Capillary Zone Electrophoresis - American Chemical Society

Department of Chemistry, University of North Carolina at Chapel Hill,. Chapel Hill, NC 27514 ... The various techniques of modern electrophoresis are ...
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Chapter 13

Capillary Zone Electrophoresis

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0335.ch013

James W. Jorgenson Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514

Capillary zone electrophoresis is a technique which permits rapid and efficient separations of charged substances in an instrumental format. Buffer-filled capillaries with typical dimensions of 50 microns i.d. and 100 cm length are used as the separation chambers. Applied potentials as high as 30 KVolts are used to drive the electrophoretic process. Such high potentials promote rapid migration of zones, while minimizing zone spreading. A simple theory of zone spreading and resolution is presented. A physical description of the system and some of its operating characteristics is provided. Separating performance of the system is described and example separations shown. Limitations of the system, particularly with regard to the separation of proteins, are discussed and future areas for research are suggested. Background The various techniques of modern electrophoresis are a powerful and versatile approach to separation and analysis of substances, e s p e c i a l l y proteins and polynucleotides. Separation modes such as i s o e l e c t r i c focusing and SDS-gel sieving electrophoresis are quite e f f e c t i v e i n t h e i r own r i g h t , and when combined i n a twodimensional format, form a technique of u n r i v a l l e d resolving power. But modern electrophoresis, as practiced, i s a rather labor intensive approach to analysis. Making gels, sample a p p l i c a t i o n , staining and destaining gels are time-consuming tasks. Furthermore, the techniques for band detection, including staining and use of a densitometer for quantitation, are characterized by l i m i t e d dynamic range and l i n e a r i t y . Indeed much of the interest i n applying HPLC to separation and analysis of biopolymers stems from the fact that HPLC i s a highly instrumental technique with autosamplers and on-line detectors connected to computers for data a c q u i s i t i o n and analysis. 0097-6156/87/0335-0182$06.00/0 © 1987 American Chemical Society

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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183

A f u l l y i n s t r u m e n t a l v e r s i o n o f e l e c t r o p h o r e s i s would seem t o be a d e s i r a b l e g o a l and s e v e r a l systems have been d e s c r i b e d . H j e r t e n ( _ l ) employed zone e l e c t r o p h o r e s i s in a g e l - f r e e tube o f t h r e e mm i n n e r d i a m e t e r . In t h i s system thermal c o n v e c t i o n was m i n i m i z e d by r o t a t i o n o f the tube around i t s l o n g i t u d i n a l a x i s . Zone d e t e c t i o n was a c c o m p l i s h e d w i t h an UV a b s o r p t i o n d e t e c t o r which scanned the l e n g t h o f the tube. KolinC2) described several f r e e zone e l e c t r o p h o r e s i s systems which used flow i n s e r p e n t i n e and h e l i c a l paths t o combat t h e r m a l l y d r i v e n c o n v e c t i o n . CatsimpoolasO) d e s c r i b e d an i n s t r u m e n t a l system w i t h scanning d e t e c t i o n f o r f o l l o w i n g the c o u r s e o f i s o e l e c t r i c f o c u s s i n g i n g e l f i l l e d tubes. These t e c h n i q u e s have not come i n t o widespread use presumably due t o t h e i r c o m p l e x i t y . An a l t e r n a t i v e approach t o i n s t r u m e n t a l e l e c t r o p h o r e s i s i s t o c a r r y out the s e p a r a t i o n i n a c a p i l l a r y tube o f s u b - m i l l i m e t e r d i a m e t e r . M i k k e r s e t a l . ( 4 ) demonstrated f r e e zone e l e c t r o p h o r e s i s i n a c a p i l l a r y tube on equipment d e s i g n e d f o r s e p a r a t i o n s by i s o t a c h o p h o r e s i s . Thormann e t a l . c a r r i e d out e l e c t r o p h o r e s i s i n g e l - f r e e chambers o f r e c t a n g u l a r c r o s s s e c t i o n (28,29). Tubes o f s m a l l d i a m e t e r o f f e r many advantages as a format i n which t o do e l e c t r o p h o r e s i s . J o u l e heat i s g e n e r a t e d u n i f o r m l y throughout the tube c r o s s s e c t i o n but i s d i s s i p a t e d through the tube w a l l s . T h i s r e s u l t s i n a temperature g r a d i e n t a c r o s s the tube w i t h the f l u i d i n the c e n t e r b e i n g warmer than t h e f l u i d at the w a l l . T h i s temperature g r a d i e n t r e s u l t s i n a d e n s i t y g r a d i e n t ( l e a d i n g t o c o n v e c t i v e flow) as w e l l as v i s c o s i t y and pH g r a d i e n t s a c r o s s the tube. C o n v e c t i v e flow may l e a d t o s p r e a d i n g o f e l e c t r o p h o r e t i c zones, w h i l e v i s c o s i t y and pH g r a d i e n t s may a l s o a c t as a cause o f zone d i s p e r s i o n . The use o f s m a l l e r d i a m e t e r tubes reduces the magnitude o f temperature d i f f e r e n c e s w i t h i n the tube. The temperature d i f f e r e n c e between the f l u i d i n the c e n t e r and at the w a l l i s r o u g h l y p r o p o r t i o n a l t o the square o f the tube's i . d . ( 5 , 6 , 7 ) D e c r e a s i n g tube diameter has o t h e r b e n e f i c i a l e f f e c t s as c o n c e r n s zone s p r e a d i n g . C o n v e c t i v e flow i s damped by the drag o f the s t a t i o n a r y tube w a l l a c t i n g on the v i s c o u s f l u i d . This e f f e c t , c a l l e d the " w a l l e f f e c t " by M i k k e r s e t a l . ( 4 ) , becomes i n c r e a s i n g l y e f f e c t i v e i n p r e v e n t i n g c o n v e c t i v e f l o w as the tube diameter i s decreased. A l s o , r a d i a l pH and v i s c o s i t y g r a d i e n t s are o n l y u n d e s i r a b l e t o the extent t h a t i n d i v i d u a l a n a l y t e m o l e c u l e s spend an i n o r d i n a t e amount o f time i n e i t h e r the tube c e n t e r o r near the w a l l ( 8 ) . D i f f u s i o n o f a n a l y t e m o l e c u l e s tends to randomize t h e i r r a d i a l occupancy a l l o w i n g a m o l e c u l e t o "sample" a l l p o r t i o n s o f the tube c r o s s s e c t i o n . T h i s d i f f u s i o n a l a v e r a g i n g l e a d s t o narrower zones. The e f f e c t i v e n e s s o f d i f f u s i o n a l a v e r a g i n g i s i n c r e a s e d as the tube diameter i s reduced. Thus use o f tubes o f decreased diameter l e a d s t o s m a l l e r temperature d i f f e r e n c e s and f u r t h e r m o r e a c t s t o m i n i m i z e the zone s p r e a d i n g e f f e c t s o f any r e s i d u a l temperature g r a d i e n t s . These compounded e f f e c t s argue s t r o n g l y f o r the use o f s m a l l d i a m e t e r capillaries. One o b v i o u s d i f f i c u l t y i n t h i s c o n c l u s i o n i s t h a t i t n e c e s s i t a t e s s m a l l sample volumes, and thus p l a c e s g r e a t demands on d e t e c t i o n s e n s i t i v i t y .

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Another important group of mechanisms of zone spreading may be collected under the heading of sample overloading. The sample, i f s u f f i c i e n t l y concentrated, may perturb the physical and chemical properties of the electrophoresis medium.(4,8,9) For instance, polymeric analytes may increase the v i s c o s i t y of the medium, and thus decrease electrophoretic mobility within the zone. Most analytes contain acidic and/or basic groups and thus can shift the pH within the zone, again affecting mobility. And probably most important, the analytes are themselves ions, and may a l t e r the e l e c t r i c a l conductivity of the medium, leading to d i s t o r t i o n of an otherwise homogenous e l e c t r i c f i e l d and resulting in zone spreading. A l l of these d i f f i c u l t i e s argue for a very low ratio of analyte concentration to concentration of supporting e l e c t r o l y t e (buffer) in order to minimize these sample overloading e f f e c t s . Again the obvious d i f f i c u l t y with this conclusion i s that i t w i l l place great demands on detection s e n s i t i v i t y , since sample concentration must be kept low. In the ultimate l i m i t i n g case, i f a l l other mechanisms of zone spreading can be rendered i n s i g n i f i c a n t , zone broadening in zone electrophoresis w i l l be dominated by a seemingly unpreventable mechanism, longitudinal d i f f u s i o n . As d i f f u s i o n in l i q u i d s is rather slow, this may result in rather narrow zones. It is possible to describe the separating power of a zone electrophoresis system in terms of theoretical plates in analogy with chromatography(10). Since an instrumental zone electrophoresis system w i l l produce data in the form of an electropherogram, closely analogous to a chromatogram, the use of theoretical plates to describe performance has a similar merit (and limitations) in electrophoresis as i t does in chromatography. Jorgenson and Lukacs(8) predicted that i f longitudinal d i f f u s i o n was the only s i g n i f i c a n t source of zone broadening, the number of theroretical plates, N, is given by: N =

\H

(1)

2D where u and D are the analyte's electrophoretic mobility and d i f f u s i o n c o e f f i c i e n t , and V i s the voltage applied to the system to drive the electrophoretic separation. Since mobility and d i f f u s i o n c o e f f i c i e n t are not easily altered in a way to increase N, high applied voltages are the most direct way to high separation e f f i c i e n c i e s . It must be remembered that this prediction is based s t r i c t l y on the assumption that longitudinal d i f f u s i o n is the dominant mechanism of zone broadening, a condition which may be d i f f i c u l t to r e a l i z e in practice. It must also be borne in mind that an i n f i n i t e l y narrow band of injected sample is assumed. It is interesting to note that tube length does not enter into this equation. Thus in p r i n c i p l e , very short tubes may be used to promote rapid analyses, while not jepardizing separation e f f i c i e n c y . However, in practice this is not e n t i r e l y true, as w i l l be shown. Description of System and i t s Basic Operating Characteristcs A schematic of the c a p i l l a r y zone electrophoresis (CZE)

system i s

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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shown i n f i g u r e 1. A c a p i l l a r y tube i s f i l l e d w i t h b u f f e r and suspended between two r e s e r v o i r s f i l l e d w i t h the same b u f f e r . The end at which h i g h v o l t a g e i s a p p l i e d i s surrounded by a p l e x i g l a s s s a f e t y i n t e r l o c k box. T h i s box must be opened to g a i n a c c e s s to the e l e c t r o d e and r e s e r v o i r , and opening the box a u t o m a t i c a l l y shuts o f f the h i g h v o l t a g e power supply w h i l e simultaneously s h o r t i n g i t s output to ground through the h i g h v o l t a g e r e l a y . T h i s r e l a y i s b u i l t i n - h o u s e , u s i n g a s o l e n o i d connected to a s w i t c h mechanism w i t h an 8 cm gap when open. A t t e n t i o n to s a f e t y i s e x t r e m e l y i m p o r t a n t , as a power supply which can d e l i v e r as much as 30 K V o l t s i s used i n t h i s system. The o t h e r end of the c a p i l l a r y i s d i p p e d i n t o a b u f f e r r e s e r v o i r i n which the e l e c t r o d e i s connected through an ammeter to ground. E l e c t r i c a l currents in CZE depend on c a p i l l a r y d i m e n s i o n s , b u f f e r c o n d u c t i v i t y and a p p l i e d p o t e n t i a l , but t y p i c a l o p e r a t i n g c u r r e n t s are between 10 and 100 microamps. Sample i s most e a s i l y i n t r o d u c e d i n t o the c a p i l l a r y u s i n g an electromigration technique(8). The b u f f e r r e s e r v o i r at the h i g h v o l t a g e end i s r e p l a c e d w i t h a r e s e r v o i r c o n t a i n i n g sample. High v o l t a g e i s a p p l i e d f o r a s p e c i f i c amount of time ( u s u a l l y a few seconds) and then turned o f f . T h i s b r i e f a p p l i c a t i o n of v o l t a g e m i g r a t e s a narrow band of sample i n t o the c a p i l l a r y . Now the sample r e s e r v o i r i s removed, and the b u f f e r r e s e r v o i r i s r e p l a c e d . H i g h v o l t a g e i s a g a i n a p p l i e d , and the e l e c t r o p h o r e t i c run b e g i n s . T h i s sample i n t r o d u c t i o n t e c h n i q u e i s simple and e f f e c t i v e , i n t r o d u c i n g sample w i t h minimal band b r o a d e n i n g . However i t does a c t to d i s c r i m i n a t e a g a i n s t the v a r i o u s components of the sample, based upon t h e i r m o b i l i t i e s . D e t e c t i o n i s u s u a l l y a c c o m p l i s h e d by "on-column" f l u o r e s c e n c e ( l l ) and UV a b s o r p t i o n ( 1 2 ) . The e l e c t r i c field, c o n d u c t i v i t y , and thermal d e t e c t e r s which are e f f e c t i v e i n c a p i l l a r y i s o t a c h o p h o r e s i s are of i n s u f f i c i e n t s e n s i t i v i t y to be g e n e r a l l y u s e f u l i n CZE(j 3). Where f e a s i b l e , f l u o r e s c e n c e d e t e c t i o n i s a t t r a c t i v e due to i t s g r e a t s e n s i t i v i t y . However, many samples, such as p r o t e i n s , do not appear to be good candidates for fluorescence detection. The i n t r i n s i c fluorescence of p r o t e i n s i s q u i t e v a r i a b l e from p r o t e i n to p r o t e i n , and i s u s u a l l y r a t h e r weak. F l u o r e s c e n c e l a b e l l i n g of p r o t e i n s i s a l s o difficult, as t h e r e i s a tendency to produce a complex assortment of m u l t i p l e ( p a r t i a l l y l a b e l l e d ) p r o d u c t s . P r o t e i n s have been d e t e c t e d most e f f e c t i v e l y by UV a b s o r p t i o n d e t e c t i o n . In both f l u o r e s c e n c e and UV a b s o r p t i o n , d e t e c t i o n i s c a r r i e d out "oncolumn", s h i n i n g the i n c i d e n t l i g h t beam onto the c a p i l l a r y and measuring e i t h e r f l u o r e s c e n c e or t r a n s m i t t e d l i g h t . In t h i s r e g a r d , c a p i l l a r i e s f a b r i c a t e d from fused s i l i c a are v a s t l y s u p e r i o r to those from g l a s s , due to t h e i r e x c e l l e n t UV t r a n s p a r e n c y and e x t r e m l y low background l u m i n e s c e n c e . L

F i g u r e 2 shows the e f f e c t of tube i . d . on the measured s e p a r a t i o n e f f i c i e n c y of d a n s y l - l a b e l l e d i s o l e u c i n e ( 1 4 ) . The tubes were a l l 100 cm long Pyrex type 7740 b o r o s i l i c a t e g l a s s c a p i l l a r i e s , f i l l e d w i t h pH 6.86, 0.05 M phosphate b u f f e r , and o p e r a t e d at a p o t e n t i a l of 15 K V o l t s . Only the tube d i a m e t e r s were v a r i e d . C l e a r l y , below 80 m i c r o n s , a performance of a p p r o x i m a t e l y 250,000 t h e o r e t i c a l p l a t e s i s o b t a i n e d , and no

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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s i g n i f i c a n t improvement i s seen at smaller diameters. Tube diameters larger than 80 microns result in a precipitous decrease in plates. This i s in general agreement with the zone broadening considerations described e a r l i e r . Figure 3 shows the effect of tube length on the separation e f f i c i e n c y of the same analyte(14). A l l tubes were 75 micron i . d . , f i l l e d with the same phosphate buffer as before, and operated at an applied p o t e n t i a l of 15 KVolts. Tubes of approximately 100 cm and longer y i e l d consistently high plate numbers approaching 230,000. Tubes longer than 100 cm do not improve separation e f f i c i e n c y , but do result i n s i g n i f i c a n t l y longer analyses. Tubes shorter than 100 cm show a dramatic loss in plates. This i s presumably due to "thermal overloading" of the system. Shorter c a p i l l a r i e s offer lower e l e c t r i c a l resistance, and thus at constant voltage, carry higher currents. Power dissipated (the product of voltage and current) by the c a p i l l a r y increases while a v a i l a b l e surface area over which to d i s s i p a t e t h i s heat decreases. Below a length of 100 cm, even a c a p i l l a r y of 75 microns i . d . cannot d i s s i p a t e heat e f f i c i e n t l y enough to prevent s i g n i f i c a n t temperature gradients and t h e i r attendant zone broadening phenomena. Figure 4 shows the effect of applied p o t e n t i a l on the separation e f f i c i e n c y of fluorescamine-labelled hexylamine(8). The analyte was run i n a pH 6.86, 0.05 M phosphate buffer. At the lower applied p o t e n t i a l s , general agreement with equation 1 i s seen, with plates being proportional to applied p o t e n t i a l . Beginning at approximately 20 KVolts, t h i s r e l a t i o n s h i p begins to f a i l . This i s again l i k e l y to be the result of thermal overloading. Figure 5 shows the r e s u l t s of an i n d i r e c t measurement of the average temperature of the buffer inside of three d i f f e r e n t c a p i l l a r i e s as a function of applied potential(15). A l l three c a p i l l a r i e s were 75 inn i . d . x 100 cm long. The buffer f i l l i n g each c a p i l l a r y was again a pH 6.86, 0.05 M phosphate buffer. Temperature was measured by f i r s t measuring the conductivity of t h i s buffer as a function of temperature. The conductance of the buffer f i l l e d c a p i l l a r y was then measured as a function of applied p o t e n t i a l , and from t h i s the temperature was i n f e r r e d . With a l l three c a p i l l a r y materials a s i g n i f i c a n t increase i n temperature i s seen as p o t e n t i a l i s increased. These elevated temperatures are not only important from the point of view of zone broadening mechanisms, but also in regard to the s t a b i l i t y of thermally l a b i l e analytes, such as many proteins. Figure 6 shows the effect of analyte concentration on separation efficiencey(14). The analyte i s dansyl-labelled i s o l e u c i n e , run i n a pH 6.86, 0.05 M phosphate buffer. The c a p i l l a r y was 75 microns i . d . , 100 cm long, and the applied p o t e n t i a l 30 KVolts. Achieving the highest separation e f f i c i e n c i e s requires the use of low analyte concentrations, due to the e f f e c t s of sample overloading, described e a r l i e r . As analyte concentration approaches 1x10 M, there appears to be some plateau in performance being approached. This i s expected, as at some point sample overloading should become i n s i g n i f i c a n t . An a l t e r n a t i v e way to minimize sample overloading i s to use higher concentrations of buffer s a l t s . This approach has i t s l i m i t s , as increased salt concentrations lead to increased e l e c t r i c a l currents, power d i s s i p a t i o n , and thus thermal overloading.

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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interlock capillary HV

DETECTOR

power

_ solvent "reservoir

f

supply

solvent reservoir

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plexiglass box

ADC

grounding device

personal computer

F i g u r e 1. Schematic o f CZE system. Reproduced w i t h from Ref. 27. C o p y r i g h t 1986 W a l b r o e h l , Y.

I

1 40

I 80

d,jjm

I 120

I

I 40

l 80

permission

I I 120 160

L,cm

Figure 2 ( l e f t ) . S e p a r a t i o n e f f i c i e n c y as a f u n c t i o n o f tube i n n e r diameter. Reproduced w i t h p e r m i s s s i o n from Ref. 14. C o p y r i g h t 1985 J . High Res. Chromatogr. Chromatogr. Commun. Figure 3 ( r i g h t ) . S e p a r a t i o n e f f i c i e n c y as a f u n c t i o n o f tube length. Reproduced w i t h p e r m i s s i o n from Ref. 14. C o p y r i g h t 1985 J . High Res. Chromatogr. Chromatogr. Commun.

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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0

10

20

30

Applied Voltage (kv) F i g u r e 4. S e p a r a t i o n e f f i c i e n c y as a f u n c t i o n o f a p p l i e d p o t e n tial. Reproduced w i t h p e r m i s s i o n from Ref. 15. C o p y r i g h t 1981 A n a l . Chem.

0

5

10

15

20

25

30

a p p l i e d v o l t a g e (kv)

Figure 5. Mean buffer temperature as a function of applied p o t e n t i a l . A l l c a p i l l a r i e s 75 micron i . d . 0 = teflon; | = fused s i l i c a ; A pyrex. Reproduced with permission from reference 15. C o p y r i g h t 1983 Lukacs, K. D. =

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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It i s evident that the operating performance, in terms of t h e o r e t i c a l plates, involves a complex interplay of c a p i l l a r y dimensions, buffer concentration, applied p o t e n t i a l , and analyte concentration. Although general trends are c l e a r l y apparent, a successful and accurate detailed quantitative theory of zone broadening as a function of these and other parameters does not yet e x i s t , and may prove to be so complex as to be beyond r e a l i z a t i o n . None the less, e f f e c t i v e operating parameters can be found r e l a t i v e l y e a s i l y by experiment.

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System Performance An electropherogram of a group of dansyl-labelled amino acids, detected by fluorescence, i s shown in figure 7(16) . Good separation e f f i c i e n c y and a r e l a t i v e l y rapid analysis time are evident. The analytes migrate in order of charge, with the more p o s i t i v e l y charged basic amino acids being detected f i r s t , and the negatively charged acidic amino acids coming out l a s t . The d i r e c t i o n of migration of a l l analytes, regardless of sign of net charge, i s from p o s i t i v e to negative. Even negatively charged analytes migrate to the negative electrode. This i s due to a strong electroosmotic flow of buffer toward the negative electrode. This flow i s strong enough at pH 7 to sweep most ions, regardless of charge, toward the negative electrode. Fortunately electroosmotic flow e x h i b i t s a v i r t u a l l y p e r f e c t l y f l a t flow p r o f i l e and thus i s i n s i g n i f i c a n t as a cause of zone spread ing (_17). Different analyte ions are s t i l l separated i n the presence of electroosmotic flow, as t h e i r electrophoretic m o b i l i t i e s are simply superimposed upon the electroosmotic flow. Electroosmosis can actually affect resolution of zones i n CZE(jJ). I f r e s o l u t i o n , Rs, i s defined in a manner analogous to that in chromatography(10), then the resolution of two zones i n CZE i s given by: Rs = 0.177

u

u

( l~ 2)

( 2 )

i>^ Ji where , and ^2 electrophoretic m o b i l i t i e s of the two analytes, D i s the average of t h e i r d i f f u s i o n c o e f f i c i e n t , is the average of t h e i r m o b i l i t i e s , and u i s the electroosmotic flow c o e f f i c i e n t (electroosmotic flow v e l o c i t y in an e l e c t r i c f i e l d of unit strength). From this equation i t may be seen that the greatest resolution may be obtained when the electroosmotic flow i s roughly equal in magnitude but opposite i n sign ( d i r e c t i o n ) to the analyte s m o b i l i t i e s . This w i l l y i e l d higher r e s o l u t i o n , but at the expense of longer analysis times. Figure 8 shows the effect of electroosmotic flow on the resolution of some dansylated amino acids(8). In the upper electropherogram the analytes were run i n an untreated glass c a p i l l a r y which e x h i b i t s r e l a t i v e l y rapid electroosmotic flow. In the lower electropherogram, t h i s same c a p i l l a r y was s i l y l a t e d with trimethylchlorosilane (TMCS) and then f i l l e d with buffer and the same set of analytes run again. TMCS, by reacting with many of the surface s i l a n o l s , eliminates some of the surface charge and thus reduces the electroosmotic flow. The result of improved resolution and longer analysis time i s obvious. a r e

D(y+u

c

t n e

o

s

m

f

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-4 log

-3

-2

Conc(M)

F i g u r e 6. S e p a r a t i o n e f f i c i e n c y as a f u n c t i o n o f a n a l y t e concentration. Reproduced w i t h p e r m i s s i o n from Ref. 14. C o p y r i g h t 1985 J . High Res. Chromatogr. Chromatogr. Commun.

B

A

TIME (min) Figure 7. E l e c t r o p h e r o g r a m o f d a n s y l amino a c i d s . A » ^ - l a b e l l e d l y s i n e ; B = d i l a b e l l e d l y s i n e ; C = i s o l e u c i n e ; D = methionine; E = a s p a r a g i n e ; F • s e r i n e ; G = a l a n i n e ; H = g l y c i n e ; I and J = unknown i m p u r i t i e s ; K » d i l a b e l l e d c r y s t i n e ; L = g l u t a m i c a c i d ; M = aspartic acid; N « cystic acid. Reproduced w i t h p e r m i s s i o n from Ref. 16. C o p y r i g h t 1984 J . High Res. Chromatogr. Chromatogr. Commun.

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JORGENSON

Figure 8: Electropherograms of dansyl amino untreated glass c a p i l l a r y . Lower: in glass with trimethylchlorosilane. A = asparagine; C = threonine; D = methionine; E = serine; F G = glycine. Reproduced w i t h p e r m i s s i o n 1981 A n a l . Chem.

acids. Upper: i n c a p i l l a r y treated B = isoleucine; = alanine;

from Ref.

8.

Copyright

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Figure 9 shows the separation of fluorescamine labelled peptides obtained from a t r y p t i c digest of reduced and carboxymethylated egg white lysozyme(18). Very high sepration e f f i c i e n c i e s for t h i s rather complex mixture are seen. I t was hoped that t h i s kind of performance would be obtained for yet larger analytes such as proteins. With t h e i r smaller d i f f u s i o n c o e f f i c i e n t s , proteins could be expected to exhibit in excess of several m i l l i o n theoretical lates in CZE i f l o n g i t u d i n a l d i f f u s i o n dominates zone broadening. Unfortunately, proteins tend to adsorb strongly to surfaces, and any adsorption leads to dramatic zone broadening in CZE. Since proteins contain so many kinds of functional groups ( c a t i o n i c , anionic, hydrophobic, polar) and are of high molecular weight, they tend to adsorb strongly to a wide v a r i e t y of surfaces. I t i s a d i f f i c u l t challenge to create a surface to which proteins w i l l not adsorb, but r e a l i z a t i o n of t h i s goal w i l l be an important development i n CZE. One approach to prevent protein adsorption i s to modify the surface of the c a p i l l a r y with a silane. A bonded d i o l silane ("glycophase"), based on a procedure of Chang et al.(19) was t r i e d . Figure 10 shows a t y p i c a l electropherogram of proteins from such a treated c a p i l l a r y . Although the separation i s f a i r l y e f f i c i e n t and the peaks show l i t t l e evidence of " t a i l i n g " , the separation e f f i c i e n c i e s obtained are more than an order of magnitude below what i s predicted based on l o n g i t u d i n a l d i f f u s i o n alone. Furthermore, t h i s surface treatment e x h i b i t s a limited l i f e t i m e , with protein adsorption and peak broadening becoming more noticeable after only a few days of use. In addition, any such s i l y l a t i o n treatments are only stable in a pH range of 2 to 7. Basic pH conditions lead to rapid loss of the silane by hydrolysis. Lauer and McManigill(20) proposed that protein adsorption could be minimized i f they are run in a buffer pH where both proteins and the surface are negatively charged. They reasoned that under these conditions the protein might be e l e c t r o s t a t i c a l l y repelled from the surface thus preventing adsorption. Figure 11 shows a separation of proteins in a pH 8.24 t r i c i n e buffer, a pH above the i s o e l e c t r i c points of a l l the proteins in the sample(21). The buffer was also 40 mM in KC1 to help minimize zone-broadening from sample overloading. Very sharp peaks are evident in the electropherogram, with peaks B, C and D e x h i b i t i n g nearly one m i l l i o n t h e o r e t i c a l plates. This approach appears highly e f f e c t i v e i n eliminating adsorption. I t s only serious disadvantage i s that i t requires working at a pH on the basic side of the i s o e l e c t r i c point of the sample proteins, and thus does not give a great deal of f l e x i b i l i t y in operating conditions. This discovery by Lauer and McManigill suggested to me that much of the protein adsorption might be due to ion exchange interactions between cationic sites i n the protein and cation exchange s i t e s (silanoate groups) on the fused s i l i c a surface. As in ordinary ion exchange chromatography, t h i s interaction could be weakened by r a i s i n g the concentration of competing ions i n the buffer. Figure 12 shows the results of CZE of proteins in a pH 9 Ches buffer with 0.25 M K^SO. added i n an e f f o r t to decrease ion exchange interactions(22). This approach apparently works since

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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6

I

i5

\6 Time

2o

(minutes)

F i g u r e 9. E l e c t r o p h e r o g r a m of f l u o r e s c a m i n e - l a b e l l e d p e p t i d e s o b t a i n e d from a t r y p t i c d i g e s t of reduced and c a r b o x y m e t h y l a t e d egg white lysozyme. Reproduced w i t h p e r m i s s i o n from Ref. 18. Commun.

C o p y r i g h t 1981

J . High Res. Chromatogr.

B

D

A

1 1

Chromatogr.

E

C

,05 AU

0

1————

s

15

30

minutes

F i g u r e 10. E l e c t r o p h e r o g r a m of p r o t e i n s t a n d a r d s run i n a g l y c o p h a s e - t r e a t e d fused s i l i c a c a p i l l a r y , i n pH 7.0 phosphate buffer. A = lysozyme; B = cytochrome c; C = r i b o n u c l e a s e ; D = chymotrypsinogen; E = h o r s e m y o g l o b i n .

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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T .0035

A U

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I

10

15

Time (min)

Figure 11. Electropherogram of protein standards run in an untreated fused s i l i c a c a p i l l a r y , in pH 8.24 t r i c i n e buffer. A = sperm whale myoglobin; B • horse myoglobin; C = human carbonic anhydrase; D = bovine carbonic anhydrase; E = B-lactoglobulin B; F = B-lactoglobulin A. Reproduced with permission from Ref.

21. C o p y r i g h t

1986 A n a l . Chem.

minutes

Figure 12. Electropherogram of protein standards run in an untreated fused s i l i c a c a p i l l a r y in pH 9 Ches buffer, with 0.25 M KC1. A = lysozyme; B = trypsinogen; C = myoglobin; D = B-lactoglobulin B; E = B-lactoglobulin A. Reproduced with p e r m i s s i o n from Ref. 22.

Copyright

1986 Trends A n a l . Chem.

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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195

lysozyme, with an i s o e l e c t r i c point of 11, i s s t i l l migrated as a r e l a t i v e l y sharp zone, even though i t i s 2 pH units below i t s i s o e l e c t r i c point. In general the peaks i n t h i s electropherogram are quite sharp. However, the time scale of the analysis i s longer than usual. This i s due to the fact that the run was done in a 25 micron i . d . c a p i l l a r y , 50 cm long, with only 5 KVolts of potential instead of the more usual 20 KVolts. The high salt concentrations required the use of lower voltages and smaller i . d . c a p i l l a r i e s in order to avoid serious thermal overloading. These electropherograms of proteins also serve to i l l u s t r a t e another s i g n i f i c a n t problem with proteins. The signal to noise r a t i o i n these separations i s not extremely high. A l l three separations were monitored by on-column UV absorption detection. For CZE to be t r u l y successful, detection l i m i t s for proteins must be improved by roughly two orders of magnitude. This i s a formidable and yet important goal. CZE i s v e r s a t i l e i n permitting unusual separation chemistries to be investigated with r e l a t i v e ease. An example i s shown i n figure 13, which i s an electropherogram of dansyl-labelled d , l amino acids separated in a buffer containing a copper complex of 1h i s t i d i n e ( 2 3 ) . Separation of the d and 1 isomers i s made possible by t h e i r d i f f e r e n t i a l association with the copper-l-histidine complex. Another unusual electropherogram i s shown i n figure 14(24). This i s the separation of neutral organic molecules by t h e i r hydrophobic i n t e r a c t i o n with tetrahexylammonium ion. In t h i s case, the more hydrophobic the analyte, the more i t "binds" to the hydrophobic cation, and the faster i t migrates through the system. This "hydrophobic i n t e r a c t i o n electrophoresis" gives a new tool to aid separation by electrophoresis. Although t h i s electropherogram shows a separation of r e l a t i v e l y small neutral molecules, a s i m i l a r effect might be used to aid i n separation of proteins based i n part on t h e i r r e l a t i v e hydrophobicities. Some of the necessary future developments i n CZE are c l e a r . C a p i l l a r i e s with surfaces non-adsorptive toward proteins are important. Perhaps more important are detection schemes for proteins which are v a s t l y more sensitive than present detectors. In my lab we are constructing and testing autosamplers and microf r a c t i o n c o l l e c t o r s for CZE, both of which function under microcomputer c o n t r o l . Hjerten(25,26) has begun to explore the p o s s i b i l i t i e s of g e l - f i l l e d c a p i l l a r i e s and of i s o e l e c t r i c focusing in c a p i l l a r i e s . A l l of this work has as i t s goal a v e r s a t i l e and powerful instrumental version of electrophoresis complementary to modern HPLC.

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

NEW DIRECTIONS IN ELECTROPHORETIC METHODS

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F i g u r e 13. E l e c t r o p h e r o g r a m s o f D, L - d a n s y l amino a c i d s w i t h upper: C u ( I I ) - L - h i s t i d i n e e l e c t r o l y t e at pH 7. lower: Cu(II)-D, L - h i s t i d i n e e l e c t r o l y t e at pH 7. Reproduced w i t h p e r m i s s i o n from Ref. 23. C o p y r i g h t 1985 S c i e n c e .

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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T

.005

0

5

AU

10

15 Time

20

25

(min)

Figure 14. Electropherogram of neutral organic compounds i n 50/50 acetonitrile/water with 0.025 M tetrahexylammonium perchlorate. A = benzo-(GHI) perylene; B = perylene; C * pyrene; D = 9-methyl-anthracene; E = naphthalene; F = mesityl oxide; G = formamide. Reproduced w i t h p e r m i s s i o n from Ref. 1986 A n a l . Chem.

24.

Copyright

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Acknowledgment Support for this work was provided by a grant from the Alfred P. Sloan Foundation, the Hewlett-Packard Corporation, and the National Science Foundation under Grant CHE-8213771. Literature Cited

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1. 2.

Hjerten, S., Chromatogr. Rev. 1967, 9, 122. Kolin, A.; In "Electrophoresis - A Survey of Techniques and Applications, Part A: Techniques"; Deyl, Z., Ed.; Elsevier: Amersterdam, 1979; Chap. 12. 3. Catsimpoolas, N., ibid., Chap. 9. 4. Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, Th. P.E.M.; J. Chromatogr. 1979, 169, 11. 5. Wieme, R. J.; In "Chromatography: A Laboratory Handbook of Chromatographic and Electrophoretic Methods 3rd Ed."; Heftmann, E., Ed.; Van Nostrand Reinhold: New York, 1975; Chap. 10. 6. Hinkley, J.O.N.; J. Chromatogr. 1975, 109, 209. 7. Brown, J. F.; Hinkley, J. O. N.; ibid, 218. 8. Jorgenson, J. W.; Lukacs, K. D.; Anal. Chem. 1981, 53, 1298. 9. Bier, M.; Palusinski, O. A.; Mosher, R. A.; Saville, D. A.; Science 1983, 219, 1281. 10. Giddings, J. C.; Sep. Sci. 1969, 4, 181. 11. Green, J. S.; Jorgenson, J. W.; J. Chromatogr. 1986, 352, 337. 12. Walbroehl, Y.; Jorgenson, J. W.; J. Chromatogr. 1984, 315, 135. 13. Everaerts, F. M.; Beckers, J. L . ; Verheggen, Th. P.E.M.; "Isotachophoresis: Theory, Instrumentation and Applications"; Elsevier, Amsterdam, 1976. 14. Lukacs, K. D.; Jorgenson, J. W.; J. High Res. Chromatogr. Chromatogr. Commun. 1985, 8, 407. 15. Lukacs, K. D.; Ph.D. Thesis, University of North Carolina, Chapel Hill, N.C. 1983. 16. Green, J. S.; Jorgenson, J. W.; J. High Res. Chromatogr. Chromatogr. Commun. 1984, 7, 529. 17. Rice, C. L . ; Whitehead, R.; J. Phys. Chem. 1965, 69, 4017. 18. Jorgenson, J. W., Lukacs, K. D.; J. High Res. Chromatogr. Chromatogr. Commun. 1981, 4, 230. 19. Chang, S. H.; Gooding, K. M.; Regnier, F.E.; J. Chromatogr. 1976, 120, 321. 20. Lauer, H. H.; McManigill, D.; Anal. Chem. 1986, 58, 166. 21. Jorgenson, J.W.; Anal. Chem. 1986, 58, 743A. 22. Lauer, H. H.; McManigill, D.; Trends Anal. Chem. 1986, 5, 11. 23. Gassmann, E.; Kuo, J. E.; Zare, R. N.; Science 1985, 230, 813. 24. Walbroehl, Y.; Jorgenson, J. W.; Anal. Chem. 1986, 58, 479. 25. Hjerten, S.; J. Chromatogr. 1983, 270, 1. 26. Hjerten, S.; Zhu, M.-D.; J. Chromatogr. 1985, 346, 265. 27. Walbroehl, Y.; Ph.D. Thesis, University of North Carolina, Chapel Hill, N.C. 1986. 28. Thormann, W., Arn, D., Schumacher, E., Electrophoresis 1984, 5, 323. 29. Thormann, W., Arn, D., Schumacher, E., Sep. Sci. Technol. 1985, 19, 995. RECEIVED

November 25, 1986

In New Directions in Electrophoretic Methods; Jorgenson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.