Anal. Chem. 1988, 60, 2193-2197
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Stopped-Flow Determination of the Parameters Affecting the Application of Peroxyoxalate Chemiluminescence to High-Performance Liquid Chromatographic Detection Nobuaki Hanaoka*
Shimadzu-Kansas Research Laboratory, 2095 Constant Avenue, Lawrence, Kansas 66046 Richard S. Givens, Richard L. Schowen, and Theodore Kuwana
Center for Bioanalytical Research, University of Kansas, Lawrence, Kansas 66046
Stopped-flow method was employed for the lnvestlgatlon of the factors affectlng the peroxyoxalate chemiluminescence (PO-CL) reactlon. Precise determlnation of the tlme course of the fast PO-CL reactions under hlghly aqueous condltlons (IO-50% H,O), which simulate those of a reversed-phase high-performance liquld chromatography (RP-HPLC) system wlth a PO-CL detector, was carrled out. The varlables examined in thls study were temperature, pH, water content, the nature of the catalyst, the solvent, and the concentratlon effect of oxalate, H202,and catalyst. From the results, the effects of these parameters on the maxhnwn intendty ( J ) and the time at which the PO-CL reactions reach the maximum (7,,,-) were evaluated. Two of these parameters were modeled to a “tlme window concept” In order to predict the effects of each parameter on the response of the PO-CL detector for HPLC measurements. The results were directly compared with the data obtained by the flow Injection analyds. The utlllty of the stopped-flow data in conjunction with the “tlme wlndow concept” was demonstrated by determlnatlon of optbnum conditions for HPLC appllcations that utliize PO-CL detectlon.
Following the discovery in the early 1980s of the sensitivity of peroxyoxalate chemiluminescence as a detection technique for high-performance liquid chromatography (HPLC) (I), various substrates have been successfully analyzed by this method (1-18). The method depends on a chemiexcitation process, in which high-energy intermediates are formed and activate a fluorophore to its f i s t excited singlet state. Subsequently, the fluorophore emits light as normal fluorescence. Chemical reactions involved in the peroxyoxalate chemiluminescence (PO-CL) can basically be written as follows (1, 19): oxalate H202 intermediates (1)
+ +
- -
fluorophore intermediates fluorophore* (2) fluorophore + hv fluorophore* (3) These reactions, especially the process of the intermediates formation (l),are rather complicated, having several steps complicated by a number of side reactions (20). For this reason, the PO-CL reactions are inherently sensitive to several environmental factors that affect the maximum intensity and the chemiluminescence lifetime. Therefore, in order to maximize the capability of PO-CL detection, it is essential to know what factors affect the efficiency in what manner. Some reseachers have elucidated the effect of a few of these factors, such as pH and solvent, either by examining the effects through solution studies or by employing a flow system (I, 6,9,15,21,22). In some cases, mechanical mixing of solutions
made it impossible to detect the initial or onset reaction rates, and, in the other case, rather hydrophobic conditions were employed which lowered the rate of the PO-CL reaction. In order to determine the optimum conditions for LC, the parameters affecting the early part of the PO-CL time course must be understood. Only then can one determine the optimum volume between initial mixing of the eluate with the reagents, i.e. the oxalate/H202mixture, and the detector to produce the maximum collectable chemiluminescence signal, while at the same time avoiding band broadening. Moreover, RP-HPLC, which is the most popular separation method, especially for biological substances, employs a mobile phase containing a high percentage of water. Water is known to increase the rate of the PO-CL reaction (6, 15). With this in mind, we have approached the study of peroxyoxalate chemiluminescence by employing a stopped-flow method for the measurement of the effects of various factors. The stopped-flow apparatus used here (23) is comprised of a pneumatic flow actuator, two driving syringes for injection of solution, a mixer, a cuvette in which PO-CL reaction is monitored, and a PMT which collects the emitted light. High pressure from the pneumatic flow actuator was applied to the solutions in the syringes, injecting them into the cuvette through the mixer which rapidly combined the two reagents (less than 2 ms). The signal from the PMT is amplified with a very short time constant (0.1 ms). The analog output of the amplifier was converted to a digital signal and stored by the digitizer in high-speed sampling time (10-200ms). By utilizing these fast measuring techniques, an accurate and complete detection of the reaction course is possible. Our first study was the thorough evaluation of various factors affecting the PO-CL reaction. The factors measured were temperature, pH, water content, catalyst, solvent, and concentration of oxalate, H202,and catalyst. Among these factors, temperature and catalyst are the ones about which accurate information has not been obtained previously. For all of the measurements of these factors, we employed mixed aqueous solvents (20% H 2 0 in final solution). Also, measurement of the effects of water content ranging from 10 to 50% in the final solution was possible. Finally, to verify the utility of the stopped-flow data for the determination of the conditions of HPLC with the PO-CL detector, the “time window concept” was applied to two of the variables to obtain the following: (1)a predicted change in the sensitivity of the assay as a function of the change in the variable; (2) prediction of the optimum value of the variable for the highest sensitivity of the detector; (3) an estimate of the error propagated by the fluctuation of the variable. The data thus acquired were compared with the FIA values. Both data agreed very well, and the application of the “time window” concept to the stopped-flow data was verified to be
0003-2700/8S/0360-2193$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
a practical method for the determination of the optimum conditions for HPLC measurements.
EXPERIMENTAL SECTION Chemicals. Dansylalanine (DNS-Ala, cyclohexylamine salt) was purchased from Sigma Chemical Co. Bis(2,4,6-trichlorophenyl) oxalate (TCPO) and imidazole were purchased from Fluka Chemical Corp. and Aldrich Chemical Co., respectively. Water was purified by the Barnstead NAN0 pure I1 system. All other chemicals were reagent grade. Stopped-Flow System. Durrum stopped-flow photometer, Model D-130, was employed with the light source turned off. The cell volume was 63 pL. Nual volumes of the two reagent solutions were introduced into the cell when a force of 3 kg/cm2 was applied on the two supply syringes. The potential applied to the PMT was 1000 V, and the time constant of the amplifier was 0.1 ms. All the operations were performed according to the manufacturer’s instruction (23). To the output of the amplifier, a SONY 390 AD programmable digitizer was connected for sampling and storage of the output signals. The sampling intervals of 390 AD could be varied from 100 ns to 200 ms, but 10-200 ms was preferable for these experiments. The data stored in 390 AD were transferred to a Zenith 2FA-161-52 microcomputer and processed. Experimental Conditions. Standard conditions adopted for the measurements were employed for all measurements. Solutions containing the chemiluminescence reagents were made up as follows: Solution 1 was composed of 0.05 mM DNS-Ala and 2.0 mM imidazole dissolved in a mixture of 400 mL of HzO and 600 mL of CH3CN. The pH was adjusted to 7.0 with HN03. Solution 2 was composed of 1.0 mM TCPO and 5.0 mM H20p in 1000 mL of CH3CN. The solutions were kept at 30 “C. Procedure. The variables evaluated in this study were (1)the temperature (over a range of 5-50 “C), (2) the pH (over a range of 5.5-8.0), (3) water content (over a range of 10-50% in final solution), (4) solvents (varying among acetonitrile, ethyl acetate, acetone, methanol, ethanol, and 2-propanol), (5) the catalysts (either imidazole, aniline, pyrazine, tris(hydroxyethy1)aminomethane, or mono-, di-, and triethylamine and tetramethylammonium salts), (6) the oxalate concentration (over a range of 0.01-2 mM), ( 7 ) the H2O2concentration (over a range of 0.1-15 mM), and (8) the catalyst concentration (over a range of 1-100 mM). For all measurements, a standard set of experimental conditions outlined above was employed except for a variable feature. For example, to measure the pH effects, only the pH of solution 1 was changed for the measurements, while the other parameters were kept unchanged. Temperature control of the solutions was accomplished by circulating thermostated water through the circuit system of D-130 which incubated the solutions in the driving syringes for 3 min. The pH of each solution was measured by Orion 811 pH meter. For the final solution, the pH of a mixture of equal amounts of solutions 1and 2 was measured since the stopped-flow apparatus injects the same volume of each solution. For the measurements of the solvent effects, acetonitrile in solution 1was replaced by other solvents. The solvent was not changed in solution 2 because of the instability of the TCPO/H2O2 mixture in hydroxylic solvents;they could not be used to prepare solution 2 (24). The imidazole added to solution 1as the catalyst also served as the buffer and could not be easily replaced by the other catalysts for evaluation, since several of them had no buffer capacity. Therefore, for measurements of the effect of each catalyst, a sodium phosphate buffer was used. Solution 1was then composed of the following: 0.05 mM DNS-Ala, 1.0 mM NAzHP04,and 2.0 mM catalyst dissolved in a mixture of 400 mL of HzO and 600 mL of CH3CN. The pH was adjusted so that the final solution was 6.7, the same as that of the imidazole-buffered solutions used in the previous studies. The TCPO/HzO2solution was prepared fresh and used within 5 min to avoid errors arising from the decomposition of the reactants. FIA Measurements. The FIA system is shown in the Figure 1A. It consists of Shimadzu HPLC pumps (P), type LC-6A,
A
:p-L7 SDI”ll0“ 2
SOlYIiO”
--
3
vi
v 2
own
Figure 1. (A) Schematic diagram of FIA system: P, pump; I, injector; M, mixer; D, detector; A, stainless-steel tube (i.d. = 0.5 mm; I = 1 m); B, stainless-steel tube (i.d. = 0.5 mm); V , , volume of M B; V p . volume of the cell of D. (B) Time window concept: t , = Vl/total flow rate (TFR); t 2 = V,/TFR; Here, V , = 30 pL, TFR = 2 mL/min, and t , = 1.0 s. The detector collects the shaded portion of time-intensity
+
profile.
0
50 Tome
100 [..SJ
Temperature dependence of PO-CL reaction of DNS-Ala: (1) 5;(2)10; (3) 15; (4)20;(5)30; (6)40;(7)50 “C. The vertical axis is the output of D-130. Flgure 2.
connected to the mixers (M), as developed by Imai et al. (W),an injector (I) (Rheodyne 7125 with a 20-pL sample loop), and a detector (D) fitted with a Hamamatsu R268UH PMT in front of a cell constructed by authors. Stainless-steel tubing (0.5-mm i.d.) was used in all flow lines. The length of A was 1m, the volume of the mixer M was 26 pL, and the volume of the detector cell, V,, was 30 pL. The standard conditions for the measurements were as follows: Solution 1contained 2 mM imidazole dissolved in a mixture of 400 mL of H20 and 600 mL of CH3CN. The pH was adjusted to 7.0 with HN03 Solution 2 contained 2.0 mM TCPO dissolved in 1000 mL of CH3CN. Solution 3 contained 10.0 mM Hz02 dissolved in loo0 mL of CH3CN. The flow rates for these solutions were 1.0 mL/min for solution 1 and 0.5 mL/min for solutions 2 and 3. The temperature was kept at 30 “C. The injected sample was 1 pM of DNS-Ala dissolved in 20 pL of solution A. The final concentrations of the reagents for this study were equivalent to those employed in the stopped-flow measurements. The effects of two parameters, the imidazole concentration and the temperature, were examined by varying them from the standard conditions. The length of line B of the FIA system was 72 cm for the measurements of the imidazole concentration effect in order to make tl = 5 s. For the temperature effect measurements, line B was 156 cm to make tl = 10 s; this correlates with the time necessary to give the highest intensity for the PO-CL reaction at 20 “C (Figure 2). The concentration range was 5-50 mM of imidazole, and the temperature range was 5-50 “C. Application of the Time Window Concept to the Stopped-Flow Data. The procedure is depicted in Figure 1B. “tin is the time required for the fluorophore to reach the detector; it
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
2195
Table I. Effects of Temperature on the Maximum Intensity ( J )and the Time at Which PO-CL Reactions Reach the Maximum (r-) temo. . I
7,-
J
O C
5
10
15
20
30
40
50
15.1 1.07
14.1 1.13
12.1 1.21
10.9 1.32
9.7 1.41
7.3 1.50
4.8 1.67
Table 11. p H Effects on the Time-Intensity Profile: p H of Solution 1 and, in Parentheses, p H of the Final Solution DH
5.5 (5.3) 6.0 (5.8) 6.5 (6.3) 7.0 (6.7) 7.5 (7.3) 8.0 (7.7)
?-
25.6 0.04
15.5 0.23
11.0 0.88
6.8 1.46
5.8 1.35
5.3 0.87 Time
Table 111. Effects of Water Content on the Time-Intensity Profile: v/v % of the Final Solution
;-
10
20
26.6 1.17
11.1 1.53
H,O (%I 30 6.3 1.77
40
50
3.1 1.98
2.1 1.67
Table V. Effects of Catalysts on the Time-Intensity Profile: (1) Imidazole, (2) Tris(hydroxymethy1)aminomethane, (3) Triethylamine, (4) Pyrazine, (5) Diethylamine, and (6) Others catalysts
Table IV. Effects of Methanol on the Time-Intensity Profile: v/v % in Solution 1
?=
0
0.5
10.6 1.42
10.0 1.07
CH,OH (%) 1.0 8.0 0.73
1.5
2.0
6.5 0.50
5.1 0.28 ~~
I.-=.)
Figure 3. Effects of imidazole concentration: (1) 1; (2) 3; (3) 5; (4) 10; (5) 30; ( 6 ) 50; (7) 100 mM In solution 1.
7,-
J
1
2
3
4
5
6
10.3 1.50
30.0 0.11
4.5 0.19
1.5 0.15
15.0 0.05
undetectable undetectable
Table VI. Effects of TCPO Concentration on the Time-Intensity Profile: Concentration Given Is That in Solution 1 ~
is given by Vl/total flow rate (TFR). "tz"is residence time of the fluorophore in the detector and given by Vz/TFR. The light collection period is indicated by the shaded portion of the PO-CL time-intensity profile. As noted earlier, tl is 5 or 10 s for the measurement of each variable, and V2 is 30 ML,which gives a 1-svalue of t2. Each set of tl and t2 (tl = 5, t2 = 1 s for imidazole concentration effects, tl = 10, t z = 1 s for temperature effects) was applied to the stopped-flow data of each variable, and the "time window" was calculated.
;ma
0.02
9.7 0.23
10.1 0.54
TCPO (mM) 0.5 1.0 9.0 1.08
2.0
6.6 1.47
8.2 2.17
Table VII. Effects of H201 Concentration on the Time-Intensity Profile: Concentration Given Is That of Solution 1
RESULTS AND DISCUSSION Selection of Conditions. In this study, DNS-Ala and TCPO were selected as fluorophore and oxalate, respectively, because they are the most popular materials for PO-CL (1, 4,11,12,21). Acetonitrile was chosen as the solvent since the TCPO/H202mixture in acetonitrile is more stable than in the other solvents (24), and this is a common solvent for HPLC. The pH was adjusted with HNOBto 7.0, in the optimum pH region (6,9,21,22). It is known that NO, does not quench the PO-CL reaction (21). Results of Stopped-Flow Measurements. Figures 2 and 3 show the changes of the PO-CL timeintensity curves as a function of the temperature and the concentration of imidazole, respectively. Tables I through VI11 show the effects of each factor on the maximum intensity, the J value, and on the elapsed time to reach the maximum intensity, the value. One of the major unknown factors that we investigated was the effect of temperature on the chemiluminescence yield. As shown in Figure 2 and Table I, raising the temperature accelerates the PO-CL reaction as well as increasing the J value and decreasing the T,, value. Since it is known that the PO-CL reaction involves a complex series of reactions (19,20), evaluation of the temperature effect on the individual steps
0.01
?-
0.1
0.5
12.7 0.39
13.5 0.48
H,O, (mM) 1.0 3.0 5.0
10.0
15.0
9.9 0.68
8.7 2.07
7.3 2.36
9.9 1.26
8.2 1.48
Table VIII. Effects of Imidazole Concentration on the Time-Intensity Profile: Concentration Given Is That of Solution 1 imidazole (mM)
;msl
1
3
5
10
30
50
100
22.0 0.46
10.6 2.27
9.7 4.40
5.5 6.90
2.8 9.32
1.4 8.18
0.9 7.34
would be very difficult and was not attempted. However, the conclusion derived from our observations is that the temperature of the solutions must be carefully controlled, especially for accurate and/or highly sensitive measurements (vide infra). The results for pH effects, Table 11, indicate an optimum pH of 6.7 which compares favorably with several reported studies, i.e. 6 (21), 7 (22), 7.5 (6),8 (9). The small variation in our results probably derives from the differences in the solvent composition and our use of the pH of the final mixture.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
Water content is widely known to change the velocity of PO-CL reactions (6, 15). It is clearly seen from Table 111that increasing the water content decreases T ~ = . At low H 2 0 concentrations (40%),the J value actually decreases. Solvent effects on the PO-CL reactions have already been reported by Imai et al. (I). We also have measured the effects of solvents such as acetonitrile, acetone, 2-propanol, ethanol, and methanol on the CL intensity-time profile. Our results with acetonitrile and acetone were nearly identical, with acetonitrile producing a higher maximum intensity than acetone. In alcoholic solvents, however, our results showed a greater loss of CL than those of Imai et al. (I). For propanol, ethanol, and methanol, the relative intensities of PO-CL reported by Imai et al. were 0.5, 0.38, and 0.12, respectively, compared with that for acetonitrile. In our experiments, relative values of 0.25 and 0.20 were obtained for propanol and ethanol. Furthermore, the emission in methanol was too weak to detect by the stopped-flow apparatus. These dkagreements probably arise from the differences in water content (2.8% in the experiments by Imai et al. vs 20% in ours); this led us to investigate in detail the effects of methanol on CL intensities under highly aqueous conditions. The results obtained are shown in Table IV. The addition of only 1% methanol decreased the maximum intensity by as much as half. This observation is very important in HPLC applications, especially RP-LC measurements, since methanol is one of the most common solvents. For PO-CL emission, catalysis by a weak base is generally necessary to obtain reproducible results (19).Triethylamine (TEA), tris(hydroxymethy1)aminomethane (Tris), and imidazole have typically been used as the catalyst (1,6,9,12,20). In our study, imidazole, Tris, aniline, pyrazine, and mono-, di-, and triethylamine and tetraethylammonium ions were examined. The remarkable enhancement noted with imidazole in contrast to the other catalysts (Table V) suggests its use for measurements requiring the highest sensitivity. Tables VI-VI11 and Figure 3 show the effects observed from varying the concentration of TCPO, H202, and imidazole. Among these results, the most noteworthy is that of imidazole (Table VI11 and Figure 3). When compared with the other two reagents which gave only a &fold increase in intensity with a 100-fold increase in concentration, the intensity increases more than 20-fold with only a 30-fold increase in the concentration of imidazole. Furthermore, in the case of TCPO and H202,a change in the concentration does not alter the Tvalues very much (a slight decrease is noted with HzOz, however, which is probably due to an increase in water content), whereas an increase in the imidazole concentration increases the reaction rate drastically. From these data, it is easily predicted that the effect of imidazole concentration for the PO-CL detection system will be much more significant than either the TCPO or Hz02 variables. The data of the imidazole concentration effects are used below to verify the utility of the stopped-flow method for the determination of the conditions of HPLC with PO-CL detection. Application of “Time Window Concept” and Comparison with the Results of FIA. To establish the utility of the stopped-flow data, the “time window concept”, shown in the portion B of Figure 1, was constructed to illustrate the effect of temperature (Figure 2) and imidazole concentration (Figure 3) on HPLC applications. For the imidazole concentration effects, tl was set at 5 s as an estimate of the mixing time necessary for the PO-CL reaction. tl for the temperature effects was 10 s ( T ~ =at 20 “C). The integrated values were compared with the peak heights of actual FIA measurements,
- 10.0 -
q-
4
0
0.50
0
a
2 v)
2a
--4 fv)
+
r
5.0
0.25
>
.% I Y
U
a
a
Uncorrected 0 Corrected
3
0
E
F O 0
a LL
I
I
25
50
0.00
Imidazole (mM) Figure 4. Comparison between time window concept and FIA (imidazole concentration effect): (0)values obtained by the application of time window concept to the data in Figwe 3; (0)mected time wlndow values with the assumption of mixing time of 0.5 s; (A)peak heights of FIA measurements.
i
J
-
0.05
E l
F
0
5
Uncorrected
LI
0 Corrected
1
I
I
I
I
1
0
10
20
30
40
50
Temperature
1
(OC)
Figure 5. Comparison between time window concept and FIA (tem-
perature effect): (0)values obtained by the application of time window concept to the data in Figure 2; (0)corrected tlme window values with the assumption of mixing time of 0.5 s; (A)peak heights of FIA measurements.
and the results are shown in Figure 4 and 5. For the imidazole concentration effects, slight differences were observed between the change of the intensity expressed by the uncorrected “time window” values and the change of the peak heights of the FIA measurements (Figure 4). The optimum value of imidazole concentration for the highest intensity predicted by the data from the uncorrected “time window” was about 17.5 mM. In contrast, the value obtained by the actual FIA measurements was 20 mM. Here, again, an assumption was made that the FIA measurements include a 0.5-s mixing time and that the PO-CL reaction started at approximately 0.5 s after the fluorophore was mixed with the admixture solution of TCPO and HzOz. In this case, the apparent tl is 4.5 s. The calculated values of the corrected “time window”, when plotted with the FIA results, show very good agreement (Figure 4). A similar comparison of the temperature effects is given in Figure 5. At the lower temperatures, the FIA results are in good agreement with the “time window” data corrected with the assumption of a mixing time of 0.5 s. At higher temperatures, the uncorrected data of the ”time window” are in better agreement. This result is probably due to the fact that at higher temperatures, the mixing efficiency increases and, consequently, the mixing time is shorter.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1968
Table IX. Error (in %/OC) Calculated for a 1 "C Uncertainty in the Measured Value for the Time Window Model (Figure 5) temp,
5 uncorrected time window corrected t i m e window FIA peak heights
10 15
"C
20 30
40
50
0.9 1.5 2.1 1.4 0.4 -0.1 -1.0 0.8 1.6 2.3 1.4 0.5 0.2 0.0
0.9 1.8 2.4 1.3 0.5 -0.2 -0.9
Utility of Stopped-Flow Data. Through consideration of the data outlined above, it was evident that several valuable predictions from the stopped-flow data can be applied to PO-CLmeasurements, that is, (1)the detector's response can be optimized according to each factor and (2) an estimate of the expected error for each factor can be derived from the "time window" analysis. The error is d e f i e d as the change of the intensity per unit of the factor divided by the intensity at the definite point of the factor. The change of the intensity per unit of the factor can be obtained from the slope of the tangent line at that point, The values of the error to +1OC change in temperature calculated from the temperature effect data (Figure 5) are shown in Table IX. Under 30 OC, all the values at each temperature coincide very well. At higher temperatures, however, the values of the corrected "time window" data are slightly different from those of the other methods, probably due to the same reasons mentioned above. From the time window data, the following conclusions are derived. (1)The error is a t most about Z.l%/deg at around 15 "C. (2) In the event that the expected error is tolerable or that the ambient temperature is sufficiently stable that the error is expected to be small, additional temperature control of the solutions would not be necessary. (3) When temperature control is necessary, the temperature should be maintained at around 35 O C where the error is the smallest. (4) The accuracy of the temperature control that is necessary to reduce the error to the tolerable limit can also be estimated from the calculated error values. These conclusions are the same as those derived from the data of the actual FIA measurements. Thus, the accurate and complete time course of PO-CL reactions, which were monitored by stopped-flow method, established the dependence of PO-CL intensity aa a function of various factors that affect the FIA CL intensity. The application of the time window concept to the stopped-flow data presents an ideal opportunity to determine the optimum
2197
conditions for HPLC measurements with PO-CL detection. The features and parameters of the PO-CL phenomenon presented here do not represent the limit of the information which can be acquired from the stopped-flow studies. We are also examining (1) the determination of the kinetic rate constants of each variable in a mechanistic evaluation of the PO-CL phenomenon, (2) computer simulation to model the PO-CL reaction intensity as a function of the reaction conditions, and (3) the stopped-flow data for the evaluation of other oxalates and fluorophores.
ACKNOWLEDGMENT We gratefully acknowledge Arjav Shah for assistance with the stopped-flow measurements. LITERATURE CITED Kobayashi, S.; Imai. K. Anal. Chem. 1980, 5 2 . 424-427. Kobayashi, S.; Sekino, J.; Imai, K. Anal. Bbchem. 1981, 112, 99. Sigvardson, K. W.; Birks, J. W. Anal. Chem. Ig83, 55, 432-435. Meiibin, G. J . LlquM Chromatogr. 1983. 6 . 1603. Kricka, L. J.; Thorpe, G. H. G. Analyst 1983, 708, 1274-1296. Welnberger, R. J . Chromatogr. 1984, 314, 155-165. Sigvardson, K. W.; Birks, J. W. J . Chromatogr. 1984. 316, 507-518. Sigvardson, K. W.; Kennish, J. M.; Birks, J. W. Anal. Chem. 1984, 56, 1096-1102. De Jong, G. J.; Lammers, N.; Spruit, F. J.; Th. Brinkman, U. A.; Frei, R. W. Chromatographia 1984, 18, 129. Kozioi, T.; Grayeski, M. L.; Weinberger, R. J . Chromatogr. 1884, 317, 355-366. Miyaguchi, K.; Honda, K.; Imai, K. J . Chromatogr. 1984, 3 0 3 , 173-176. Miyaguchi, K.; Honda, K.; Imai, K. J . Chromatogr. 1984, 316, 501-505. Meilbin. G.; Smith. B. E. F. J . Chromatogr. 1984, 312, 203-210. Imai, K.; Weinberger, R. Trends Anal. Chem. 1985, 4 , 170-175. De Jong, 0. J.; Lammers, N.; Spruit. F. J.; Frei, R. W.; Th. Brinkman, U. A. J . Chromatogr. 1988, 353, 249-257. Zoonen. P. V.; Kamminga, D. A.; Oooljer, C.; Velthorst. N. H.; Frei, R. W. Anal. Chem. 1886, 58, 1245-1248. Weber, A. J.; Grayesky, M. L. Anal. Chem. 1987, 59, 1452-1457. Weinberger, R.; Mannan, C. A.; Cerchio. M.; Grayeski, M. L. J . Chromatogr. 1984, 288, 445-450. Rauhut, M. M.; Bollyky, L. J.; Roberts, 8. G.; Loy, M.; Whitman, R. H.; IannOtta, A. V.; Semsei, A. M.; Clarke, R. A. J . Am. Chem. Soc. 1987, 89, 6515-6522. Aivarez, F. J.; Parekh, N. J.; Matuszewski, B.; Givens, R. S.; Higuchi, T.; Schowen, R. L. J . Am. Chem. Soc. 1986, 708, 6435-6437. Honda, K.; Sekino, J.; Imai. K. Anal. Chem. 1983, 55, 940-943. Honda, K.; Miyaguchi, K.; Imai, K. Anal. Chim. Acta 1985, 177, 103-110. Operation ami Maintenance Menual of Durrum RapM Kinetics Systems Serles 0 - 1 0 0 ; Durrum Instrument Carp.: Palo AHo. CA. Kawasaki, T. (Showa University); Hayakawa, K. (Kanazawa University). personal communication, 1987. Kobayashi, S.; Imai, K. Anal. Chem. 1980, 52, 1548-1549.
RECEIVED for review February 2, 1988. Accepted June 24, 1988.
