Anal. Chem. 1986, 58,1187-1192
1187
Sample Cleanup Procedure for Polynuclear Aromatic Compounds in Complex Matrices D. L. Karlesky, M. E. Rollie, a n d I. M. Warner* Department of Chemistry, Emory University, Atlanta, Georgia 30322 Chu-Ngi H o
Department of Chemistry, East Tennessee State University, Johnson City, Tennessee 37614
This paper describes a procedure for fractionating polynuclear aromatic compounds (PNAs) from complex matrices according to number of aromatic rings. The procedure uses cartridges packed with amino polar bonded phase packing materials to achieve chromatographic separation of the polynuclear aromatic compounds. Data are provided demonstrating the applicability of the approach for isolating PNAs from cornpiex matrices. This procedure is compared to a fairly well-known solvent extraction scheme for the isolation of PNAs. Finally, the utility of the approach is further demonstrated by applying the method to an extract of air particulate sample acquired from an oil refinery in the Lake Charles, LA, area.
Polynuclear aromatic compounds (PNAs) are an important class of chemical pollutants commonly found in urban air. Many PNAs are on the Environmental Protection Agency (EPA) priority pollutants list because they are known or suspected to be mutagenic and/or carcinogenic ( 1 , 2 ) . These PNAs are derived from diverse sources such as gasoline and diesel exhausts, power plant emissions, and as byproducts of crude oil refining. Furthermore, the potential hazards of PNAs increase significantly when they react with other common pollutants in the environment. Thus, two pollutants that may not have any known mutagenic or carcinogenic effect can become hazardous when they react to form a new compound. Josephson (3) has summarized many of the known examples of this type and the known mechanisms for the mutagehicity and carcinogenicity of PNAs. Because of the above-mentioned facts, the analysis of PNAs in the environment is very important. Depending upon the source of pollution, environmental samples containing PNAs can be extremely complex and difficult to analyze. In addition to the parent PNAs and their isomers, substitution, alkylation, and hydrogenation of the molecules can all contribute to increase the complexity of the samples. Moreover, in environmental samples, PNAs are often present only as a small fraction of the total organic matter. Hence, besides the normal separation and extraction steps, it is also necessary to separate the PNAs from the other classes of organic compounds. Many procedures based on chromatographic separation and spectroscopic methods have been developed for the determination of PNAs. Interested readers can consult some of the extensive reviews available (4-8). The extensive and time-consuming separations, however, tend to increase the chances of loss of compounds of interest and contamination, thus reducing the detectability of the analyte and reproducibility of the analysis. Therefore, these separation procedures are extremely important and must be carefully conceived and prudently executed. Natusch and Tomkins (9) have developed an extraction procedure using dimethyl sulfoxide and pentane to separate
the PNAs from the aliphatic compounds in the samples. An alternative approach employing HPLC using a polar amine bonded phase to separate PNAs according to ring size has been shown by Wise and co-workers ( l o ) ,and this principle has been automated and applied by Sonnefeld et al. ( 1 1 ) . Chmielowiec and George (12) have evaluated the performance of these polar bonded phases for HPLC separation of PNAs. They found that the diamine sorbent offers some advantages over the monoamine and other polar sorbents in terms of better ring-size resolution. They can separate up to four fused aromatic rings, However, PNAs of higher ring number are not necessarily eluted in ring number sequence. In this paper, we present our experience with the separation procedure of Natusch and Tomkins and our preliminary evaluation of an alternate procedure based on cartridges packed with polar sorbents described by Chmielowiec and George. EXPERIMENTAL SECTION Instrumentation. All separated fractions of the samples were analyzed on a Hewlett-Packard 5880 gas chromatograph (Avondale, PA) using a flame ionization detector. A 30-m, DB-5 (J & W Scientific, Sunnyvale,CA) fused silica capillary column coated with SE-54 of 0.25 mm i.d. was used. The samples were injected via Grob's splitless injection. The injection port temperature was 250 OC; column carrier gas was helium with a column linear velocity of 50 cm/s at 270 OC. The column was temperature programmed in two steps: initial temperature was 50 "C at injection of sample and for elution of solvent, then it was heated at 100 OC at 15 OC/min, finally to 270 "C at 10 OC/min. Chemicals and Reagents. The PNA standards were obtained from various sources at 95% purity or better and were used as received. Cyclohexane, pentane, and methylene chloride were d l HPLC grade (Burdick and Jackson, Muskegon, MI) and used without purification. The water was HPLC grade (J. T. Baker, Phillipsburg, PA), and the dimethyl sulfoxide (Me2SO)and dimethylformamide were reagent grade (Fischer,Fairlawn, NJ) and all were used as received. The cartridges used were Bond Elut cartridges obtained from Analytichem Int. (Harbor, City, CA) and were used with slight modification. Each cartridge was prepacked with approximately 0.5 g of a bonded-phase primary-secondary amine packing material. After some experimentation, we decided to combine the material from three cartridges into one single cartridge containing approximately 1.5 g of the 30-pm material. The glass fiber filters were obtained from Andrew McFarlane of the Civil Engineering Department at Texas A&M University. These filters do not have a binder and were treated by heating to 800 "C for approximately 10 h before use in this experiment. Sample Cleanup Procedures. For the Soxhlet extraction, the filters were either cut into strips or folded to fit inside the Soxhlet extractor. Between 200 and 300 mL of cyclohexane was added and the extraction carried out for 24 h with the solvent recycled approximately every 15 min. At the end of Soxhlet extraction, the extract was concentrated by slow evaporation in a rotary evaporator to near dryness and finally redissolved in 10 mL of cyclohexane. After this, two different extraction procedures were evaluated for their suitability in further separating the PNAs
0003-2700/86/0358-1187$01.50/00 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
Table I. List of Priority Pollutants and Approximate Concentrations" peak no.
compd
1 2
naphthalene acenaphthylene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene 1,2-benzanthracene chrysene benzo[b]fluoranthene benzo [ k ]fluoranthene benzo[a]pyrene 1,2,5,6-dibenzanthracene indeno[l,2,3-c,d]pyrene benz[ghr]perylene perylene
3 4 5
6 7 8 9
10 11 12
13 14
15 16 17
abbreviation
5-component std, M 4 x 10-4
Nap ACY
Ace Flo Phe Ant Flu Pyr 1,2-BA Chr BbF BkF BaP DBA IPY BPe Per
2 x 10-3
perylene plus 16-component std, M 4 x 5x 5x 5x 2x 3x
2 x 10-4
4x 4x 3x
1 x 10-4
3x 1x 1x 1x 6X 6
1 x 10-4
10-4 10-5 10-5 10-5 10-4 10-5 10-5 10-6 10-5 10-5 10-4 10-4 10-4
X
3 x 10-5 2 x 10-4
"The original stock solutions of PNAs were prepared by Chem Service (West Chester, PA) with each component at approximately 100 ppm concentration. To atmosphere
t
Figure 1. Apparatus for the cleanup system using Analytichem cartridge.
from the other organic compounds present in the extract. Me2SO/PentaneExtraction, This method has been worked out in detail by Natusch and Tomkins (9) and is applied here for comparison. An aliquot of the Soxhlet extract was combined with an equal volume of Me2S0 for extraction of the PNAs. This was repeated until the fluorescent compounds (monitored by a hand-held uVlamp) were transferred to the Me2s0* The Me2s0 layers were then combined and diluted with 2 volumes of water. This Me,SO/water solution was then back-extracted 3 times with equal volumes of water to remove traces of Me2S0. The pentane solution containing the PNAs was filtered through Pyrex wool, which was pretreated by heating to 800 "C for 8 h to remove trace organics. The extract was finally concentrated by rotary evaporation for final analysis by capillary GC. Bond Elut Cartridges. The experimental apparatus for the Bond Elut procedure is shown in Figure 1. The cartridge fits into a stainless steel needle inserted through a rubber stopper into a test tube that fits in the bottom of the filter flask. This arrangement allows an aspirated vacuum to be applied across the cartridge with the eluent from the cartridge collected in the test tube. The cartridge was first wet with cyclohexane and any gaps in the packed bed filled. Then about 10 mL of cyclohexane was passed through to wash and prepare the cartridge for use, The c h i d g o was then filled with cyclohexane to the top of the packing material. A 1OO-fiL aliquot of the Soxhlet extract was pipetted onto the top of the column. Eluting solvents were then added in 1-mL addition and the eluents collected. The elution scheme for the standard solution containing 16 priority pollutants plus perylene was as fo~~ows: 1-3 mL, 100% cyclohexane, no vacuum; 4-10 mL, 100% cyclohexane, vacuum; 11-15 mL, 3% methylene chloride in cyclohexane,vacuum; 16-20 mL, 6% methylene chloride in cyclohexane, vacuum; 21-30 mL, 100%methylene chloride. Each of these fractions was analyzed by use of the gas chromatograph. Real Sample. The real sample, which was analyzed by using the Bond Elut procedure described here, was a Soxhlet extract
of particulates obtained from a high-volume air sample obtained in a coke cutting plant within an oil refinery in the Lake Charles, LA, area. This sample was collected after passing air through the high-volume filter at a rate of 40 ft3/min for 48.8 h. The extract of this sample was obtained by the following scheme: (1) Soxhlet extraction of a high-volume air filter sample with 200 mL of cyclohexane for 26 h with 12-min cycle time; (2) the Soxhlet extract was concentrated to 10 mL and then extracted with N,N-dimethylformamide/ H20 and subsequently back-extracted with cyclohexane (13);(3) the cyclohexane extract was dried with Na2S04,filtered, and concentrated to 1mL; (4) an aliquot of this sample was used in the procedure outlined previously.
RESULTS AND DISCUSSION As discussed before, the environmental samples containing PNAs are extremely complex, and the PNAs are only a minor fraction Of the Organics present. Soxhlet extraction has been the preferred method for the extraction of organic compounds from particulates collected by high-volume sampling, although sonification has also been applied (14). After extraction, to separate the PNAs from the other organic compounds present, the method of Me2SO/pentane of Natusch and Tomkins (9) has been a popular choice. The alternate cleanupprocedure is one involving small cartridges packed with suitable liquid chromatographic packing materials, This kind of introduced by Analytichem as Bond Based cartridge upon the work done by Chmielowiec and George (12), who showed that the diamine polar bonded-phase packings could be used to separate PNAs according to the number of condensed rings, we decided to pack these cartridges with the same kind of packing material and follow a somewhat similar elution scheme to separate the PNAs from the complex matrix of the environmental samples. To compare the two extraction procedures, Le., that of a Natusch and (') and the Bond standard solution containing 17 PNAs identified in Table I was used. An aliquot of this standard solution was pipetted onto a glass fiber filter and Soxhlet extracted for 24 h. Figure 2 shows the gas chromatogram of a standard solution of identical concentrations, but containing only the 16 priority pollutants and not perylene. Figure 3 is a gas chromatogram of the standard solution containing perylene and which had undergoneSoxhlet extraction with cyclohexane and was reconcentrated to the original volume. The increase in the number of peaks in this extracted solution is most likely due to residues on the filter, which were not completely eliminated in the Soxhleting process. It is also probable that photo and thermal degradation products of some PNAs may have been
ANALYTICAL CHEMISTRY, VOL. 58, 1
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NO. 6, MAY 1986
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11 12 13
L
L
Minutes Flgure 2. Gas chromatogram of a standard solution of 16 priority PNA pollutants in acetonltrile. 34
7
56
8
13
16
34
Minutes ~
Flgure 3. Gas chromatogram of an aliquot of a standard solution of PNAs on the glass fiber filter and Soxhlet extracted for 24 h.
formed during the sample handling process. A filter without any PNA solution added had previously been taken through the same Soxhlet extraction and was found to give a similar background, though with a lower number of peaks in the gas chromatogram. An identical aliquot of the standard PNAs solution was similarly treated. However, the extract was further processed by using the Me2SO/pentane extraction. Figure 4 is the gas chromatogram of the final pentane extract. The background has been eliminated to quite an extent. However, there are still quite a number of extra peaks remaining. Also the amount of some PNAs seems to have diminished indicating
some loss of sample components. This is especially true of the more volatile compounds such as naphthalene, acenaphthene, acenaphthylene, and fluorene. Some of these may have been lost in the reconcentration/evaporation process also. We experimented with both the amine and diamine polar bonded-phase packing materials in the Bond Elut cartridges for their utility in separating the PNAs from these samples. We found that both the amine and diamine packing materials would separate the PNA by ring size, but the monoamine material did not retain PNAs strongly enough to give as well-resolved a separation as one would desire. So the diamine polar bonded phase, which retains PNAs stronger, is used and
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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34
Minutes
Figure 4. Gas chromatogram of the Soxhlet extract after Me,SO/pentane extraction.
Table 11. Summary of Peak Areas Found for Each PNA in Each Fraction
fraction compd
Nap ACY
Ace Flo Phe Ant Flu PYr
1,2-BA Chr
BbF BkF BaP DBA IPY BPe
A
B 10.63 189.24 335.19 268.37 71.25 44.81 85.09
C
6.74 452.70 125.72 935.82 1170.83 150.49 98.21 3.06 3.98 3.99
D
23.74 17.58 181.10 190.44 24.17 35.94 39.29
evaluated further. A five-component mixture of PNAs of different ring sizes was prepared and an aliquot of it placed on the cartridge. (It was first eluted with 9 mL of 100% cyclohexane under gravity. Then 10 mL of 1%methylene chloride in cyclohexane was eluted with vacuum applied. Finally, 10 mL of 3% methylene chloride in cyclohexane was eluted, also with vacuum applied.) Figure 5 shows the results of the separation. The x axis represents the volume fractions. The height of each bar represents the fraction of the total for the specific PNA eluted in that milliliter. The results indicate discrimination between ring sizes, although the two- and three-ring compounds partially coelute. This experiment was repeated using the standard solution containing the 16 priority pollutants plus perylene. The resolution of PNAs by ring size is, as expected, not as good as that for the same material in HPLC (12). This is because the amount of bonded-phase material and column length are less in the Bond Elut cartridges, the particular size is larger, and the column is not as uniformly packed as in a regular HPLC column. Moreover, as reported by Chmielowiec (12),
E
7.02 4.07 15.81 23.32 122.31 94.80 161.30 11.35 60.64 3.57
F
27.64 178.49 22.92
G
14.7
H
total 10.63 189.24 341.93 721.07 196.97 1011.39 1277.57 347.40 311.97 149.54 134.72 204.58 38.99 253.30 26.49
the diamine polar bonded phase can only resolve PNAs of up to four rings. Even then, if we combine fractions, the resulting solution usually contains no more than two ring sizes. This procedure using the Bond Elut cartridges was applied to an aliquot of the Soxhlet extract of 16 PNAs standard solution described previously. In this experiment, no attempt was made to quantitate the individual PNA in each milliliter fraction. Rather, the identification was done to determine the contents of each fraction and how they could be combined for further analysis. Similar results to that of the original 17 PNAs standard solution were obtained. Then, using visual fluorescence from the fractions monitored with UV light, these fractions were combined into eight total fractions, A-H. Each of these final fractions was concentrated down to 100 wL. From the chromatograms, as we progressed to later fractions, we observed more of the higher ring PNAs indicating that we did have the desired separation trend. This is more clearly discernible in Table I1 where the peak areas of individual components found in each fraction are tabulated. Except for fluoranthene and pyrene, which are found in four fractions, the others are found in only two or three fractions. Even then,
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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PI
Table 111. Comparison of the Percent Recovery from the Soxhlet Extracts for Each PNA for Each Cleanup Technique % recovery Me2SO/pentane
compd Nap ACY Ace Flo Phe and Ant Flu PYr 1,2-BA Chr BbF and BkF BaP DBA IPY BPe
3 4
9 17 31 32 33 36 31 27 26 24 28
PI
E e
Bond Elut 11 28 39 41 50 68 78 75 76 67 51 62 42
disproportionate amounts of each PNA are found in one particular fraction. It seems that the background from the filter seen in the plain Soxhlet extract (Figure 3) appears in fractions, A, E, G, and H. A summary of the percent recoveries from the Soxhlet extracts for each PNA using the different cleanup procedures is given in Table 111. The recovery of PNAs using the liquid-liquid extraction scheme is unusually low when compared to the original study of Natusch and Tomkins (9). This may be attributed to an activation of the glass wool used to filter the pentane solution. Active sites may have been formed when the glass wool was heated to 800 “C to remove trace organics. In addition, we did not add salt in our procedure when Me2SO/water was partitioned with pentane, since we did not have serious emulsion problems. This may have resulted in a lower separation efficiency of the desired PNAs. At this point, we evaluated the applicability of our approach to a real sample. On September 28, 1984, we obtained a high-volume air sample in a coke cutting plant within an oil refinery in Lake Charles, LA. This sample was prepared as outlined in the Experimental Section of this paper. A 1OO-pL aliquot of the reconstituted extract was fractionated by use of a scheme similar to that outlined in our Experimental Section for the standard PNA solutions earlier in this paper. Each fraction was collected and reconstituted to 100 pL. However, due to the apparently small quantity of PNAs present as well as slight differences in the cartridges, satisfactory separation was not obtained. Therefore, another aliquot of the original extract was concentrated twofold and the elution procedure modified as outlined in Table IV. Each fraction was then analyzed by using “selected ion monitoring” on a Finnigan Model 4000 GC/MS system. As indicated in Table IV, the three-, four-, and five-member ring PNAs were isolated in three distinct fractions. Thus, the applicability of this approach to real samples is very apparent from these results. Based upon these experiments, we observed that, as have others (15,16), Soxhlet extraction of glass fiber filters causes
1 11 x
I
J
5
10
,
PI PI
PI0
15
20
n
c
1
T
Figure 5. Elution profile of a five-component PNA mixture using Bond Elut packed with a diamine polar bonded phase.
contamination from dissolution of compounds of the filter and reactions or decomposition of PNAs. For further cleanup of these Soxhlet extracts, Bond Elut seems to be a viable alternative approach to Me,SO/pentane procedures. Solvent consumption is small in the Bond Elut procedure. Moreover, optimization of the Bond Elut procedure can be carried out by using different packing materials, cartridge length, and elution scheme, depending on the kind of samples encountered. It is thus a more flexible procedure to achieve the kinds of separation one desires. Finally, we recognize that different cartridges will have to be individually calibrated due to differences in packing material, particle size, and packing efficiency. However, this is not an insurmountable problem when one considers that it is a relatively simple process to completely clean each cartridge after use. Thus, many sample cleanups are possible for a single cartridge.
Table IV. Fractionation of PNA Extract from Oil Refinery Sample fraction no. 1 2 3 4
5
eluting solvent 1-5 mL, 100% cyclohexane (no vacuum) 6-8.5 mL, 100% cyclohexane (vacuum) 8.6-14 mL, 3% methylene chloride in cyclohexane (vacuum) 15 mL, 6% methylene chloride in cyclohexane (vacuum) 16-25 mL, 100% methylene chloride (vacuum)
PNA present anthracene/phenanthrene pyrene l,2-benzanthracene chrysene perylene none detected none detected
no. of rings 3 4 4 4
5
1192
Anal. Chem. 1986, 58, 1192-1194
ACKNOWLEDGMENT We gratefully acknowledge the technical assistance of Judy Sophianopoulos and Matthew Tarr for acquisition and preparation of the oil refinery sample discussed in this paper. Registry No. Nap, 91-20-3; Acy, 208-96-8;Ace, 83-32-9;Flo, 86-73-7;Phe, 85-01-8;Ant, 120-12-7;Flu, 206-44-0;Pyr, 129-00-0; 1,2-BA,56-55-3;Chr, 218-01-9;BbF, 205-99-2;BkF, 207-08-9 B e , 50-32-8;DPA, 53-70-3;IPy, 193-39-5;BPe, 191-24-2;Per, 198-55-0.
LITERATURE CITED Merller, M.; Alfhein, J. Atmos. Environ. 1980, 14, 83. Salamone, M. F.; Heddle, J. A,; Katz, M. Environ. I n t . 1979, 2 , 37. Josephson, J. Environ. Sci. Techno/. 1981, 15,20. "Chemical Analysis and Biological Fate: Polynuclear Aromatic Compounds"; Cooke. M., Dennis, A. J., Eds.; Battelle: Columbus, OH, 1980. (5) "Polycyclic Hydrocarbons and Cancer"; Gelboin, H. V., Ts'o, P. 0. P., Eds.; Academic Press: New York, 1978. (6) Lee, M. L.; Novotny, M.; battle, K. D. "Analytical Chemistry of Polycyclic Aromatic Hydrocarbons"; Academic Press: New York, 1981. (1) (2) (3) (4)
(7) "Polynuclear Aromatic Hydrocarbons"; Jones, P. W., Leber, P., Eds.; Ann Arbor Sclence: Ann Arbor, MI, 1979. (8) "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Bjorseth, A., Dennis, A. J., Eds.; Battelle: Columbus, OH, 1980. (9) Natusch, D. F. S . ; Tomkins, B. A. Anal. Chem. 1978, 50, 1429. (10) Wise, S. A.; Cheder, S . N.; Hertz, H. S . ; Hilpert, L. R.; May, W. E. Anal. Chem. 1977, 4 9 , 2308. (11) Sonnefeld, W. J.; Zoller, W. H.; May, W. E ; Wise, S . A. Anal. Chem. 1882, 5 4 , 723 (12) Chmieiowiec, J.; George, A. Anal. Chem. 1980, 52, 1154. (13) Bjerrseth, A. Anal. Chim. Acta 1977, 9 4 , 21. (14) Swanson, D. H.; Walling, J. F. Chromatogr. Newsi. 1981, 9 , 25. (15) Swartz, G. P.; Daisey, J. M.; Lioy, P. J. Am. Ind. Hyg. Assoc. J . 1981, 42, 258. (16) Lee, F. S.-C.; Pierson, W. R.; Ezike, I . "Polynuclear Aromatic Hydrocarbons: Chemlstry and Biological Effects"; Bjerrseth, A,, Dennis, A. J., Eds.; Battelle: Columbus, OH, 1980, 543.
RECEIVED for review July 12, 1985. Resubmitted January 9, 1986. Accepted January 14,1986. This work was supported by the Department of Energy Grant DE-AS05-82ER60100.
Derivatization Technique for the Determination of Peroxides in Precipitation Gregory L. Kok,* Kathleen Thompson,' and Allan L. Lazrus
National Center f o r Atmospheric Research, P.O. Box 3000, Boulder, Colorado 80307 Scott E. McLaren
Atmospheric Science Research Center, State University of N e w York at Albany, Albany, N e w York 12222
A derivatization technique has been developed for the combined determination of hydrogen peroxide and some organic hydroperoxidesin precipitation samples. A fluorescent dimer is formed via the reaction of peroxides with p-hydroxyphenylacetic acid and horseradish peroxidase. The resulting dimer Is stable for a minimum of 5 days, eliminatlng dlfficulties caused by the decomposition of peroxides in stored samples. The detection limit is 3 X lo-* M, based on 3 times the standard deviation of the blank.
The accurate determination of hydrogen peroxide (H,O,) in cloud and precipitation samples is important for determining the amount of H202available for the oxidation of bisulfite ion (1,2). Measurements of H20zin cloud water at Whiteface Mountain, NY, show a strong seasonal variation, with winter concentrations on the order of M and summer concentrations 2 orders of magnitude higher ( 3 ) . However, a major difficulty in obtaining reliable analytical data is the rapid decomposition of H,Oz in collected samples. Decomposition rates of up to 5% per hour have been measured ( 4 ) . Under these conditions it is important that HzOzbe determined immediately or derivatized for accurate analytical results. In this paper a technique is presented to rapidly derivatize peroxides to the stable p-hydroxyphenylacetic acid dimer, which can be quantified by fluorescence several days after prepararation. 'Present address: 1529 Seymour Ave., North Chicago, IL 60064. 0003-2700/86/0358-1192$01.50/0
Table
I. P e r o x i d e R e a g e n t C o m p o s i t i o n o
components
concn, M
tris(hydroxymethyl)aminomethaneb(Tris)
0.5 Na2EDTAc 0.005 formaldehyde (HCHO)d 0.26 p-hydroxyphenylacetic acid' 0.15 horseradish peroxidase' g aAdjust the final pH to 9.0 using concentrated HC1. bFisher Scientific. Mallinckrodt Chemical. Baker Chemical, 37% stock solution. e Fairfield Chemical, Co., Blythewood, SC. {Sigma Chemical Co., P-8250, Type 11. 9150 units/100 mL solution.
THEORY The analytical procedure for peroxides is based on the peroxidase enzyme fluorescence technique developed by Lazrus et al. (4). This technique uses the formation of a fluorescent dimer from the reaction of hydroperoxides, p hydroxyphenylacetic acid, and peroxidase for quantitation. The reaction chemistry is as follows: CHzCOOH CHzCOOH
CH2COOH
PEROXIDASE
OH
OH
OH
Extensive studies have shown the technique to be reliable and free from interferences typically present in precipitation samples. The original implementation of the peroxidase analytical technique utilizes a dual-channel flow system 0 1986 American Chemical Society
