Anal. Chem. 1881, 63,2207-2208
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Preparation of Polyacrylamide Gel-Filled Fused-Silica Capillaries by Photopolymerization with Riboflavin as the Initiator Tianlin Wang,' Gerard J. Bruin, Johan C. Kraak, and Hans Poppe* Laboratory for Analytical Chemistry, University - of. Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands High-efficiency electrophoretic separations of proteins and oligonucleotides by capillary gel electrophoresis (CGE) of polyacrylamide gel filled fused-silica capillaries with 150 pm internal diameter have been introduced by Hj6rten (1)for the first time and a few years later by Cohen and Karger for smaller inner diameters (2). A gel provides a unique mode of selectivity in electrophoresis. It is needed for separation according to the size of large molecules, especially DNA fragments. Therefore, CGE has drawn considerable interest in the biochemical community (3). However, the practical application of CGE is not without difficulties. It appears hard to prepare bubble-free polyacrylamide gels inside fused-silica capillaries in the conventional manner, with ammonium persulfate as initiator, N,N,N'fl'-tetramethylethylenediamine (TEMED) as catalyst, and the usual wall binding reagent, [3-(methacryloxy)propyl]trimethoxysilane. Bubbles in the gel can lead to diminished resolution, decreasing current during operation, and changing of separation patterns. Several modifications in the preparation of capillary polyacrylamide gel columns have been proposed to cope with these problems. For example, one patented method was to compress the monomer solution to 7.0 X lo7P a and maintain the high pressure until completion of the polymerization (4). Another method was using y radiation from a 8oCosource to initiate the polymerization (5). Recently, the surface pretreatment with non-cross-linked polyacrylamide, which was originally invented by Hj6rten (6) for the elimination of electroosmotic flow in free zone electrophoresis, was also found to give an improvement in this respect (7). However, it is still an interesting challenge to find a simple way for the reliable preparation of reproducible gel-filled capillaries. This paper describes a method for the preparation of bubble-free polyacrylamide gels with minimal instrumental requirements. It consists of photopolymerization with riboflavin as the photoinitiator carried out at decreased temperature. Separation performance of the gels was checked with samples of oligodeoxyadenylic acids, pd(A).
EXPERIMENTAL SECTION Apparatus. The pressure reservoir as described by Tock et was used to fill solutions into the capillaries. A UV light al. (8), source (Philips, TLD36W/08, Eindhoven, The Netherlands) was employed to initiate the polymerization. The experimental setup for capillary electrophoretic separations has been described in detail elsewhere (9). Use is made of a UV detector, adapted for CE detection (Applied Biosystems, Model 757, Foster City, CA). The temperature during the separations was kept at 25.0 f 0.2 OC. Materials. [3-(Methacryloxy)propyl]trimethoxysilanewas purchased from ABCR (Karlsruhe, Germany). Boric acid and urea (BDH Ltd, Poole, England) and Tris and EDTA (Merck, Darmstadt, Germany) were of analytical grade. Acrylamide and N,N'-methylenebis(acry1amide)were purchased from AldrichChemie (Steinheim,Germany). Riboflavin was a product of BDH Ltd. Water used in all the experiments was purified by passage through a PSC filter assembly (Barnstead, Boston, MA). Oligodeoxyadenylic acid (pd(A))samples were supplied by Isogen
* To whom correspondence should be addressed.
On leave from the Department of Chemistry, Shandong Normal
University, Jinan, China.
0003-270019 1/0363-2207$02.50/0
Bioscience (Amsterdam, The Netherlands) and Pharmacia (Woerden, The Netherlands). The samples were stored at -20 "C. UV transparent fused-silica capillaries of 53 pm i.d. were a kind gift of Philips Research Laboratories (Eindhoven, The Netherlands). These capillaries have UV-cured acrylate polymers such as used for optical fibers as an outside coating. Procedure. The fused-silica capillaries were flushed with 1 mol/L potassium hydroxide solution (1 h), water (1/2 h), 0.03 mol/L hydrochloric acid (1/2 h), and water (1/2 h). A mixture of methanol and [3-(methacryloxy)propyl]trimethoxysilane(50/50, v/v) was introduced into the capillary and left there for at least 3 h (3). A certain length (usually 30-40 cm) of the treated capillary was cut off. To provide an on-column optical detection window, about 2 mm of the acrylate coating was burned off with an electrically heated wire (3,lO). Then, a mixture of l-mL degassed (10 min) gel-forming solution of acrylamide (T) and bis(acry1amide) (C) (11) and 20 pL of an aqueous saturated riboflavin solution (0.008% w/v) was used to fill the capillary, after which both ends were sealed with rubber septa. Next, the capillary was moved into a 3-L glass beaker containing an ice-water mixture. The capillary was illuminated by the UV source through the bottom of the beaker overnight. Sample solutions were injected electrokinetically. Injection conditions normally were 1-3 s at 1-4 kV for aqueous solutions of the samples, containing about 1pg/pL pd(A). Electric field strengths ranging from 150 to 200 V/cm were applied during electrophoresis. The buffer used for preparing the gel-filled capillaries and for the electrophoreticsamples during electrophoresis contained 100 mmol/L boric acid, 100 mmol/L Tris, 2 mmol/L EDTA, and 7 mol/L urea and was measured to have a pH of 8.7.
RESULTS AND DISCUSSION Some authors considered the bubble formation, observed in the common preparation procedure with persulfate and TEMED, mainly as a result of shrinkage during polymerization, since the polymer gel is substantially more dense than the original prepolymer solution (4, 7). The success of the method to prevent shrinkage by application of high pressure appeared to confirm this (4). Without such methods, the shrinkage cannot be made up by an inward flow of the gelforming solution from both ends of the capillary, especially because of the binding to the wall. Indeed, we observed less bubble formation when the binding reagent, [3-(methacryloxy)propyl]trimethoxysilane, was not used. Dissolved air in the gel-forming solution is considered as a minor reason since oxygen has an inhibitory effect on the polymerization and has to be removed with helium anyway. The original investigation of photopolymerization of acrylamide with riboflavin as the initiator was reported by Oster et al. (12). This method was used to prepare polyacrylamide gel in disc electrophoresis (13). We adapted the method for the preparation of the gel-filled capillaries. Initiating the polymerization with riboflavin has some advantages: it can be used a t very low concentrations and allows control at the start of the polymerization, since gelation is only initiated by exposure to light. During photopolymerization the capillary filled with the gel-forming solution was placed in an ice-water mixture. Under these conditions bubble formation was observed to be less pronounced. It was also found that degassing of the solution with helium for 10 min was sufficient to remove oxygen, as monitored by the rate of polymerization. In separate vial experiments, carried out 0 199IAmerlcan Chemlcal Soclety
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 19, OCTOBER 1, 1991
are not reported in most of the papers. Figure 1 shows a electrophoretic separation of pd(A)16-ao on a 7.5% T, 3.3% C gel. Plate numbers for this sample amounted to about 200000 for capillaries of about 22-cm effective length. For an even larger DNA fragment sample pd(A)ll,, an approximate plate number of 2 X 106 was observed (electropherogram not shown). No significant loss in performance was observed after 50 injections. However, sometimes bubbles formed at the injection end of the capillary gel column after several injections. The separation power could be restored by cutting 1 or 2 mm (3). At electric field strengths higher than 200 V/cm, bubble formation occurred, similar to other experiences (14).
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ACKNOWLEDGMENT The technical assistance of G . Feenstra is acknowledged.
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time (minutes) Flgure 1. Separation of oligcdeoxyadenylic acids P ~ ( A ) , ~spiked -~, with pd(A),,. Conditions: capillary IhWt = 21.7 cm, L = 30.0 cm, 53 pm 1.d. polyacrylamide gel, 7.5% T, 3.3% C; injection voltage 3020 V; Injection time 5 s; buffer (see text): separation voltage 173 V k m ; UV detection at A = 260 nm. The numbers in the electropherogram refer to the number of bases in the oligonucleotides.
under these conditions, the first visible gel forming occurred after about 10 min of illumination. Success rate of preparation of bubble-free polyacrylamide gelfilled capillaries was about 80% for the 7.5% T, 3.3% C gels discussed here. For other types of gels (not reported here) we likewise found much better success rates with photopolymerization as compared to conventional methods; linear gels (0% C) giving the best and gels with high values for T and C still giving the worst results. It is difficult to compare the success rate percentage for this method with that of other methods, because these percentages
LITERATURE CITED Hj6t?en, S. J . Chromatogr. 1989, 270, 1. Cohen, A. S.; Karger, B. L. J . Chrmrogr. 1987, 397, 409. Paulus, A.; Ohms, J. I. J . Chromatogr. 1990. 507, 113. Bente, P. F.; Myerson, J. Eur Pat. EP0272925 A2 (Hewlett-Packard Co.), June 29, 1988. Lux, J. A.; Hausig, U.; Schomburg. G. J . High Resolut. Chromatogr. 1990, 13, 436. HjBrten, S., J . Chromatogr. 1985, 347, 191. Yin, H. F.; Lux, J. A,; Schomburg, G. J . High Resolut. Chromatogr. 1990, 13, 625. ToCk. P. P. H.; Stegeman, 0.; Peerboom, R.; Poppe, H.; Kraak, J. C.; Unger, K. K. Chromatographia 1987, 24, 617. Bruin. G. J. M.; Tock, P. P. H.; Kraak, J. C.; POOW, . . H. J . Chrometm. 1990, 517,557. Lux, J. A.; %usig, U.; Schomburg, G. J . High Resolut. Chromatogr. 1990. 1.3. 373. . Hi&&, S. Arch. Blochem. Bbphys. Suppl. 1962, 1 , 147. Oster, G. K.; Oster, G.; Prati, G. J . Am. Chem. SOC.1957, 79, 595. Davis, B. J. Ann. N . Y . Aced. Sci. 1964, 121, 404. Drossman, H.; Luckey, J. A.; Kostichks, A. J.; D'cunha, J.; Smith, L. M. Anal. Chem. 1990, 62, 900.
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RECEIVED for review March 11,1991. Accepted July 3,1991.
CORRECTION Composite Multivariate Quality Control Using a System of Univariate, Bivariate, and Multivariate Quality Control Rules S. J. Smith, S. P. Caudill, J. L. Pirkle, and D. L. Ashley (Anal. Chem. 1991,63, 1419-1425). The second equation on p 1420 is incorrect. It should read
+
[log [ E d 2 K ] ] / n
for 0
