2434
Anal. Chem. 1992, 64, 2434-2437
Acrylamide Polymerization Kinetics in Gel Electrophoresis Capillaries. A Raman Microprobe Study Tracey L. Rapp, Will K. Kowalchyk, Kevin L. Davis, Elizabeth A. Todd, Kei-Lee Liu, and Michael D. Morris' Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055
The formatlon of 3.5 %TI 3.3 %C crosdlnkedpolyacrylamlde is monitoredIn 75-pm4.d. electrophoresk caplilarlesby Raman mlcroprobe spectroscopy. The disappearance of the acrylamide 1292cm-' band b followed with 80-8 t h e rewlutlon for 30 mh, and 2-4 mln reodulknfor upto 10 h. Polymerlratkn Ir 98% complete In 1.5 h and greater than 99% complete after 2 h. I n the 900-1700-~m-~regionno bands attrlbutable to cross-linking are observable. Reactlon in the capliiary foNows secondorder kinetics. The reactlon is faster In the bulk system because heat disdpatlon is not sufficient to maintaln a constant temperature.
INTRODUCTION Capillary gel electrophoresis (CGE) is an attractive complement to slab gel electrophoresis. CGE was first developed by Cohen and Karger for the separation and molecular weight determination of peptides and proteins.' The capillary inner diameter dimensions of less than 100 pm permit efficient heat removal and facilitate the use of high field strengths. The technique is increasingly used in gene sequencing by the Sanger dideoxy chain extension Pulsed field capillary gel electrophoresis for separation of large nucleic acid fragments has been demon~trated,~?~ and the benefits of capillary gel affinity electrophoresis, with separation in a buffer containing an intercalator, have also been shown.6 While the utility of CGE is clear, column lifetime is a significant problem. At high field strengths (typicallygreater than 300 V/cm), or after repeated use, gel performance deteriorates. Analyte mobilities increase, resolution decreases, and sometimes the gel fails c~mpletely.~J Bubble formation in the capillary during polymerization of the gel or during later use is one catastrophic failure mode. However, little is known about the gel structure in the capillaries and the processes which cause the slow deterioration. As the first step in a study of capillary gel structure and dynamics, we report the intracapillary polymerization kinetics of crosslinked polyacrylamide. In doing so, we also investigate the utility of Raman microprobe spectroscopy for measurement of intracapillary gel properties. The Raman microprobe provides spectra with 1-10-pm spatial resol~tion.~,~ The microprobe retains the well-known strengths of Raman spectroscopy,includingeasy applicability (1)Cohen, A. S.;Karger, B. L. J. Chromatogr. 1987,397,409-417. (2)Drossman, H.; Luckey, J. A.; Kostichka, A. J.; D'Cunha, J.; Smith, L. M. Anal. Chem. 1990,62,900-903. (3)Swerdlow,H.;Zhang, J.Z.;Chen,D.Y.;Harke,H.R.;Grey,R.; Wu, S.; Dovichi, N. J.; Fuller, C. Anal. Chem. 1991,62,2835-2841. (4)Heiger, D. N.;Cohen, A. S.;Karger, B. L. J. Chromatogr. 1990,516, 33-48. (5)Demana, T.; Lanan, M.; Morris, M. D. Anal. Chem. 1991,63,27952797. (6)Guttman, A,; Cooke, N. Anal. Chem. 1991,63,2038-2042. (7)Karger,A. E.; Harris, J. M.; Gesteland, R. F. Nucl. Acids Res. 1991, 19,4955-4962. (8)Clark,R. J.H.,Hester,R. E.,Eds. AduancesinZnfraredandRaman Spectroscopy;Heyden and Son: London, 1980;Vol. 7,pp 223-282. 0003-2700/92/0364-2434$03.00/0
to aqueous systems. Raman microscopy is routinely used in polymer film and fiber production for the identification of impurities. It has also been successfully used to identify local differences in crystallinity and local concentration of copolymers in polymer proces~es.~ As evidenced by its use for these applications, Raman microprobe spectroscopy could also provide a direct method for monitoring gel structure and chemistry within an electrophoresis capillary. The Raman spectra of polyacrylamide gels have been reported by B a n d and Gupta,'OJ1 who assigned most bands. SERS spectra of polyacrylamidehave been reported by Suh and Michaelian.12 Ahern and Garrell13used SERS to probe polymerization on a silver colloid. There is generalagreement on the assignment of major bands in the region 900-1700 cm-1, but there is disagreement on whether Raman spectroscopy can detect polybisacrylamide sequences in crosslinked polyacrylamidegels, especially with a low percentage of cross-linker (%C). Gupta and Bansil attribute bands at 1035, 1063, and 1075 cm-' to the skeletal (-C-C-C-C-) stretches of the cross-linked polymers and 418,441, and 462 cm-l to the (-C-C-C-) bending vibration of the gel." Ahern and Garrell could not find any of these bands but did observe a band at 705 cm-' which could possibly be attributed to the polybisacrylamide. They also observed that monomeric bisacrylamide itself has a peak at 705 cm-l and left this band unassigned.13 Polymerization of cross-linked and linear polyacrylamide gels has been monitored in bulk by various methods such as viscosity measurements,14J5titration of unreacted monomer,16 UV ab~orption,'~ Tyndall effect (increase in light scattering due to gel formation),18and NMR spectros~opy.~~ However, there have been no attempts to study the polymerization kinetics of linear or cross-linked polyacrylamide gels in the environment of the microbore fused-silica capillary.
EXPERIMENTAL SECTION Raman spectra were obtained with a modified Spex 1877triple spectrograph fitted with either an 1200-groove/mm or 1800 groove/" grating in the spectrograph stage and a Photometric5 series 200 cryogenicallycooled CCD detector.20The light source (9)Gardiner,D.J., Graves, P. R., Eds. PracticalRaman Spectroscopy; Springer-Verlag: Berlin, 1989;pp 119-151. (10)Gupta, M. K.; Bansil, R. J. Polym. Sci.: Polym. Phys. Ed. 1981, 19,353-360. (11)Gupta, M. K.; Bansil, R. J.Polym. Sci.: Polym. Lett. Ed. 1983, 21,96-77, (12)Suh, J. S.;Michaelian, K. H. J . Raman Spectrosc. 1987,18,409414. (13)Ahern, A. M.; Garrell, R. L. Langmuir 1988,4,1162-1168. (14)Gromov, V. F.;Galperina, N. I.; Osmanov, T. 0.; Khomikovskii, P. M.; Abkin, A. D. Eur. Polym. J. 1980,16,529-535. (15)Feng, X. D.;Guo, X. Q.; Qiu, K. Y. Makromol. Chem. 1988,189, 77-83. (16)Bosisio, A. B.; Loeherlein, C.; Snyder, R. S.; Righetti, P. G. J. Chromatogr. 1980,189,317-330. (17)Pegon, Y.;Quincy, C. J. Chromatogr. 1974,100, 11-18. (18)Gelfi, C.; Righetti, P. G. Electrophoresis 1981,2,213-219. (19)Gromov, V. F.;Bogachev, Y. S.; Bune, Y. V.; Zhuravleva, I. L.; Teleshov, E. N. Eur. Polym. J. 1991,6,505-508. 0 I992 American Chemlcai Society
ANALYTICAL CHEMISTRY. VOL. 64, NO. 20, OCTOBER 15, 1992
was a frequency-doubled CW Nd-YAG laser, 532 nm (ADLAS DPY 305 c/315) providing 30-40 mW of power at the sample. The spectrograph slit width was adjusted to maintain 5- or 8-cm-1 resolution. Illumination and back-scatteredlight collection were through an Olympus, IMT-2inverted research microscope using a 20X/0.4 NA ultralong working distance objective for bulk measurements or a 20X/0.7 NA objective for intracapillary measurements. Capillaries were held stationary by a locally constructed Delrin stage to prevent heat loss to the metal of the microscope frame. A 3.5%T/3.3%Cpolyacrylamide gel was prepared by combining 1.4 mL of 50 % stock solution made from solid acrylamide and NJV-methylenebisacrylamidemonomer (Sigma) with 4.00 mL of 5 X Tris-borate-EDTA (TBE) and 420 pL of 3% ammonium persulfate (BioRad) diluted to 20.00 mL. The stock solutionwas deoxygenated by bubbling Nz through for 2-3 min. The TBE was prepared by mixing 0.5 M EDTA (Mallinckrodt), Tris base (Boehringer),and boric acid (Baker). Fluorocarbon-coated fused silica capillary (Polymicro Technologies) with 75-pm i.d. and 360-pm 0.d. was cut to 30-cm total length. The inner capillarywalls were cleaned with 1M HC1,l M KOH, and methanol. To bind the gel to the walls, a 1:lmixture of methanol and 3-(trimethoxysily1)propyl methylacetate (Aldrich) was pumped into the capillary and allowed to stand for a minimum of 4 h. Polymerization was initiated by mixing 5 pL of TEMED (Life Technologies,Inc.) with the 20 mL of monomer solution describedabove. The mixture was then pumped through the capillary for 1min, after which time the pumping was stopped and Raman spectra were collected. The time between addition of the catalyst and the start of Raman data collection did not exceed 2 min. Bulk solutionswere prepared in the same manner as the solutions used in the capillaries. The reaction was monitored in a 20-mL aliquot contained in a 25-mL glass sample vial placed on the Delrin microscope stage. Reaction-monitoringspectrawere obtained with an integration time which varied during the course of the reaction. During the f i s t 15 min, a 30-8 integration was used. For the second 15min a 2-min integration time was employed. For later spectra (30 min to 10 h) a 5-min integration time was used. By increasing the integration time as the reaction proceeded, we were able to monitor small amounts of starting material late in the reaction without undue compromisein time resolution. All reactions were performed at room temperature (approximately 24 O C ) . Temperature inaide the bulk reaction vessel was monitored using a type k thermocouple (Omega). The signal was amplified with a low-noise amplifier constructed locally. To derive kinetic information,band intensities were taken as areas under bands, calculated with the integration routines in Spectra-Calc (Galactic Industries). Rsactiona were followed as the attenuation of the acrylamide C-H bending vibrationat 1292 cm-l. For presentation, all spectra were subjected to a SavitakyGolay quadratiecubic smooth (n = 9) using the routines in Spectra-Calc. For the extraction of kinetic data, however, only simple background subtraction or ratioing was employed. Second-order kinetic fits were performed using DeltaGraph (DeltaPoint)
.
RESULTS AND DISCUSSION Figure 1shows the Raman spectra of bulk solution samples of the reaction starting materials: 5.0 7% by weight acrylamide and bisacrylamide. The band positions agree well with those reported by others.'OJ2 Figure 2 showsspectra obtained during the bulk and capillaxypolymerization of a 3.5 7% T/3.3 7% C gel. Representative curves early (2 min) and late (32 min) in the reaction are shown. For the bulk sample, a spectrum of completely polymerized (10.5 h) monomer is alsoshown. There is little difference between the corresponding spectra of the bulk and capillary samples. The corresponding band poeitions are identical within experimental error (fl-2 cm-9, and in both cases the observed band paeitions are in agreement with literature values. (20) McGlashen, M. L.;Davis, K.L.;Morris, M.D. A w l . Chem. 1990, 62, 846-849.
2435
I N
.-Y
1
N r
E
.e 4
N
500
800
7W
BOO
gM)
1WO
11W
1200 1300 1400
1500
1700
1600
1800
Ramen Shift (Wavenumber, cm-1) Figuro 1. Raman spectrum of 5 % monomer solutions. (A) Bisacryc amide. (B) Acrylamlde. Integration times were 60 8.
B
800
900
1000
1100
1200
1300
1400
1500
1600
1700
'
10
Ramen Shift (Wavenumber, cm-I) Flgm 2. Raman spectrum of T = 3.5%, C = 3.3% polyacrylamide gels. (A) Buk spectrum: 2 mln, 32 mln, and 10.5 h after Inltietbn. (B) CaplHaryspectrum: 2 and 32 min after inltietion. Integrationtlm were 60 s.
We have deviated slightly from common capillary electrophoresis in two ways. First,fluorocarbon-coated capillaries were used instead of the more common polyimide-coated capillaries. The fluorocarbon coating is nonfluorescent, but strong her-excited fluorescence from the polyimide coating obscures the much weaker polyacrylamide Raman spectra. Second, via the method of Feng and co-workers,l6 we have degassed solutions with nitrogen rather than helium. Although helium is used because of ita low water solubility, we have chosen to maintain consistencywith the polyacrylamide polymerization kinetics literature, where nitrogen or vacuum degassing are common. Early in the course of the reaction, bands of monomeric arylamide and bisacrylamide are still clearly visible, although the bands of the polymer are already measurable. The monomer bands observed 2 min after initiation (Figure 2) are identical to their positions in the individual monomer spectra (Figure 1). At 32 min after initiation, the spectrum largely resembles the spectrum of completely polymerized acrylamide. The shift in frequency of some bands and the
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
disappearance or growth of others can be understood by comparison of the observed bands with literature band assignments. At the high-frequency end of the monomer spectrum (or the spectrum 2 min after initiation), the three bands at 1680, 1639, and 1602 cm-I are assigned to C=O stretching (amide I), C=C stretching, and NH2 bending (amide 11)vibrations, respectively. The 1639-cm-1 band disappears as the C=C double bond is broken during the course of the polymerization. The 1680- and 1602-cm-I amide bands undergo small to moderate frequency shifts to 1680 and 1615 cm-' in response to the changing environment. An intense peak at 1442 cm-l in the monomer spectrum is assigned to the superposition of a CH2 bending mode and a C-N stretching mode (amide 111). After polymerization, these two bands are observed at the shifted frequencies of 1462 and 1435 cm-l, respectively. Another intense monomer peak in the spectrum is the vinylic C-H bending mode at 1292cm-l. Thisband disappears during the course of the reaction. In its place, a broad aliphatic C-H bending vibration in the polymerized gel appears at 1329 cm-l. This is an expected frequency shift when a C=C bond is broken. Two partially unresolved peaks a t 1223and 1187cm-' grow in as the polymerization progresses. These two bands are assigned to a NH2 wag and a polymer skeletal vibration. A strong, broad peak around 1128 cm-l from a C-C skeletal stretching mode in the acrylamide monomer can also be observed. This asymmetric band moves to a lower frequency of 1114 cm-1 upon polymerization. Weak bands at 1083 and 1064 cm-l in the initial polymerization spectra (2 min after initiation) can still be observed after 32 min in the polymerized gel spectrum. These two weak bands are no longer visible approximately 2 h ( i 1 5min) after the initiation of the reaction when polymerization is 99 % complete. Another weak band in the monomer spectrum a t 979 cm-' which is assigned to an out of plane HC-CH bend disappears during the initial stages of polymerization. During the later stages of the polymerization, a new band a t 987 cm-l may be attributed to the same vibration in the polymer. In the region between lo00 and 1100 cm-', Gupta and Bansilll have assigned three bands (1075,1063,1035 cm-9 to different C-C skeletal stretching vibrations which may arise from structurally different forms of highly-branched polyacrylamide. These bands were absent in the spectra of completely reacted cross-linked polyacrylamide reported by Ahern and Garrell.13 There are several reasons why Gupta and Bansil may have observed these unconfirmed bands. Percent conversion (polymerization efficiency) drops as the concentration of TEMED (reaction catalyst) is increased. Feng et al.15 reported only a 95% conversion for the concentration of TEMED used by Gupta and Bansil. Also, Bosisio et al.16found that a t high bisacrylamide concentrations (>20% C) only an 80% polymerization was observed after 1 h. However, if the system was left standing overnight, 96% conversion was achieved. Gupta and Bansil reported spectra after 8 h of polymerization. Therefore, it is possible that they may have been observing incompletely polymerized gels containing free monomer. We observe only two bands, at 1083 and 1064 cm-l, in the 1000-llOO-cm-l region. The 1083-cm-l band is from the initiator ammonium persulfate and is easily observed in solutions even as dilute as 3 X l(r3M. The 1064-cm-l vibration is found in the monomer spectrum of acrylamide (Figure 1B). These assignments are reasonable because both bands eventually disappear from the completely polymerized gel spectrum. We concur with Ahern and Garrell,13who concluded that a t low cross-linkerconcentration, this region of the Raman
io
"$1
c
:1
v
/ I
Flgurr 3. Reaction kinetic plots for T = 3.5%, C = 3.3% polymerizatlons (A) in bulk and (B) in an electrophoresis capillary. Acrylamide reciprocal concentration is plotted in units of l/(% T).
spectrum is not especially sensitive to the presence of crosslinking. We have investigated the feasibiity of time-resolved Raman microscopy for kinetic studies of the polymerization of acrylamide. We have chosen to follow the disappearance of the acrylamide C-H bending vibration a t 1292 cm-l. The intensity of this bending vibration and other weaker bands should not be affected by the potential problem of the Tyndall effect. Gelfi and RighettP reported only a 5 % transmission loss by scattering for gels containing 3%C. Therefore the Tyndall effect in our gels should be negligible. The use of an array detector allows us to simultaneously monitor several bands in the spectrum, but we are constrained by the length of the spectral window (approximately 280 cm-' with an 1800 grooves/" grating). The bands chosen for kinetics should be intense and wellresolved from nearby strong bands. In the 800-1700-~m-~ interval, there are several strong monomer bands, including those at 1292,1442, and 1639cm-l. The 1442-cm-' band was excluded because of the presence of polyacrylamide bands a t 1435 and 1462 cm-l which appear as the reaction proceeds. Similarly, the 1639-cm-l region was rejected because of the proximity of amide I and I1 bands at 1680 and 1602 cm-1. Therefore, the C-H bending region was employed. The C-H bending mode of acrylamide a t 1292 cm-' was monitored for the disappearance of monomer. The growth of the 1329-cm-l polymer band could also be monitored in this spectral window, but because this band is weaker than the monomer band, intensities could not be measured with adequate accuracy for kinetic studies. The lowest detectable amount of unreacted monomer was determined by measuring the integrated peak areas under the 1292-cm-' band for a series of unpolymerized acrylamide/ bisacrylamide solutions. The total monomer concentrations (5% T) of the solutions were varied while the bisacrylamide content (%C) remained constant. We obtained a linear calibration curve (r = 0.9995) with a detection limit of 0.035%T, or 1.0% of the 3.5%T initial concentration used in the kinetics experiments. Second-order kinetics for acrylamide polymerization have been reported by Gelfi and Righettila and by Chen and Crambach.21 Figure 3A,B demonstrates that the reactions in the bulk and capillary have different behaviors. After an initial induction period of about 7 min, the capillary polymerization follows the expected second order kinetics. We can fit to a second order rate constant of 1.5 X 10-3%T1s-1 out (21)Chen, B.;Chrambach, A. J . Biochem. Biophys. Meth. 1979, 1 , 105-116.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
to about 28 min. Beyond this time the acrylamide concentrations are too low for a reliable fit. In the bulk, however, we find a limiting rate constant of about 8.1 X 10-3%T-1s-l over the period 16-30 min, which corresponds to the interval of maximum temperature (see below). After the induction period of 5 min, short sections of earlier parta of the curve can be fitted to second-order kinetic equations, but we find such agreement fortuitous because the system is not at constant temperature. We do observe an induction period of about 6 min before measurable decrease in acrylamide Raman intensity begins. Thirty minutes after initiation,the polymerizationwas 97 5% complete in the bulk and 88 % complete in the capillary. The reaction was >98% complete after 1.5 h in both the bulk and the capillary. These times are longer thanthe times reported by Gelfi and RighettP for 5%T/3%Cgels (95% complete after 12-15 min). However,slower reaction rates are expected for gels with total monomer content below 5 % T because of the lower availability of the acrylamide monomer to the growing polymer chain.18 The identity of the Raman spectra at intermediate points during the reaction strongly suggest that the reaction mechanisms and final products are similar in the 75-pm4.d. capillary and bulk solution. However, the kinetics differ significantly. It has been reported that as the polymerization temperature is increased, the reaction rate increases and the induction period decreases.22*aTherefore, the rate constants and induction periods for the bulk and capillary polymerizations should be identical only if the reactions proceed at the same temperature. We have made thermocouple measurementa of the temperature rise in the bulk system during the course of the polymerization (Figure 4). For the first 8-9 min, i.e. during the induction period, the temperature remains at the initial temperature, 23-24 "C. It then rises 5.0-5.5 OC to about 29 "C over 6-7 min and then drifts slowly downward as the reaction rate declines. The laser wavelength (532 nm) is far removed from any acrylamide absorption bands; therefore, laser-induced heating should be negligible. To test this assumption, we have made temperature measurements with and without laser illumination. Comparison of Figure 4A (laser on) and B (laser off) shows that laser heating does not contribute to the observed temperature change. In the bulk polymerization, the induction period is 2 min shorter and the final rate constant is 5 times larger than observed inside the capillary. The entire edifice of capillary electrophoresis is built upon the observation of good heat dissipation in 100-pm-i.d. and smaller capillaries. The 5 "C temperature rise accompanying the exothermic reaction in the bulk polymerization system would be expected to be smaller in the capillary. This difference in thermal environments accounts for the apparent difference in reaction rates between the bulk and the capillary. The good adherence to second-order kinetic equations in the capillaryimplies that the temperature change is under 1"C. It has been suggested that the resulting cross-linked polymer structure is a function
-" J
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Time (Min)
Flguro 4. Temperature rlse during bulk polymerization of T = 3.5 % , C = 3.3%(A) with 30 mW of 532-nm Illumination and (B) wtthout laser
lllumlnation. of polymerization temperature.18 We propose that conclusions about polyacrylamide chemistry and structure from experiments in bulk solution cannot be applied uncritically to the chemistry and structure of the stoichiometrically equivalent gels in electrophoresis capillaries.
CONCLUSIONS Raman microprobe spectroscopy has been shown to be a useful tool for probing structures of molecules within an electrophoresiscapillary. Our system is based on a modified f/9 spectrograph. An optimized instrument could easiy have 10timea better throughput. In that case, it should be possible to observe changes in Raman spectra under electrophoresis working conditions with an acquisition time of 1-3 s. Even more information should be obtainable at low (50-500-cm-1) frequencies,where vibrationalmodes of the polymer backbone are observed. Recentlydeveloped holographic beam splitters allow high throughput Raman microscopy in this region" and should allow Raman probes of polyacrylamide conformation under field-freeor biased conditions. Finally we note that anti-Stokes Raman intensities measure thermal population of vibrational excited states and have long been used for temperature measurement. Anti-Stokes Raman microscopy may prove useful as a direct probe of intracapillary temperatures during electrophoresis. Experiments toward these goals are underway in our laboratories.
ACKNOWLEDGMENT This work was supported in part by NIH Grant GM-37006 and in part by DOE Grant DE-FG02-89ER13996.
RECEIVED for review April 1, 1992. Accepted July 24, (22) Gelfi, C.; Righetti, P. G. Electrophoresis 1981, 2, 220-228. (23) Greseel, J.; Rosner, A.; Cohen, N. AM^. Biochem. 1975,69,84-91. (24) P&ter,D.;Liu,K.-L.;Morris,M.D.;Owen,H.AppZ. Speetrosc., in prese.
1992. Registry No. Acrylamide, 79-06-1; Nfl-methylenebisacrylamide, 110-26-9.
