Anal. Chem. 1990, 62, 2193-2198
not achieved for all the peaks, the separation efficiency for the most intense peak in Figure 4B corresponds to approximately 200 000 theoretical plates. Shown in Figure 5 is the electropherogram for Jeffamine ED-2001. The mass spectrum for the polymer (not shown) indicates a broad distribution of oligomers with molecular weights ranging from 1400 to 2400. Although the mass spectra peaks are also separated by 14 amu, it is apparent from Figure 5 that most of the oligomers were eluted together in the CZE experiment. Better resolution of these higher molecular weight components might be possible through the use of gels, which can more easily discriminate between higher molecular weight species (32). ACKNOWLEDGMENT The loan of a capillary electrophoresis system (Model 270A) by Applied Biosystems, Inc., and helpful conversations with Michael Albin and Steve Moring are gratefully acknowledged. LITERATURE C I T E D (1) Bear, G. R. J. Chromatogr. 1988,459, 91-107. (2) Desbene, P. L.; Desmazieres, B.; Basselier, J. J.; DesbensMonvernay, A. J. Chromatogr. 1989,461, 305-313. (3) Aserin, A.; Gartl. N.; Frenkei, M. J. Li9. Chromatogr. 1984, 7 (E), 1545-1557. (4) Ernst, J. M. Anal. Chem. 1984,56, 834-835. (5) Tsygankov, A. Y.: Motorin, Y. A.; Wolfson, A. D.; Kirpotin. B. D.; Oviovsky, A. F. J. Chromatogr. 1989,465,325-329. (6) Okada, T. Anal. Chem. 1990,62, 327-331. (7) Chester, T. L. J. Chromatogr. 1984,299, 424-434. (8)Richter, B. E. HRC CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1985,8 , 297-300. (9) Ashrafkhorssani, M.: Taylor, L. T. LC-GC 1990,8 (4), 314-320. (10) Paveiich, W. A.; Livigni, R. A. J. Po/ym. Sci., Part C 1968, 21, 215-223. (11) Garcia-Rubio, L. H.: McCregor, J. F.; Hamielec, A. E. Po/ymer Characterization;Craver, C. D., Ed.; American Chemical Society: Washington, DC, 1983;pp 31 1-344.
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(12) Giddings, J. C.; Graff, K. A.; Caldwell, K. D.; Myers, M. N. Po/ymer Characterization; Craver, C. D., Ed.; American Chemical Society: Washington, DC, 1983;pp 257-269. (13) Otsuki, A.; Shiraishi, H. Anal. Chem. 1979,57, 2329-2332. (14) Shiraishi, H.; Otsuki, A.; Fuwa, K. Bull. Chem. SOC.Jpn. 1982, 55, 1410- 1415. (15) Nuwaysir, L. M.; Wilkins, C. L.; Simonsick. W. J., Jr. J. Am. SOC. Mass Spectrom. 1990, 1, 66-71. (16) Siegel, M. M.; Tsao, R.; Oppenheimer, S.; Chang, T. T. Anal. Chem. 1990,62,322-327. (17) Jorgenson, J. W.; Lukacs. K. D. Science 1983,222, 260-272. (18) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981,278,209-216. (19) Kuhr, W. G. Anal. Chem. 1990,62, 404R-414R. (20) Lukacs, K. D.; Jorgenson, J. W. HRC CC, J. High Res. Chromatogr. Chromatogr. Commun. 1985,8 , 407-411. (21) Kuhr, W. G.; Yeung, E. S. Anal. Chem. l98& 6 0 , 1832-1834. (22) Vanorman, B. B.; McIntire, G. L. J. Microcolumn Sep. 1989, 1 (6), 289-293. (23) Pretka, J. E. (E. I. du Pont de Nemours & Co.) U S . Patent 3,021,232, Feb. 13, 1962. (24) Technical Report, Texaco Chemical Co.,Texas USA. (25) Grossman, P. D.; Lauer, H. H.; Moring, E. S.; Mead, D. E.;Oldham, F. M.; Nickel, J. H.; Gouberg, J. R. P.; Krever, A.; Ranson, D. H.; Coiburn, J. C. Am. Biotechnol. Lab. 1990,8(2), 35-43. (26) Moring, E. S.; Colburn, J. C.; Grossman, P. D.; Lauer, H. H. LC-GC 1990. 8 .111. 34-46. (27) Perrin, D. b.: Dempsey, B. Buffers for pH and Metal Ion Control; ChaDman and Hall: London. 1974. (28) Roach, M. C.; Harmony, M. 'D. Anal. Chem. 1987, 59, 411-415. (29) Carlson, R. G.; Srinivasachar, K.; Givens, R. S.; and B. K. Matuszewski, B. K. J. Org Chem. 1986,51,3978-3987. (30) Dissociation Constants of Organic Bases In Aqueous Solutions ; Perrin, D. D.. Dalzeli, D., Eds.; Butterworths: London, 1965. (31) Rose, J. D., Jr.; Jorgenson, J. W. J. Chromatogr. 1988, 447, 117-131. (32) Cohen, A. S.; Najarian, D. R.; Pauius, A.; A. Guttman, A,; Smith, J. A.; Karger, B. L. Proc. Natl. Acad. Sci. U . S . A . 1988,85,9660-9663.
....
RECEIVED for review May 10, 1990. Accepted July 16, 1990. This work was supported, in part, by the Society for Analytical Chemists of Pittsburgh, the National Science Foundation (Grant No. CHE-8957394), and the Office of Naval Research (Grant No. N00014-89-5-3071).
Absorption Detection in Capillary Electrophoresis by Fluorescence Energy Transfer Tommy W. G a r n e r a n d E d w a r d S.Yeung*
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011
Normally, only highly fluorescent materials can be detected at low concentratlons In the small detectlon volumes typical of caplllary electrophoresls In a laser-based fluorometer. We report here the detectlon of absorbing but nonfluoresclng analytes by laser-excited fluorescence. This Is possible If the excited analytes transfer thelr energy to a fluorescent addltlve In the running buffer to Increase the backgroundfluorescence level. Two different fluorophores and four different absorbing analytes were tested In thls detectlon scheme. Concentrations as low as 6 X lo-' M and amounts as small as 4 amol at lnjectlon are detectable. The data support a long-range energy-transfer scheme, but the transfer efflclency Is much larger than those reported for other donor-acceptor palrs.
Recent research efforts in microcolumn separations, particularly in capillary electrophoresis (CE) (1,2) and in open tubular capillary liquid chromatography (OTCLC) (3-8),have
* To w h o m correspondence should b e addressed.
shown that very high separation efficiencies can be obtained. I t appears that 2-10 pm i.d. open tubes provide the best performance. In CE, these small diameters restrict the current flow and thus reduce the amount of band broadening induced by joule heating. In OTCLC, small diameters are needed to maximize the interaction with the stationary phase. On the other hand, the small elution volumes and the short optical path lengths available across these columns put severe restrictions on the selection of detection schemes for the analytes. The easiest way to detect analytes in capillary columns is by UV absorption (9-12). At 210 nm or so, most organic analytes have reasonable molar absorptivities. Indeed, the many commercial capillary electrophoresis systems on the market all rely on this detection mode. However, if one assumes an absorbance detection limit of 5 X for the detector, for an analyte with a fairly high molar absorptivity of lo4 L mol-l cm-', the concentration limit of detection (LOD) at the detector can be estimated to range only from 5 X to 1 X M for path lengths of 10-50 pm, respectively. In fact, as one goes to small columns, the decreasing amount of light available through the column degrades the absorbance
0003-2700/90/0362-2193$02.50/00 1990 American Chemical Society
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LOD. The sensitivity of absorption detection can be enhanced by sophisticated techniques such as photothermal deflection spectrometry (13,14). The LOD is in the range 5-50 x M for dabsyl-amino acids injected into 50-pm columns (14). Very impressive detection limits have been achieved by laser-excited fluorescence (1.516) and by electrochemistry (17, 18). The order of M seems to be quite typical. One requires that the analyte fluoresce or be electrochemically active, respectively. For more universal applications, conductivity (19) and indirect fluorescence (20-23) have been developed. Conductivity has been shown to work in 50-pm capillaries, and indirect fluorescence has been shown to work in even 5-pm capillaries. The latter is based on the displacement of a fluorescing ion present in the electrophoretic buffer to create a negative-going peak. Concentrations down to loe7M or amounts to 50 amol have been detected. The relative ease with which laser-excited fluorescence can be implemented for on-column detection in small capillaries raises the question whether there exist other detection schemes that are variations of direct fluorescence detection. The simplest idea is to monitor the fluorescence intensity of an additive rather than the transmitted light intensity for absorption detection. As long as the analyte absorbs at either the excitation or the emission wavelength, a signal is generated. This is similar to the use of fluorescent thin-layer chromatographic plates ( 2 4 ) ,but the difficulties in achieving stable fluorescence levels and the extremely short path lengths prevent one from taking advantage of this scheme in capillaries. A special situation arises when the analyte quenches the fluorescence of the additive (25).but the interaction is nonlinear and not very general. In this work, we explore the use of energy transfer as a detection scheme in capillary electrophoresis. A fluorophore is incorporated in the electrophoresis buffer a t low concentrations. A steady fluorescence background is thus present at the detector all the time. When an absorbing analyte passes into the detector region, additional light energy will be extracted from the excitation laser beam. Normally, this extra excitation will be lost if the absorbing analyte does not fluoresce. However, energy (excitation) transfer can occur between the absorbing analyte and the fluorophore in the buffer. The fluorophore therefore receives additional excitation to produce a higher level of fluorescence, i.e. a positive peak. Detection is actually based on absorption by the analyte and not by converting the analyte to a fluorescent derivative. This then allows one to use the identical laser detector (21) to monitor fluorescing analytes (direct fluorescence), charged analytes with no absorption or fluorescence (indirect fluorescence or displacement), or absorbing but nonfluorescing analytes (energy transfer).
EXPERIMENTAL SECTION The CZE (capillary zone electrophoresis) system is similar to that described previously (21). A high-voltage power ( S P e l h " Plainview, NY; Model UHR50PN50) was used to supply the electromotive force across the capillary. The anodic high-voltage end of the capillary was isolated in a Plexiglas box for operator safety, while the cathodic end was held at g m . d potential. Injections were made for 1 s at 30 kV,and the voltage was held at 30 kV throughout each run. The capillary columns (Polymicro Technologies, Phoenix, AZ) varied in length from 63 to 70 cm. The inner diameter of the columns were all 21 pm, while the outer diameters were 150 pm. The polymer coating was burned off 10 cm from the cathodic end of the capillary t o form the observation region. An argon ion taser (Control Laser Corp., Orlando, CA: Model 554143 operating at 488 nm with 15-mw power was used for excitation. The laser beam was stabilized to within 0.04% with a laser power stabilizer (Cambridge Institute, Cambridge, MA; Model LS100). The laser beam was focused into the capillary with a 1 cm focal length lens. The capillary was mounted at
I CqOH
Fluorescein
Riboflavin
(Jb2 / Orange
Cresol Red
G
Dabsyl Amino Acids
Figure 1. study.
Structures of absorbers (A) and fluorophores (F) used in this
Brewster's angle to reduce scattered radiation. The fluorescence was collected at 90" with a 20X microscope objective. The fluorescence image was focused onto a PMT photomultiplier tube (Hamamatau, Middlesex, NJ; Model R928). Stray and scattered radiation were rejected by two spatial filters preceding the PMT. The fluorescence was further isolated with two color filters (Corning Glass, Corning, NY; Model 3-69). The PMT voltage was adjusted to maintain a 0.25-pA background current, which was monitored with a picoammeter (Keithley, Cleveland, OH; Model 417). The output from the picoammeter was recorded on a chart recorder (Fisher Series 5OOO) or on a personal computer after analog to digital conversion (Data Translations, Marborough, MA; Model DT2827). Digital data were smoothed with a smoothing routine based on the Savitsky-Golay smoothing algorithm. A 2-s time constant was added at the picoammeter. Reagents. The fluorescein used was laser grade (Eastman Kodak Co., Rochester, NY), while all other chemicals were reagent 1 were prepared in deionized water (Millipore grade. ~ 1 buffers Gorp., Bedford, MA; Mil1i-Q System) and purged with nitrogen to carbon dioxide. The two dabsyl-amino acids were prepared by a standard method (26).AU analytes were prepared in buffer solution. The fluorophores used in this work are fluorescein and riboflavin, and the absorbing used are cresol (CR), orange (oG), dabyl-phenyl&ine (DAB-phe) and dabsyl-glutamine (DAB-Glu). Their chemical structures are shown in Figure 1.
RESULTS Normally, we use fluorescence spectrometry for the determination of analytes that "fluoresce". Clearly, every electronic transition that allows absorption also allows fluorescence. It is merely a matter of how much competition there is for the excited state to relax nonradiatively. When the fluorescence quantum yield, aPn, is less than we normally will not select fluorometry as the detection method.
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a, 0
1
C a, 0 v)
;
-3 LL
3
7
5
9
Time (min) Figure 3. Separation of DAB-Phe and DAB-GIu with and without fluorescein in the buffer: (a) 10 mM PO, buffer (pH 9.0);(b) 10 mM PO, and 1 X lo-' M fluorescein buffer (pH 9.0).The amino acids are at approximately 1 X lo-' M.
Also, in the standard fluorometer, it is difficult to distinguish between the observation of a weak fluorescence signal and a strong resonance Raman signal. The fact is, one can often detect "nonfluorescent" analytes by laser-excited fluorescence. This is illustrated in Figure 2a, which is the capillary electropherogram of the dyes CR and OG. For comparison purposes, a trace of fluorescein is also added to the sample. The native fluorescence yield of CR and OG is therefore some 4 orders of magnitude lower than that of fluorescein. The limits of detection for the two dyes are 1.5 X lo4 and 5.6 X lo4 M, respectively, which is comparable to or slightly better than the best one can do by monitoring absorption across these narrow capillaries. Since the resonance Raman spectra of CR and OG are not known, we cannot estimate what fraction of the signal in Figure 2a is actually due to fluorescence. In any case, this represents an unusual scheme for detecting nonfluorescent analytee in small capillaries. On the addition of a small concentration of fluorescein in the running buffer, the same analytes produced the electropherogram in Figure 2b. Note that the concentrations of CR and OG have been lowered by a factor of 200. Depicted is a substantial enhancement in the fluorescence signal from CR and OG. The baseline is more noisy because there is now a constant large background fluorescence due to the fluorophore added to the buffer. An offset has also been applied to the electropherogram in Figure 2b. The enhancement of the native emission from absorbing but nonfluorescing compounds opens up the possibility of detecting them in small capillaries with good sensitivity. An important question is how general this phenomenon is and therefore how broadly applicable the detection scheme might be. We tested four different analytes and two different
Table I. Influence of Fluorophore on Enhancement Factors and Detection Limits enhancement factor fluorescein riboflavin
phore cresol red orange G DAB-Phe DAB-Glu a Buffer:
280" 330" 290b 310b
40' 140~
LOD,M fluorescein
riboflavin
6X 3 x io-sa 3.4 x 10-7* 2.5 x 10-7*
2X 5 x 10-7c
10 mM PO4 and 2 X
PO4 and 1 X
fluorescein. bBuffer: 10 mM fluorescein. cBuffer: 10 mM PO, and 2 X lo6
riboflavin.
fluorophores in such a detection mode. Figure 3 shows the electropherogram of two dabsyl-amino acids in a plain buffer (Figure 3a) and then in one with a trace of fluorescein added (Figure 3b). Note that the samples are at the same concentration but there is a scale change in the two parts of Figure 3. The results are essentially identical with those in Figure 2. The dabsyl derivatives normally are used for the absorption detection of the amino acids but are detected here by the enhanced fluorescence emission. Similar results are obtained when riboflavin is used instead of fluorescein as the trace additive. A direct comparison is given in Table I. The enhancement factor is defined to be the ratio of peak heights for each analyte in the presence vs in the absence of the added fluorophore. Peak areas were not used because of the influence of retention times (migration velocity past the detector) and because of baseline noise. Table I shows generally large enhancement factors, which are beneficial to analytical applications. The enhancement factors for the analytes are surprisingly similar for a given fluorophore. This must be con-
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sidered fortuitous since the native emission efficiencies (weak fluorescence or resonance Raman) of the analytes are highly variable. A different comparison among the test compounds is also given in Table I, which lists the LOD for each analyte at the individually optimized fluorophore concentrations. Optimization was not universal because larger fluorescence background levels produce larger absolute noise levels, even though the dynamic reserve (baseline stability, refs 20 and 21) is held constant. The enhancement factors, on the other hand, depend on the fluorophore concentration; vide infra. The best LOD is for CR with fluorescein as the additive. The 6 x M (injected) level is substantially better than that detected with standard absorption and is comparable to that from photothermal methods in larger capillaries (14). The mass LOD is 4 amol on the basis of an estimated injection volume of 9.6 nL. The LOD is not nearly as impressive as regular fluorescence detection (e.g. dansyl-amino acids), but there is no reason to expect comparable LODs. The LOD is also better than indirect fluorescence detection via charge displacement (21)by 1 order of magnitude. There is no simple relationship between the LOD and the molar absorptivities of the analytes. We independently measured these as dissolved in the running buffer at 488 nm and found that t(CR) = 1.15 x lo4, t ( 0 G ) = 2.45 X lo4, and c(dabsy1) = 2 x lo4 L mol-' cm-'. By direct injection of a mixture of fluorescein and riboflavin in a clear 10 mM PO4 buffer, the relative excitation-fluorescence efficiency was found to be 5 times higher for the former. This is consistent with the poorer LOD when riboflavin was used as the fluorophore.
DISCUSSION The analytes and fluorophores were selected for this work on the basis of their similarity in molar absorptivities and fluorescence characteristics, respectively. However, they represent very different structural classes (Figure 1) and electronic configurations. Included are both ionic and neutral species at the pH used. This indicates that the observations are not peculiar to certain chromophores or specific resonance structures. Naturally, more studies are needed to confirm the generality of this fluorescence enhancement effect. The difficulty in finding relatively nonfluorescent but strongly absorbing analytes at the wavelength used limits the present data set. Extension of these results to the UV region will shed new light on the structural requirements for the analytes and the fluorophores for this phenomenon. Even though it will require extensive work to establish a clear mechanism, there is sufficient information to draw some conclusions based on the observations presented here. The enhanced emission is a direct result of absorption by the analyte, since the injection of nonabsorbing analytes did not produce any peaks, even at high concentrations. The mechanism is not related to indirect fluorescence detection (20,21) although the experimental arrangement is almost identical. The reason is that both neutral fluorophores and neutral analytes can produce enhancement. Displacement by charge was necessary in indirect fluorescence detection. The running buffer used here is also at substantially higher concentrations and the fluorophores are at substantially lower concentrations than those needed for sensitive indirect fluorescence detection. A corollary is that one does not need to deviate from the standard electrophoresis conditions to produce enhanced emission. The scheme should therefore also be applicable to other systems, such as open tubular capillary liquid chromatography. We can also rule out specific compound formation or ion pairing between the analytes and the fluorophores on the basis of the applicability to diverse structural classes. The retention times of the analyte peaks also remained constant (taking into account the slight variability
of electroosmotic flow rates for different capillaries and different pH) regardless of whether the fluorophore is present or not. Furthermore, the enhancement was found to be a monotonic function of concentration, with no noticeable stoichiometric constraints. Finally, for samples containing a mixture of CR, OG, and fluorescein, the latter produces peak heights and retention times identical with those of the samples containing fluorescein alone. The data are consistent with an energy-transfer mechanism whereby the excitation in the analyte, A, is transferred to the fluorophore, F, enhancing its fluorescence. This is analogous to fluorescence quenching, in which a nonfluorescing additive competes for the excitation of a fluorescing analyte. Here, a fluorescing additive competes for the excitation of a nonfluorescing analyte. Energy transfer has been studied extensively before (ref 27 and references therein). Almost all reported work involves molecules in the millimolar range and up. The potential for using energy transfer to detect nonfluorescing species at the submicromolar level has not been suggested previously. This is because instrumentally it is difficult to maintain a steady background fluorescence so that small enhancements can be recorded. In fact, it is our ability to stabilize the large background fluorescence level (created by the added fluorophore) that led to the impressive detection limits listed in Table I. Clearly, one cannot simply insert and replace a sample cell in a fluorometer to look for small differences in signal levels. Also, if the fluorophore is a t the millimolar level, one cannot expect to detect micromolar levels of an analyte even if the energy transfer is 100% efficient. The fractional change in signal will be too small. There is one literature precedent for energy transfer at micromolar concentrations (28), but that is due to complexation between the donor and the acceptor, drawing them much closer together than the concentrations would suggest. In the diffusion-controlled regime one has the equivalence of Stern-Volmer kinetics (29). A possible scheme is k
A &k-1 A*
-
+ A 2A A* + ~ k 3F* . +A A*
k2
(2)
(3)
where the asterisks represent the excited forms of the two species. kl is the rate of excitation of the analyte, k..l is the nonradiative decay rate of A*, k2 is the self-quenching rate of A, k 3 is the rate of excitation transfer to the fluorophore. On the basis of our experience with fluorescein as a mobilephase additive in indirect fluorescence detection, quenching of F* by analytes (reverse of k 3 ) is unlikely at concentrations below 1 mM. So, the rate of formation of the excited analyte is d[A*]/dt = k,[A] - k-,[A*] - k,[A*][A] - k,[A*][F] (4) Using the steady-state approximation, one obtains
What is observed is proportional to [F*]. Equation 3 gives relative signal = k,[A*][F]
For a fiied fluorophore concentration, one can see that at high analyte concentrations &[A] >> (k3[F] + k1)), the enhanced emission reaches a maximum value. At low analyte concen-
ANALYTICAL CHEMISTRY, VOL. 62, NO. 20, OCTOBER 15, 1990
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Flgure 4. Peak height as a function of analyte concentration in 10 mM PO, and 2 X lo-’ M fluorescein buffer (pH 9.0): (top) CR; (bottom) OG. The curves extend to lo4 M, where the intensities are CR = 2.0 and OG = 1.13. 70
~-
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Figure 5. Peak height as a function of fluorescein concentration in 10 mM PO, and fluorescein buffer (pH 9.0). M of CR (0)and 3 X M of OG (+) were Injected in each case.
trations ((k,[F] + k - J >> k,[A]), the enhanced emission intensity is proportional to the analyte concentration. Figure 4 shows the experimentally observed concentration dependence of the enhanced emission for the two dyes CR and OG. To provide a valid comparison, a fixed concentration of fluorescein in the sample mixture is used as an internal standard. For clarity, we did not plot the data points corresponding to 1 X M CR and OG, which are at 2.0 and 1.13, respectively. Figure 4 shows that the data fall within the nonlinear region between the two extremes, although the lowest values are in the linear region and the highest value of CR is approaching a maximum. The above energy-transfer scheme also predicts that for a fixed analyte concentration, the enhanced emission intensity should be independent of the fluorophore concentration as long as it is large (k,[F] >> (k2[A]+ k-J). When the fluorophore concentration is low (k,[F]
