Anal. Chem. 1995, 67,4255-4260
On=LineRaman Spectroscopy of Ribonucleotides Preconcentrated by Capillary Dsotachophoresis Patrick A. Walker, 111, Will K. Kowalchyk,t and Michael D. Mods* Department of Chemistry, The University of Michigan, 930 North University, Ann Atbor, Michigan 48109-1055
Normal Raman spectroscopy is used as an on-line detector for capillary isotachophoresis (I”€’) of adenosine 5 triphosphate, adenosine 5-&phosphate, and adenosine 5-monophosphatein phosphate buffers. PreconcentraM phosphate buffer (PH 7.5) into tion is from a 1 x a leading electrolyte of 0.1 M KCl or Na2S04, with a terminating electrolyte of 0.1 M Q-morpholinepropanesulfonic acid. The ribonucleotides are concentrated to above M at the detectionwindow, allowing measurement of Raman spectra with 1 s integration, from starting M or higher. concentrations of 5 x The familiar capillary electrophoresis (CE) advantages of rapid, high-resolution separations in an inherently simple and rapid experiment’ are balanced by the lack of widely applicable detection methods with a high content of qualitative information. The most popular detection methods, UV absorbance: laser-induced fluore~cence,~ and electrochemistry,4offer only quantitative information about the separation. Positive identification of the eluted analytes usually requires extensive prior knowledge of the sample and comparison to appropriate mobility standards. The soft ionization techniques, electrospray mass spectrometry and fast atom bombardment MS6 have been successfully interfaced to CE. These provide molecular weight information, but with little or no fragmentation. There have been promising reports of successful low-frequency proton nuclear magnetic resonance spectroscopy (H NMR) in electrophoresis capillarie~.~ On-capillary resonance, preresonance, and normal Raman spectroscopywere demonstrated by our group several years ag0.83~ We have also used intracapillary normal Raman spectroscopy to follow polymerization of acrylamide’O and to measure capillary ’ Current address: Kaiser Optical Systems, Inc., P.O. Box 983, Ann Arbor, MI 48106. (1) Foret, F.; Krivhkova, L.; Bocek, P. In Capillay Zone Electrophoresis;Radola, B. J.. Ed.; Cambridge University Press: New York, 1993. (2) Foret, F.; Deml, M.; Kahle, V.; Bocek, P. Electrophoresis 1986,7, 430432. (3) Pentoney, S. L., Jr.; Sweedler, J. V. In Handl~ookofElectrophoresis; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1994; Chapter 7, pp 147-183. (4) Wallingford, R. A; Ewing, A G. Anal. Chem. 1987,59, 1762-1766. ( 5 ) Fang, L.; Zhang, R.; Williams, E. R.; Zare, R. N. Anal. Chem. 1994,66, 3696-3701. (6) Olivares, J. A; Nguyen, N. T.; Yonker, C. R; Smith, R D. Anal. Chem. 1987, 59, 1230-1232. (7) Wu, N.; Peck, T. L.; Webb, A G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994,66,3849-3857. (8) Chen, C.-Y.; Moms, M. D. Appl. Spectrosc. 1988,42, 515-518. (9) Chen, C.-Y.; Moms, M. D., j. Chromatogr. 1991,540, 355-363. (10) Rapp, T. L.; Kowalchyk, W. K; Davis, K L.; Todd, E. A; Liu. K-L.; Moms, M. D. Anal. Chem. 1992,64, 2434-2437. 0003-2700~95/0367-4255$9.00/0 0 1995 American Chemical Society
operating temperatures.”J2 These more recent experiments were performed on essentially unmodified Raman microprobes. The only “interfacing” necessary was provision of rigid mounting of capillaries in the light path and electrical insulation from the microscope stage and frame. Apart from the interest of the results, these experiments demonstrate the inherent simplicity of oncapillary Raman spectroscopy. In the absence of preconcentration, on-capillary normal Raman spectroscopy requires unrealistically high analyte concentrations. Typically, detection limits are near M largely because of the background Raman from water or buffer components. Without resonance enhancement or surface enhancement, an analyte concentration of least M is necessary for good signal/noise ratios at short acquisition times. Electrophoretic preconcentration is a more general strategy than resonance or surface enhancement of the Raman spectrum. In a recent communication, we showed that field-amplified injection into an electrophoresis capillary can be used to preconcentrate ions at subppm concentrations to levels at which intracapillary normal Raman spectroscopy is p~ssible.’~Field amplification, however, is not applicable to samples in which the analyte is in a matrix containing a high concentration of background electrolyte. In the presence of a concentrated background, electrolyte isotachophoresis (IT€’) is the preferred preconcentration technique.I4 ITPrequires a leading electrolyte (LE) of higher mobility and a trailing electrolyte OT) of lower mobility than any of the ions of interest. In a simple three-ion system the ultimate analyte ion concentration, Ca, is described by the Kohlrausch equation.
In eq 1, CLis the concentration of the leading electrolyte,pa,p ~ , and p~ are the electrophoretic mobilities of the analyte, leading electrolyte, and counterion, and ZLand 2, are the number of the elementary charge of the ion. In general, isotachophoresis concentrates an ion to nearly the leading electrolyte concentration. When multiple analytes are separated, the concentration of each analyte zone is determined by that of the preceding zone. ~~
~
(11) Davis, IC L.; Liu, IC-L.; Lanan, M.; Moms, M. D. Anal. Chem. 1993,65, 293-298. (12) Liu, K-L.; Davis, K L.; Moms, M. D. Anal. Chem. 1994,66, 3744-3750. (13) Kowalchyk, W. K; Walker, P. A, 111; Moms, M. D. Appl. Scpectrosc. 1995, 49, 1183-1188. (14) Schwer, C.; Lottspeich, F. j. Chromatogr. 1992,623, 345-355.
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Therefore, the stacking efficiency of the analytes decreases from the higher mobility analytes to the lower mobility ones. Field-amplified introduction occurs if the leading electrolyte is at a much higher concentration than any analyte or the background electrolyte containing the analytes. To a 6rst a p proximation, the stacking caused by field ampli6cation will be given by eq 2.15In eq 2, y is the stacking factor and CBis the y = C&
concentration of the sample background electrolyte, assumed to be in excess. During field-amplified introduction, the electric field seen by the sample is higher than the mean elechic field by y. The amount of material introduced is higher by the same factor than in the absence of this stacking. The width of the stacked zones, but not the final analyte concentrations, is affected by stacking. Concentrations are governed by the isotachophoretic environment Capillary lTP alone has long been used in analytical chemistry. As a preconcentration technique for electrophoretic separation, transient ITP has shown better than lWfold concentration increasesL6Many variants of the ITP principle are possible, and analytically important cases have been reviewed.I7 In this paper, we demonstrate isotachophoreticseparation and on-line Raman spectroscow of the nucleotide adenosine 5'triphosphate (ATP), adenosine Ydiphosphate (ADP), and adenosine 5'-monophosphate (AMP). ATP is of interest to neurochemists because of its role both as an energy source and as a possible neurotransmitter. The hydrolysis of ATP to ADP or AMP releases energy which is used to drive processes such as muscle contraction and to regulate the sodium/potassium pump mechanism in resting and firing chromaffin cells. The separation/ spectroscopy of these inherently interesting ions is a useful demonstration of the previously unreported ITP/Raman spectros COPY.
Micmsqm Stape
Figure 1. Block diagram of the capillaly isotachophoresidRaman spectroscopy system. Component details in text.
A
i
m
I_
Ik,
MIP
I_
,a0
,-I
R a m ~ nShift (em-I)
Flgun?2 Raman microprobe spectra of 0.1 M ATP, ADP, and AMP contained in 50pm i.d. capillaries: 400 mW 532 nm excitation, 1 s integration, and resolution 16 cm-'.
EXPERIMENTAL SECTION
Isotachophoresis. Figure 1is a block diagram of the ITP/ Raman system. The locally constructed isotachophoresis a p of an upright microscope (Olympus, BH-2) with a 20x/0.46 NA paratns consisted of a -30 kV higbvoltage power supply (Glassmicroscope objective (Olympus) for illumination and scattered man PS/LG-30R-5) which was connected to the injection end of light collection. A 85 mm axial transmissivespectrograph (Kaiser the capillary. The injection end was contained in a Plexiglass Optical Systems, Inc., HoloSpecf/l.8i)18 fitted with a holographic safety enclosure. The capillary was held in a V-groove machined transmission grating and a cryogenically cooled CCD camera (CHinto an aluminum block, which also provided some heat sinking. 270, PhotometricsLtd.) equipped with a 1024 x 256 chip (Em15 To insulate it from the microscope stage, a Dehin spacer was 11) for detection. A 50 or 100pm spectrograph entrance slit was placed between the aluminum block and the stage and secured employed to give a resolution of about 8 and 16 cn-',respectively. with Nylon machine ScTews. The exciting laser was an arc lamp pumped frequencydoubled The 50 pm id., 365 pm 0.d. fused-silica capillaries (Polymicro Nd-YAG laser, operating at 532 nm (Quantronix C o p , 416), Technologies, Inc.) were coated with 3%Tlinear polya~rylamide'~ which delivered between 400 and 700 mW to the capillary. Spectra to minimize electroosmotic flow. In most runs, the distance from were acquired using CCD goo0 software (Photomehics Ltd.) and the capillary entrance to the detection window was 20 cm and processed in GRAMS 386 (Galactic Industries). The spectra were the total length was 42 cm. In these capillaries, samples were obtained with 1 s integration times, with a 200 ms reset and electrokinetically introduced at -4.2 kV (-100 V/cm) for 30 s. storage delay between acquisitions. The running voltage was -8.4 kV (-200 V/cm). For lowconcentration samples, a capillary with a 30 cm entrance to (15) Bur@. D.S.: Chien, R-L. Anal. Cham. 1991.63,2042-2041. (16) Stegehuis. D. S.: Irth, H.: ljjaden. U.R Van Der Greet J. J. Chmnotogr. detector length and 45 an overall was used. For these experi1991.538.393-402. ments, samples were electrokinetically introduced at -9.0 kV (17) Bocek. P.: Deml. M.: Gebauer, P.: Dolnik. V. InA n o I ~ t i ~ ~ l I s o t n c h ~ p h ~ , ~ ~ ~ (-200 V/cm) for 80 s. The running voltage were initially -9.0 Fadola. B. I.. Ed.: Cambridge University Press: New York, 1988.
Raman Instrumentation. The Raman microprobe consisted
(18) Battey, D. E.: Slater. J. B.: Wludyka, R Owen. H.: Pallister, D.M.: Morris, M. D.ADD/. Sgectrosc. 1993.47.1913-1919.
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(19) Hjerten. S. J.J. Chronatogr. 1988.347,191-198.
I
,
,
,
,
,
1100
1200
1300
1400
1500
,$00
/'%
Time(Sec)
Raman Shift (cm-1) Flgure 3. On-capillary Raman spectra of 5 x M ATP, ADP, and AMP preconcentrated by isotachophoresis: injection time 30 s at -4.2 kV; 400 mW 532 nm excitation; 1 s integration; resolution 16 cm-I. The spectra have been ratioed to the background electrolyte spectrum and subjected to seven point Savitsky-Golay quadratic smooth.
kV (-200 V/cm) and after 5 min was reduced to -2.3 kV (-50 V/cm) . Reagents. All solutions were prepared from ACS reagent grade materials using Type I deionized water. Potassium chloride, sodium sulfate (Baker), 4morpholinepropanesulfonicacid (MOPS; Sigma), sodium phosphate monobasic (Fisher), and sodium phosphate dibasic heptahydrate (Malliickrodt) were used as received. All solutions were adjusted to pH 7.5 with 0.1 M sodium hydroxide. A phosphate buffer of pH 7.5 was used to ensure full ionization of adenosines. Serial dilutions of ATP, ADP and AMP (Sigma) were used to reach the working sample concentration M. range, 5 x 10-4-5 x RESULTS AND DISCUSSION
Figure 2 shows the 1 s integration Raman spectra of 0.1 M solutions of ATP, ADP, and AMP contained in electrophoresis 50 pm i.d capillaries. We discuss the strongest bands, using the assignment of Lanir and Yu.zO All three ribonucleotides have adenine ring breathing bands at 720 cm-I and sugar bands at 1340, 1415, and 1510 cm-'. The differences in the spectra arise from the different degrees of condensation in the phosphate groups. The phosphodiester band appears at 1131 cm-' in ATP and at 1115 cm-I in ADP. It is absent in AMP. The 1100-1500 cm-' region can be used to distinguish between the otherwise spectrally similar ATP and ADP. AMP is readily identified by the wellresolved triplet in the 1300-1400 cm-' region. Figure 3 shows the sequence of Raman spectra (1.2 s intervals) during elution of an ITP-concentrated mixture of 5.0 x M ATP/ADP/AMP in 0.01 M phosphate buffer (PH 7.5). The LE (20) Lanir, A; Yu, N.-T. j . Biol. Chem. 1979,254, 5882-5886.
1
3
s .-,x
Time (Sec)
Raman Shift (cm-1) Figure 4. Expanded view of the phosphodiester peaks of ATP (1131 cm-l) and ADP (1115 cm-I) from Figure 3.
was 0.1 M KCl and the TE was 0.1 M MOPS, both adjusted to pH 7.5 with 0.1 M sodium hydroxide. The order of elution is ATP, ADP, AMP. The Raman spectra of the three nucleotides are clearly visible, as comparisonwith Figure 2 demonstrates.The boundary between ADP and AMP at 345 s is visually striking. Because of the limitations of the stacked plot format, the boundary between ATP and ADP at 340 s is less evident on casual inspection. The ATP/ADP boundary at 340 s is clear in the expanded view of Figure 4. With the limited temporal resolution of our instrument, we find no spectrum in which bands of the two nucleotides are present together. This finding implies that the transition regions are short compared to the 600 pm traveled in 1 s. Analytical Chemistry, Vol. 67, No. 23, December 7, 1995
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Table 1. Ribonucleotide Detection Window Concentrations and Stacking Factors
concn (M) time (s)
measd stacking factor
(A)Initial Concentrations: 5 x ATP ADP AMP
330-340 340-345 345-361 1072- 1108 1108- 1113
1.7 x lo-* 4.5 x 10-2 1.1x 10-2
295-307 380-315
1iw
5 10-3 1.5 x lo-*
360x 980x
l3iM
1
4w
3.3 x 10-2 4.7 x 10-2 8.5 x lo-*
M ATP,ADP and A M P
1000x 3000x
(C) Initial Concentration: 5 x 10-j M ATP and ADP, 2 x
ATP ADP
theor
M ATP,ADP,and A M P
340 x 900x 220x
(B) Initial Concentrations: 5 x
ATP/ADP AMP
measd
3.3 x 10-2/4.7 x lo-* 8.5 x
M Fluorescein 1.8 x 4.9 x 10-2
6.6 x 9.4 x 10-2
15w
Raman Shift (cm-1) M ATP, ADP, and AMP preconcentrated by isotachophoresis: injection time 80 s at -9.0 Figure 5. On-capillary Raman spectra of 5 x kV; 680 mW 532 nm excitation; 1 s integration; resolution 8 cm-I. Other conditions as in Figure 3.
To estimate the ion concentrations in the stacked zones, we calibrated our on-line spectra against Raman spectra of known solutions of each nucleotide taken in the measurement capillary. The concentrations of each nucleotide and stacking factor at the detection window are shown in Table 1. The theoretical stacking factors are calculated using the mobility data of Pospichal et aLZ1 The concentration of ADP, 4.5 x M, is 900 times greater than its starting concentration. The agreement with the 4.7 x M predicted by eq 1is striking, but probably fortuitous. More typical is the stacking of ATP to 1.7 x M, almost half the theoretical concentration and AMP and 1.1x M, stacked to only -13% of the theoretical concentration. To some extent these anomalies may be a consequence of errors in the measured mobilities. In the case of AMP, the stacking is almost certainly incomplete at the time that the ion passes under the microscope objective. Incomplete preconcentration is also inferred from the fact that the time/concentration products of the three zones are equal to within -20%. The cylindrical capillary acts as a strong diverging cylinder lens. Cylindricity causes loss of approximately half of the Raman (21) Pospichal, J.: Gebauer, P.: Bocek, P. Chem. Rev. 1989,89,419-430.
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scatter which might otherwise be captured by the microscope objective. The spectra we observe in rectangular capillaries are approximately twice as strong as those we observe in the conventional cylindrical capillaries. We have continued to use the cylindrical capillaries simply because they are more convenient than rectangular capillaries despite the optical inefficiency. Figure 5 shows the sequence of Raman spectra obtained (1.2 s intervals) during elution of an ITP-concentrated mixture of 5.0 x M ATP/ADP/AMP. The data were taken at a field strength of -50 V/cm. As noted in the Experimental Section, running voltage was reduced 5 min after sample introduction to make the zone widths large enough to be easily measurable. The combination of lower electric field strength and a longer capillary increased the running time from -6 to -18 min. In addition, the distance from the capillary entrance to the detection window was 30 cm. A 45 cm capillary was used to obtain the data. The boundary between ADP and AMP is readily visible in Figure 5. But at the achieved signahoise ratio and resolution, the 1131cm-' band at ATP and the 1115cm-' band of ADP cannot be reliably distinguished. It is most likely that the ribonucleotide concentrations are too low in the stacked zones. With our existing
Raman Shift (cm-1) Figure 6. On-capillaryRaman spectra of 5 x 10-5 M ATP, 5 x 10-5 M ADP, and 2 x 10-8 M fluorescein preconcentrated by isotachophoresis. Conditions as in Figure 2.
equipment, good signal/noise ratio with 1s integration requires that ribonucleotides be stacked to close to 2 x 10-2 M. The detected nucleotide concentrations and the preconcentration factors at the detection window are shown in Table 1. At the detection window, AMP is concentrated by a factor of 3000. The concentration and stacking factor for ATP/ADP is a p proximate. Even so, it is clear that ITF' preconcentration allows normal unenhanced Raman spectroscopy of adenosine phosphates from starting concentrations normally associated with surfaceenhanced Raman spectroscopy. Capillary isotachophoresis is also a useful and convenient cleanup technique for obtaining Raman spectroscopy of samples containing luminescent impurities. When working with slightly contaminated ribonucleotides or other materials, we have routinely observed that luminescence is spatially segregated from Raman scatter. Typically one or more zones of featureless luminescence spectra of contaminants appear, while the analyte spectra themselves have flat baselines. To provide a more rigorous test of spatial rejection of fluorescence,we spiked an ATP/ADP mixture with fluorescein. Figure 6 presents the sequence of Raman spectra and fluorescence spectra obtained from a solution originally 5.0 x M ATP, 5.0 x M ADP, and 2.0 x M fluorescein in 0.01 M phosphate buffer @H 7.5). The LE was 0.1 M Na2S04,and the TE was 0.1 M MOPS. The inset shows the full-scaleresponse of the detector, using the same data. Saturation of the detector is at 64K counts. The fluorescein emission must therefore be more than 1000 times more intense than the strongest Raman bands in the ATP or ADP spectra. Despite this fact, fluorescence contaminates only three ADP Raman spectra immediately precedingthe main fluorescence spectrum, and on only two is fluorescence more intense than
Raman scatter. On no other ADP or AMP Raman spectrum was fluorescein emission even measurable. CONCLUSION We have demonstrated that with ITP preconcentration normal Raman spectra are obtainable from solutions of adenosines originally at micromolar concentrations. Similar results can be expected with other ions. Even lower concentrations may be detectable if the isotachophoretic separation is refined. Of course, surfaceenhanced Raman spectroscopy and resonanceenhanced Raman spectroscopyprovide routes to nanomolar concentrations, perhaps lower. The spatial resolution of a Raman microprobe is better than 1 pm. In the systems we have examined, the zones are sufiiciently long that spatial separation was not needed. If necessary, transient isotachophoresis and subsequent zone electrophoresis can he used to spatially separate zones containing ions whose spectra are similar. Even in cases where multicomponent separationsare not needed, ITP/Raman spectroscopy or CE/Raman spectroscopy may provide a convenient alternative to near-infrared excitation for Raman spectroscopy of ionic materials containingfluorescent impurities. Experimentally, ITP and CE are easily coupled to a Raman microprobe. It is only necessary to keep the capillary stationary and, as in any electrophoresis experiment. to isolate the highvoltage portions of the system. There are no restrictions on buffer composition or concentration. ITP/Raman spectroscopy and CE/ Raman spectroscopy appear to be well-adapted to the emerging microchip formats?* The microchip is essentially a modified (22) Harrison, D. J.: Manz. A Fan. Z.: Ludi, H.: Widmer, H. M.Anal Chem. 1992.64. 1926-1932.
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microscope slide. Its flat surfaces would automatically double the collection efficiency observed in cylindrical capillaries. That improvement alone would allow operation with reduced laser power, better time resolution, or both. ACKNOWLEDGMENT
Financial support was provided by NIH Grant GM-37006. We thank Kaiser Optical Systems, Inc. for the consignment of the
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Raman spectrograph. Michael Navin provided many useful insights into the electrophoretic aspects of this work. Received for review August 2, 1995. Accepted September 13, 1995.e AC950776W Abstract published in Advance ACS Abstracts, November 1, 1995.
