424
Anal. Chem. 1980, 52, 424-427
(8) O'Hare, J. J.; Nice, E. C. J. Chromatogr. 1979, 171,209-226. (9) Larsen, 6.; Viswanatha, V.; Chan, S. Y.; Hruby, V. J. J. Chromatogr. Sci. 1978. 16. 207-210. (IO) Meyers, C: A.; Coy, D. H.; Hwng, W. Y.; Schally, A. V ; Reeding, T. W. Biochemistry 1978, 17,2326-2331. (11) Larsen, 6.; Fox, B. L.; Burke, M. F.; Hruby, V. J. Int. J. Pept. Protein Res. 1979, 13, 12-21. (12) Sawyer, W. H. Fed. Proc. 1977,36, 1842-1848. (13) Merrifield, R. 8. J. Am. Chem. SOC. 1983,85,2149-2154. (14) Hruby, V. J.; Muscio, F.; Groginsky, C. M.; Gitu, P. M.; Saba, D.; Chan, W. Y. J. Med. Chem. 1973, 16,624-629. 115) UDson. D. A.: Hrubv. V. J. J. Ora. Chem. 1976. 41. 1353-1358. H;uby,'V. J.; Upson,*D. A.; A g a r w i , N. S. J. Org.'Chem. 1977, 42, 3552-3556. Yamamoto, D. M.; Upson, D. A.; Linn, D. K.; Hruby, V. J. J. Am. Chem. SOC. 1977, 99, 1564-1570. Hruby, V. J.; Barstow, L. E.; Linhart, T. Anal. Chem. 1972,44, 343-359. Yamashiro, D. Nature (London) 1984, 207, 76-77. Hruby, V. J.; Groginsky, C. M. J. Chromatogr. 1971, 63, 423-428. Smith, C. W., Ph.D. Dissertation, University of Arizona, 1973. Gitu, P. M., Ph.D. Dissertation, University of Arizona, 1974. A. F. . . Hrubv. V. J.: Smith. C. W.: Aaarwal. N. S.: Powers.. S.:. SDatola. . Unpiblished' work, University i f Arizona, 1977. (24) Eckhardt, J. G.; Stetzenbach, K.; Burke, M. F.; Moyers, J. L. J. Chromatogr. Sci. 1978, 16,510-513. (25) Bakalyar, S. R.; McIlwrich, R.; Roggendorf, E. J. Chromatogr. 1977, 142,353-365. (26) Karger, B. L.; Snyder, L. R.; Horvath, C. "An Introduction to Separation Sciences"; John Wiley and Sons: New York. 1973; pp 129-138. (27) Bakalyar, S. R . Am. Lab. 1978, IO, 43-61. (28) Tanaka, N.; Goodell, H.; Karger, B. L. J. Chromatogr. 1978, 158, 233-248. (29) Cline. S.M.; Stetzenbach, K. J.; Burke, M. F. Anal. Len. 1979,in press. (30) Stetzenbach, K. J., Ph.D. Dissertation, University of Arizona, 1980. (31) Hildebrand, J. H. Proc. NaN. Acad. Sci. U . S . A . 1979, 76, 194. (32) Brewster, A. I.R.; Hruby, V. J. R o c . Natl. Acad. Sci. U . S . A . 1973, 70, 3806-3809. (33) Meraldi. J. P.; Hruby. V. J.; Brewster, A. I. R. Proc. Nafi. Acad. Sci. U . S . A . 1977, 74,1373-1377. (34) Boicelli, C . A.; Bradbury, A. F.; Feeney, J. J. Chem. Soc., Perkin Trans. 2 1977,477-482.
(35) Krishna. N. R.; Huang, D. H.; Glickson. J. D.; Rowan, R.; Walter, R. Siophys. J. 1979, 26, 345-366, and references therein. (36) Von Dreele, P. H.; Brewster. A. I.; Dadok, J.; Scheraga. H. A,; Bovey, F. A.; Ferger, M. F.; du Vigneaud, V. Proc. Natl. Acad. Sci. U . S . A . 1972,69, 2169-2173. (37) Wyssbroad, H. R.; Fishman, A. J.; Live, D. H.; Hruby, V. J.; Agarwal, N. S.; Upson, D. A. J. Am. Chem. SOC. 1979, 101,4037-4043. (38) Waiter, R.; Prasad, K. U. M.; Deshuriers, R.; Smith, I.C. P. Proc. Nat/. Acad. Sci. U . S . A . 1973, 70,2086-2090. (39) Hruby, V. J.; Deb, K. K.; Spatola, A. F.; Upson, D. A,; Yamamota, D. J . Am. Chem. SOC. 1979, 101,202-212. (40) Beychok, S.; Breslow, E. J. Biol. Chem. lS88, 243, 151-154. (41) Urry. D. W.; Quadrifoglio. F.; Walter, R.; Schwartz, I.L. Proc. Natl. Acad. Sci. U . S . A . 1988, 6 0 , 967-974. (42) Fric, I.; Kodicek, M.; Flegel, M.; Zaoral. M. Eur. J. Biochem. 1975,56, 493-502. (43) Maxfield, F. R.; Scheraga, H. A. Biochemistry 1977, 16,4443-4449. (44) Hruby, V. J.; Deb, K. K.; Fox, J.; Bjarnason, J.; Tu, A. T. J . Bio/. Chem. 1978. 253,6060-6067. (45) Tu, A. T.; Lee, J.; Deb, K. K.; Hruby, V. J. J. B i d . Chem. 1979,254, 3272-3278. (46) Tu, A. T.; Bjarnason, J. 6.; Hruby, V. J. Biochim. Siophys. Acta 1978, 533,530-533. (47) Glasel, J. A.; Hruby, V. J.; McKelvy, J. F.; Spatola, A. F. J. Mol. Biol. 1973, 79,555-575. (48) Deslauriers, R.; Smith, I. C. P.; Walter, R. J. Am. Chem. SOC. 1974, 96,2289-2291. (49) Wyssbrod, H. R.; Balhrdin, A.; Schwartz, I. L.; Walter, R.; Van Binst, 0.; Gibbons, W. A.; Agosta. W. C.; Field, F. H.; Cowburn, D. J. Am. Chem. SOC. 1977, 99,5273-5276. (50) Rekker, R. F., "The Hydrophobic Fragmental Constant"; Elsevier: A m sterdam, New York, 1977, p 301.
RECEIVED for review September 28,1979. Accepted December 10,1979. This work supported in part by U.S. Public Health Grant AM-17420 (V.J.H.) and by the National Science Foundation (V.J.H.).
Determination of Fluorescent Compounds by High Performance Liquid Chromatography with Chemiluminescence Detection Shin-ichiro Kobayashi and Kazuhiro Imai * Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Univerity of Tokyo, Hongo 7-3- 1, Bunkyo-ku, Tokyo 113, Japan
chemiluminescence (CL) of dansyl amino acids generated with bis( 2,4,6-trichiorophenyI)oxaiate (TCPO) and hydrogen peroxide (H202)was studied for the application to the detection system for high performance liquid chromatography. Not only the concentration of TCPO and H202but also the constkuents of the medium both the intern adle time of CL. The combination of 0.51 mUmln of 5 mM TCPO in ethyl acetate, 1.2 mL/min of 0.5 M H202 in acetone, and 0.18 mUmin of eluent (0.05 M Tris HCI buffer (pH 7.7)-acetonltriie, ""9 'Iv) from a column Of pBondapak '18 was for the fmoi detection Of the fiuorophores. The peak heights of dansyiated alanine, glutamic acid, methionine, and norleucine were proportional to the quantities more than 50 fmoi.
High performance liquid chromatography (HPLC) has been a common tool for the measurement of substances in biological materials. In the determination of minute amounts of substances such as biogenic amines (1-3) and amino acids ( 4 ) in body fluids, fluorescence detection is usually adopted. Generally speaking, the fluorescence detection system for HPLC consists of (a) a light source for the excitation of fluorophores, (b) a flow cell through which the eluate passes and (c) a photomultiplier for the detection of the emitted light from the fluorophores. There are problems to be encountered 0003-2700/80/0352-0424$01 .OO/O
for increasing the sensitivity of the detection system. First, a Part of the Stray radiation comes through the flow cell from the light Source into the PhotomultiPlier to raise the background level. Second, fluctuation of the light source causes variation of the sum of the scattered light and the emitted fluorescence to lower the signal-to-noise ratio. In order to diminish the stray radiation, a droplet of the eluate from a column was directly irradiated by laser beam (5, 6). On the other hand, as a way to excite the fluorophores instead of irradiation, reaction of oxalic esters with hydrogen peroxide (H,O,) has been investigated by several workers (7-9). The proposed mechanism of the chemiluminescence (CL) reaction was as follows: 0
0
1,2-dioxetanedione
0
0
/I IC1 +
r-
it 3-0
fluorophor
-
fluorophor*
fluorophor*
light
(excited state)
+
+
2CO2
fluorophor
It has been used mainly for the determination of H,Oz in solution (10-12), and only two papers have appeared on its 0 1980 American
Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
425
~~
Table I. Effect of Combination of Solvents on Chemiluminescence Reactiona
bufferb
0.2 0.2 0.2 1.0 1.0
solvent, mL ethyl acetoacetate nitrile 4.3 0.5 3.8 1.0 2.0 2.8 3.5 0.5 1.0 3.0
relative CL intensity
-
i
0.1
i
0.2
t
0.1 0.1
80 s 26.1 = 0.1 32.6 3 0 . 1 41.0 I 0.2 14.7 i 0.3
0.3
17.5 i 0.1
20 s
38.9 47.1 49.0 26.8
I
27.3
I
T , I 2 ,min 1.73 i 0.02 1.89 t 0.03 3.13 I0.09 1.15 i 0.05 1.59z 0 . 0 5
dansyl alanine,
Results are the average of two experiments. 0.05 M Tris HC1 buffer (pH 8.0). Final concentrations: 2.4 x i o 4 M : TCPO. 1 x 10-3 hi: H,o,. 1 x 10-3 M. a
use for the detection of fluorophores, such as dansyl amino acids, on thin-layer chromatograms (13, 14). Since the CL reaction seemed to be suitable t o avoid the fluctuating light source mentioned above, its applicability to the detection system for HPLC was examined. As the oxalic ester, bis(2,4,6-trichlorophenyl)oxalate (TCPO) was used because it is stable and easy to prepare (9) and high quantum yields can be obtained with its use (B), and as the representative fluorophores, dansyl amino acids were used in this work. The conditions for CL of the fluorophores were studied both in a simple static system and in a flow system, and the resultant suitable conditions were applied to the determination of dansyl amino acids by HPLC.
I t
Column
Solution TCPo
Mixing \,---
- 8
FI PM MC
EXPERIMENTAL Reagents. Dansyl alanine, dansyl glutamic acid, dansyl methionine, and dansyl norleucine were purchased from Sigma Chemical Company. H202(30%) was purchased from Mitsubishi Gas Kagaku Co. TCPO was prepared by the method of Mohan and Turro (9). All the other chemicals were of reagent grade. Procedure. Experiment in the Static System. Dansyl amino acid was dissolved in an aqueous buffer solution, which was the main constituent of the eluent for HPLC. TCPO and H202were dissolved in organic solvents. Aliquots of the solutions were combined in the order of dansyl amino acid, TCPO, and H202,then mixed vigorously by hand shaking. Since the spectrum of CL was the same as that of fluorescence, the CL intensity was measured at 510 nm with the band width of 40 nm using a Hitachi MPF-2A fluorescence spectrophotometer with the light source off. The time course of relative CL intensity was recorded following the addition of H202. The intensity increased rapidly and then decreased apparently according to a first-order rate equation. The period required to was determined from the slope of the reach ' / 2 intensity linear plots of log intensity vs. time. All the experiments were performed at room temperature (25 f 1 "C). Experiment in the Flow System. The apparatus shown in Figure 1 was used. The temperature of a column, pBondapak CI8(4 x 300 mm, Waters Associates Inc.), was maintained at 35 "C. A model 1720 Syringe Loading injector (Rheodyne Inc.) with a 20-pL loop and two DAM type dampers (Umetani Seiki Co.) were used. A detector, Schoeffel FS-970 LC fluoromonitor (Schoeffel Instrument Corp.) with a flow cell (3 X 3 X 7 mm), was adopted with the light source off. Stainless tubings with 0.5-mm diameter were used in all flow lines except a mixing coil which was made from the 0.8-mm diameter tubing. The eluent was the mixture of acetonitrile and 0.05 M Tris HCl buffer (pH 7.7). Ethyl acetate and acetone were used as the solvents for TCPO and H202,respectively. The eluate from the column and the reagent solutions were mixed by passing through the mixing coil which was connected to the flow cell. In order to promote the mixing of three solutions in the mixing coil, ethyl acetate containing TCPO and acetone containing H20, were first mixed in a mixing vessel and then the pre-mixed reagent solution was mixed with the eluate with use of four pumps (reciprocating type, Kyowa Seimitsu Co.).
RESULTS Conditions for CL in the Static System. Soluents Effect. Among the solvents tested for TCPO, dioxane and ethyl
1
Figure 1. Schematic diagram of the apparatus in the flow system. D, damper; F, flow cell; I, injector; MC, mixing coil; P, pump; PM, photomultiplier: R , recorder
.$:IC
r
\
7n
"I
'
~
\Acetonitrile \ Acetone
\\
}
\
n\A -\n ryl
\
alcohol n-Butyl alcohol
n - Propanol
'Ethanol
Methanol
I
0
1
2 3 4 5 Time (min)
6
Figure 2. Solvent effect on the intensity and life time of chemiluminescence. Dansyl alanine (0.1 M Tris HCI buffer, pH 8.0), TCPO (ethyl acetate), and HO , , (above each solvent) were mixed by volume 1:25:10. M, TCPO 1.7 X Final concentrations: dansyl alanine 5.7 X M, H,O, 6.9 x 10-4 M
acetate gave the highest CL intensity. Ethyl acetate was selected since TCPO was more stable in ethyl acetate than in dioxane. As the solvent for H 2 0 2 ,alcohols, acetone and acetonitrile, which promote the mixing of an aqueous buffer with ethyl acetate, were tested. As shown in Figure 2, acetone and acetonitrile yielded the longer life time, while the highest intensity was attained with acetonitrile, which was used for the subsequent experiments in the static system. Considering the results shown in Table I, the contents of aqueous buffer and acetonitrile in the reaction medium should be reduced as little as possible in order to obtain higher intensity and longer Concentration and p H of the Buffer Solution. When the concentration of the buffer (pH 8.0 Tris HCI) increased from 0.05 to 0.2 M, the initial CL intensity increased but the lifetime decreased. As the p H of the buffer (0.05 M phosphate) increased, the initial intensity increased but the lifetime decreased. The effect of p H of the Tris HCl buffer (0.05 and
426
ANALYTICAL CHEMISTRY, VOL. 5 2 , NO. 3, MARCH 1980
Table 11. Effect of Damper o n Reproducibility of Peak Height
.240-
damper
VI
eluent
re agent solution
peak height," c m
-b
-
+c
-
i
+
17.5 t 1.4 1 6 . 6 I1 . 5 1 6 . 5 t 0.4
+
1 6 . 7 i 0.3
5
20-
aJ
10-
+ C
-
% of S.D.
8.0 9.0 2.4 1.8
>
5 -
'z
CI
d
" Values are means * S.D. ( n = 3).
?t
Without damper. With damper. 0.05 M Tris HC1 buffer ( p H 7.7)-acetonitrile ( 7 : 3 ,v / v ; eluent, 0 . 4 m L / m i n ) , 5 mM TCPO in e t h y l acetate ( 0 . 4 m L / m i n ) , 1 0 mM H,O, in acetone ( 1 . 2 m L / m i n ) . Three p m o l of dansyl alanine were injected o n t o t h e column.
2.5T
L
0
60 90 Time ( s e c )
120
30
Figure 3. Effect of H,02 concentration on the intenslty and life time of chemiluminescence. Dansyl alanine (0.1 M Tris HCI buffer, pH 8.0), TCPO (ethyl acetate), and H,O, (acetonitrile) were mixed by volume 2:1:5. Final concentrations: dansyl alanine 2.9 X M, TCPO 6.2 x 10-4 M
0.1 M) was similar to that of phosphate buffer. Concentration of Reagents. The CL intensity increased with the increment of the concentration of TCPO from 3.9 X to 1.3 X M in the reaction medium containing 2.9 X lo4 M dansyl amino acid and 1.8 X M H202,while was unchanged. The results in Figure 3 indicate that the concentration of H202below 7.8 X M was insufficient in the reaction medium containing 2.9 X M dansyl amino acid and 6.2 X lo4 M TCPO, and the increment of the concentration above 1.6 x M increased the initial intensity whereas the lifetime rapidly decreased. Response to Dansyl Amino Acids. Under the conditions selected from the above data, the CL intensities of dansyl alanine, a t definite periods (20 and 80 s) after the initiation of the reaction, were proportional to the concentrations from 2.5 to 12.5 WM(the coefficient of variation was less than 4.4%). Conditions for CL in the Flow System. Effect of Damper. In order to increase the sensitivity, it is necessary to attain a stable base line (background) and reproducible peak height (CL intensity). For this purpose, syringe-type pumps are desirable because they yield a constant flow, but they were unavailable and reciprocating pumps were used in this study. T o diminish the pulse, two dampers were used (Figure 1). The one which was connected to the flow line before the injector did not improve the reproducibility of the peak height because the column itself functioned as a kind of damper, while the second damper which was connected to the flow line from the mixing vessel was more effective as shown in Table 11. Flow Rate of Each Solution. Acetonitrile used as the solvent for H 2 0 2in the static system was not suitable in the flow system because of the unstable base line and irreproducible peak height. Acetone gave much improved results, probably because of the faster mixing with the other solutions. Similar disturbances in the base line and the peak height were observed when the flow rates of the solutions were not proper as shown in Table 111, in which the best reproducibility was
obtained when the flow rates of the eluent, TCPO solution, and H202solution were 0.2,0.4, and 1.6 mL/min, respectively. Higher flow rates of the eluent caused the precipitation of TCPO on account of water in the flow line, which disturbed the flow rates. Mixing of the Reaction Solution. Table IV demonstrates the effect of the mixing coil (duration of mixing). A mixing coil of 70 cm gave the best results, while a shorter one gave larger variation and a longer one gave smaller peak height when the flow rates of the eluent, TCPO solution, and HzOz solution were fixed tentatively a t 0.4, 0.3, and 1.3 mL/min, respectively. Concentration of H202. The suitable concentration of H202 to produce the maximum peak height was found. With higher concentration, the peak height decreased because of the short life time of CL compared with the duration of mixing (cf. Figure 3). Determination of Dansyl Amino Acids by HPLC. For the adequate separation of dansyl-alanine, glutamic acid, methionine, and norleucine, a column of FBondapak CISwas operated with the mixture of 0.05 M Tris HC1 buffer (pH 7.7)-acetonitrile (16:9, v/v) a t the flow rate of 0.18 mL/min. On the basis of the data of flow rates in Table 111, the flow rates of the reagent solutions were modified to be suitable for the CL detection. Figure 4 shows the chromatogram of 400 fmol of each dansyl amino acid. The peak heights of those four dansyl amino acids were proportional to the quantities from 50 fmol to 40 pmol injected onto the column. In the working range, the coefficient of variation was less than 3%.
DISCUSSION In the determination of H202by the CL reaction, Williams and Seitz used methanol for mixing the two phases of solutions ( I I , I 2 ) . However, our data in Figure 2 indicate that acetone
Table 111. Effect of Flow Rates on Peak Height and Base Line flow rate, m L / m i n eluenta 0.2 0.2 0.2 0.2 0.4 0.4 0.4 0.4
5 mM TCPO in ethyl acetate
1 0 mM H , O , in acetone
peak height,b c m
% of S.D.
base line
0.4 0.4 0.8 0.8 0.4 0.4 0.8 0.8
0.8 1.6 0.8 1.6 0.8 1.6 0.8 1.6
1 4 . 8 i 0.3 8 . 0 i 0.1 21.6 I1 . 2 1 4 . 5 z 0.3 7.8 I 0.4 7.5 i 0.2 1 4 . 3 z 1.1 1 3 . 1 r 0.7
2.0 1.3 5.6 2.1 5.1 2.1 7.7 5.3
S' S USd
0.05 M Tris HCI buffer ( p H 7,7)-acetonitrile ( 1 3 : 7 , v i v ) . Three p m o l of dansyl alanine were injected o n t o the column.
Values are means
T_
S.D. ( n = 3).
S
us us
S
us Stable.
Unstable.
ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980 ~
_
_
Table IV. Effect of Length of Mixing Coil o n Peak Height' length coil,
of
duration of mix-
cm
ing, s
nb
4.5 10.6 19.6
4 3 3
30 70 130
peak height, cm
alanine
norleucine
9.3 I 0.8
10.5 i 0.8
0.1 I 0.1
1 4 . 2 t 0.2 10.5 i 0 . 2
12.1 8.9
I
' 0.05 hl Tris HCI buffer ( p H 7.7)-acetonitrile (13:7, v / v ; eluent, 0.4 mlimin), 5 mM TCPO in ethyl acetate ( 0 . 3 mL/min), 12.5 mM H,O, in acetone (1.3 mL/min). The mixture of 3 pmol of dansyl alanine and 4 pmol of dansyl norleucine was injected onto the column. ber of measurements.
15
c
Num-
Met
n
E
U
10-
ECT, .-
aJ
I
_
427
Excess concentration of TCPO causes precipitation in the flow line to disturb the flow. Then there is a limitation of the usable concentration of TCPO according to the flow rates of eluent and acetone. Next is the selection of suitable length of the mixing coil to obtain reproducible peak heights. When the above conditions are fixed, the most suitable concentration of H,Oz for peak height should be adjusted last since HzOz resolved well in any composition of the solution. The sensitivity of the present method, in which 10 fmol of dansyl amino acids are to be detected, is far more than that of the conventional fluorescence detection method in which the detection limit for dansyl derivatives is 100 pmol (15)and is comparable to that of the laser-induced fluorescence detection method in which 2.5 fmol of aflatoxins (5) and l pmol of zearalenone (6) can be detected. The sensitivity of the present method will be increased by changing the shape of the flow cell so as to be placed more closely to the photomultiplier. In this work, the effort was focused on the applicability of CL reaction to the detection system for HPLC using artificial samples of dansyl amino acids. However, the proposed method might be applied to biological samples, for example, to the determination of N-terminal amino acid of some protein a t the level of 10 ng, assuming that the molecular weight is loo00 and the recovery of the amino acid through the whole procedure of derivatization, extraction, and hydrolysis is about 10%. On this subject, further investigations are in progress in our laboratory. This work opened the way to high sensitive determinations of fluorophores by HPLC with the use of the CL reaction. Fluorophores other than dansyl derivatives, which are suitable for the generation of CL (IO),should be determined in the femtomole range by the present method with some modifications for reaction conditions.
ACKNOWLEDGMENT
Time (rnin) Flgure 4. Chromatogram of dansyl amino acids. The mixture of 400 fmol of each dansyl amino acid was injected onto the column. 0.05 M Tris HCI buffer (pH 7.7bacetonitrile (16:9, v/v; eluent, 0.18 mL/min), 5 mM TCPO in ethyl acetate (0.51 mL/min), 0.5 M H,O, in acetone (1.2 mL/min). Glu, dansyl glutamic acid; Ala, dansyl alanine; Met, dansyl methionine; Nor-Leu, dansyl norleucine
and acetonitrile are more suitable to give higher intensity. The data in the static system (Figure 2 and 3) demonstrate that the CL intensity decays apparently according to a first-order rate equation and so the measurement should be done as soon as possible. In fact, care should be taken on the CL detection for thin-layer chromatography (13,14). On the contrary, in the flow system, reproducible peak heights, which are proportional to the concentration of fluorophores, are obtained on the chromatogram since the detector measures the CL intensity during the fixed stage of the reaction. How to obtain the optimum conditions in the flow system can be explained as follows: As shown in the results in the static system, the increments of both the percent of ethyl acetate in the reaction medium (Table I) and the concentration of TCPO led to the increase of CL intensity. In the flow system for the purpose of thorough mixing of the solution, the increment of the flow (which means the increment of volume ratio) of ethyl acetate was restricted to some extent.
The authors thank Zenzo Tamura of University of Tokyo for the valuable suggestions and support. Thanks are also due to Hiroaki Tanaka for his advice. The authors are also grateful to Kyowa Seimitsu Co. (Tokyo) for the use of pumps and Atto Corp. (Tokyo) for the use of the detector.
LITERATURE CITED (1) K. Imai and 2 . Tamura, Clin. Cbim. Acta, 85, 1 (1978). (2) K. Samejima, J . Chromatogr., 96, 250 (1974). (3) K. Mori, Jpn. J. Ind. Health, 17, 116 (1975). (4) J. C. Dickinson, H. Rosenblum, and P E. Hamilton, Pediatrics, 36, 2 (1965). (5) G. J. Diebold and R. N. Zare, Science, 196, 1439 (1977). (6) G. J. Diebold, N. Karny, and R. N. Zare, Anal. Cbem., 51, 67 (1979). (7) M. M. Rauhut, L. J. Bollyky, E. G. Roberts, M. Loy, R. H. Whitman. A. V. Iannotta, A. M. Semsel, and R. A. Clarke, J. Am. Cbem. SOC.,89,
6515 (1967). (8) M. M. Rauhut, Acc. Cbem. Res., 2, 80 (1969). (9) A. G. Mohan and N. J. Turro, J. Cbem. Educ., 51, 528 (1974).
(IO) P. A. Sherman, J. Holzbecher, and D. E. Ryan, Anal. Chim. Acta. 97, 21 (1978). ( 1 1 ) D. C. Williams 111, G. F. Huff, and W. R. Seitz, Anal. Cbem., 48, 1003
(1976). (12) D. C. Williams I11 and W. R . Seitz, Anal. Cbem., 48, 1478 (1976). (13)T. G. Curtis and W. R. Seitz. J . Chromatogr., 134, 343 (1977). (14) T. G. Curtis and W. R. Seitz, J. Cbromatogr., 134, 513 (1977). (15) T. Seki and H. Wada. J . Chromatogr., 102, 251 (1974).
RECEIVED for review May 30, 1979. Accepted September 27, 1979. A preliminary report of the work was presented at the 27th annual meeting of Japan Society for Analytical Chemistry, Kanazawa, October 11-13, 1978, Abstract p 528.
