple equation (eq. 2.14),

~ 0 =s b

xA

(2.14)

where A represents the observed current. The electro-osmotic flow-rate is slow below pH 3.0 and increases up to pH 5, then gradually reduces again. This type of curve was observed when the pH was changed from alkaline to acidic conditions, at the same ionic strength [3 11. While in the case presented the flow-rate at higher pH values was practically constant when a Pyrex capillary tube was used, it was slightly pH-dependent even at higher pHs 1321. The reduction in the flow-rate at high pH was explained as resulting from a reduced zeta-potential [33,34]. This sigmoid curve can be roughly expressed by eq. 2.15:

(2.15)

References pp. 91 -93

Chapter 2

66

I:

cathode

+

e

-1

1

0

f

I

2

+

+ +

I

4

Analysis time

I

6

8

hour

Fig. 2.8. pH Change of pH 4.00, 50 mM sodium phosphate solution at 35 PA. Conditions: column, SO cm x 50 pm I.D.; separation length, 30 cm; +, cathode; x, anode.

where c is the slope related to the osmotic flow-rate and applied current (A), Ch is the highest slope - measured at pH 6.0 - and cl is the lowest slope measured at a pH below 3. The values of H+ can be derived from the given pH, and Ks can be obtained from the value obtained at pH 4 where the curve varies. The reproducibility of the migration depends upon the condition of the inner capillary wall, while the stability depends on the pH change of the buffer electrolyte. The pH of the carrier at both the anode and cathode changes slowly with time. Thus, if the pH change (ApH) was measured over 4 h at 35 pA, the typical profile (at pH 4, see Fig. 2.8) was obtained. The migration time of the marker (fructose) can be expressed by eq. 2.16, which relates the change of the endo-osmotic flow-rate

(Apes) to the analysis time (at) at pH 4:

Apes = 3.22e'2 x at + 13.29

(2.16)

The constant of eq. 2.16 depended on the initial pH. The change in the endo-osmotic flow-rate could be neglected between pH 7 and 10 under the experimental conditions. The slope of the relationship between ApH and pH is shown in Fig. 2.9. The larger are the values of slope related to pH change, the larger is the pH change. The change depends upon the strength of the buffer, pH did not significantly change at pH 2 and 7. As an example, ApH at pH 7.00 and 4.00 for 50 mM sodium phosphate

67

Strategies for Electromigration Separations

+

0 l-

+

X

X

0 m

-

0

0

3

.

6

4

9

1

2

PH

Fig. 2.9. pH Change upon working hours: 50 mM sodium phosphate solution at 35 PA. Conditions:column, 50 cm x 50 pm I.D.; separation length, 30 cm; +, cathode, x, anode

buffer was 0.015 and 0.137 pH units, respectively, after 100 min. The electrophoretic migration of cations such as H+ affects the ApH of background electrolyte solutions in both reservoirs. The volume change of solutions in both reservoirs related to the electro- osmotic flow, will be negligible. However, the change of the hydrogen-ion concentration depends upon the applied current and not on the pH of the buffer. The total hydrogen ion produced by electrolysis can be calculated by Faraday’s equation (Q = At, where Q is in Coulombs, A is the applied current, and t is the time applied, in seconds). The total concentration of the hydrogen ion is 0.67.10-4, when 100 pA are applied for one hour. This amount is quite large for a small-volume background electrolyte solution. This means that when a small volume of electrolyte solution is used, containing diluted buffer components, the analysis time should be considered. When the volume is 700 pI - one-tenth of that in the above experiment - a longer analysis time should be avoided, even if a SO mM phosphate buffer of pH 7.00 is used, unless the pH gradient is well designed. The pH will change by 0.15 pH unit after 100 min’s operation. The reproducibility therefore depends on both the buffer effect and the working hours for one background electrolyte. Usually, a low concentration of buffer solution is employed in capillary electrophoresis, and the buffer effect and working hours are critical. The shortening of the migration time can be measured in solutions of pH above 5 . As a typical example, the effect of pH on the relative migration time of phenols is shown in Fig. 2.10. The relative migration time of the anions was calculated from eq. (2.4). The pH effect upon the relative migration time of anions is References pp. 91-93

Chapter 2

68

x 0

3

6

+

9

!

PH Fig. 2.10. Relative migration time (Rmt) of phenols related to pH of carrier. Conditions: column, 50 cm x 50 pm I.D.; separation length, 30 cm; carrier, 50 mM sodium phosphate solution; injection, hydrostatic method; x, 2,4-dichlorophenol; +, 4-chlorophenol; 0,3-chlorophenol.

similar to that found in reversed-phase liquid chromatography, but it is shifted toward the higher-pH side. When the pH change of each buffer was considered from the viewpoint of total operation hours of one anode reservoir, the pH shifted toward lower values during the operation, as shown in Figs. 2.8 and 2.9. Thus, for example, a buffer starting at pH 10 shifted to pH 9.5 after 3 h of operation. This means that the relative migration time can be predicted, as in reversed-phase liquid chromatography. If phenols are used as examples, the difference is that phenols in their ionic form are eluted faster than in their molecular form, in reversed-phase liquid chromatography, while the ionized phenols migrate more slowly than their molecular forms, in capillary electrophoresis. The reproducibility is significantly influenced by pH changes in the electrically-driven separations [35]. 2.3.4 Selection of the background electrolyte Details of composition of the background electrolytes are dealt within specific chapters of this book. The most important point is to select good buffer solutions whose pH are not easily changed by electrolysis during the operation, as is explained in Section 2.3.3. Otherwise, a reproducible migration time cannot be obtained, even if the electrolyte solutions in the anode and cathode reservoirs are replaced before every analysis. The pH change cannot be avoided in electromigration methods. This is a fundamental problem in the use of capillary electrophoresis and capillary electrochro-

Strategiesfor Eiectromigration Separations

69

matography, even when the theoretical plate number is extremely high at more than 100,000 plates per column. When the pH change is carefully managed, the separating power solves the difficulty of separation of very similar compounds. The composition of the background electrolytes in free-solution-capillary-electrophoresis and electro-endo-osmotic chromatography are basically buffer solutions, salts, and organic solvents and resembles to a certain extent the eluents for ion-exchange liquid chromatography. The main components of the liquid used for micellar electrokinetic chromatography are the buffer and the surfactant - whose concentration must exceed the critical concentration for micelle formation (CMC) - as in micelle liquid chromatography. In contrast to reversed-phase ion-pair liquid chromatography, it is possible in micellar electrokinetic chromatography to vary the concentration of the pseudophase in the background electrolyte, influencing thereby the separation conditions: in ion-pair reversed-phase liquid chromatography the amount of the stationary phase available for the partition process is constant. The components of the background electrolyte used in capillary electrophoresis with colloidal sols are similar to those in slab-gel electrophoresis, although they are used at lower concentrations; buffer solutions with or without a surfactant can be used. In host-guest electrophoresis, by which general term bio-affinity separations and chiral separations are described, the background electrolyte must contain a modifier to ensure the bio-affinity interaction, or a chiral selector, as described in detail in Chapter 5 . 2.3.5 Sample loading

The sample-loading technique and sample-loading capacity are important in the handling of a narrow-bore tube used as the separation column. The smaller the column size, the smaller the sample-loading capacity. Usually, the injection volume is 2 nl for a regular sized column (50 pm i.d.), in capillary electrophoresis and capillary electrochromatography. With an increase in the injection volume from 2 to 9 nl, the theoretical-plate-number declines to less than one-third of its original value. For improving the sensitivity of detection, the techniques used in capillary liquid- and gas-chromatography are applied. These include pre-concentration, column-switching, pre-derivatization, and post-column reaction detection. These are generally known to chromatographers, and are not repeated here.

2.3.5.1 Selection ofcolumn size The separation capability of capillary electrophoresis and capillary electrochromatography is expanding from polar- to non-polar-, and from small to large molecules. References pp. 91-93

Chapter 2

70

100

50

250 pm1.D.

1+2

3

L

Fig. 2.1 1. Selection of column bore size. Conditions: column, 50 cm x 50, 100 and 250 p n I.D.; separation length, 30 cm; carrier, 50 mM sodium phosphate buffer @H 7.0); injection, hydrostatic method; Sample 1, dansylisoleucine;2, dansylalanine; 3, dansylvaline.

Expanding the separation power to the preparative scale is, however, very difficult, because increasing the internal diameter of the capillary tube for a large-sample capacity markedly reduces the theoretical-plate number, as seen in Fig. 2.1 1, where SO-, loo-, and 250 pm i.d. columns were used. The injection volume was roughly related to the ratio of the squares of the internal diameters. The mixture of dansyl-isoleucine, dansyl-alanine and dansyl-valine was separated in a 50 pm i.d. column. The separation of dansyl-isoleucine and dansyl-alanine was difficult in a 100 pm i.d. column, and these two compounds were co-eluted from a 250 pm i.d. column.

2.3.5.2 Multi-channel columns One method for increasing the sample loading capacity is to use multi-channel columns [36].The accuracy of the internal diameter of fused silica capillary tubes is very high. In order to scale up the sample-loading capacity, multi-channel columns were made, as shown in Fig. 2.12. The resolution of every column is identical, owing to the high quality of the fused silica tubes. Increasing the number of columns multi-

--

Strategies for Elechomigration Separations

71

splitter

n

A

A Fig. 2.12. Multi-channel column. Conditions: A, direct current; CR, carrier reservoir; D, detector (From [36] with permission).

plies the sample loading capacity and the current at the same voltage, as shown in Fig. 2.13. The maximum number of columns is therefore dependent upon the capacity of the power supply, as expected from the basic equations:

V=AxR

(2.17)

1 , =~ l R l + 1R2 + ----+ l/Ri

(2.18)

Ri

ai x Li x & (i

=

1,2,....n)

(2.19)

where V and A are the applied voltage and current, respectively. The quantity R is the resistance of the column which, in turn, depends on a, the cross-sectional area of the column; L is the length of the column and Rc is the resistance of the electrolyte used. Since ai, Li and & of a multi-channel column are constant, R is related to the number of columns. The higher the voltage is, the shorter is the analysis time: however, the power supply has a maximum current limitation. This is one feature of a preparative scale-up Reference3 pp. 91-93

Chapter 2

72

300 20 10 I

I

I

I

I

I

L

1 2 3 4 5 6 7

Number of columns

Fig. 2.13. Relation between number of columns and current, and migration time of dansylalanine (DnsAl) and dansylisoleucine (DnsIle) (From [36]with permission).

multi-column with splitter

without splitter

A Fig. 2.14. Monitoring with and without splitter in multi-channel column (six columns). Conditions: one column, 50 cm x 50 pm I.D.; separation length, 30 cm; carrier, 50 mM sodium phosphate buffer (PH 7.0); injection, hydrostatic method; applied voltage, 8 kV, Sample I , dansylisoleucine; 2, dansylalanine.

Strategies for Electromigration Separations

73

operation. In one example, where one 50 cm x 50 pm column gave a current of 39 pA at 8 kV in a 50 mM sodium phosphate buffer (pH 7.00) with methanol (1005, v/v), up to fourteen columns could be combined, and one-tenth of an injection (split injection) was possible. The results is shown in Fig. 2.14. The maximum injection volume was 120 nl, and it depended upon the sample concentration. More than 130 ng of each ffaction can theoretically be collected, and the detection limit of dansyl-amino acids was a few pg. Zero-dead-volume connectors are required for successful separations. 2.3.5.3 Selection of injection method

The construction of a small-volume injector is not easy. Several types of injection techniques are in use, e.g., physical injection (hydrostatic method, vacuum method, pressure method, slide-type method) [37] and the electro-migration injection shown in Fig. 2.15. A quantitative injection could not be achieved by the electromigration method as seen in Fig. 2.16 where the xanthine peak is not observed while theobromine was selectively injected. Increasing the concentration of albumin in the background electrolyte (for drug-binding studies), and hence increasing the viscosity of the sample solution, makes the quantitative injection of sample solution by hydrostatic injection difficult (Fig. 2.16). The reduction in the peak height of xanthine was caused by its adsorption onto albumin. The effect of viscosity on injections is naturally found in both pressurized- and vacuum injection modes. The electromigration injection is simple and easily automated, but the amount of injected compounds may vary according to the molecules’ electromobility. Thus in a model system where theobromine, theophylline and xanthine were injected, the indication was that only about 30% of theophylline was injected, as compared to theobromine, and only a trace amount of xanthine reached the column. This is a situation to be stressed. Certainly, electro-injection is now widely used and is the method of choice when working with colloidal sols (because the use of pressurized vacuum - injection is difficult for this column type). 2.4 ANALYSIS OF BIOLOGICAL MACROMOLECULES

Primary biological macromolecules include DNA, proteins, glycoproteins, and polymeric sugars. Electrophoresis is widely used for the separation of these solutes, for the elucidation of their structures (e.g., the base-sequence of DNA), and in a number of clinical tests based on serum protein analysis. For detailed information the reader is deferred to the respective specialized chapters in this book.

References pp. 91-93

Chapter 2

14

Hydrostatic method

Electro-migration method n

S

1-

I

6 I

S

Vacuum method

D

C

Pressure method D r ,

1

n I

,

D

V

Mechanical injection A: direct current C: carrier solution CR: carrier reservoir D: detector P: pressure S: sample solution Sy: syringe V vacuum Fig. 2.15. Sample injection methods. Conditions: A, direct current; C, carrier solution; CR, carrier reservoir; D, detector; P, pressure; S, sample solution; Sy, syringe; V, vacuum.

-

75

Strategies for Electromigration Separations

Hydrostatic

Electromigration

I-----

-0

ID

P,

x

5

E. ca

2

0

20

40

0

20

Albumin

Vacuum

I

3

40

0

20

40

mg/mL

Fig. 2.16. Sample injection volume by different injection methods. Conditions: one column, 50 cm x 50 prn I.D.; separation length, 30 cm; carrier, 50 mM sodium phosphate buffer (pH 7.0); sample 1, theobromine, 2, theophylline, 3 , xanthine. 2.4.1 Separation of DNA

The purpose of analysing DNA and its fragments is directed to revealing its primary structure and assessing variations in genetic material. The resolution of separation required for DNA sequencing is within one base as a single-strand DNA, and that required for genetic diagnosis using polymerase chain reaction (PCR) is within a few bases as base-pairs of double-stranded DNA kagments. The dissociating functionality is primarily the phosphate group, whose pKa is about 1. Therefore, the electric charge of DNA per unit molecular mass is nearly the same with all species analysed, so they cannot be separated by their electronic properties alone. Therefore, molecular sieving effects using gels and dynamically introduced polymers have to be exploited (for details see Chapter 14). 2.4.1. I Gels used for separation and nucleic acid sequencing

The gels used for electrophoresis include polyacrylamide and agarose. Factors that affect the separation of DNA are the pore size of the gel and the mobilization voltage: the former is, however, of primary importance. The relationship between the pore size of the gel and the DNA size has been described by Slater et al. [38]. The pore size of the gel and the degree of cross linking can be easily adjusted by the gel concentration. These parameters are selected according to the size of the target DNA fragment. The

References pp. 9I-93

16

Chapter 2

polyacrylamide gels are prepared in the conventional way by polymerization using

N,N,N',N'-tetramethylethylenediamine and ammonium persulfate as catalysts. The pore size of the gel is expressed as total acrylamide concentration (T%), i.e., as a percentage of the sum of acrylamide and N,N'-methylenebisacrylamide(Bis)

T% =

acrylamide (8) ml

+ Bis @ .

The degree of cross-linking (C) is expressed as the percentage of Bis:

C% =

Bis (P) ' 100 acrylamide (g) + Bis (9)

The gel can be bonded to the capillary wall through 3-(methacry1oxy)propyltrimethoxysilane. Acrylamide gel thus polymerized and immobilized in capillaries can be used several times for sequencing and more than ten times for analysis of doublestrand DNA in PCR products. The other alternative is to use agarose-filled capillaries. The preparation of the agarose is easy, because it readily dissolves on heating and gels on cooling. Linear polyacrylamide (non-cross-linked) cellulose derivatives and polyethylene oxide (PEO) can also be used as entangled polymers. The advantages of using such polymeric structures (as additives to the background electrolyte) are that polymerization in capillaries is unnecessary, and the capillaries, once mounted into the instrument, can be used repeatedly and dynamically replaced by changing the polymer at each analysis. More detailed information can be found in other chapters of this book and in the review by Heller [39]. 2.4.I . 2 Size separation of single-strand DNA for DNA sequencing Separation of single-strand DNA serves for DNA sequencing and revealing DNA polymorphism. As mentioned above, separation within one base difference in length is required in DNA sequencing. In order to prevent the formation of double strands in this case the DNA is separated in electrolytes containing urea. The acrylamide gel concentration varies from 3% to 6% T, with C from 3% to 5% depending on the size of DNA to be separated.

77

Strategies for Electromigration Separations R

IVI

yo 2IV

I

317

I

Fig. 2.17. Electropherogram of chain-termination sequencing reaction product (ddTTPterminated M13mp 18) (From [40] with permission).

Fig. 2.17 shows an example of separation for DNA sequencing [40]. Linear polyacrylamide [41] and PEO [42] can also be used for this purpose.

2.4.I . 3 Analysis of single-strand DNA-conformation polymorphism (SSCP) Single-strand DNA can exhibit various conformations depending on the complementary hydrogen bonding of the bases in the DNA. Its conformation is known to differ widely according to differences in the base composition, and single-strand DNA-conformation polymorphism (SSCP) of DNAs of the same size can be reversed using non-denaturing gel electrophoresis [43]. This approach can detect single-base differences and is used today as an effective method for genetic diagnosis. This method is based on the amplification of the mutation site and its neighbourhood by PCR. At this stage, wild-type DNA fragments without mutations, as well as mutanttype DNA fragments with a single base mutation, are formed. Next, the wild-typeand mutant-type fragments are dissociated into single strands by heating, and are separated by electrophoresis according to differences in their conformations (which References pp. 91-93

Chapter 2

78

(1)

PCR

+

Wild

Mutant

+

famplification (-

x 106)

(2) Denatured

+ Single strand

(3) Gel electrophoresis

Wild

Mutant

Fig. 2.18. The principle of analysis of single strand DNA conformation polymorphism (SSCP).

are capable of reflecting even single base replacements). Consequently, electrophoresis (unlike separation for sequencing) must be done in a non-denaturating gel, i.e., without the addition of urea (Fig. 2.18) [44-511. The ability of gel electrophoresis to detect SSCP is affected by the concentration of polyacrylamide, the running temperature, the size of the DNA fragment resulting fiorn PCR, and the base-composition of

79

Strategies for Electromigration Separations

1

PCR

SSCP by Laser-Induced Fluorescence Capillary Electrophoresis

Fig. 2.19. PCR o f K ras mutations and electropherogram of SSCP. SSCP (71 base) obtained from a mixture of seven kinds of k ras codon 12 normal (Glyj and muted (Ala, Arg, Cys, Ser, Val, Asp)n by CE with laser-induced fluorescence detection. Each peak show sense sscp obtained from seven kinds of k ras codon 12. Conditions: gel. 8%T, capillary, 100 pm 1.D.; 50 cm in total length and effective length -30 cm; runnning buffer, 100 mM Tris-borate (pH 8.3); temperature, 25°C; field. 200Vicm; laser-induced fluorescence detection, Ex, 590 nm (He-Ne), Em, 61 5 nm.

the DNA. A higher gel concentration and a lower running temperature are considered to be desirable for separation and stabilization of the conformation. In contrast to Referetice~pi?. 91 -93

80

Chapter 2

traditional (flat bed) electrophoresis, in CE the heat dissipiation is more favourable and the separation can be done at relatively high temperatures. Fig. 2.19 shows the SSCP of seven point mutations [Val (-GTT-), Arg (-CGT-), Ala (-GCT-), Cys (-TGT), Asp (-GAT-), Ser (-AGT-), Gly (-GGT-)I in codon 12 of K-ras gene, with detection by fluorescence. Fluorescent labelling was done by labelling 71 bp fragments during PCR amplification using a sense-primer of PCR labelled with Texas Red. This method is about 1,000 times more sensitive than that with LW absorption detection (at 260 nm) and permits the analysis of 1 pg samples of human DNA. 2.4.1.4 Separation of double strand DNASfagments

It has been mentioned already that acrylamide and agarose are used as gels for filling in the capillaries, and linear polyacrylamide and cellulose derivatives are used as polymeric modifiers of the background electrolyte. The size of separated DNA fragments depends on the concentrations of the polymer, as shown in Table 2.1 [52]. As would be expected, small DNA can be separated at higher polymer concentrations, while large DNA needs gels with lower concentrations. The gels used are more stable when Bis is added to polymerize the acrylamide. Fig. 2.20 shows examples of separation using polyacrylamide gels 3% T-0.5% C. DNA fragments with a difference of 10 bp (271 bp and 281 bp) could not be separated by this method. The use of an intercalation reagent for double-strand DNA is known to increase the resolution of its separation. This effect is considered to be a result of structural changes in the DNA caused by its unfolding by binding with the intercalation reagent, and a reduction in

TABLE 2.1 PROPOSED CONCENTRATON RANGE OF THE POLYMER-TYPE SEPARATON MATRIX FOR THE SEPARATIN OF DNA FRAGMENTS (From [52],with permission). Effective DNA size range of separation (bp)

Concentration of polymer (YO, w/v) LPAA HEC, HPMC, MC

PEG, PEO

1 - 100

8 -12

1.O - 3.0

6.0 - 8.0

100 - 300

7.0 - 8.0

0.7 - 1.0

3.0 - 6.0

300 - 1000

5.0 - 7.0

0.5 - 0.7

2.0 - 3.0

1000-10000

3.0 - 5.0

0.3 - 0.5

0.5 - 2.0

10000-30000

2.0 - 3.0

0.01 - 0.3

-

Strategies for Electromigration Separations

81

1353

281

5

10

15

20

(mln)

Fig. 2.20. Electropherograms of 4x174 RF DNA/Hae 111 digest obtained using polyacrylamide. Conditions: gel, 3%T-0.5%C; capillary, 100 pm I.D.; 50 cm in total length and effective length 30 cm; runnning buffer, 100 mM Tris-borate (pH 8.3); field, 200Vicm. UV detection, 260 nm. the effective electric potential of the DNA itself [53].Moreover, as these intercalation reagents emit fluorescence by binding with DNA, the sensitivity of detection can be increased by laser excitation. Fig. 2.21 shows an electropherogram of a 4x174 RF DNA-Hinc I1 digest obtained by using thiazole orange as an interaction reagent. Base-line separation of 291 bp and 297 bp can be achieved by the addition of thiazole orange; the sensitivity is increased about 100 times compared to that given by UV detection at 260 nm. Other intercalation reagents such as TOTO-1 and YOY03 can be used as well. The separation of large DNA fragments ( 0 . 1 4 Mbp) can be effected by pulse-field electrophoresis with the reversal of the electric field. Fig. 2.22 shows an example of a separation of large DNA (0.21-1.6 Mbp) by this technique. However, several methods other than the pulse-field method for the separation of large DNA fragments (< 25 Kbp), based on the adjustment of the polymer concentration, have been reported. It is easy to understand that very low concentrations are used in this case. Table 2.2 summarizes reports concerning these techniques [ 54-6 11. Referencespp. 91-93

Chapter 2

82

I1.P.U

400

i

1057

770

300

345 495 34 I 335 392 297 ,

200

I

1

1

100

0

, 5

I0

1s

Time (min.)

Fig. 2.21. Electropherogram of 4x174 RF DNAMinc I1 digest obtained by using thiazole orange as an intercalation reagent. Comditions: gel, 3%T-O.S%C; capillary, 100 pm I.D.; 37 cm in total length and 30 cm effective length 30 cm; runnning buffer, 100 mM Tris-borate @H 8.3) cotaining 0.1 mgiml Thiazole Orange; Field, 200Vicm. Fluorescence detection, Ex at 488 nm, Em 530 nm.

2.4.2 Separation of proteins By convention, peptidic structures possessing molecular masses of 10,000 and above are categorized as proteins. As with traditional electromigration procedures there are three major approaches for the separation of proteins by CE - namely capillary zone electrophoresis (based on both the electric charge and molecular mass), capillary isoelectric focusing (based on the electric charge of the proteins alone), and separation in the presence of sodium dodecyl sulfate (SDSkgel electrophoresis (based on the molecular mass differences). In this section we deal with the general strategies used for protein separations: a more thorough insight is presented in Chapter 13. The main problem in capillary-electrophoretic separations is that the analytes stick to the capillary wall. Operations at low pH or high pH, resulting in reduced dissociation of the silanol groups on the capillary surface or of the free amino groups of the separated proteins, are used to prevent this phenomenon to variable extents.

Strategies for Electromigration Separations

83

1

10

11

12

14

13

nme(rnh)

Fig. 2.22. Electropherogram of megabase 1 DNA standard in OSxTBE buffer containing 0.00375% hydroxyethyl cellulose/0.002% polyethylene oxide, pulse-field condition: 100 V/cm + 14 Hz square wave, 250% modulation. Peak identification: ( I ) 0.21, (2) 0.28, (3) 0.35, (4) 0.44, (5) 0.55, (6) 0.60, (7) 0.68, (8) 0.75, (9) 0.79, (10) 0.83, (11) 0.94, (12) 0.97, (13) 1.10, 1.12, (14) 1.6 Mbp(From [56] with permission).

TABLE 2.2 CONCENTRATION OF THE POLYMER-TYPE SEPARATON MATRIX AND METHODS USED FOR SEPARATION OF LARGE DNA FRAGMENTS

Gel, polymer, Yn

Method

DNA

Pulsed-field

8.3kbp - lMbp 0.4 - 0.6% LPAA

54

75bp

0.01

55

0.21

0.00375% Hydroxyethyl cellulose/0.002% polyethylene oxide

56

1% Hydroxypropyl cellulose

57

0.0033% Hydroxyethyl cellulose/ 0.004% polyethylene oxide

58

<0.002% I-iydroxyethyl cellulose

59

electophoresis

1

- 23kbp - 1.6Mbp

- 50kbp

- 1.9Mbp Ultra-dilute polymer solution

- 23.1 kbp 2 - 23.1 kbp 72bp - 23.1 kbp 2

~-

References pp. 91-93

Ref.

- 0.4% Methyl cellulose

0.00125%, 0.25%, 0.15% Hydroxyethyl cellulose 60

-

0.00125% 0.4% LPAA, Hydroxyethyl cellulose, 61 hydroxypropyl cellulose ~

..

~~

Chapter 2

84 2.4.2.I Separation by capillary zone electrophoresis

This type of separation is accomplished mostly in the positive mode (with a high voltage at the anode). Both alkaline and acidic buffers can be used but the migration times with acidic buffers are always long (up to 1 h). A typicaI example is shown in Fig. 2.23 [62] where the separation of a peptidoglycan-associatedprotein is demonstrated. There are numerous other examples in the literature, such as separations of basic proteins [63], human serum proteins [64], monoclonal antibodies [65], and the recombinant proteins, plasminogen activator and human growth hormone [66,67] etc.

I

I

K

Sam

swim

V IS

IV

L

M

I

0

TIME (MIN)

Fig. 2.23. Electropherogram of the separation by capillary-zone electrophoresis of peptidoglycan-associated proteins. Conditions: fused-silica capillary, 50 pm I.D.; 65 cm in total length and 45 cm effective length; buffer, 50 mM disodium tetraborate, pH 9.1; detection at 205 nm; separation voltage 20kV. IS=internal standard; I-VI= protein numbers (From [62] with permission).

Strategies for Electromigration Separations

85

2.4.2.2 Capillary isoelectric focusing Capillary isoelectric focusing (CIEF) separates proteins according to their isoelectric points (pl). Proteins with a difference of 0.005 p l can be separated by CIEF. CIEF differs from traditional gel-based IEF separations in that the focused protein zones must be mobilized to pass the detector in order to be detected. There is a two-step method which focuses and mobilizes in separate steps, and a one-step method which focuses and mobilizes simultaneously. In the one-step method, focusing takes place between the detector and the anode, and the separated proteins are mobilized to the detector by the electro-osmotic flow. In the two-step method, mobilization is perfomed by the electrophoretic or pressure-driven method, after focusing. CIEF is useful for separating hemoglobin variants [68], immunoglobulin [69], human plasma or serum proteins [70], and can also be used for determining the pZ of a protein. The technique offers several advantages over traditional gel-based IEF, including eliminating laborious staining and destaining, and giving rapid and quantitative analysis.

E

Migration time (min)

Fig. 2.24. Electropherogram of hemoglobins A, F, S and E by capillary isoelectric focusing (From [68] with permission). Referencespp. 91-93

86

Chapter 2

However, proteins which tend to denature and to reduce in solubility when they reach their p l value cannot be analysed by this method: this is a common disadvantage in CIEF and IEF. Fig. 2.24 shows an example of the separation of haemoglobins by CIEF [68]. An excellent review on protein separation by CIEF is now available [71]. 2.4.2.3 Capillary sodium dodecyl sulfate-gel (SDS-gel) electrophoresis

In SDS-gel electrophoresis, the size-separation of proteins is done using a molecular sieving matrix after the electrical properties of the proteins have been equalized. (Nearly all proteins bind the same mass proportion of SDS which gives the arising aggregate a negative change, while the charge proper to the protein molecule can be neglected.) Cross-linked polyacrylamide, as well as linear polyacrylamide (as a dynamically introduced sieving matrix [72,73]) and, more recently, PEO [39,74,75], pullulan [76] and dextran solutions [77,78] (used also for dynamic molecular sieving), are widely used for the separation of proteins. Dynamic introduction of the sieving matrix is a technique which is being used more and more frequently as it offers a change of the sieving matrix with every single analysis, thus preventing the clogging problems encountered with gel-filled capillaries. A typical example of a separation is shown in Fig. 2.25. A recent review by Guttman provides a source of detailed information [79].

-0.0005 6.00

a20

8.00

10.00

12.00

16.00

16.00

Migration Time (ruin)

Fig. 2.25. Electropherogram of myoglobin molecular mass markers by capillary SDS electrophoresis.Conditions: buffer, 0.4 M Tris-borate, 0.1% SDS, 10% glycerol, 12% dextran (Mr 2,000,000), pH 8.3; voltage, 12 kV; detection, 220 nm. Peak identification: (1) Mr 2512, (2) Mr 6214, (3) Mr 8159, (4) Mr 10701, ( 5 ) Mr 14404, (6) Mr 16949 (From [77] with permission).

Strategies for Electromigration Separations

87

2.5 BINDING OF LOW MOLECULAR MASS SOLUTES TO PROTEINS Capillary columns without packing materials are suitable for the separation of mixtures of small and large molecules, for example when a mixture of albumin and drugs is separated [80]. This technique can be used for the measurement of drug-protein binding constants, especially drug-human-serum albumin binding because albumin is the major plasma and tissue protein to usually be responsible for the nonspecific binding of most drugs. Although liquid chromatographic separations are still widely used to reveal binding of low-molecular-mass solutes to proteins (mainly serum albumin, see Chapter l), capillary electrophoresis widens the possibilities in this respect. In liquid chromatography and capillary electrophoresis the HummelDryer method, the vacant peak method, and the frontal analysis method can be applied. These are discussed later in this Section. In liquid chromatography the differences in elution of the free- and bound-drugs are exploited by using the differences in molecular mass and in the molecular mass and charge of the solutes involved. Because the protein molecule is much larger, and carries a much larger charge, than any drug, there is a justified assumption that neither the molecular mass nor the charge of the protein will be changed upon binding to a drug or any other small molecule. Consequently, the protein itself and its complex with the drug will exhibit equal electrophoretic mobilities. The only assumption to be fulfilled is that there is a different electrophoretic mobility for the protein and the low molecular ligand or drug. In Hummel-Dryer’s method the capillary is filled with a background electrolyte containing the drug, which results in an increased detection background. The sample containing the drug and protein dissolved in background electrolyte is introduced. The concentration of the drug in this sample must be the same as in the background electrolyte that was used to fill the capillary. A part of the drug in the sample is, however, present as the drug-protein complex. Upon applying the voltage the drugprotein complex migrates towards the cathode - if the proteins are negatively charged - while the drug itself migrates to the anode - if the drug is positively charged. Both the drug-protein complex and the free drug migrate to the end of the capillary as a result of electro-osmotic flow. This results in a local reduction in the drug concentration, which yields a negative peak on the high background. The position of the peak corresponds to the drug’s mobility, while its area is related to the amount of drug complexed with the protein. The protein-drug complex is in equilibrium with the free drug present in the background electrolyte. Therefore, the drug-protein complex yields a positive peak on the electropherogram. If the absorbance of the protein involved were zero and if the molar absorption coefficients of the

References pp. 91-93

88

Chapter 2

free drug and the drug-protein complex were identical, then the areas of the positive and negative peak would also be identical. In the vacant peak method, the capillary is filled with the background electrolyte containing both the binding protein and the drug. Background electrolyte devoid of these components is used as the sample. Assuming that the mobility of the free protein, and of its complex with the drug, are higher than the mobility of the drug in its free form (an assumption that is nearly always met in practice), both the protein and its complex will migrate towards the anode faster than does the free drug (after the voltage is applied). In the front of the injected sample (free buffer) the drug migrates more slowly than the protein, and is retarded. At the end of the zone the protein migrates more rapidly than the injected sample of the free background electrolyte. Both these processes proceed until the zone of the drug and the zone of the protein merge and, because the process is reversible, this re-adsorption proceeds until the drug concentration reaches its original level. From this moment on, two negative peaks can be observed on the electropherogram. The first corresponds to the bound drug, the other corresponds to the drug-free zone. In the frontal analysis method the capillary is filled with the background electrolyte only and then a broad zone containing buffer, protein, and the drug investigated is introduced. Owing to the differences in electrophoretic mobility the free drug starts to escape from the sample zone: at the end of the sample zone a region containing free drug is created. At the beginning of the sample zone a second plateau appears - this contains free protein. The overall result is a three-zone profile in which the free-protein zone comes first, then the zone of protein-bound drug, followed by the area which contains free drug only. In practice, as a result of the wavelengths used (in most cases the detection is by UV absorbance) the zone of free protein cannot be distinguished on the electropherogram Practical applications can be exemplified by the following. Honda et al. determined the association constant of the monovalent-mode protein-sugar interaction by capillary electrophoresis, using a carrier solution containing sugar. The association constants of Ricinus communis agglutinin, peanut agglutinin, and soy bean agglutinin to lactobiotic acid were 3.3.103, 9.1.102 and 1.1-102mol-’, respectively [81]. Kraak et al. measured the warfarin-bovine serum albumin association constant using the Hummel-Dreyer method, the vacancy peak method, and frontal analysis, and the values obtained varied from 0.70 to 1.61.10’ [82]. Shibukawa et al. used frontal analysis to measure the binding parameter of verapamil and a1-acid glycoprotein, and observed the value 1.13.106 [83]. Kostiainen et al. analysed naproxen-lysozyme conjugate by capillary electrophoresis-ion-spray mass spectrometry, and found that lysozyme conjugated up to three naproxen molecules [84]. F r ~ k i z ret al. studied and determined the molar cou-

Strategies for Electromigration Separations

89

pling ratios of hapten-Kunitz soybean trypsin-inhibitor conjugates, using p-nitrophenyl-a-D-galactoside as a model compound of hapten, using micellar electrokinetic capillary chromatography with sodium cholate as the micellar phase [8S]. Capillary electrophoresis is a useful tool for the measurement of drug-protein interactions, since it requires only a small amount of test solutions. However, no single technique can handle all experimental requirements, and a suitable method must be selected for each drug-and-protein pair to be examined; this is determined mainly by their solubilities. Further development of such techniques is required because of the poor solubility of drugs in water and the poor detection-sensitivity of the presently available techniques. In addition, advances in the quantitative analysis of proteins by capillary electrophoresis are necessary for the elucidation of protein-drug binding. Proteins, including human serum albumin, are usually adsorbed onto the walls of the fused silica tubes. The use of coated fused silica capillary tubes is feasible [86]. However, such coated tubes are not suitable for the measurement of the migration time of non-ionic compounds, owing to their lack of osmotic flow. Therefore, the further development of electrophoresis for proteins is required. Highly sensitive detectors are also required, owing to the poor solubility of drugs. The measurement of protein-drug binding using liquid chromatography is described in Chapter 1 .

2.6 FUTURE OF CE AS A METHOD FOR THE SEPARATION OF BIOLOGICAL MATERIALS’ CONSTITUENTS. CONCLUSIONS Capillary electrophoresis allows high-speed, high-resolution, and high-sensitivity analysis of DNA and proteins. Also, this category of methods has excellent quantitative precision and reproducibility. Furthennore, as the sample can be introduced automatically in the electrokinetic mode or by a pressure gradient, the method is suitable as a routine procedure, especially in clinical fields. A considerable disadvantage of CE is that each analysis is run with a single capillary so that only one sample can be separated in every run. Recently, a capillary array method, in which several hundred capillaries were used simultaneously, was reported [87,88]. Another technique, in which capillaries are placed on microchips, can be considered as an alternative solution [89,90]. The apparatus for multiple sheath-flow capillary-array electrophoresis is shown in Fig. 2.26. Standard gel electrophoretic methods have not been discussed in detail as they represent a well established, well known category of methods. For their applications the potential reader is deferred to the specific chapters of this book.

References pp. 91-93

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gel-filled capillary

computer Fig. 2.26. Schematic view of the multiple sheath-flow capillary-array electrophoresis system.

2.7 ABBREVIATIONS A a at b Bis C

CIEF E HEC HPMC IEF k K; P K ~ KK L LPAA log P mt mt0 mt, PCR PEG

current slope related to the buffer or sodium chloride concentration analysis time ratio of the electroosmotic flow rate divided by the current N,N'-methy lenebisacrylamide slope related to osmotic flow rate capillary isoelectric focusing electric field strength hydroxyethyl cellulose hydroxypropylmethyl cellulose isoelectric focusing retention factor dissociation constant a magnitude derived from pKa (eq. 2.12) column length linear polyacrylamide octanol-water partition coefficient migration time migration time of neutral compound migration time of ionized solute polymerase chain reaction polyethylene glycol

Strategies for Electromigration Separations PEO PI R R* Rmt SDS SSCP V

vwv W

Pos Pep

91

polyethylene oxide isoelectric point resistence selectivity relative migration time sodium dodecyl sulfate single-strand DNA-conformation polymorphism applied voltage van der Waals volume a magnitude related to pK, (eq. 2.13) electroosmotic flow-rate electrophoretic flow-rate

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