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Anal. Chem. 1092, 64, 2465-2468
Synchronization of Timing in Chemiluminescence Thin-Layer Chromatographic System by Coupling Pneumatic Nebulization with Optical Fiber-Based Detection Nian Wu and Carmen W. Huie' Department of Chemistry, State University of New York at Binghamton, Binghamton, New York 13902-6000
INTRODUCTION The peroxyoxalate chemiluminescence (CL) reaction involving the oxidation of oxalate derivatives, typically bis(2,4,6-trichlorophenyl)oxalate(TCPO) or bis(2,4dinitropheny1)oxalate (DNPO),in the presence of a sensitizer is among the most efficient nonenzymatic reactions known and has been extensively investigated for the postcolumn detection of various fluorophores or compounds derivatized with fluorescent labels in high-performance liquid chromatography (HPLC). For example, peroxyoxalate CL detection in HPLC has been demonstrated to be highly sensitive and selective for the determination of catecholamines,' secondary and tertiary amines,2J polycyclic aromatic amines and hydrocarbons,4,6 steroids? and carboxylic acids.7 In addition, HPLC determination of compounds such as choline and acetylcholine using an immobilized enzyme reactor followed by peroxyoxalate CL detection has also been reported.8 Recently, a number of dansyl amino acids have been used as model systems for the optimization of CL detection in HPLC based on the oxidation of TCPO or DNPO, and the average detection limit and relative standard deviation on reproducibility were found to be about 200 fmol(O.07 ng) and 4 ?6 , respectively.9 In contrast, very little work has been reported on the use of CL detection methods for the determinationof analytes after separation by thin-layer chromatography (TLC). The reason for this is perhaps in large part related to experimental difficulties encountered in the synchronization of timing between the introduction of the CL reagents onto the TLC plates and detection of the rapidly decaying CL emission generated from the analytes adsorbed on the plate surface. This particular timing problem, however, can be overcome with relative ease in HPLC since all experimental parameters can be controlled with good accuracy.9 The use of CL detection in TLC was first demonstrated by Curtis and Seitz involvingthe measurement of various dansyl amino acids by successively spraying the plate with TCPO and HzOz solutions and subsequently collecting the CL emission with an optical fiber-based detection system.10 Although in general this CL detection method was shown to be comparable to conventional fluorescence detection, it was found that the CL intensity changed rapidly with time under optimized conditions, leading to loss in sensitivity during a complete scan of the TLC plate. To minimize this problem, (1)Kobayashi,S.; Sekino,J.; Honda,K.; Imai, K. Anal. Biochem. 1981, 122,99-104. (2) DeJong, G. J.; Lammers, N.; Spruit, F. J.; Brinkman, U. A. Th.; Frei, R. W. Chromatographia 1984,18,12%133. (3)Kwakman, P.J. M.; Brinkman, U. A. Th.; Frei, R. W.; DeJong, G. L.; Spruit, F. J.; Lammers, N.; Van Den Berg, J. Chromatographia 1987, 24, 395-399. (4)Sigvardson, K. W.; Birka, J. W. Anal. Chem. 1983,55,432-435. (5) Sigvardson, K. W.; Kennish, J. M.; Birks, J. W. Anal. Chem. 1984, 56. 1096-1102. ~ - - ~ ~ (6)Koziol, T.; Grayeeki, M. L.; Weinberger, R. J. Chromatogr. 1984, - - !
---. (7) Grayeski, M. L.; DeVasto, J. K. Anal. Chem. 1987,58,1203-1206.
31 7. 355-366. ---
(8)Van Zoonen, P.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. J.Pharm. Biomed. Anal. 1987,5,485-492. (9)Baeyens, W.;Bruggeman, J.; Dewaele, C.; Lin, B.; Imai, K. J. Biolumin. Chemilumin. 1990,5 , 13-23. (10)Curtis, T.G.; Seitz, W. R. J. Chromatogr. 1977,134,343-350.
0003-2700/92/0364-2465$03.00/0
CL detection was coupled with a vidicon rapid scanning spectrometer, since this detection system is capable of simultaneously measuring CL emission from all areas of the plate, thus reducing errors associated with the measurements of the rapidly changing CL signal." However, this modification introduced complexity and cost to the CL instrumentation, making it difficult, for example, in the application of CL detection methods for analyses in routine clinical laboratories. In this note a novel experimental setup involving the use of a pneumatic nebulizer to provide relatively uniform and reproducible spraying of the premixed peroxyoxalate CL reagents (TCPO and H202) onto the TLC plates and its coupling with an optical fiber to synchronize the timing between chemical excitation and measurement of CL emission is reported. Experimental parameters such as nebulization conditions, positioning and size of the optical fiber, microenvironment of the sample spot, scan speed, and particle size of the TLC plate were optimized to achieve sensitive and reproducible detection of three dansyl amino acids.
EXPERIMENTAL SECTION Apparatus. Figure 1 shows a diagram of the experimental setup. A commercially available TLC scanner (CAMAG) was modified to serve as a precision two-dimensional stage for scanningthe TLC plate in the x-y directions. Situated at a fixed position directly above the translational stage was a pneumatic nebulizer mounted in series with an optical fiber along the x-direction using an aluminum plate (dimensions 1.5 X 6 cmz), which was designed to allow for adjustment of distance between the nebulizer and optical fiber and of their respective heights from the TLC plate. Pneumatic nebulization provides a means by which a steady flow of reactive aerosol is sprayed onto the sample spots that are traveling directly beneath the nebulizer along the x-direction; the optical fiber allows for CL emission generated from the sample spots to be rapidly and reproducibly transported to the detection system in synchronization with chemical excitation. A concentric pneumatic nebulizer obtained from an atomic absorption spectrophotometer (Model 82-810, Jarrell Ash) was used for the generation of aerosol. As shown in Figure 1, appropriate concentrations of TCPO and HzOz solutions were placed in separate containers and the solutions were forced to move toward the direction of the nebulizer under the influence of pressurized argon gas. A gas flow controller obtained from a gas chromatograph (Model 376000, Varian) and a fine metering valve (Part No. 180502, PGC Scientifics) were placed between the gas flow controller and the CL reagent containers to allow for fine adjustment of the liquid flow rates of TCPO and HzOz. These two CL reagents were mixed within a stainless steel tee (V16-in. Swagelok),and this premixed solution was then delivered to the nebulizer via a 50-cm-long X 75-rm-inner-diameterfusedsilica capillary (CatalogNo. Tsy-075375,PolymicroTechnologies). After the premixed solution entered the nebulizer, an argon gas stream (nebulizinggas) with flow rate controlled by another gas flow controller interacted with the liquid stream of premixed CL solutions,resulting in the formation of aerosolwhich was sprayed onto the TLC plate. It should be noted that one or more reactive (11)Curtis, T.G.; Seitz, W. R. J. Chromatogr. 1977,134,513-516. 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
Flow
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Flgurs 1. Experlmental setup used for the CL detection of dansyl amlno acMs separated on HPTLC or TLC plates.
intermediates may be formed due to reaction of TCPO with H202.l' Two types of optical fibers were evaluated for the transport of CL emission to the detection system: an optical fiber bundle, 61-cm long X 1.6-mmdiameter with a numerical aperture of 0.55 and an acceptance angle of 68" (Part No. 77520, Oriel), and a single optical fiber, 50-cm long X 0.4-mm diameter with a numerical aperture of 0.22 and an acceptance angle of 47.2" (Superguide G, Fiberguide). One end of the optical fiber was placed adjacent to the nebulizer as shown in Figure 1 and the other end was interfaced to the detection system. The CL emission was isolated by a 10-nm band-pass filter centered at 520 nm (Corion) and was then detected by a photomultiplier tube (Model9558B,EMI) operated at a voltage between 700 and 800 V. The photocurrent was fed to a picoammeter (Model 7080, Oriel) and the signal was recorded on an integrator (Chromjet, Spectra-Physics). Optimization of Detection Sensitivity. A TLC plate was sprayed successively with a diluted dye solution of rhodamine B to completelywet the plate surface. The dye molecules adsorbed on the silica gel surface were then excited chemically using the aerosol aspirated from the nebulizer to determine the optimum flow rates for the CL reagent solutions and the nebulizing argon gas stream by adjusting the corresponding gas flow controllers and/or fine metering valve and monitoring the characteristics of the CL emission. It was found that at a pressure reading of 20 psi (flow rate: 6 L/min) and 3 psi (flow rate: 0.03 mL/min) for the nebulizing argon gas and CL reagent solution flows, respectively, the aerosol aspirated from the nebulizer appeared to produce a relatively strong and steady electronic signal. This CL emission can also be observed visuallyfrom the plate surface as very well-defined luminescence zones. Using this set of optimized flow conditions, the optimum distance between the nebulizer and optical fiber and their respective heights from the plate surface which give the highest CL intensity while maintaining good chromatographic resolution were also briefly investigated. It was found that, for the CL detection of three dansyl aminoacidsseparated on HPTLC plates (vide infra),the optimum distances between these experimental components were as follows: nebulizer and optical fiber = 8 mm; optical fiber and plate surface = 4 mm; tip of nebulizer and plate surface = 5 mm (corresponding to 15 mm from the aerosol exit orifice). Procedures. Development was carried out in either an acid solvent system of chloroform-ethyl acetate-methanol-acetic acid (9154.50.2) or a basic solvent system of methyl acetate-2(12)Alvarez, F. J.;Parekh,N. J.;Matuszemki,B.; Givens, R. S.; Hizuchi, T.; Schowen, R. L. J. Am. Chem. Sac. 1986,108,6435-6439.
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'Time (seconds) Flgurs 2. CL intensity-tlme curves of five sample spots a. equal amounts of dansylglycine (3.1 ng/spot) separated on the same HPTLC plate uslng the acM solvent system. The spots were detected (a) without prespraylng with any organic solvent and alter prespraylng with (b) ethyl acetate, (c) dioxane, (d) Trlon X-lOO/chloroform mixture (1:4 v/v), and (e) llquld paraffin/chloroform mixture (3:2 vlv). TCPO and H202concentrations were 3 mM and 0.88 M, respecthdy.
propanol-aqueous ammonia (9:7:2). The plates were air-dried for a few minutes at room temperature after development. If added, CL enhancers, i.e., various organic solvents, were presprayed onto the sample spots using a compressed gas sprayer (Catalog No. 14654,Alltech)prior to CL measurements. A handheld UV lamp was used to generate visible fluorescence from the sample spots to aid in the alignment of the sample spots separated on the plate along the x-direction with respect to the position of the nebulizer and optical fiber. Afterward, the plate was sprayed with the aerosol while being scanned along the x-direction at an optimum scan speed of 2 mm/s. Chemicals and Materials. TCPO was prepared by the procedure described by Mohan and Turro.13 Dansylglycine, dansyl-L-arginine,and dansyl-L-leucine were purchased from Sigma. Hydrogen peroxide (30%) and all other chemicals were obtained from Aldrich. The dansyl amino acids were dissolved in a 20% 0.04 M LiZCOs-HCl buffer of acetonitrile and kept at below 0 "C. Stock solutions of TCPO and HzOz with concentrations of 3-5 mmol/L in ethyl acetate and 0.88-1.2 mol/L in acetonitrile,respectively, were used. Separations were performed on either conventional silica gel TLC plates (Whatman LK6) or 5-pm silica gel HPTLC plates (Whatman LHP-K) which were purchased from Alltech.
RESULTS AND DISCUSSIONS Figure 2a shows a CL emission intensity-time profile of dansylglycine spotted on a HPTLC plate. It can be seen that the CL intensity reached ita maximum within - 5 s after the sample spot had been sprayed with the aerosol, followed by a rapid decay of the CL signal. This observation is consistent with those obtained by Curtis and Seitz who investigated the effects of TCPO and Hz02 concentrations on the CL intensity of dansylglycineadsorbed on silica gel TLC surfaces and found that at high peroxide concentrations, Le., 0.8-1.2 M, the CL intensity was the highest but lifetime was very short, and the converse was true for low peroxide concentrations.1° However, (13)Mohan, A. G.;Turro, N. J. J. Chem. Educ. 1974,51, 528-530.
ANALYTICAL CHEMISTRY, VOL. 84, NO. 20, OCTOBER 15, 1992
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Flguro 4. H p n C chromatogams of two dansyl amlno acMs (Arg = 4.9 ng of dansyk-arginkreand Qly = 4.3 ng of dan8yiglycIne)devebped In the basic solvent system and detected uelng (a) an optical fiber bundle and (b) a single optical fiber. TCPO and H202 concentrations were 3 mM and 0.88 M, respectlvely.
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Distance (cm) Flgwo S. HPTLC chromatograms of three dansyl amino aclds (Arg = 4.9 ng of dannyykarginine, Qly = 4.3 ng of dansylglyclne, and Leu = 3.8 ng of dansyk-buclne) developed in (e)the acld solvent system and (b) the bask solvent system. TCPO and H202 concentrations were 3 mM and 0.88 M, respectlvely.
variation in TCPO concentration in the range above 10-3 M appeared to have little effect on the CL intensity-time behavior. Using the present experimental setup, rapid and reproducible measurements of the CL emission at the peak maximum region as shown in Figure 2a is possible, thus allowing for significant enhancement in sensitivity and precision for the quantitation of analytes using the peroxyoxalate CL reaction in TLC. At a distance of 8 mm between the nebulizer and optical fiber, a scan speed of 2 mm/s was found to produce the best sensitivity while good resolution was maintained for the detection of three dansyl amino acids separated on a HPTLC plate using an acid solvent system, as shown in Figure 3a. At this scan speed, each sample spot travela past the light collection zone of the optical fiber at 4 s after it has been sprayed with the aerosol, which happens to fall within the time period at which the peak maximum of CL emission occurs, as shown in Figure 2a. The average limit of detection (LOD) based on a signal-to-noiseratio W N )of 3 for the three dansyl amino acids was found to be -0.45 ng, which is about 1order of magnitude lower than that reported by Curtis and Seitz (LOD for dansylglycine -7 ng) using their optical fiber-based detection system.10 Relative standard deviation (n = 6) for dansylglycine (3.1 ng/spot) was found to be -6%, which could arise from inconsistency in the manual spotting of the small volume (0.10-0.50pL) of samples onto the HPTLC plates using a 1-pL syringe and/or variations related to chemical excitation, e.g., CL efficiency. Calibration plots of dansylglycine indicated linear response from LODs up to0.lpg (r = 0.995). Figure 3b shows a HPTLC chromatogramof the three dansylamino acids obtained under the same experimentalconditionsas in Figure 3a except using
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a basic solvent system for development. It can be seen that significant peak overlap occurs between dansylglycine and dansyl-L-arginine. To minimize the extent of this overlap, higher resolution was obtained by employing an optical fiber with significantly smaller core diameter as the light guide while other experimentalconditionswere kept the same. Parts a and b of Figure 4 compare the resolution and sensitivity of the two dansyl amino acids separated on the same HPTLC plate using an optical fiber bundle and a single optical fiber, respectively. It is clear that significant enhancement in resolution was achieved by employing the optical fiber with the smaller core diameter, but this gain in resolution was accompanied by a loss in sensitivity. It has been demonstrated that luminescence intensity of certain amino acid derivatives generated by conventionalTLC fluorescence detection methods can be enhanced by 100fold after the plate has been sprayed with a viscous and nonvolatileorganic solvent system containing liquid paraffin or Triton X-100.14 Thus, it seems that further gain in sensitivity for the CL detection of dansyl amino acids separated on TLC plates may be achieved by exploiting the capabilities of the present experimental setup to detect CL signals enhanced by prespraying the samplespots with organic solvent systems having appropriate physical properties. Interestingly, parta a and b of Figure 2 indicate that the CL intensity of dansylglycinewas slightlyhigher when the sample spot has been presprayed with ethyl acetate; however, when compared to Figure 2c which shows the effect of prespraying dioxane onto the sample spot, enhancement in CL intensity is even higher. This observation was surprising since it is well-known that CL intensity is greatest in ester and ether solvents. A similar observation has also been reported by Curtis and Seitz, who suggested that when compared to ethyl acetate, the stronger interaction of dioxane with the TLC plate may play an important role in providing greater CL intensity for the dansyl amino acids.10 Perhaps more interestingly, parta d and e of Figure 2 show that a CL intensitytime profile of dansylglycine changed significantly, i.e., increased in peak intensity and lifetime, after the sample
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(14)Uchiyama, S.Uchiyama, M.J. Liq. Chromatogr. 1980,3,681-691.
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ANALYTICAL CHEMISTRY, VOL. 84, NO. 20, OCTOBER 15, 1992
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of three equal amounts of dansylglycine samples eluted simultaneously on the same HPTLC plate and detected with the present experimental system at identical Rfvalues. It can be seen that the Triton X-lOO/chloroform and liquid paraffidchloroform solvent systems enhanced the CL intensity 5- and 7-fold, respectively, which yield corresponding average LODs (S/N = 3) of about 0.10 and0.08ng, respectively. The average relative standard deviation (n = 6) on reproducibility using both solvent systems was -4 % (3.1ng/spot). Lastly, the detectability of dansyl amino acids separated on HPTLC plates as compared to conventional TLC plates was ala0 briefly inveatigated. It was found that without the influence of any organic enhancers, the average LOD (S/N = 3) obtained for the CL detection of the three dansyl amino acids separated on TLC plates was -0.80 ng, which is only slightly higher than that found on HPTLC plates (-0.45 ng) More detailed studies are necessary for the further optimization of various experimentalparameters to achievebetter analyticalfigurea of merit. To this end, a better understanding of the fundamental processes involved in the chemical excitation of analytes adsorbed on the plate surface and of the physicochemical basis for the increase in CL observed when plates treated with a viscous and nonvolatile solvent are used is essential; of particular interest would be a comparison study of CL intensity-time behavior of analyte and background signals generated from the plate surface, since the kinetics of these signals have been shown to be different when CL detection is performed in HPLC, which leads to significant improvement of detection limits of the analyteS.’6
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f bur. 5. Reletbe CL Intenskyof threesample spots of equal amounts of dansylglyclne (3.1 ng/spot) separated on the same HpnC plate uslng the acld solvent system. The spots were detected (a) wlthou! preapraylng w h any organic solvent and after preepraylng w h (b) Trlton X-lOO/chloroform mixture (1:4 v/v) and (c) liquid paratfin/ chloroform( 3 2 vlv). TCW end H202concentrations were 5 mM and 1.2 M, respectlvely.
spots were presprayed with two different viscous and nonvolatile organic solvent systems. To evaluate the gain in detectability of dansylglycineafter treatment with the viscous and nonvolatile organic solvent systems, parts a-c of Figure 5 show the relative CL intensity (15) Hnnaoka,N.;Tanaka,H.;Nakamoto,A.;Takada,M. A w l . Chem. 1991,63,2680-2685.
RECEIVED for review April 13, 1992. Accepted July 23, 1992. Registry No. Dansylglycine, 1091-85-6;daneyl-L-arginine, 28217-22-3; dansyl-L-leucine, 1100-22-7.
