Anal. Chem. 1991, 63,2038-2042
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Capillary Gel Affinity Electrophoresis of DNA Fragments Andras Guttman* and Nelson Cooke Beckman Instruments, Inc., Palo Alto, California 94304
The incorporation of an affinity band wlthln a polyacrylamide gel provides a general means of manipulating the seiectivlty of capillary gel electrophoresis separations. As an example of thb approach, high resolution of DNA restriction fragments by capliary gel a " y electrophoresis has been achieved by addlng a soluble intercalating agent, ethidium bromlde, to the gel-buffer system. A migration model has been developed that can be used for seiectlvlty optimization. Various parameters, such as llgand concentration and applied electrk field, have been examined in terms of their influence on retention and Selectivity of different-size DNA molecules. From this study, high-resolutlon separations have been developed with efficiencies as high as lo' theoretical plates per meter.
18),and in capillary zone electrophoresis (19) as a selective
intercalating agent for double-stranded DNA molecules in order to increase selectivity. Since the ethidium bromide is positively charged, it causes a reduction in the electrophoretic mobility of DNA when intercalating into the two strands (20). In addition to the charge effect, the mass of the molecule is increased (up to 12%) due to complex formation.
THEORY Ethidium bromide, as a complexing ligand (L+),intercalates between the strands of double-stranded DNA (Pn-),and since it is positively charged, it decreases the electrophoretic mobility of the DNA-ligand complex (PLm(n-mF) by reducing the ionic charge:
p n - + mL+ + P Lm (n-m)-
INTRODUCTION Capillary electrophoresis is fast becoming an important separation technique in biochemistry and molecular biology (1-5). With narrow-bore fused-silica capillaries filled with polyacrylamide gels, high resolving power can be achieved in the separation of oligo- and polynucleotides (6-10). Capillary electrophoresis offers the capability of multiple injections onto the same gel-filled column with on-line detection and automation (11). As in high-performance liquid chromatography, the versatility of capillary gel electrophoresiscan be extended via the incorporation of special selectivity into the migration process. Affinity electrophoresis is a method in which chemical/biospecific interaction between the analyte and the affinity ligand occurs, causing a change in electrophoretic mobility (12). Affinity-type ligand molecules may be either soluble or immobilized in the gel-buffer system (13). With the use of soluble ligands (Figure lA), analyte complexes having a broad range of physical or chemical properties can be formed. If the ligand is very small compared to the electrophoresed analyte, the change in mobility of the complex will be relatively small or negligible, especially if the ligand has a small or zero charge. A substantial effect on the electrophoretic mobility is expected if the ligand and the complex are comparable in size or if the ligand is small but highly charged. In this case the interaction may result in a strong decrease or increase in mobility. When the ligand is immobilized, it can be physically entrapped into (Figure 1B) or chemically bonded (Figure IC) to the gel matrix (14). The entrapping method was used in our earlier work for the separation of chiral compounds by incorporating cyclodextrins into polyacrylamide gel-filled capillary column (15). This paper reports capillary affinity electrophoresisresults with polyacrylamide gel columns using the soluble ligand method in order to achieve enhanced resolution of DNA restriction fragments. As an example of selectivity manipulation, we have explored the use of ethidium bromide, a small positively charged molecule, as an affinity ligand in capillary gel electrophoresis. Ethidium bromide has been used previously in affinity chromatography (16),in affinity electrophoresis (17, *To whom correspondence and reprint requests should be ad-
dressed.
K = [PLm(n-m)-]/[P"-][L+]m
(1)
(2)
where K is the formation constant of the complex, m is the number of the positively charged ethidium bromide molecules in the complex, and n is the total number of negative charges on the DNA molecule (i.e., the number of phosphate groups). According to LePecq et al. (21) the double-stranded DNA molecule has approximately one ethidium bromide binding site for five base pairs depending on the dye/polymer ratio, salt concentration, etc., and there is no base composition selectivity of the binding. The velocity (u) of the polyion complex in capillary gel electrophoresis can be expressed as (15) u = l / t M = ppERp (3) where 1 is the effective length of the gel-fied capillary column, tM is the migration time of the solute (from injection to the detection point), pp is the electrophoretic mobility of the polyion, and E is the applied electric field. The molar ratio of the free polyion, Rp is given by
R p = - [pn-I = CP
1 1
+ K[L+]"
(4)
where cp is the total concentration of the polyion, P. Combining eqs 2 and 3 gives eq 5, which expresses the resultant
(5) velocity of the negatively charged polyion complex in the presence of the positively charged complex ligand (ethidium bromide). This equation shows that an increase in the constant of the complex formation and/or in the concentration of the ligand leads to decreasing migration velocity of the DNA-ligand complex. When K[L+]" >> 1, eq 6 expresses the electrophoretic velocity of the complex. u = ppE-
1
K[L+Im
EXPERIMENTAL SECTION Apparatus. In all these studies, the Pf ACE System 2000
capillary electrophoresis apparatus (Beckman Instruments, Inc.,
0003-2700/91/0363-2038$02.50/00 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15, 1991
2039
L
A
0
C
Fburr 1. Schematic representation of techniqk used for solubilized and immoblllzed ligands In polyacrylamide gels. (A) Solubilized ligand method: The ligand can move freely in the *buffer system. (E) The macroiigand method: The solution of acrylamide contains the macroligand that becomes entrapped within the gel matrix after polymerization. (C) Chemically bound ligand: Direct copolymerlzatlon of polyacrylamide gel wRh the copoiymerirabie derivative of the ligand.
Palo Alto, CA) was used with the cathode on the injection side and the anode on the detection side. The separations were monitored on-columnat 254 nm. The temperature of the gel-fded capillary columns was maintained in all experiments at 25 i 0.5 O C , even at high field strengths, by the Peltier device controlled cooling system (22) of the P/ACE instrument. The electropherograms were acquired and stored on an Everex 386/33 computer using the System Gold software package (Beckman Instruments, Inc., San Ramon, CAI. Procedures. Polymerization of the different-concentration (high and low viscosity) linear non-cross-linked polyacrylamides was accomplished within fused-silica capillary tubing (Polymicro Technologies, Inc., Phoenix, AZ) in 100 mM Tris-boric acid, 2 mM EDTA (pH 8.5) buffer according to the procedure of Heiger et al. (8). For stabilization, the high viscoSity linear polyacrylamide gel was covalently bound to the wall of the column by means of a bifunctional agent, (methacryloxypropy1)trimethoxysilane (Petrach Systems, Bristol, PA) (6). Polymerization was initiated by ammonium persulfate and catalyzed by tetramethylethylenediamine (TEMED). In a second column type, the use of low-viscosity linear polyacrylamide without binding to the capillary wall permits replacement of the gel-buffer system in the capillary column by means of the rinse operation mode of the P/ACE apparatus (Le., replaceable gel). The ethidium bromide was dissolved in the running buffer in the required concentration and loaded onto the polyacrylamide gel-filled capillaries electrokinetically (ethidium bromide is positively charged) after the complete polymerization of the gel. The samples were injected either electrokinetically (typically: 0.015-0.15 W s) into the high viscosity or by pressure (typically 5 s, 0.5 psi) into the replaceable gel-filled capillary column. Chemicals. The DNA restriction fragment mixtures, 4x174 DNA-Hae I11 digest and pBR322 DNA-Msp I digest were purchased from New England Biolabs (Beverly, MA). The 123-bp DNA ladder was obtained from Gibco BRL (Gaithersburg, MD). Polydeoxyadenylic acids, p(dA) 25-30,40-60 were purchased from Pharmacia (Piscataway,NJ). The samples were diluted to 50 pg/mL with water before injection and were stored at -20 O C when not in use. Ultra-pure electrophoresisgrade acrylamide,Trk, boric acid, EDTA, ammonium persulfate, and TEMED were employed in the experiments (Schwartz/Mann Biotech, Cambridge, MA). All buffer and acrylamide solutions were filtered through a 0.2 pm pore size filter (Schleicher and Schuell, Keene, NH) and carefully vacuum-degassed.
RESULTS AND DISCUSSION Migration and Separation. Initial efforts were focused on achieving high-resolution separations of DNA restriction fragments using high-performance capillary polyacrylamide gel electrophoresis. Our f i t results showed leas than desirable resolution for various DNA fragments (23). However, Dingman et al. (18) showed that the resolving power of polyacrylamide gels can be increased if a complexing additive, such as ethidium bromide, is used. While it is possible to attach the ethidium bromide covalently to the polyacrylamide gel we decided that the simplest and most reproducible approach would be to incorporate the complexing ligand di-
(In,
Figure 2. Capillary gel electrophoreticseparation of 6x174 DNAHae 111 digest restriction fragment mixture in the absence (A) and in the presence (E) of ethidium bromide (1 pg/mL) in the gel-buffer system. Peaks were identified by their increasing area, which correlates to the chain length (base pairs): 1 = 7 2 2 = 118; 3 = 194; 4 = 234 5 = 271; 6 = 281; 7 = 310 8 = 603; 9 = 872; 10 = 1078; 11 = 1353. Condltlons: isoelectrostatic (constantapplied electrlc M), 250 V/cm; replaceable polyacrylamide gel column effective length = 40 cm, total length = 47 cm; buffer, 0.1 M Tris-boric acid, 2 mM EDTA (pH 8.5) (TEE); Injection 5 8, 0.5 psi.
rectly into the porous gel matrix by simply adding ethidium bromide to the buffer system. Since the ethidium bromide is positively charged it migrates oppositely to the DNA molecule in the gel-filed capillary and therefore substantially decreases the mobility due to the formation of DNA-ethidium bromide complex (eqs 1, 5, and 6). Figure 2 compares the separations of the 9x174 DNA-Hae I11 digest restriction fragment mixture on polyacrylamide gel-fied capillaries with and without ethidium bromide. For this study, a replaceable polyacrylamide gel was used in the capillary column, using the pressure injection mode of the automated capillary electrophoresis system. The peaks were identified on the basis of their area, which correlates to the lengths of the fragments, assuming that there are no anomalies in the elution order. The electrophoretic behavior of DNA in polyacrylamide gels in which ethidium bromide was incorporated (Figure 2B) was quite different from that observed in normal gels (Figure 2A). It can be seen that for any DNA fragment, migration is slower in the presence of ethidium bromide, primarily due to the retardation caused by complex formation. This retardation increases with increasing m0lecular weight of the DNA (eqs 1 and 3). The W absorbance values in the presence of ethidium bromide are 2-%fold higher, because of the significantly higher absorbance of the DNAethidium bromide complex (24). The three small broad peaks between fragments 2 and 7 appeared only in the first run when the running buffer was changed for the one with ethidium bromide and are probably impurities. The enhanced absorbance dominates over the higher background (0.025 AU) due to the presence of ethidium bromide in the gel-buffer system, resulting in improved detectability. The complex formation with ethidium bromide also increases the size selectivity by increasing the time window of the separation (from 6 to 12 min). This effect can be followed in Figure 3, where the migration times of the different-size DNA fragments are plotted as a function of their molecular weight. The slopes of the curves are dependent on the concentration of the complexing ligand in agreement with eq 5. Figure 4 compares the separationsof the pBR322 DNA-Msp I digest restriction fragment mixture in the absence (Figure
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15, 1991
MIGRATION TIME (mlnl
YI
no EtEr
+ A-
0.1 u ~ h EtBr 1 .--Q--.
0 . 1 u P h l EtEr
......D.....
i . 0 u g h 1 EtBr
--A-
100
300 BASE PAIR NUMBER
1000
Fbure 3. Relationshipbetween the migration time and the molecular weight of the $C X174 DNA-Hae 111 digest regtrictkn framnt mlxtue. Lines correspond to different ethidium bromide concentration in the gel-buffer system. Conditions: isoelectrostatlc, 300 V/cm; hlgh-vlscosity polyauylamlde gel column effective length = 7 cm, total length = 27 cm; buffer, 100 mM TBE; injection 2 s, 50 mW DNA concentration, 50 pg/mL.
I
Lo7 O Y
.a=
q BY
3c :
-
f
?
8
0
9
8
s 0
FWr 5. Effect of the ethidium bromide on the capillary gel eiectrophoretic separation of the 123bp DNA ladder: (A) no ethidium b r m ide; (B) 1 pg/mL ethidium bromide in the gel-buffer system. Peaks (base pairs): 1 = 123; 2 = 246 3 = 369; 4 = 492; 5 = 615; 6 = 738; 7 = 861; 8 = 984; 9 = 1107; 10 = 1230; 11 = 1353; 12 = 1478; 13 = 1599; 14 = 1722. Condltbns: isoelectrostetlc300 V/cm (A), 200 V/cm (B); highviscosity polyacrylamide gel column effective length = 20 cm (A), 7 cm (B), total length = 27 cm; buffer, 100 mM TBE; Injection 5 8 , 150 mW.
Ill '
1
Flguro 4. Effect of the ethidium bromide on the capillary gel electrophoretlc separatlon of pBR322 DNA-Msp Idigest restriction fragment mixture: (A) no etMdlvn bromide; (B) 1 p g / d ethidium bromide in the gel-buffer system. Peaks (base pairs): 1 = 26; 2 = 34; 3 = 67; 4 = 76; 5 90; 6 = 110; 7 = 123; 8 = 147; 9 = 147; 10 = 160; 11 = 160; 12 = 180 13 = 190 14 = 201; 15 = 217; 16 = 238; 17 = 242. Conditions: isoeiectrostatic 100 V/cm (A), 200 V/cm (B); tll#l-Polyaaylamide gel cdunneffecthre length = 20 cm,total length = 27 cm; buffer, 100 mM TBE; injection 3 8 , 30 mW.
4A) and in the presence (Figure 4B) of ethidium bromide. The peaks were identified in the same way as in Figure 2. There is an unquestionable improvement in separation in the case of added ethidium bromide (Figure 4B) over the entire molecular weight range, specially for the 140-200-bp fragments (peaks 8-14). Because of the decreased mobility for the DNA-ethidium bromide complex (W), a !&foldhigher applied electric field was used in the presence of ethidium bromide in order to achieve faster migration. It should also be pointed out that the capillary gel technique allows a separation of the two 147-bp fragments and the two 160-bp fragments; conventional polyacrylamide gel electrophoresis does not separate the 160-bp fragments. The resolution for the higher base pair range (up to 1700 bp) was evaluated by testing the separation of the 123-bp DNA ladder on a 20-cm column in the absence (Figure 5A) and on a 7-cm column in the presence (Figure 5B) of ethidium bromide. By use of the intercalating ligand and shorter effective column length, the higher members of the 123-bp ladder still can be differentiated in significantly shorter sep-
FiQm8. CepVlerygelelectrophoreticseparatknofthe~tranded polyadenylic acid test mlxture in the absence (A) and In the presence (B) of ethMium bromide (1 pg/mL) In the gel-buffer system. Peaks: p(dA) 25-30,40-60. Conditions: isoelectrostatic, 400 V/cm; highviscosity polyacrylamide gel column effective length = 30 cm, total length = 37 cm; buffer, 100 mM TBE; injection 2 s, 15 mW.
aration time in spite of the overlapping peaks. The reason for the appearance of the small peaks near the main peaks in Figure 5B is unknown and under further investigation. The electropherogramsin Figures 2,4, and 5 clearly demonstrate that the use of ethidium bromide as an intercalator additive results in increased resolution and efficiency so that higher peak capacity can be achieved. As an example, in Figure 2 the resolution between the 271-bp (peak 5) and 281-bp (peak 6) fragments increases from 1.3 to 2.1 and the theoretical plate numbers of the 1353-bp fragment increases from 1.18 X lo6 to 3.61 X lo6 in the presence of ethidium bromide. In the theoretical plate number calculation, appropriate equations were used to take account of slight asymmetry of the peaks (7).Typically l@-107 plates/m are obtained in the separations shown in Figures 4 and 5.
2041
ANALYTICAL CHEMISTRY, VOL. 63,NO. 18, SEPTEMBER 15, 1991 MOBILITY IlOE-51
K’
MOBILITY IlOE-51
I
72bp
+ 194bP
194bP
---e--. 60
-a%.--................... ............................... a‘8. ..... ......................................... m........................................................
- - - - _ _- - - 0-- - - - - - - - - _ - - - -
...... .....
-
0
-60%P &-
I
Q
*
o
.
i
_0 . 2 t
0.4
0.6
l-#
310bP
......D.....
................n
I
107RbP
A------L7 . 1353bP O
D... ............B................ ................n ...............
0.8
2ot
-*--
-
I
0
I
100
200
Since ethidium bromide intercalates between the base pairs of double-stranded DNA molecules, there is no, or very limited, complex formation with single-stranded DNA molecules (21, 26). This phenomenum is demonstrated in Figure 6, which shows that ethidium bromide has no effect on the separation time of the single-stranded homooligomer test mixture. It is worth noting that the UV absorbance values are significantly lower with the presence of ethidium bromide. Because there is no complex formation with the poly dA,the UV absorbance of the solute is not increased; however, the background absorbance is increased and, therefore, the detectability of the solute decreased, as observed in Figure 6.
Factors Influencing the Mobility of DNA Molecules. Figure 7 shows plots of mobility versus concentration of ethidium bromide in the gel-buffer system, with mobility values approaching a plateau at higher concentrations of the complexation ligand. The greatest curvature occurs with the higher molecular weight DNA molecules, which have a higher number of binding sites for the ligand (eq 1). Maximum binding of ethidium bromide occurs at a concentration of approximately 0.8 pg/mL. Further increase of the ligand concentration beyond this point will not increase the complex formation significantly but will however increase the conductivity and therefore the Joule heating (22) as well as the background absorbance of the gel-buffer system, as discussed above. In general, the electrophoretic mobility is independent of field strength. However, it was found that high field density in the high molecular weight range leads to field-dependent mobilities in the electrophoresis of DNA (27-29). These effeda are believed to arise from the orientation and stretching of the coiled configuration of the DNA by the increased applied voltage (30). Figure 8 shows the mobilities of some DNA fragments in capillary gel electrophoresis as a function of the applied electric field. Under our experimental conditions, there is an apparent linear relationship between mobility and field strength. In agreement with the results of Heiger et al. (B), we also observed that in capillary gel electrophoresis an increase in field strength also causes an increase in mobility of the DNA molecules with chain lengths higher than 1000 bp. Moreover, we have found that below 600 bp the mobility of the double-stranded DNA molecules decreases with field strength. This result might be due to the apparent increase in the size of the low molecular weight fragments, which leads to anomalous migration (partial melting) (31) or DNA-gel interactions (17). Both can be caused by the high electric field used (600 V/cm). Any effect of the temperature (Le., Joule heating) on the separation was minimized within the capillary even at high voltages with the use of the cooling system in the capillary electrophoresis apparatus (22).
400
500
600
700
ELECTRIC FIELD lV/cm)
ETHIDIUM BROMIDE CONCENTRATION (Up/nIl)
Flgue 7. Effect of the ethidium bromkle concentratbn on the mobHlty of the 4x174 DNA-Hae I11 dlgest mixture. Lines correspond to different-slze DNA fragments. DNA concentration in the Sample was 50 pg/mL (injected amount: approxlmately 0.1 ng).
300
Flguro 8, Relationship between the field strength and the mobility of the 4 X174 DNA-Hae III dlgest mixture. Lines correspond to different-size DNA fragments. MOBILITY l10E-51 72bp
+ 194bp ---e--. 310bp
......0...... W3bP
--A1078bp
-e1353bp
-e-. 0
100
200
300
400
500
600
700
ELECTRIC FIELD (V/cnI)
Figure 9. Mobility values of the 4x174 DNAHae I11 digest restrlctlon fragments as a function of the applied electric field in the presence of 0.5 pg/mL ethidium bromide in the gel-buffer system. Lines correspond to different-size DNA fragments.
We have observed however that DNA migrates differently in the presence of an intercalating ligand, aa can be seen in Figure 9 where the mobility of all the fragments i n c d with the field strength. It was previously found (32) that the apparent length of the DNA molecule increases with complexation of ethidium bromide, and that this intercalating ligand increases the intrinsic viscosity of DNA. Therefore the increased apparent length and rigidity of the DNA would certainly tend to change migration properties of the complex. The intercalation probably prevents the end-melting effect that may occur in the lower molecular weight range, resulting in a reversed field strength dependence in mobility.
CONCLUSIONS Analysis of DNA fragments, a common activity in the molecular biology laboratory, can be performed rapidly by using capillary gel electrophoresis. Using affinity ligands in the polyacrylamide gel expands the separation potential for DNA restriction fragments. It was shown that the migration velocity of the free DNA and the complex differ significantly from one another and the binding equilibria can be utilized for achieving high resolutions. The complex formation increases the UV absorbance of the DNA molecules, resulting in increasea in the sensitivity of the detection. When ethidium bromide is incorporated into polyacrylamide gel capillary columns, high resolution of closely eluted DNA fragments was achieved in the broad molecular weight range of 1 6 1 0 ’ (20-2000 bp). The mechanism whereby DNA migrates in polyacrylamide gels in the presence of ethidium bromide appears to be very complex. It was found that while the ethidium bromide affects the separation of double-stranded DNA molecules, it has no effect on the separation of singlestranded homooligomers, such as poly dA. The field strength
2042
Anal. Chem. 1991, 63,2042-2047
dependence of the mobility of DNA was found to be different in the absence and in the presence of ethidium bromide, especially for the lower molecular weight fragments.
ACKNOWLEDGMENT We acknowledge Prof. Barry L. Karger for his stimulating discussions. We further thank Drs. Anita Costa, Richard Palmieri, and Herb Schwartz for reyiewing the manuscript before submission. The help of Phyllis Browning in the preparation of the manuscript is also highly appreciated. Regiatry No. Ethidium bromide, 1239-45-8.
LITERATURE CITED Jorgenson, J. W.; Lukacs, K. D. sckmce 1089, 222, 266-272. Hjerten, S.; mu, M. D. J . Wnwmtop. 1085, 347. 191-198. WaUkrgfOrd, R. A.; Ewbrg, A. 0. Anal. Clwm. 1087, 5 9 , 1762-1766. -don, M. J.; Hueng, X.; Pentoney, S. L., Jr.; a r e , R. N. S c h c e 1088, 242, 224-228. Karger, 8. L. Netwe 1980, 339, 641. Cohen, A. S.; Najarlan, D. R.; Paulus, A.; Guttman, A.; Smith, J. A.; Karger, 8. L. Proc. Net/. A a d . S d . U.S.A. 1988. 8 5 , 9660-9663. Guthen, A.; Cohen, A. S.; Hew, D. N.; Karger, B. L. Anal. C b m . 1900, 62, 147-141. Hew,D. N.; Cohen, A. S.; Karger, B. L. J . chrometogr. 1900. 516, 33-46. Lux, J. A.; Yln, H. F.; Shombwg, 0. J . H@h Resdut. Chromtogr. 1000, 73, 436-437. Smith, L. M. ~ e t u e1001, 349,812-813. K a w , B. L.; Cohen, A. S.: " a n , A. J . Chromatogr. 1089, 492, 585-614. Horeisi, V.; Tlcha, M. J . clwomefcgr. 1081, 216, 43-62. Wejsi, V.; Tlcha, M. J . Chromtogr. 1080, 376, 49-67.
(14) Nekamura, K.; Kuwahara, A.; Takeo, K. J . Chfomatw. 1080, 796, 85-99. (15) " a n , A.; Paulus. A.; Cohen, A. S.;Grinberg, N.; Karger, B. L. J . ChromatogV.1088, 448. 41-53. (16) Vacek. A.; Bourque, D. P.;Hewlett. N. 0. Am/. Blochem. 1082, 724. 414-420. (17) Flint, D. H.; Harrington, R. E. Blochemkrby 1072, 1 1 , 4858-4864. (16) Dlngman. C. W.; Fisher, M. P.; Kakefuda, T. 6kchemishy 1972, 7 1 , 1242- 1250. (19) Nathekarntkooi, S.; Oefner, P.;Chin, A. M.; Bonn, 0. Budapest Chromatography Conference, Budapest, Hungary, Aug 14-17, 1990 L-2. (20) Maniatis, T.; Fritsch, E. F.; Sambrook, J. In Molecukr Cloning: A lebwatw Manuel; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1982. (21) LePecq, J. B.; Paleotti, C. J . Md. Hol. 1967, 27, 87-106. (22) Nelson, R. J.; Paulus, A.; Cohen, A. S.; Guttman, A.; Karger. B. L. J . chrometogr. 1080, 480, 111-127. (23) . . Gurrmen. A. Beckman Instttute Inc. Research and DevebDment Laboratory, 1990. (24) Kasper, T. J.; Melera, M.; Gozel, P.;bownlee, R. G. J . Chromtugf. 1088, 458, 303-312. (25) Waye, J. S.; Fourney, R. M. Appl. Theor. Ehxtrophoressls 1000, 1 , 193-196. (26) Ukk%;,-K.; Schwerh. H. E.; Sunzeri, F. J.; Busch, M. P.; Brownlee, R. G. Thlrd Internationel Meeting on HI!& Performance Capillary Electrophoresis, Sen Diego, CA, Feb 3-6, 1991; PT-5. (27) Swthern, E. M. Anal. Blochem. 1070. 100, 319-323. (28). Stellwagen, N. C. B(0chemlStry 1989, 2 2 , 8186-6193. (29) Lumpkh, 0. J.; Dejardln, P.; Zimm, B. H. B i o ~ n w s1985, 2 4 , 1573- 1593. (30) Cantor, C. R.; Smkh, C. L.; Mathew, M. K. Annu. Rev. Siophys. Biophys. Chem. 1088, 17, 267-304. (31) Lennan, S. L.; Frisch, H. L. ekporvmers 1982, 21, 995-997. (32) Freifelder, D. J . Mol. W .1071, 60, 401-403.
RECEIVED for review April 5,1991. Accepted June 19,1991.
Optimization in Sample Stacking for High-Performance Capillary Electrophoresis Dean S. Bur& and Ring-Ling Chien* Varian Research Center, 3075 Hansen Way, Pa10 Alto, California 94304-1025
A simple modd for the opthrlzaUon of peak variance in sample stacking with a gravlty4nJect.dsample has been devdoped. I n sample stacklng, a long plug of low-concentration buffer containing a sample for separation lo introduced into a column filled with a buffer of the same wmpodtion but of a higher concentration. The sample lono then migrate very r a m underthe ed"d applied elecbicfleld Intlwcwnple plug untH they reach the concentration boundary. Once they cross the boundary, the ions slow down and stack Into a narrow band. Theoretlcally, the peak width in sample stacking b proportknd to the ratb, 7, ol buffer concentration In the odglnai cwnple SdUHon to that In the column. However, this difference in the concentratlons indde the capillary column a h generates an .kctrooemotk pressure orlglnatlng at the concentration boundary. The laminar flow resuiting from the electroosmotic pressure causes extra peak broadening. Sample stacking and laminar broadening work against each other to yield an optlmai point rdatlng to the sample buffer concentration, the separation buffer concentratlon, and the sample plug length. The predlcations of the model are in agreement with experimental results.
INTRODUCTION One area of concern in high-performance capillary electrophoresis (HPCE) since its inception has been the short 0003-2700/9 1/0363-2042.$02.50/0
optical path associated with on-column detection ( I ) . Conventional W detectors have detection limits between 1X lob and 1 X lo4 M analyte for a 75-pm4.d. column. An increase in sensitivity usually is obtained through improvement of the detector. However, it is possible to obtain enhanced detection limits by performing on-column concentration. Several techniques have been reported to perform on-column concentration to enhance the detectability in HPCE (2-8). For example, Everaerts and other groups used isotachophoresis to achieve a high sample concentration within a pre-CE column (2,3,6).However, a careful choice of leading, terminating, and background electrolytes was required to perform this isotachophoretic preconcentration step. A simpler concentration technique called sample stacking has been well-known in electrophoresis (7-10). An increase of a factor of 10 in detectability using sample stacking in HPCE was reported by Lauer's group (5). In our application of sample stacking, the compositions of the buffer in the sample plug and in the column are the same. A long plug of sample, dissolved in a lower concentration buffer, is introduced hydrostatically into the capillary containing the same buffer but with a higher concentration. Then the separation voltage is applied across the column. Because the buffer in the sample plug has a low concentration of ions, the resistivity in the sample plug region will be higher than the resistivity of the rest of the column. Consequently, a high electric field is set up in this region. As a result, the ions will migrate rapidly under this high field toward the steady-state 0 1991 American Chemical Society
