2880
Anal. Chem. 1991, 63, 2680-2685
Kinetic Study of Background Emission from Peroxyoxalate Chemiluminescence Reaction and Application to the Improvement of Detection Limits in Liquid Chromatography Nobuaki Hanaoka,* Hiroshi T a n a k a , A k i r a Nakamoto, a n d Michinosuke T a k a d a Analytical Instrument Research Laboratory, Shimadzu Corporation, Nakagyo-ku, Kyoto 604,J a p a n
Time-intensity curves of Iight-emltting reactions in a mlxture of imidazole buffer, bls(2,4,6trkhbrophenyI) oxalate (TCPO), and H202 without fiuorophore were examlned by the stopped-flow method. For this purpose, a modified normal highperformance liquid chromatography (HPLC) system with peroxyoxalate chemiluminescence (PO-CL) detectlon was used to record actual background emlsslon In the HPLC system. Background emlssion occurred much sooner than that from fluorophores. The influence of environmental factors such as temperature, pH, water content, and the concentration of reagents on lntenstty and kinetic rates of emlsslon was assessed. Under condltlons of less water and lower temperature, simple rise/faii curves of background emisslon split Into two pulses. The effects of an optical fitter and quenchlng materlais, as well as the Influence of the above factors, on these pulses were examlned for clarlflcatlon of the background sources and reaction mechanisms of emission. Finally, conditlons for measurement of dansylated (DNS) amino acids by HPLC wlth the PO-CL detector were optimized on the basis of experlmental data and results from flow InJectlon analysis (FIA).
INTRODUCTION Peroxyoxalate chemiluminescence (PO-CL) is a very sensitive detection method for high-performance liquid chromatography (HPLC) (1-11). For maximum performance of this detector, the nature of analytes for generating high signals and background emission features for the supression of intensity must be understood thoroughly. Regarding PO-CL reactions of fluorophores, the kinetic profiles, reaction mechanisms, and influence of various factors have been extensively studied (1-3,6-8,12-16). On the basis of the results, optimum conditions for various compound measurements by HPLC with PO-CL detection have been determined and PO-CL is now a well-established detection method in HPLC. Background sources of PO-CL detection have also been studied to develop means for decreasing the background level. PO-CL impurities in reagents were initially considered to emit background light (2, 3). The purification of reagents and placing an optical filter in front of a photomultiplier tube (PMT) were shown to be effective for supressing background emission. P M T dark current was not negligible, being nearly the equivalent of the P M T current generated by background emission (5). The signal to noise ratio (SIN) of detection was improved by applying the proper voltage to the P M T and eliminating baseline noise by the filtration of P M T signals with an amplifier. In 1983, Birks et al. observed weak emission from a mixture of oxalate and H202without fluorophore ( 4 ) and concluded background signals of PO-CL detection to be produced not by emission from reagent impurities but by the light they had observed, since its spectra were identical in different solvents and no fluorescence could be detected in any reagent.
The spectra and kinetic rates of emission from reactions of various oxalates and Hz02were analyzed by Grayeski et al. (17). They found impurities not to be the source of background emission but rather direct phosphorescence from PO-CL intermediates as the main source. Two light-emitting species were also detected in background reactions. Reaction mechanisms are discussed in the following on the basis of these findings. Background signals from reagent impurities, the PMT, and PO-CL intermediates have been reported independently (2-5, 17). Although PO-CL intermediates have recently been shown to produce nearly all background signals they decrease as a result of reagent purification (3). Different rates of P M T dark current in background signals have also been reported ( 4 , 5). An effective technique for suppressing background emission levels is still not available. Suppression should be possible through an appropriate optical filter (2,3), but in many cases, wavelengths of emission from analytes are almost the same as those of background emission and a filter would decrease the background level as well as the peak height of an analyte. I t is quite difficult and impractical to further purify commercially available reagents whenever used. A change in P M T voltage also causes a t the same time variation in the signal intensity of an analyte and background emission. Hence, high background is observed in HPLC measurements with PO-CL detection and detectability is limited due to associated noise. This study was conducted for further clarification of the sources of emission and reaction mechanisms of PO-CL background and to find means for improving the detection of minimal quantities of analytes based on differences in the nature of emission from fluorophores and the background. EXPERIMENTAL SECTION Chemicals. DNS-amino acids (cyclohexylaminesalts) were purchased from Sigma Chemical Co. Bis(2,4,6-trichlorophenyl) oxalate (TCPO) was from Wako Pure Chemicals Co. Water was purified by the Yamato WG-25 system. All other chemicals were of reagent grade. Measurements of TCPO Purity. Impurities in the TCPO reagent were examined by HPLC using a Shimpac-CLC ODS column (4.6 X 150 mm, Shimadzu Corp.) and a Shimadzu UV detector, Type SPD-6A. The wavelength for the detection was 290 nm. The mobile phase was pure acetonitrile, and the flow rate, 1 mL/min. TCPO (200 nM) dissolved in 20 pL of ethyl acetate was measured. The chromatograms were recorded by a Shimadzu chromatopac C-R6A instrument. The measurements were made at room temperature. Stopped-Flow System. In our previous study (15),a Durrum stopped-flow photometer was used for measurements, in which two solutions were introduced into the cell by two supply syringes. In this study, the stopped-flow apparatus shown in Figure 1was a modification of a standard HPLC system with a PO-CL detector to record the time-intensity curves of background reactions in PO-CL detection. It was composed of Shimadzu HPLC pumps (P), Type LC-SA, connected to the mixers (M), premixers for Shimadzu LC-6A, a six-portion valve (V), Rheodyne 7000, and a flow cell (C) made by the authors. Mixer and cell volumes were about 30 and 70 pL, respectively. T was a Hamamatsu photo-
0003-2700/91/0363-2880$02.50/0 0 1991 American Chemical Society
(In,
ANALYTICAL CHEMISTRY, VOL. 63,NO. 23, DECEMBER 1, 1991 2681 V
3 A
Drain
Table I. Effects of Temperature on Background Intensity at the Start of Measurement ( I ) and Half-Life of Background Emission (rL11) temp, OC 20 25
B TM,
s
I , nA
10
15
11.8
7.9 41.6
29.7
5.8 55.4
4.5 67.9
30
40
3.8
2.8
70.6
44.8
V
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A
Flgure 1. (A) Schematic diagram of the stopped-flow system: P, pump; V, valve; M, mixer; C, flow cell; T, photomultiplier tube; D, amplifier; R, recorder; A, stainless-steel tube (i.d. = 0.3 mm; I = 1 m); B, stainless-steel tube (i.d. = 0.3 mm; I = 20 cm). (6) The position of the valve (V) for stopped-flow measurements.
multiplier tube (PMT), Type R268UH, and D, an amplifier designed by the authors. The applied voltage to the PMT was 700 V. Output signals were recorded by a Shimadzu chromatopac C-R6A (R) instrument. Solution and PMT temperatures were controlled by installing the parts surrounded by the dotted line in Figure 1 into an aluminum block followed by thermostating with a Peltier element. The temperature ranged from 10 to 40 OC, and accuracy of the control was within f0.1 "C. A and B were stainless steel tubes (i.d. = 0.3 mm) 100 and 20 cm in length, respectively. Stainless-steel tubing (0.5 mm id.) was used in all other flow lines. Procedure. Stopped-flow measurements were made by rotating the six-portion valve (V) to the position indicated in Figure 1. Lag time for the measurement, defined as the volume of the mixer (M) + the tube (B)/total flow rate, was ca. 1.3 s since M and B were 30 and 14 r L in volume, respectively, and the total flow rate was 2.0 mL/min. For evaluation of the variables, solutions 1-3 and their flow rates were made the same as those for the FIA measurements in the previous report (15). The final solutions were thus the same as those for the stopped-flow measurements of DNS-Ala. All procedures, experimental conditions, and evaluated parameters were the same except as follows: (1)measurements besides that of temperature dependency were made at 23 OC, since at 30 OC, reactions occurred too fast to permit assessment of the influence of each variable; (2) temperature control of the solutions was as mentioned above. For measurements of the two pulses of background emission, solution 1 consisted of 0.5 mM imidazole dissolved in a mixture of 300 mL of HzO and 700 mL of CH3CN. The pH was adjusted to 7.0 with HNO,. Solutions 2 and 3 were not changed. The flow rate of each solution was 1.5 mL/min so that the lag time would be shorter (ca. 0.6 s). The temperature of the aluminum block was kept at 10 "C, at which reactions would occur more slowly and parameter influence could be evaluated easily. The variables were determined in the same manner as above. The optical filter used was a HOYA Y-48 by which light was cut off at less than 470 nm. The quenching effect of 2,4,64richlorophenol (TCP) was evaluated by adding 1 mM of this reagent to solution 2. The influence of COz was examined by bubbling it through solution 1 for ca. 15 s. Conditions for the Separation of DNS-Amino Acids. The mobile phase for separating dansylated alanine (Ala), phenylalanine (Phe), isoleucine (Ile), and valine (Val) was determined using a normal isocratic HPLC system provided with a UV detector. The column was 4 mm i.d. X 150 mm ODS (type STR ODS-M, Shimadzu Techno-Research, Inc.), and the detector, a Shimadzu UV detector, SPD-6A. The flow rate of the mobile phase was 0.8 mL/min, as recommended by the manufacturer (18). The wavelength for detection was 340 nm (19). A 10-ng amount of each DNS-amino acid dissolved in 20 WLof mobile phase was injected into the column. All measurements were conducted at room temperature (ca. 23 O C ) . FIA Measurements. The FIA system used for optimization of detection conditions was the same as in the previous report (15). A Teflon tube (i.d. = 0.5 mm) was used as tube B and tl
Table 11. pH Effects on Background Emission (pH of Solution 1 and, in Parentheses, Time at Which the Background Reaction Reaches a Maximim (7-) and the Maximum Intensity ( J ) ) PH
5.5
6.0
6.5
7.0
7.5
8.0
s 16.5 (5.8)
12.3 (4.7) 8.9 (2.8) 5.6 4.9 4.0 11.9 (13.5) 29.0 (30.0) 44.0 (45.5) 55.0 57.1 52.3
q,df ( T , , , ~ ) ,
I(J), nA
Table 111. Effects of Water Content on Background Intensity (v/v % of the Final Solution) % HzO
ha,
s
I , nA
10
20
30
40
50
6.9 45.5
5.7 55.3
4.6 66.6
3.7 64.2
3.2 46.1
Table IV. Effects of Methanol on Background Intensity (v/v % in Solution 1) 0
1.0
CHSOH 2.0
3.0
4.0
5.8 55.7
5.1 47.7
3.8 41.3
3.4 30.8
2.8
%
half,
s
I. nA
23.3
was set at various values by adjusting its length. (For the definition of tl, see ref 15.) Standard conditions for measurements were as follows: Solution 1was the optimized mobile phase for HPLC measurements. Solution 2 contained 0.5 mM TCPO dissolved in 1000 mL of CH3CN. Solution 3 contained 10.0 mM HzOz dissolved in 1000 mL of CH3CN. The temperature was maintained at 23 "C. HPLC Measurements. The HPLC system was made by replacing tube A of the FIA system with the HPLC column specified above. The parts surrounded by the dotted line in Figure 1 were also temperature-controlled by an aluminum block. A 50-fmol amount of each DNS-amino acid dissolved in 20 pL of mobile phase was measured under two sets of conditions, one of which being the same as that for the FIA measurements. The other set was that optimized for greater detection, as follows: The mobile phase was composed of 1.8 mM imidazole dissolved in a mixture of 750 mL of HzO and 250 mL of CH3CN. The pH was adjusted to 7.0 with HNO,. The flow rate was 0.8 mL/min. Solution 2 was composed of 0.5 mM TCPO in lo00 mL of CH,CN. The flow rate was 0.5 mL/min. Solution 3 was composed of 40 mM HzOzin lo00 mL of CH3CN. The flow rate was 1.2 mL/min. The length of tube B was 4 m so that tl would be 20 s. The temperature of the aluminum block was kept at 30 OC.
RESULTS AND DISCUSSION P u r i t y of TCPO. One of the major impurities in the TCPO reagent is TCP. On the obtained chromatogram for TCPO, one small peak in front of a large peak was observed and was considered a T C P peak since its retention time (4.6 min) was identical with that of TCP. The calculated rate of T C P in TCPO reagent was 1.2 wt %. This amount of T C P had no effect on the background intensity or kinetics (vide infra). Results of Stopped-Flow Measurements. Parts A and B of Figure 2 show changes in the time-intensity curves of
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 23, DECEMBER 1, 1991
Table V. Effects of TCPO Concentration on Background Intensity'
7h.l~
I, nA
0.01
0.1
3.0 0.97
4.8 22.4
ITCPOl, mM 0.25 0.5 5.3 30.8
A
B
1.0
5.8 54.2
5.9 112.9
The concentration given is that in solution 2.
Table VI. Effects of H202 Concentration on Background Intensity' 1 7m
s
I(J), nA a
IH7071, mM 10 20
5
50
150
12.0 (4.3) 8.6 (3.2) 5.8 4.4 3.6 2.5 12.2 (12.8) 32.7 (33.6) 54.7 82.6 94.5 66.6
The concentration given is that in solution 3.
Table VII. Effects of Imidazole Concentration on Background Intensity' 0.5 7half ( T - ) ,
I(J), nA
s
limidazolel, mM 1.0 2.0 5.0 10.0 50.0
14.0 (4.4) 9.8 (3.2) 6.1 4.9 3.2 2.5 12.6 (30.9) 37.7 (39.2) 55.7 74.9 63.5 46.4
'The concentration given is that in solution 1. background emission as a function of imidazole concentration and temperature, respectively. Tables I-VI1 show the effects of each parameter on background intensity a t the start of measurements, I, and half-life of falling reactions (8),T M In the previous study on DNA-Ala ( 1 3 , maximum intensity, J, and the time to reach maximum intensity, T-, were measured to evaluate parameters affecting fluorophore intensity. In this study, reactions occurred very fast and in most cases, T- was less than the lag time of the measurements (Figure 2). For cases in which they occurred slowly and maximum emission could be detected, J and T~~ are shown in the parentheses in the tables. A comparison of the data with the results for DNS-Ala (15) clearly indicates background reactions occur much faster than those of DNS-Ala. Under standard conditions, only ca. 30% the total emission from DNS-Ala was generated during a period of 20 s, while nearly all background light was emitted during the same period. This should be considered for clarification of emission sources and mechanisms of background emission. The conditions for the maximum S I N in HPLC using a PO-CL detector were determined on the basis of kinetic differences (vide infra). Environmental factors contributing to emission from DNS-Ala (15) were also examined for their effects on background emission. As shown in Figure 2 and Table I, an increase in temperature caused background reactions as well as emission from DNS-Ala to accelerate. It should be noted that background intensity increased with temperature up to 30 OC,but beyond this, I decreased. This has also been noted for DNS-Ala with imidazole higher than 30 mM. Water and imidazole also accelerated the reactions, and I was noted to decrease at higher concentrations. A pH of 7.0 corresponded to the highest background intensity, as was also the case for fluorophores. This value agreed well with the optimum pH for DNS-Ala. Background reactions were retarded by an increase in [H+],as also observed with DNS-Ala. Methanol strongly quenched background emission. The TCPO concentration had virtually no effect on the kinetic rates of background emission but only increased its intensity
1
o
i
m
4
0
6
Tlme (wc)
0
0
M
Q
W
Tlme (sec)
Flgure 2. (A) Effects of imidazole concentration on the background emission: (1) 1, (2)2,(3)5 mM in solution 1. The vertical axis is the output of the PMT. (B) Temperature dependency: (1) 10, (2)15, (3) 20 OC.
when it was more than 0.1 mM. The effects of these factors on background emission were essentially the same as those on emission from DNS-Ala. H202 exerted different effects on background reactions. Raising the H202concentration did not significantly change the reaction rates of DNS-Ala but accelerated background reactions remarkably (Table VI). The H z 0 2used to prepare solution 3 was dissolved in water, and its addition increased the water content of the reaction solution. However, changes in the kinetic rates considerably exceeded those expected from an increase in water content. This may be an inherent feature of background emission. Background Emission Sources. Fluorescent impurities in reagents were initially considered background emission sources ( 2 , 3 ) . Studies by Birks et al. ( 4 ) and Grayeski et al. (17) indicated direct phosphorescence from intermediates formed by reaction of oxalate and H202 to be the main background light source, and this has been confirmed by spectrometric methods and by quenching by 02. The data presented above also appear as evidence for this, since the kinetic rates of PO-CL reactions were essentially the same for different types of fluorophores (13). Also, that only reactions of fluorescent impurities would be much faster than those of other fluorophores appears quite unlikely. No slow reaction curves were obtained from the present data. Observation of Two Emission Pulses. That PO-CL reactions of background emission were found to occur much more quickly than those of fluorophores prompted further investigation of their reaction mechanisms. In the case of fluorophore PO-CL, two pulses of light emission were observed under hydrophobic conditions (14). The same conditions except for the catalyst used, i.e. imidazole, were applied for stopped-flow measurements of background emission, and only peak of emission was observed. However, with a small amount of water (10% fiial solution) and more catalyst (more than 0.1 mM imidazole), the reaction curve was clearly seen to separate into two peaks. The conditions for this observation are summarized in the Experimental Section. The reaction solutions had to be cooled so as to slow both reactions and facilitate their observation. The flow rate of each solution was made 1.5 mL/min to decrease the lag time to 0.6 s. Time-intensity curves measured at different concentrations of imidazole are shown in Figure 3. Background emission involved at least two reactions having different kinetic rates. By spectrometric analysis of the light produced by reactions of oxalates and H202,two emitting compounds were shown to be present in the background emission (17). These data
ANALYTICAL CHEMISTRY, VOL. 63,NO. 23, DECEMBER 1, 1991
2883
Scheme I TCPO + H202
t
Et3N
t
rx-2-
I I hv
0
30
60
90
Time (sec) Flgwe 3. Imidazole concentration effects measured with less water content (10% Rnal solution)and at lower temperature (10 OC): (1) 0.1, (2) 0.25, (3) 0.5, (4) 1 mM in solution 1.
also indicate the possibility of two different species in the background reactions. The influence of the factors mentioned above was determined to clarify the features of these pulses. Emission in each case was affected in the same way by temperature, water content, imidazole concentration and HzOz: they all accelerated both reactions. At pH 7.0, the intensities of both reactions were highest. Methanol also strongly quenched the reactions. TCPO concentration had almost no effect on kinetic rates. Emission in both cases is thus concluded to have a feature in common with the environmental factors specified above. In their study of light produced in a mixture of bis(2,4,5trichloro-6-carbopentoxyphenyl) oxalate and H 2 0 2 (17), Grayeski et al. found one of the emissions with a longer maximum wavelength (Amm = 620 nm) to occur faster than the other with a A,, of 450 nm. A,, of the emission from a TCPO/H2OZmixture was 450 and 540 nm. Thus, an optical filter which cuts off light at less than 470 nm was used in this study for measurements of the TCPO/HzO2reactions. Slow reactions were shown to have lost more than 70% their intensity, while more than half the light from faster reactions passed through the filter into the PMT. Slow reactions thus have shorter wavelengths than those that are fast, being less than 470 nm. This agrees well with the observations of Grayeski et al. Mechanisms of Background Reactions. In 1986, a model of PO-CL reactions of a fluorophore was proposed by Givens et al. (14), in which TCPO/HzO2 generates successively two intermediates,X and Y. These in turn excite fluorophores and at least one intermediate, Z, not directly responsible for the excitation, is situated between them (Scheme I). The observation by Grayeski et al. that there are at least two light-emitting species in background reactions appears consistent with this reaction model (17). They mixed HzO, with various oxalates and measured the spectrum of light from each mixture. Each spectrum was found to have a featureless broad emission band around 450 nm, while the intensity and wavelength of light at a longer wavelength was specific to oxalate features. They considered 450-nm emission to be
phosphorescence due to COz from the decomposition of 1,2dioxetanedione and light of longer wavelengths to be from compounds containing phenolic residues. These considerations appear consistent with the hypothetical structures of intermediates proposed by Givens et al. The different kinetics of these emissions also appear to support the conclusion that X and Y are light-emitting spieces. The most characteristic feature of background emission as noted in this study is that kinetic rates differ considerably from those of fluorophores. A comparison of the data in Figure 2 with those for DNS-Ala obtaied under the same conditions (15) indicated emission from fluorophores to persist even after background emission decreased to less than the detectable level. Thus, at least one intermediate which excites the fluorophore, Y, according to the Givens' model, is still present after the background has blackened. If, as speculated by Grayeski et al., Y emits light by itself, there is the question as to why emission from Y ceases so early. One possibility may be that the reaction products of Y quench emission. If compounds produced by the decomposition of Y quench light directly emitted from Y, quenching should occur as the PO-CL reaction of Y proceeds and this emission may have ceased before all of Y decomposed. For confirmation of such a possibility, quenching by two possible products of PO-CL reactions, TCP and COz, was investigated. No quenching could be detected following the addition of 1 mM TCP to the TCPO solution. This is also indicated in ref 17. COz may quench the reaction in two different ways. One is that the generated COz may change solution pH to decrease light intensity. However, measured pH in the final solution was 6.8, a t which the intensity of background emission was still high (Table 11). There is the second possibility that COz itself quenches emission. The ability of C02to do this was assessed by bubbling gas through solution 1. After 15 s, the pH was 6.4, but the observed decrease in light did not exceed that calculated from the data in Table 11. These two PO-CL reaction products thus do not quench background emission. Quenching may thus possibly be caused by some other compound produced during the Y reaction. Also, intermediate Y may not emit light but other spieces, which form and decompose rapidly, may do so. X' in Givens' model would be one such species since its kinetic features appear consistent with the present data. Otherwise, a new emitting intermediate would have to be present in the model. None of the present data rules out the possibility that intermediate X is an emitting source, since the reaction of X takes place quite rapidly (14). It should be noted here that these speculated reactions have yet to be confirmed since neither PO-CL intermediates nor their reaction pathway has been identified. On the basis of the present data, background is concluded to involve at least two emissions differing in wavelength and kinetic rate. Both apparently occur much faster than those from fluorophores. Enhanced Detection Limits of DNS-Amino Acids. In our previous study ( 2 6 ) , application of the time window concept to the stopped-flow data of PO-CL reaction of a fluorophore was shown to be an ideal means for estimating '
2684
ANALYTICAL CHEMISTRY, VOL. 63,NO. 23, DECEMBER 1, 1991
A
11 i
d
in
5min
in
5min
Figure 4. Chromatograms of dansyl amino acids. Each peak represents 50 fmol of DNS-amino acid: (1) Ala; (2) Val; (3) Iie; (4) Phe. (A) The moblie phase contained 2 mM imidazole dissolved in a mixture of 750 mL of H,O and 250 mL of CH3CN. The pH was adjusted to 7.0 with HNO,, and the flow rate was 0.8 mL/min. The TCPO solution contained 0.5 mM TCPO dissolved in 1000 mL of CH,CN. The flow rate was 0.5 mL/min. The H,O, solution contained 10 mM H,02 dissolved in 1000 mL of CH,CN. The flow rate was 1.2 mL/min. t , was set at 3 s, and the temperatue was kept at 23 "C. (B) The mobile phase contained 1.8 mM Imidazole dissolved in a mixture of 750 mL of H,O and 250 mL of CH3CN. The pH was adjusted to 7.0. The TCPO solution was the same as in (A). The H,O, solution contained 40 mM H,O, dissolved in 1000 mL of CH3CN. The flow rates of ail solutions were the same as in (A). t , was set at 20 s, and the temperature was 30 "C.
their intensities in HPLC measurements. It was also applied to the present data for determining background emission levels under definite conditions. In this manner, factors determining these levels were clarified as well as optimal conditions to obtain low background levels. The final set of conditions for the best SIN was established by measuring DNS-Ala by FIA. Before examination of stopped-flow data, factors affecting HPLC measurements were determined. Separation conditions for DNS-amino acids were determined as described in the Experimental Section. The mobile phase was composed of 2 mM imidazole dissolved in a mixture of 750 mL of H 2 0 and 250 mL of CH3CN. The pH was adjusted to 7.0, optimal for the highest signal of DNA-Ala. Following application of this mobile phase and standard conditions for stopped-flow analysis to FIA, the pump pressure in all cases was found to have increased to more than 200 kg/cm2. The mobile phase may thus have been too hydrophilic so that TCPO precipitated in tube B. The flow rate of the H2O2solution was consequently increased from 0.5 to 1.2 mL/min, following which no increase in pressure could be detected. The flow rates of mobile phase and TCPO and H202solutions for assay of FIA and HPLC were 0.8,0.5, and 1.2 mL/min, respectively. T o obtain better detection limits, analyte intensity must be increased and/or background noise decreased. Analysis of stopped-flow data indicated one very effective technique to improve S I N in PO-CL detection: use of high values for factors such as temperature and concentration of imidazole and H202,by which the intensity of analyte signals will increase, leading a t the same time to much faster background reactions. When fluorophores strongly emit light and background emission has already lost its intensity, it should be possible at the same time to obtain high analyte signals as well as low background emission levels. The problem is, however, that these factors accelerate not only background reactions but also those of fluorophores. For
example, the data of DNS-Ala clearly show that an increase in imidazole concentration to more than 10 mM enhances the intensity of emission from fluorophores but makes the reaction so fast that emission virtually ends at less than 10 s. The present data show background emission to still be high before 10 s have passed. For confirmation of the above, the effects of temperature and concentration of imidazole and H202on DNS-Ala signals and background were investigated by FIA, with tl set a t 5 s. The value range of each of these factors was determined by time window analysis of the stopped-flow data of DNS-Ala as 23-40 "C for temperature and a concentration of 2-50 mM imidazole and 10-100 mM H20z The highest analyte intensity was obtained with 5 mM imidazole, 20 mM H202and 30 "C. But, under these conditions, background and associated noise were very high (-65 and 0.2 nA, respectively). It was possible to decrease the background level to ca. 20 nA by making the above factors maximum, but under such conditions, the analyte peak became less than one-fifth. As expected from analysis of stopped-flow data, good SIN with this tl value was difficult to obtain. tl was subsequently increased to 10 s, at which the background level should be rather low. The best results were obtained at 2 mM imidazole, 40 mM HzOz,a t 30 "C. The detection limit of DNS-Ala as determined under these conditions by HPLC was ca. 4 fM ( S I N = 2). Increasing HzOz from 10 to 40 mM decreased the background level to almost half, but with H 2 0 2higher than 40 mM, a slight increase in background was observed, possibly due to impurities in the H 2 0 2reagent. Raising the temperature from 23 to 30 "C lowered the background level from 25.6 to 10.4 nA. However, the baseline became noisy above 30 "C despite a decrease in this level. A ca. 50% increase in the noise level was observed on raising the temperature from 30 to 35 "C. The next method for improving detectability was to use a long tl, at which the background emission would become only slight. t , values of 15and 20 s were used while the temperature was varied from 23 to 30 "C, the imidazole concentration from 1 to 3 mM, and the H202concentration from 10 to 40 mM. tl values longer than these were not used since slight band broadening (ca. 20% increase of peak width) occurred at a tl of 25 s. SIN was largest at 1.8 mM imidazole, 40 mM HzOz, 30 "C, and tl = 20 s. Under these conditions, the peak heights of DNS-amino acids decreased to ca. 60% of those measured under optimum conditions with a t , of 10 s. But the background level also decreased to ca. 3.5 nA. Consequently, SIN improved by a factor of 2. The dark current level of the PMT was ca.0.2 nA. The chromatograms of four DNS-amino acids taken under the above conditions and nonoptimal conditions are shown in Figure 4. S I N was improved by more than 10 times, and the minimal detectable amount of DNS-Ala was ca. 2 fM ( S I N = 2). Registry No. TCPO, 1165-91-9;DNS-Ala, 35021-10-4; DNSVal, 1098-50-6DNS-Ile, 1100-21-6DNS-Phe, 1104-36-5;CH,OH, 67-56-1; imidazole, 288-32-4. LITERATURE CITED (1) Kobayashi, S.; Imai, K. Anal. Chem. 1980, 52, 424-427. (2) Kobayashi, S.; Sekino, J.; Honda, K.; Imai, K. Anal. Blochem. 1981, 172, 99-104. (3) Meiibin, G. J . Liq. Chromatogr. 1983, 6 , 1603. (4) Sigvardson, K. W.; Birks, J. W. Anal. Chem. 1983, 55, 432-435. (5) Weinberger, R.; Mannan, C. A.; Cerchio, M.; Grayeski, M. L. J . Chrom a t q r . 1984. 288, 445-450. (6) Weinberger, R. J . Chromatogr. 1984, 314, 155-165. (7) Honda, K.; Miyaguchi, K.; Imai, K. Anal. Chim. Acta 1985, 177, 103-110. (6) De Jong, G. J.; Lammers, N.; Spruit, F. J.; Frei, R. W.; Th. Brinkman, U. A. JT Chromatogr. 1986, 353, 249-257. (9) Grayeski, M. L.; DeVasto, J. K. Anal. Chem. 1987, 59, 1203-1206. (10) . . De Jona. G. J.: Kwakman. P. J. M. J . Chromatoor. 1989. 492. 319-345. Miyazaki, M. J . (1 1) Hayakawa, K.: Hasegawa, K.; Imaizumi, N.; Wong, 0.: Chromatogr. 1989, 464, 343-352. I
Anal. Chem. 1991, 63, 2685-2688 (12) Schuster, G. B. Acc. Chem. Res. 1979, 72, 366-373. (13) Catherell. C. L. R.; Palmer, T. F.; Cundall, R. B. J . 0”.Soc., Faradey Trans. 1984, 80, 837-849. (14) Alvarez, F. J.; Parekh, N. J.; Matuszewski, B.; Givens, R. S.; Higuchi, T.; Schowen, R. L. J . Am. Chem. SOC. 1988, 108, 6435-6437. (15) Hanaoka, N.;Givens, R . S.; Schowen, R. L.; Kuwana, T. Anal. Chem. 1988, 60, 2193-2197. (16) Hanadta, N. J . Chrometog. 1990, 503, 155-165. (17) Mann, 8.: Grayeskl, M. L. Anal. Chem. 1990, 62, 1532-1536.
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(18) Instructlon Manual ot HPLC column. STR ODS-M; Shlmadzu Techne Research: Kyoto, Japan. (19) Bayer, E.; Grom, E.; Kaltenegger, 8.; Uhmann, I?.Anal. Chem. 1976, 48, 1106-1109.
RECEIVED for review April 24,1991. Accepted September 12, 1991.
Direct Comparison of Two-Photon and One-Photon Excited Fluorescence Detection in Liquid Chromatography Using an Excimer-Pumped Dye Laser Ronald J. van de Nesse, Arjan J. G. Mank, Gerard Ph. Hoornweg, Cees Gooijer,* Udo A. Th. Brinkman, and Ne1 H. Velthorst
Department of General and Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
An excimer-pumped (XeCi) dye laser has been used to study two-photon excited (TPE) fluorescence detection in conventional-sire column liquid chromatography (LC) with aromatic compounds as test solutes. Excitation was performed at three different wavelengths, 1.e. 514, 586, and 650 nm, while emlssion light was collected around 410 nm. The results obtained with TPE at 586-nm excitation have been compared with onephoton excitation (OPE) at 293 nm; since frequency doubling was utilized, these measurements were performed under exactly the same experimental conditions. The relative peak heights observed for the various analytes with both modes of detection are distinctly different, demonstrating a noticeable difference In selectivity between TPE and OPE. Detection with TPE will in general be less sensitive than with OPE because of the Inherently less efficient excitation of the former technique; stili a detection limit as low as 1.0 nM was obtained in LC-TPE for the dye 4,4’-diphenylstilbene.
INTRODUCTION In two-photon excited (TPE) fluorescence, visible laser light is generally used for excitation of UV-absorbing analytes and the fluorescence spectrum is on the short-wavelength side of the excitation light. This implies that problems due to Rayleigh and Raman scatter and to reflection and refraction of laser radiation, as encountered in conventional laser-induced fluorescence (LIF) detection (based on one-photon excitation, OPE), are more easily solved in the TPE fluorescence detection mode. At first sight the analytical potential of T P E fluorescence detection seems to be limited, since the excitation efficiency of T P E as compared to OPE is extremely low. However, interesting detection limits can be obtained by invoking high-power pulsed lasers because the TPE efficiency is proportional to the square of the excitation power. As early as 1977, Sepaniak and Yeung published the first paper on laser T P E fluorescence detection for column liquid chromatography (LC) (I). Utilizing a 4-W continuous-wave argon-ion laser operating at 514 nm, chromatograms of oxadiazoles at concentration levels of 10-5-104 M were shown.
* To whom correspondence should be addressed. 0003-2700/91/0363-2685$02.50/0
The selectivity of T P E fluorescence detection was obvious: the highly fluorescent compounds phenol, fluorene, anthracene, and chrysene, also present in concentrations of M, did not show up. Unfortunately, the selectivity of the system could not be fully exploited because of the limited availability of laser output frequencies. The lowest limit of detection (LOD) of 3 x IO-’ M (SIN = 3;time constant, 0.5 s), obtained for 2-phenyl-5-(4-biphenylyl)-1,3,4-oxadiazole (PBD), obviously was not low enough for on-line coupling to LC. Considerable improvement has been achieved since then by applying lasers which provide high peak powers. The most recent achievement in combination with LC is in a paper by Pfeffer and Yeung (2) who used a copper-vapor laser (510/578 nm) with an average power of 3 W and a peak power of 20 kW as an excitation source for T P E fluorescence detection in a micro-LC system. For the same test solute as used before, the oxadiazole PBD, now an LOD of 9 X M ( S I N = 3; time constant, 3 s) was recorded. Recently, Wirth and Fatunmbi (3)reported the in-batch detection of 2.3 X M bis(methylstyry1benzene) ( S I N = 1; time constant, 1s) in a 1-cm cuvette using a Nd:YAG synchronously pumped dye laser tuned at 600 nm with an average power of 245 mW and a peak power of 600 W. Until now a systematic study of the analytical potential of laser-induced T P E fluorescence detection in LC has not been available. In this context the difference in wavelength dependences of T P E and OPE will be especially interesting. In the present paper an excimer-pumped (XeC1) dye laser was used, which provides an average power of about 300 mW with 1-MW pulses for efficient excitation. Since the absorbed power and, hence, the fluorescence signal are linear with the product of the peak power and average power (31,these characteristics imply that the system should allow for sensitive T P E fluorescence detection. Furthermore, the presence of a dye laser enables wavelength tuning. In our setup the dye-laser output also can be frequency-doubled, so that LC detection can be performed using either OPE (conventional LIF detection) or T P E fluorescence with exactly the same experimental assembly. That is, a direct comparison can be made between the two modes. Mixtures of polycyclic aromatic hydrocarbons (PAHs) and some other model compounds were studied in conventional-size LC at three excitation wavelengths, 514, 586, and 650 nm. 0 1991 American Chemical Society
