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Anal. Chem. 1985, 57, 2071-2074
from a plant, but the high relative abundance, and high relative molar ellipticity coefficients, of nicotine in tobacco (9),and of the cannabinoids in this case, produce the necessary signal intensity for analytical determinations.
ACKNOWLEDGMENT We wish to acknowledge the assistance of Robert Kroutil, U.S. Army, Aberdeen Proving Grounds, for developing the BASIC programs used in the analyses. We are also indebted to personnel at the forensic laboratories associated with the Oklahoma City Police Department and the Oklahoma State Bureau of Investigation for providing the marihuana specimens used in this work and to C. B. Skotland, Washington State University, for the samples of hops.
Registry No. trans-Ag-THC, 1972-08-3; CBD, 13956-29-1; 4-C02H-trans-A9-THC,23978-84-9; 4-C02H-CBD,1244-58-2.
LITERATURE CITED Waller, C. W.; Johnson, J. J.; Buelke, J.; Turner, C. E. "Marihuana: An Annotated Bibliography"; Collier Macmillan Publishing Co.: London, 1976. Clarke, E. G. C. "Isolation and Identification of Drugs"; The Pharmaceutical Press: London, 1978. Manno, J.; Manno, B.; Walsworth, D.; Herd, R. J. Forensic Sci. 1974, 19, a84. Ek, N.; Lonber, E.; Maehly, A. C.; Stromberg, L. J. Forensic Sci. 1972, 17, 456. Mechoulam, R., Ed. "Marihuana: Chemistry, Pharmacology, Metabolism and Clinical Effects"; Academic Press: New York, 1973. Small, E.; Beckstead, H. D. Lloyda 1973, 36 (2),144. Peel, H.; Perrigo, B. J. J. Anal. Toxicol. 1981, 5 ( 4 ) , 165. Timmons, J. E. J. Southwest Assoc. Forenslc Sci. 1984, 6 (3), 62. Atkinson, W. M.; Han, S. M.; Purdle, N. Anal. Chem. 1984, 56, 1947. Smith, R. N. J . Chromatogr. 1975, 715, 101. Crone, T. A.; Purdie, N. Anal. Chem. 1981, 5 3 , 17.
RECEIVED for review February 25, 1985. Accepted May 17, 1985.
I mmobiIized FIuorophores in Dynamic ChemiIuminescence Detection of Hydrogen Peroxide Gerald Gubitz* Institute for Pharmaceutical Chemistry, University of Graz, Universitatsplatz 1, A-8010 Graz, Austria
Piet van Zoonen, Cees Gooijer, Ne1 H. Velthorst, and Roland W. Frei* Department of General and Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
It Is shown that the peroxy oxalate chemllumlnescence system for the detectlon of H20, can be greatly slmpllfled by using lmmoblllzed fluorophores. Various lmmoblllzatlon procedures for 3-amlnofluoranthene are discussed. The chemiluminescence properties of materials based on cellulose, silica gel, and glass beads are compared. Controlled pore glass (CPG-10) was found to be the most suRable carrler. The material, packed In a quartz cell, Is applied In a flow ln)ectlon sydem In comblnatlon with a bed reactor contalnlng bls(2,4,t)-trlphenyI) oxalate (TCPO) In solid form. For the determination of hydrogen peroxide In rain water samples, detection limits of - 1 X IO-' M HzO, (0.3 ppb) were obtained using a homemade luminescence detector. Llnear callbration curves up to fO-' M were observed.
The immobilization of fluorophores has drawn much attention in recent years. Seitz ( 1 ) reported the use of chemical sensors based on immobilized fluorophores in a recent review. A simple pH sensor was obtained by immobilizing fluoresceinamine on cellulose (2). The principle of dynamic fluorescence quenching of immobilized fluorophores was used for the development of sensors for O2 ( 3 , 4 )and chloride (5). In view of their relatively long lifetimes, immobilized phosphors can also have potential for a promising detection technique based on phosphorescence quenching (6). Fluorescence studies of a ligand-metal ion complex immobilized on silica gel have been described by Ditzler et al. (7). Lochmuller et al. immobilized pyrene on silica gel for studies 0003-2700/85/0357-2071$01.50/0
of the distribution of silanol groups on the surface (8, 9). The application of immobilized fluorophores for chemiluminescence measurements has not yet been introduced. Chemiluminescence, used with the peroxy oxalate system, represents a simple and sensitive approach for the determination of H202(10-13). However, the instrumental setup used in such a system is relatively complex equipment. Two additional pumps are needed for delivering the oxalate and the fluorophore. Mixing problems may also occur in such an arrangement. A simplification has already been introduced in a flow injection system by using the bis(2,4,6-trichlorophenyl) oxalate (TCPO) packed in solid form in a flow-through reactor and adding the fluorophore (perylene) to the mobile carrier phase (14). It is thus possible to eliminate the reagent pumping and mixing systems. Nevertheless, having to add the fluorophore to the carrier phase still imposes certain complications and restrictions such as solubility limitations, toxicity, cost, compatibility with the mobile phase, and flow dependence of the signal (requiring a nonpulsating solvent delivery system). The idea of using immobilized fluorophores seems therefore promising for a further simplification of the instrumental setup and for an improvement of the detection properties through choice of more efficient fluorophores and with the reaction taking place directly in the detector cell in front of the photomultiplier. In this paper the immobilization of 3-aminofluoranthene to solid carriers like cellulose, silica gel and glass beads is described. This highly efficient fluorophore cannot easily be used in a homogeneous system (14) for solubility and also 0 1985 American Chemical Society
2072
ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 19a5
Figure 1. Support I: 3-amlnofluoranthene lmmoblllzed on cellulose. rLI
rLL
m a IJIb
Carrier Carrier
:
Silica .pi
:
CPG
Figure 3. Supports IIIa and IIIb: immoblliratlon of 3-aminofluorantheneon silica gel (IIIa)and glass beads (IIIb) folkwing method B (see text). 'NHR
Eo. IIb
Carrier Silica gel Carriw CPG
Flgve 2. Supports IIa and IIb: immoblllzation of 3-amlnofluoranthene on slllca gel (IIa) and ghs beads (IIb) following method A (see text).
toxicity reasons (mutagenic and carcinogenic properties). The goal of these studies is the development of simple field monitors for the determination of Hz02in surface water and rain water in connection with the acid rain problem. A further prospective application is the detection of HzOzproduced by postcolumn enzymatic or photochemical reactors in HPLC. EXPERIMENTAL SECTION Chemicals. Microcrystalline cellulose (for column chromatography) and silica gel (60,230-400 mesh) were purchased from Merck (Darmstadt, FRG). Controlled pore glaas (CPG-10,2W400 mesh, pore size 350 A), (3-aminopropyl)trimethoxysilane,and (3-glycidoxypropyl)trimethoxysilane were obtained from Serva (Heidelberg, FRG). 3-Aminofluoranthene and 2,4,6-trichlorotriazine (cyanuric chloride) were purchased from Janssen (Beerse, Belgium). Tris buffer (trizma base, reagent grade) was obtained from Sigma (St. Louis, MO). HPLC-grade methanol or acetonitrile (Baker, Deventer, NL) was used for flow injection analysis. The acetonitrile was purified on an alumina (Merck 70-230 mesh, ASTM) column prior to use. Methanol was used without prior purification. Bis(trichloropheny1) oxalate (TCPO) was prepared according to the method of Mohan and Turro (15) and recrystallized from uvasol benzene (Merck, Darmstadt, FRG). Immobilization of 3-Aminofluoranthene to Cellulose (Support I). Cellulose was activated with cyanuric chloride according to a known procedure (2, 16) and the resulting intermediate was shaken with 3-aminofluoranthene for 2 h. The product (Figure 1)was washed several times with acetone and water. Immobilization of 3-Aminofluoranthene to Silica Gel and Glass Beads. Method A (Supports IIa and IIb, See Figure 2). Silica gel (230-400 mesh) or CPG-10 glass beads (200-400 mesh) were refluxed with 5% nitric acid for 1 h, washed with acid-free water and dried at 110 "C for 1 h. One gram of the material was placed under vacuum to remove air from the pores and refluxed with 0.5 mL of (3-aminopropy1)trimethoxysilane in 20 mL of toluene for 8 h. The aminopropylanized support was then dispersed in 20 mL of a 5% sodium hydrogen carbonate solution. The suspension was cooled to 0-5 OC and 200 mg of cyanuric chloride dissolved in 10 mL of acetone was added dropwise while keeping the temperature below 10 OC during the reaction (7). The mixture was evacuated and shaken for 1 h at room temperature. The product was collected on a glass sinter filter and washed with water, acetone, and methanol. One gram of the intermediate was shaken with 200 mg of 3-aminofluoranthene in 20 mL of acetone for 4 h at room temperature. The resulting support was washed with acetone and
A
b
3
I 4
1
Figure 4. Block diagram of the experimental setup: 1, eluent vessel; 2, pump; 3, Injection valve; 4, TCPO reactor; 5, CL cell; 6, lighttight box with PMT; 7, amplifier; 8, recorder.
methanol until no fluorescence was observed in the filtrate. Method B (Supports IIIa and IIIb, See Figure 3). One gram of silica gel (230-400 mesh) or CPG-10 glass beads (200-400 mesh) pretreated as described in method A was suspended in a solution of (3-glycidoxypropyl)trimethoxysilanein 20 mL of toluene. The suspension was first kept at room temperature for 1 h and then refluxed for 8 h. The reflux condenser was kept at 70 "C in order to remove the methanol formed by the reaction. The resulting intermediate was collected on a glass sinter filter and washed with toluene and acetone. One-gram of the intermediate was shaken with 200 mg of 3-aminofluoranthene in 10 mL of a mixture of acetonemethanol 1:l for 25 h at room temperature. The final product was washed with DMF, methanol, and acetone until no fluorescence was detectable in the filtrate. Apparatus. A block diagram of the apparatus is given in Figure 4. Solvent delivery was carried out with a Kontron 410 pump (Kontron, ZClrich, Switzerland). Samples were injected by wing a Rheodyne 7010 injection valve (Rheodyne, Berkeley, CA) with Teflon injection loops (lOQ-600 pL). The solvent was either methanol-water (80:20, v/v) or acetonitrile-water (80:20,v/v) in which 0.01 M of Tris buffer (pH 8) was dissolved. The flow injection system operated at flows from 0.5 to 3 mM min-'. As a solid-statereactor for the TCPO, a Teflon-coated cartridge with an internal diameter of 4.6 mm and a length of 22 mm as previously described (14) was used. TCPO was mixed with glass beads in the ratio 2:l and dry packed by hand. The immobilized fluorophore was dry packed in a quartz tube of 3 mm i.d. and 30 mm length, provided with a plastic inlet and outlet frit (Figure 5). The connection between the TCPO reactor and the flow cell should be kept as short as possible. All connections were made with Teflon capillaries. Chemiluminescence detecton was performed with a homemade apparatus equipped with a RCA IP 28 photomultiplier tube in conjunction with a homemade high-voltage supply, operated at 1250 V (14). The signals were recorded with a Kipp BD 5 strip-chart recorder with a time constant of 5 s. Fluorescence measurements were carried out in transmission as well as in reflectance mode with a Perkin-Elmer MPF 44 spectrofluorometer equipped with a Hitachi 612 thin-layer chromatography accessory. RESULTS AND DISCUSSION Cellulose, Cellulose, silica gel, and glass beads were compared for their suitability as carriers for the immobilization of a fluorophore. 3-Aminofluoranthene was chosen as fluorophore since it was reported to have excellent chemiluminescence characteristics in the peroxy oxalate system (17).
ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985
350
EX
Ex
400
450
2073
Em
Em
500
550
nm
Figure 6. Fluorescence spectra of supports I1 and 111: dotted lines, support 11; solid lines, support 111.
Flgure 5. Design of the packed flow cell. The figure shows a quartz tube equipped with Teflon caps. The outlet cap (lower one) is equipped with a frit. The cell is mounted in the holder by means of a screw drawn at the upper part of the figure.
Table I. Elemental Analysis and Surface Coverage of Different Supports re1 carrier
C
H
N
I cellulose IIa silica gel IIb CPG IIIa silica gel IIIb CPG” IIIb CPBb
41.6
5.6 1.6 0.4 1.4 0.4 0.6
2.4 3.1 0.25 0.1 0.2
0.5
0.15
0.6
0.2
0
11.1
3.2 8.7 1.9 3.8 3.3 3.5
1.1
surface coverage, mmol/g 0.34 0.22 0.15 0.17 0.07 0.14 0.14 0.14
chemiluminescence
intens 1.5 0.7
2.5 5.7
32.0 69.0 59.6 65.0
Silanization in water. * Silanization in toluene.
Surface Coverage. Table I presents the elemental analysis and surface coverage data for the different supports. Support I based on cellulose contains a relatively high amount of bonded aminofluoranthene. It shows, however, significant disadvantages regarding mechanical stability and chemiluminescence yield. As can be seen from Table I, the surface coverage of the supports of types I1 and I11 are comparable; the chemiluminescence given off by the products of type I11 is, however, considerably more intense. Although the surface coverage is, in comparison to silica gel, generally slightly lower on glass beads, a higher chemiluminescence yield is observed on glass which is probably due to its better transparency. The silanization step was carried out either in aqueous medium (18) or in toluene (19). The supports prepared in aqueous medium are less transparent, probably due to the formation of polymer layers on the surface. The surface
coverage and the chemiluminescence intensity is higher with products prepared from intermediates silanized in toluene. The batch to batch reproducibility is demonstrated by means of the elemental analysis of three batches of IIIb prepared by the same procedure (Table I). The comparison of the batches was also made by measuring the fluorescence intensity on the surfaces. Spectroscopic studies regarding the surface distribution are in progress. Fluorescence Spectra (See Figure 6). The fluorescence spectra of I and I1 show a large hypsochromic shift in excitation as well as in emission wavelength in comparison to the spectrum of the supports of type 111. Pure 3-aminofluoranthene in solid state shows a similar shape of the spectra as 111. These are very similar to the spectra of aminofluoranthene solutions. Apparently, the spacer in I11 simulates a “pseudo” free fluorophore with little influence from the support. On the contrary, in I and I1 there is a strong electronic interaction between aminofluoranthene and the triazine ring. This is in line with the considerable difference of chemiluminescence yields (Table I). Support IIIb yields 30-100 times the chemiluminescence intensities of I and 11. Flow Injection Studies. The chemiluminescence properties of the different supports packed in quartz cells were compared by injecting a M solution of HzOz (in acetonitrile/water and methanolJwater solvents) in the flow injection system shown in Figure 4. Attempts to inject completely aqueous samples proved to be unsuccessful. The signal is highly dependent on the mobile phase composition; hence inhomogeneous mixing in the TCPO reactor causes bad reproducibility. The injection plugs are therefore adjusted to the mobile phase compositiqn before injection into the system. The cellulose material (I) is the least suitable since it swells in the above solvent systems and causes back pressure problems. For the same reason gradient systems are not applicable with cellulose. The CPG based material (IIIb) was found to be the most suitable one, even compared to the silica gels, because of its transparency and mechanical stability. In addition, less band broadening is observed with CPG. To ensure a good permeability and to avoid high back pressure in the cell, CPG glass beads with a particle size of 200-400 mesh were chosen. Experiments with this support showed good stability over several weeks against hydrolysis in the pH range between 5 and 8. We have no data on the long-term stability at pH lower than 5 since the chemiluminescence system does not operate under these conditions. In alkaline medium (pH -10) prolonged use may cause hydrolysis of the bonded fluorophore. One cell with the same packing was used for about 1month without loss in sensitivity. The cell should be washed with water and acetone prior to storage. The TCPO in the reactor column was mixed with glass beads in order to decrease the back pressure, to reduce band braodening, and to avoid the formation of major dead volumes
2074
Anal. Chem. lQS5, 57,2074-2079
principle with dynamic systems generating H202such as enzymatic and photochemical reactions in HPLC, bio- and immunoassays, etc. is also under investigation. Registry No. TCPO, 1165-91-9; Hz02, 7722-84-1; HzO, 7732-18-5.
LITERATURE CITED
--+ time
Flgure 7. Repetitive injection of
M of H,02.
after the TCPO has been deleted. One packing can continuously be used for about 8 h. To test the reproducibility of the system, H202solutions were repeatedly injected. The relative standard deviation for 10" M H202 was 0.9% (Figure 7) and 2.5% for M. Detection limits were about l X M H202 (0.3 ppb). Calibration curves were linear up to M.
CONCLUSION The use of immobilized fluorophores in conjunction with a solid-sate TCPO reactor permits a considerable simplification of the peroxy oxalate chemiluminescence system for H202detection. Thus no additional pumps for reagent delivery are needed and mixing problems are also eliminated. The use of glass beads as support material has advantages over other supports such as silica gel or cellulose. A marked reduction of band broadening and a higher sensitivity are obtained by ths approach and, in addition, the choice of suitable fluorophores is widened since parameters such as solubility, costs, and toxicity do not play as much of a role. Optimization with regard to the instrumentation and working conditions for an application to field analysis of rain water is in progress. The combination of this detection
(1) Seitz, W. R. Anal. Chem. 1984, 16A. (2) Saari, L. A.; Seltz, W. R. Anal. Chem. 1982, 5 4 , 821-823. (3) Wolfbeis, 0. S.; Offenbacher, H.; Kroneis, H.; Marsoner, H. Mlcrochlm. Acta 1984, 1 , 153-158. (4) Peterson, J. I.; Fitzgerald, R. V.; Buckhold, D. K. Anal. Chem. 1984, 56, 62-67. ( 5 ) Urbano, E.: Offenbacher, H.; Wolfbels, 0. S. Anal. Chem. 1984, 5 6 , 427-429. (6) Donkerbroek, J. J.; Veltkamp, A. C.; Gooljer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1983, 55, 1886-1893. (7) Ditzier, M. A.; Doherty, G.; Sleber, S.; Allston, R. Anal. Chim. Acta 1982, 142, 305-311. (8) Lochmuiler, C. H.; Colborn, A. A.; Hunnlcutt, M. L.; Harrls, J. M. Anal. Chem. 1983, 1344-1343. (9) Lochmuller, C. H.; Coiburn. A. S.; Hunnlcutt, M. L.; Harris, J. M. J . Am. Chem. SOC. 1984, 106, 4077-4082. (10) Williams, D. C.; Huft, 0. F.; Seltz, W. R. Anal. Chem. 1978, 48, 1003-1006. (11) Williams, D. C.; Seltz, W. R. Anal. Chem. 1978, 5 0 , 1478-1481. (12) Scott, 0.; Seltz, W. R.; Ambrose, W. R. Anal. Chlm. Acta 1980, 115, 221-228. (13) Rauhut, M. M. Acc. Chem. Res. 1969, 2 , 80-87. (14) Van Zoonen, P.; Kamminga, D. A.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chim. Acta 1985, 167, 249-258. (15) Mohan, A. G.; Turro, M. J. J . Chem. Educ. 1974, 5 1 , 528-529. (16) Kay, 0.; Crook, E. M. Nature (London) 1967, 216, 514-515. (17) Sigvardson, K. W.; Kennish, J. M.; Blrks, J. W. Anal. Chem. 1984, 56, 1096-1102. (18) Chang, S. H.; Gooding, K. M.; Regnler, F. E. J . Chromatqr. 1976, 120, 321-333. (19) Gubitz, G.; Juffmann, F.; Jelienz, W. Chromatographia 1982, 16, 103-106.
RECEIVED for review February 8, 1985. Accepted April 18, 1985. We greatly acknowledge financial support of the Cultural Exchange Program between Austria and the Netherlands.
Redox Chemiluminescence Detector: Application to Gas Chromatography Stefan A. Nyarady, Robert M. Barkley, and Robert E. Sievers*
Department of Chemistry and Cooperative Institute for Research i n Environmental Sciences, Campus Box 215, University of Colorado, Boulder, Colorado 80309
A new chromatographlc detector is based on redox reactions coupled with measurement of chemllumlnescence. Measurements Involve the catalyzed postcolumn reduction of nitrogen dioxlde by anaiytes that can readily be dehydrogenated or oxldlzed, followed by subsequent downstream oxldation of the formed nltrlc oxlde by reactlon wlth ozone. The redox chemiluminescence detector (RCD) responds to compounds that are not sensitively detected by flame ionization detectors (FID) such as ammonia, hydrogerr sulflde, carbon dlsulflde, sulfur dloxide, hydrogen peroxide, hydrogen, carbon monoxide, formaldehyde, and formic acid. The RCD Is also sensltlve to alcohols, aldehydes, ketones, aclds, amlnes, olefins, aromatlc compounds, sulfides, and thlols. Sensltlvlty of the RCD Is comparable wlth that of the FID. The RCD Is not sensltlve to the major constltuents In the matrlces of many samples, such as alkanes, chlorlnated hydrocarbons, water, nitrogen, oxygen, and carbon dioxide. Relative molar response factors range over 6 orders of magnitude.
Advances in chromatodraphic analysis often evolve from increased sensitivity that allows detection of lesser amounts of materials, or from selective detection of only certain components of interest in a complex mixture of many compounds. The goal is often to detect and quantify certain components such as, nitrogen-, oxygen-, or sulfur-containing species, in the presence of much higher concentrations of less significant compounds that constitute the matrix, such as alkanes, water, or the major constituents of air. Several recent, successful approaches to selective detection involve detection of postcolumn reaction products, especially for liquid chromatographic applications (1-4). After chromatographic separation, a reactant is mixed with the column effluent and a reaction occurs which produces a more readily detectable compound-either a derivatized analyte or other reaction product. Chemiluminescence has been used to achieve very sensitive detection of compounds that react to form light-emitting
0003-2700/85/0357-2074$01.50/00 1985 American Chemical Society
