420
Anal. Chem. l W 3 , 55,428-432
tractive forces is less for DL-tryptophan (Figure 6) than for the almost totally ionized phenylethylamine and p-methylaniline (Figures 4 and 5).
(15) Melander, W. R.; Kaighatgi, K.; Horvath, C. J . Chromafogr. 1980, 201, 201-208. (16) Melander, W. R.; Horvath, C. J . Chromatogr. 1980, 207, 211-224. (17) KonlJnendiJk,A. P.; van de Venne, J. L. M. I n "Advances In Chromatography 1979"; Ziatkls, A., Ed.; Chromatography Symposium: Houston, TX, 1979; pp 451-462. (18) van de Venne, J. L. M.; Hendrlkx, J. L. H. M.; Deelder, R. S,J , Chromatogr. 1978, 767, 1-16. (19) Cantwell, F. F.; Puon, S. Anal. Chem. 1979, 57, 623-632. (20) Iskandarani, 2.; Pietrzyk, D. J. Anal. Chem. 1962, 54, 1065-1071. (21) Bldiingmeyer, B. A.; Deming, S. N.; Price, W. P., Jr.; Sachok, B.; Petrusek, M. J . Chromafogr. 1979, 786,419-434. (22) Stranahan, J. J.; Deming, S.N. Anal. Chem. 1982, 54, 1540-1546. (23) Stranahan, J. J.; Deming, S. N. Anal. Chem. 1982, 54, 2251-2256. (24) Locke, D. C. J . Chromatogr. Scl. 1974, 72, 433-437. (25) Everett, D. H. Trans. Faraday SOC. 1984, 6 0 , 1803-1813. (26) Everett, D. H. Trans. Faraday Soc. 1985, 61, 2478-2495. (27) Langmuir, I. J . Am. Chem. SOC.1917, 39, 1848-1906. (28) Szyszkowskl, B. 2.Phys. Chem. 1908, 64, 385-414. (29) Kong, R. C.; Sachok, 8.; Deming, S. n. J . Chromatogr. 1980, 199, 307-316. (30) Knox, J. H.; Hartwick, R. A. J . Chromatogr. 1981, 204, 3-21. (31) Wahiund, K.-G.; Beijersten, I. Anal. Chem. 1982, 54, 128-132. (32) O'Neiil, R. Appl. Statist. 1971, 20, 338-345. (33) Chambers, J. M.; Ertei, J. E. Appl. Statlst. 1974, 23, 250-251. (34) O'Neili, R. Appl. Sfafisf. 1974, 2 3 , 252. (35) Benyon, P. R. Appl. Statist. 1978, 2 5 , 97. (36) Hill, I. D. Appl. Staflst, 1978, 2 7 , 380-382. (37) Addison, C. C. J . Chem. SOC. 1945, 98-106. (38) Rosen, M. J. "Surfactants and Interfacial Phenomena"; Wiiey: New York, 1978. (39) Draper, N. R.; Smith, H. "Applied Regression Analysis"; Wiiey: New York, 1966. (40) Econ, C.; Novosel, B.; Guiochon, G. J . Chromatogr. 1973, 83, 77-89.
ACKNOWLEDGMENT We thank J. J. Stranahan and We-Y.Lin for helpful discussions. Registry No. I-Hexanol, 111-27-3;I-pentanol, 71-41-0; nvaleric acid, 109-52-4.
LITERATURE CITED (1) Farulla, E.; Iacobelii-Turl, C.; Lederer, M.; Salvetti, F. J . Chromatogr. 1983, 72, 255. (2) Horvath, C.; Lipsky, S. R. Nature (London) 1986, 27 7 , 748-749. (3) Wlttmer, D. P.; Nuessie, N. 0.; Haney, W. G., Jr. Anal. Chem. 1975, 47, 1422-1423. (4) Sood, S.P.; Sartori, L. E.; Wittmer, D. P.; Haney, W. G. Anal. Chem. 1978, 48, 796-798. (5) "Paired-Ion Chromatography, an Alternative to Ion-Exchange"; Waters Associates: Milford, MA, 1975. (6) Knox, J. H.; Lalrd, G. R. J . Chromatogr. 1978, 722, 17-34. (7) Knox, J. H.; Jurand, J. J . Chromatogr. 1976, 125, 89-101. (8) Fransson, B.; Wahlund, K.-G.; Johansson, I.M.; Schill, G. J . Chromatogr. 1978, 725, 327-344. (9) Kraak, J. C.; Jonker, K. M.; Huber, J. F. K. J . Chromatogr. 1977, 142, 67 1-688. (IO) Terweij-Groen, C. P.; Heemstra, S.; Kraak, J. C. J . Chromafogr. 1978, 161, 69-82. (11) Horvath, C.: Melander, W.; Moinar, I.; Moinar, P. Anal. Chem. 1977, 49, 2295-2305. (12) Hoffman, N. E.; Liao, J. C. Anal. Chem. 1977, 49, 2231-2234. (13) Tomiinson, E.; Jefferies, T. M.; Riley, C. M. J . Chromatogr. 1978, 159 - - , 315-358. - .(14) Hearn, T. W. I n "Advances in Chromatography"; Giddings, J. C., Grushka, E., Cazes, J., Eds.; Marcel Dekker: New York, 1980; Vol. 16, pp 59-100.
RECEIVED for review August 30, 1982. Accepted November 16, 1982; This work was supported in part by a grant from Chevron Research Co.
Elution Volume Measurement in Size Exclusion Chromatography T. A. Chamberlin" and H. E. Tuinstra Central Research -Chemical
Products, Dow Chemical Company, Midland, Michigan 48640
in elution volume measurement. For the types of columns mentioned above, this means that the elution volume should be reproducible to within 10-25 pL. The most common technique for attaining this degree of precision is the use of highly sophisticated pumping systems. Even these, however, suffer from day-to-day variations and must be regularly recalibrated, or, alternatively, "known" systems must be routinely subjected to analysis in order to check the system performance. While these techniques may lend an amount of solace to the analyst, there is really no guarantee that the previous or subsequent run is an exact duplicate of the calibration run. Other techniques (siphon dump counters, direct weighing, internal standards, etc.) have been used to correct for variations in the performance of the system, but none of these have been shown to be much more satisfactory than using time and calibrated pump setting (with frequent checking). Recently, Miller and Small (7) described a flowmeter which appears to be ideally suited for elution volume measurement in size exclusion chromatography (SEC) experiments. Since we were concerned with the problems related to precise elution volume measurements, we obtained one of their flowmeters and examined its utility in this particular application. The flowmeter has proven to be even more useful than we had
A detailed study of the performance of a thermal pulse timeof-flight flowmeter for measuring elutlon volume in size exclusion chromatography has been carrled out. Use of such a flowmeter allows both minor and major flow variations to be accommodated. Preclsions of molecular weight dlstrlbutlon calculatlons based on thls form of elutlon volume measurement have been found to be wlthln 4 % under all flow conditions studied.
In any chromatographic determination of the molecular weight distribution of a polymeric system, the most critical measurement is that of elution volume. This is especially true when the analyst is using modern small particle (and consequently small active volume) high-performance columns. The effects of variations in the value of the elution volume-either controlled or uncontrolled-have been calculated (1,2) and measured (3-6). As a general rule, the accuracy of the calculated molecular weight at any particular elution volume is about 10 to 20 times the precision of the corresponding volume measurement. Since modern columns generally possess active volumes in the range of 2-5 mL, a precision in derived molecular weight of 5% would require a t least 0.5% precision
a
0003-2700/83/0355-0428$01.50/0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
initially hoped. It operates by the simple timing of the paasage of a thermal pulse through a well-defined volume, generates microprocessor compatible output, is simple to understand and use, and has proven to be very reliable. The utility of this flowmeter in terms of precision in elution volume measurement constitutes the major portion of this report. EXPERIMENTAL SECTION Chromatographic System. The pump used in this work is a Waters Model 45 LC pump, usually connected directly to a Rheodyne Model 7125 sample injection valve. In a few instances, a modest damping arrangement consisting of an Altex llOA damper followed by a set of two SEC columns is placed between the pump and the injection valve. The valve is connected directly to the first of four Brownlee Aquapore-OH (4000,1wO, 500, and 100) SEC columns. The effluent is then led into an LDC Spectromonitor 111 variable-wavelength UV detector, to a Waters Model 401 differential refractive index detector, and finally into the flowmeter (available from Molytek Corp., Pittsburgh, PA). The effluent from the system is passed through a septum in the side a n n of a 10-mLautozeroing precision buret. This last portion of the system is used only during cell calibration experiments. Polymer. As both a probe system and as a broad standard, we used a highly characterized polystyrene available locally. This system (polystyrene 1683) has been characterized by SEC, ultracentrifugation, light scattering, and coupled SEC/low angle laser light scattering (LALLS).The polymer is identical with that used in ASTM method D 3536-76. By use of the preceding method, the polymer has been shown to have weight average molecular (M.) weight of 250000 4000 (IC,30 determinations) and number average molecular weight (MJof 100000 3000. Similar determinations measured by other techniques are as follows: ultracentrifuge, M, = 250000, light scattering, M, = 256000; and SEC/LALLS, M, = 252000. The polymer is dissolved to approximately 1.0 w t 5% in THF (Burdick and Jackson) which is also used as eluent throughout all of this work. Injection sizes are used such that 200 mg of polymer are introduced into the system during each experiment. Software. All experiments are monitored by a Digital Equipment Corp. MINC-11 based system. The MINC monitors either of two analog signala originating from the detection system (DRI or UV). In addition, a digital signal from the injection valve is used to initiate collection of data. Another digital signal generated by the flowmeter is used to determine the time of flight of the thermal pulse through the flow detector. This signal is timed to an accuracy of 0.1 ms and is used for either calibration or measurement of elution volume, depending upon the type of experiment being conducted. When the experiment is one of measurement, the time of fight is converted into volume (see Discuesion section), and this value is stored in an appropriate array along with the corresponding number of counts and digitized value from the analog detector being used. At the end of the run, a graphic display of the raw data is presented enabling the operator to set base line limits. Base line normalized data rn then used to cany out either a broad standard calibration (8)or an analysis. Before an analysis, the data array is modified to contain 1024 equal volume increments by means of linear interpolation. Operator options include the display or plotting of various depictions of the flow profile during the experiment. The results from a calculation of the molecular weight distribution (MWD) for any particular set of data can also be presented in several different formats. One useful format is comprised of distribution profiles (differential and integral) as well as a listing of the more usual MWD parameters. Calibration of the column is carried out with a fifth order polynomial relationship between log (molecular weight) and elution volume. A minor modification of a DEC supplied (9) multiple regression algorithm is used to generate the appropriate coefficientsfor thia relationship. The coefficients are stored, along with other pertinent information, in a header portion of the file containing the data for each run.
*
*
DISCUSSION Performance of t h e Thermalpulse Precision Flowmeter. In order to determine whether or not the flowmeter
429
1. a
.e -
>
L I.
Y
"I rnl
Elution Volun.
(11)
Figure 1. Comparlson of thermalpulse precision flowmeter (A) and dltterentlal transducer measurements (E) of flow rate, moderately damped system. I
A
'-1
*=I
--..Elutlon Volum.
(nl)
Flgura 2. Comparlson of thermalpulse precision flowmeter (A) and dlfferentlal transducer (6) measurements of flow rate. highly damped system was sensitive to changes in flow rates, we compared its output to that of a differential pressure transducer connected across a 30 cm X 0.15 mm stainless capillary located at the outlet of the SEC columns. The pump was set a t 0.90 mL/min, and the output of the transducer (Validyne DP-15,50 psig diaphragm) adjusted to an output of 0.5 V with a simple voltage divider. During the course of the experiment (400 "counts", about 10 mL), the flow was intentionally changed three times at the pump. Figure 1 shows the output from both the flowmeter (A) and the transducer (B).Although the scales are not the same, it is easily seen that the response of the flowmeter nicely parallels that of the more conventional flow measurement. In the figure, the flow rate for each point along the digital plot has been derived as discussed below. Figure 2 is an analogous plot using a more effectively damped pumping system. On the basis of these results, we concluded that the flowmeter did indeed function as a digital flow monitoring system. Cell Constant Measurement for the Thermalpulse Precision Flowmeter. In order to use the flowmeter, it is necessary to determine values for the two constants whichalong with the flow rate-control the elapsed time between thermal pulses in the system (7). This is accomplished by collecting a number of these elapsed times while at the same time, carrying out accurate measurements of the flow rate. A convenient technique for generation of this type of data is to direct the effluent from the system into the side arm of an autozeroing buret of well-characterizedvolume. In our system, a 10.WmL buret was used. The MINC was directed to collect the elapsed time between each of approximately 200 pulses, and to subject the resulting data to standard regression analysis, yielding a statistically defined thermal pulse time of flight. During this same period, the time to fill the buret
430
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
Table I. Reproducibility of Molecular Weight Measurementsa run no. Mw Mrl Mz 419500 246980 95180 TEST50 99100 425500 TEST51 248700 TEST52 246800 94200 424400 TEST53 246500 98300 421400 96700 422700 average 247200 deviation 1000 2400 2700 0.4% 2.4% 0.7% precision a 200 p g PS-1683 injected at 1.0 mL/min nominal flow rate. from the 10.00-mL mark to the autozero point was measured with an electrical timer accurate to 0.1 s. By carrying out such experiments over a variety of pump settings, we collected values for observed flow rate and time of flight (T(eff)). A series of 15 experiments a t six different flow rates (1.3-0.8 mL/min pump setting) were carried out. The results were analyzed according to the reported relationship (7) T(eff) = V / f
+K
where T(eff) is the time between pulses, V is the cell volume (wL), f the observed flow rate (,uL/ms), and K is a constant (ms) related to the performance of the power thermistor which imparts the heat pulse to the stream. On the basis of 1,and the collected data, values for V (12.28 wL) and K (882.96 ms) were found to correlate the data with a correlation coefficient of 0.9987. The relative standard deviation ( l a ) observed for experiments carried out at a constant pump setting was about 0.2% for both observed flow rate and T(eff), for all of the pump settings examined. Use of the Flowmeter. Once the cell constants have been measured as discussed above, the flowmeter is used to measure flow rate by using the time between pulses in a rearranged form of eq 1
Table 11. hlolecular Weight Distribution of Polystyrene 1683 a thermal pulse conventional runno. :Ifn M, M\\, :)In &IL TEST60 242.7 98.0 421.2 232.2 90.8 404.7 TEST61 241.5 97.0 418.8 210.2 96.7 416.2 TEST62 242.0 97.1 414.3 242.1 96.1 415.6 TEST63 251.1 104.3 429.5 238.6 98.4 408.1 calib 247.9 99.1 421.3 247.9 99.1 423.4 TEST65 242.1 97.3 417.7 244.8 98.6 423.6 TEST66 249.1 101.0 425.0 244.8 104.1 436.8 TEST67 251.5 98.3 435.6 255.3 98.2 441.3 TEST68 250.1 101.2 427.9 251.9 101.3 434.7 TEST69 233.5 103.5 432.3 252.9 102.7 432.8 av 247.2 99.7 424.6 245.1 98.6 423.7 deV 4.6 2.7 7.2 3.7 12.5 1.6 RSD 1.9% 2.7% 1.6% 2.9% 3.S% 3.0% Comparison of conventional and thermal pulse techniques (MWD x 1000). Table 111. Effect of Minor Flow Discrepancies on Calculated Molecular Weight Distributions ( M , X 1000) flow rate conventional volumetric thermal run no. (nominal) (1.0 mL/min) corrected pulse TEST34 0.94 246.9 247.0 89.1 TEST35 0.96 131.2 250.0 248.6 TEST36 0.98 165.6 251.1 254.8 TEST37 1.00 214.3 256.5 256.6 TEST38 1.02 257.0 258.3 267.0 TEST39 1.05 323.8 253.6 254.5 TEST41 1.07 619.6 248.9 251.0 av 1.00 258.6 252.0 253.0 dev 0.05 178.1 3.8 4.2 68.8% 1.5% 1.7% RS D 4.7%
flow rate = cell volume/(T(eff) - K ) followed by conversion to volume through the relationship volume = T(eff) X flow rate This manipulation is done immediately after collection of the analog and T(eff) data. Also, the incremental calculated volume is added to all of the preceding volumes, resulting in a total measured elution volume. The result from this is an array containing “counts”, digitized analog values from the mass detector, incremental volumes, and total volume. The array is then converted to 1024 equal volume increments by linear interpolation and this data set used for all subsequent calculations. Reproducibility. Several experiments were carried out on the system as described in order to determine what precision might be obtained by using this technique to monitor elution volume. Use of TEST51 as calibration sample (Le., the results from this run were used to generate a calibration equation based on the known distribution of polystyrene 1683) led to the results shown in Table I. Another ten replicate injections of PS-1683 were analyzed in the usual manner. This time, however, the software had been modified so that MWD parameters could also be calculated in the conventional (i.e., calibrated pump setting and time) manner using the same data base. These data are shown in Table 11. Effect of Small Flow Rate Errors. The ability of the flowmeter to handle small errors in flow rate, such as might be encountered because of check valve failure, lack of pump
6
a
10
12
Elution V o l u m e (ml)
Figure 3. Effect of minor flow rate offsets in time-based profiles of polystyrene 1683. Data are from Table 111.
resettability, or undetected minor leaks in the system, was examined by connecting the pump to a Waters solvent programmer, adjusting the final flow rate to 1.10 mL/min (nominal), and carrying out experiments at various offsets from this value. Additionally, the software was further modified so that the final volume (as measured by the flowmeter) could be used in an equivalent manner to the elution time of an internal standard. The data collected for any given run were then analyzed by all three techniques. The results from these experiments are shown in Table 111. Examination of the data in Table 111leads to the conclusion that the flowmeter is at least as precise as using an internal standard for determining MWD of polymeric systems. Additionally, there is no interference in the mass detector when using this technique of elution volume measurement. A much more graphic demonstration of the ability of the flowmeter to track minor flow variations is shown in Figures 3 and 4. Figure 3 contains the base line corrected data for the runs
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
431
1.20.
a
6
12
10
Elution V o l u m e (ml)
Flgure 4. Effect of minor flow rate offsets in the flowmeter based profiles of polystyrene 1683. Data are from Table 111. Flgure 7. The flowmeter-derived flow rate profile for solvent programmed from 0.80 to 1.20 to 0.80 mL/min over a 12-min period. :.a1
I
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+
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+
+
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Figure 5. The flowmeter-derived flow rate profile for solvent pro-
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0
E
,
lZ
E
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.
,
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Figure 8. Elution profiles for dramatically changing flow rates, conventional time-based profile.
grammed from 0.80 to 1.20 mL/min over a 12-min period.
--~-7,a.
a
8
1
a
4
(mi>
Figure 6. The flowmeter-derived from flow rate profile for solvent programmed from 1.20 to 0.80 mL/min over a 12-min period.
listed in Table 111plotted according to conventional (Le., time vs. detector output) methods. Figure 4 shows exactly the s a n e data plotted according to elution volume as measured by the flowmeter. Effect of Major Flow Perturbations. Since the flowmeter has the ability to account for minor discrepancies in constant flow rate, we decided to examine how it performs under changing flow rate conditions. Using the Waters flow programmer, we made dramatic changes in flow rate during analyses. The nature of these changes is shown in Figures 5-7. Figure 5 shows the flow profile (as measured by the flowmeter)-during an analysis-for a programmed change from 0.80 to 1.20 mL/niin over a 12-min period. Figure 6 shows the flow profile for the reverse of the program used to generate Figure 5. Figure 7 shows the rather bizarre flow rate change from 0.80 to 1.20 to 0.80 mL/min over a similar 12-min period. It is rather to difficult to imagine such flow changes actually occurring during ;an SEC experiment, except perhaps for the gradual failure of one pump head in a dual-headed
8
12
10
Elution Volume
(ml)
Flgure 9. Elution profiles for dramatically changing flow rates, volume corrected (analogous to internal standard: based profiles.
Table IV. Effect of Major Flow Changes on Calculated MWD Parameters ( M , X 1000) volume thermal conventional run no. (1.O mL/min) corrected pulse 98.8 123.1 TEST42 252.3 (0.8-1.2) 753.3 TEST43 416.4 263.8 (1.2-0.8) TEST44 257.8 54 2.9 261.8 (0.8-1.2-0.8) 265.8 465.0 av 259.3 334.1 146.8 dev 6.1 55.2% RSD 71.9% 2.4% pump. Carrying out the three different types of elution volume measurements as before (Le., conventional, volume corrected and thermal pulse) leads to the results collected together in Table IV. Graphic displays of the elution profiles corresponding to the three different approaches to elution volume measurement
Anal. Chem. 1083, 55, 432-435
432
an SEC experiment. We have carried out many variations of flow perturbations during typical analyses and have yet to find conditions that the flowmeter cannot accommodate.
LITERATURE CITED
Flgure 10. Elution profiles for dramatically changing flow rates, the
flowmeter-derived profiles.
are shown in Figures 8-10, The use of the flowmeter for elution volume measurement comdetelv accommodates the dramatic flow changes occurring d&ng these experiments and also quite adequately accounts for any concievable (and even some inconcievable) variations in flow rate occurring during
Bly, D. D.; Stoklosa, H. J.; Kirkland, J. J.; Yau, W. W. Anal. Chem. 1975, 4 7 , 1810-1813. "Flow Precislon in a New HPLC Pumping System"; Du Pont Liquid ChromatographTechnical Report; Analytical Instrument Division, Concord Plaza, McKean Bldg.: Wllmlngton, DE. Schuiz, W. W. J . L i 9 . Chromatogr. 1980, 3, 941-952. Letot, L.; Lesec, J.; Qulvoron, C. J . L l 9 . Chromatogr. 1980, 1637-1655 Baker, D.-R.; George, S. A. Am. Lab. (Falrfield, Conn.) 1980, 42-46. Mori, S.; Suzukl, T. J . Li9. Chromatogr. 1980, 3, 343-351. , Miller, T. E., Jr.; Small, H. Anal. Chem. 1982, 5 4 , 907-910. (8) Yau, W. W.; Kirkland, J. J.; Bly, D. D. "Modern Size-Exclusion Liquid Chromatography"; Wiley: New York, 1979; p 294. (9) "Scientific Subroutines Proarammer's Reference Manual". AA-1101D. Digital Equipment Corp., h h n l c a i Documentation Center, Cotton Road, Nashua, NH 03060, revised, June (1980). ~
RECEIVED for review October 15, 1982. Accepted December 3, 1982.
Peroxyoxalate Chemiluminescence Detection of Polycyclic Aromatic Hydrocarbons in Liquid Chromatography Kenneth W. Slgvardson and John W. Birks" Department of Chemistry and Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, Colorado 80309
Peroxyoxalate chemlluminescence is applied to the hlghperformance llquld chromatographlc detectlon of polycyclic aromatlc hydrocarbons (PAH). PAH are excited by energy transfer from the decomposition products of the reactlon between hydrogen peroxide and bls(2,4,6-trichlorophenyl) oxalate. These reagents are Introduced by postcolumn mlxlng, and the emission Is observed by uslng a conventlonal fluorescence detector wlth Its source turned off. The method yields llnear reponses to PAH over 3 orders of magnltude, and In some cases detectlon llmlts are better than those determlned by fluorescence uslng the same fluorometer. Chemllurnlnescence detectlon Is compared to ultravlolet absorbance and fluorescence detectlon of PAH In terms of sensltlvlty and selectlvlty
the most complex PAH mixtures. HPLC detection of PAH, on the other hand, is aided by the selectivity of ultraviolet absorbance and/or fluorescence detectors. In this paper the application of a new chemiluminescence method to HPLC analysis of PAH is described and evaluated. Peroxyoxalate chemiluminescence is the most efficient nonenzymatic chemiluminescent reaction known, having quantum yields as high as 25% (2). The chemiluminescent reaction is illustrated by the reaction of an oxalic ester, such as bis(2,4,6-trichlorophenyl) oxalate (TCPO), with hydrogen peroxide and a fluorescent compound:
.
Polycyclic aromatic hydrocarbons (PAH) represent the largest class of chemical carcinogens occurring in the environment. The sources of environmental PAH are due to a variety of processes-both natural and anthropogenic. High-temperature combustion processes have become the major contributors in most urban areas (1). Due to the large number of chemical events which can take place in a combustion process, the mixtures of PAH produced are extremely complex. Currently, there is no single analytical method capable of providing the complete analysis of complex PAH mixtures. High-resolution gas chromatography and highperformance liquid chromatography (HPLC) are the two most commonly used methods of separating PAH mixtures. High-resolution gas chromatography provides the highest separation efficiencies and is necessary in the separation of 0003-2700/83/0355-0432$01.50/0
FLUOROPHOR*+
FLUOROPHOR r'
hu
Hydrogen peroxide and the oxalic ester react, forming intermediates capable of transferring as much as 106 kcal/mol of energy to the fluorescent acceptor (3). The postulated key intermediate is 1,Z-dioxetanedione ( 4 ) . Once excited, the fluorescer emits its characteristic light, as it does in fluorescence detection. The first analytical application of peroxyoxalate chemiluminescence was reported in 1976 by Seitz and co-workers (5). Hydrogen peroxide was measured in a flow injection system and was found to have a linear working range of more than 4 orders of magnitude. This system has since been improved C 1983 American Chemlcal Society
