Photocatalytic chemiluminescence detection of quinones in high

Anal. Chem. 1990, 62, 1242-1251

1242

Photocatalytic Chemiluminescence Detection of Quinones in High-Performance Liquid Chromatography James R. Poulsenl and John W. Birks* Department of Chemistry and Biochemistry and Cooperative Institute for Research in Environmental Sciences (CIRES), Campus Box 216, University of Colorado, Boulder, Colorado 80309

A reactlon dotedon scheme for quinone anatytes that utl&es thelr unique photIs descrW. The analytes sensltke the photooxidation of the hlgh-performance Uquld chromatography moblle phase In a postcolumn photoreactor. A chemical ampllflcatlon of the signal occurs because many molecules of H202are produced by each qulnone molecule during this catalytic reactbn sequence. H202Is detected by peroxyoxalate chemlluminercence. The oxalate ester, bls(2,4,6-trlcMorophenyl) oxatate (TCPO), and the fborophore, rubrene, are delivered In solutlon by podcolumn addltlon pumps. Detectlon lbnb are In the low- to SubplcOmOre range for many qulnone derlvatlves wlth very hlgh selectlvlty. The p h o i o c ~ r of y the photocatalytic ChemlumlneQcence detectlon scheme (PCCL) and optknlzatlon of the system are described. The selecllvlty of PCCL also Is characterized and the probable photochemlcal reactlons causlng the response of some compounds that are not qulnones are discussed. A cardboard extract Is used to demonstrate the advantages of PCCL over UV absorption for the detectlon of anthraquinone residues from the pulping process.

INTRODUCTION Quinones may be detected with high sensitivity and selectivity in high-performance liquid chromatography (HPLC) by means of two different postcolumn photochemical reaction schemes. In the absence of molecular oxygen and in the presence of a good hydrogen atom donor (HAD) such as methanol, quinones are photoreduced to highly fluorescent dihydroquinones. In an earlier paper, we described results for the anaerobic “photoreduction fluorescence detection of quinones” (PRFQ) ( I ) . In the presence of oxygen, quinones act as a photocatalyst for the production of hydrogen peroxide. The H20, product is detected by peroxyoxalate chemiluminescence in the “photocatalytic chemiluminescence” (PCCL) detection scheme described and evaluated here. As discussed here and in our previous paper ( I ) , the optimal detection scheme depends on the specific quinones to be detected, their concentrations, and the nature of the sample matrix. Many modern wood pulping processes add anthraquinone derivatives to catalytically enhance deligninification (2, 3). Because of environmental-decreased emissions to both air and water-and economic considerations-increased pulp yields from scarce forest resources-the use of anthraquinone additives is expected to increase ( 4 ) . Detection and determination of these compounds in finished paper products, or even at intermediate stages of the pulping process, are challenging due to their low concentrations and/or complex matrices. A large amount of residual phenolic material will be coextracted with the quinone analytes.

* Corresponding author. ‘Current address: Henkel Research Corp., 2330 Circadian Way, Santa Rosa, CA 95407.

Quinones also are intermediates in the environmental oxidation of polycyclic aromatic hydrocarbon (PAH)compounds. For example, it has been shown that up to 19% of anthracene emitted on particulate matter is oxidized to anthraquinone (5). In seawater, the photooxidation of anthracene to anthraquinone is an important step that allows subsequent biodegradation to occur (6). However, the levels of quinones in natural waters and urban air are extremely low, on the order of 10-s-lO-ll g/L and 10-8-10-10g/m3, respectively (6,7). Once again, the determination of quinones at these levels in environmental samples is difficult. While bacterial degradation of anthracene is enhanced by the photooxidation to anthraquinone (6),the photooxidation of fuel oil spills (8)and of organics extracted from particulate matter (9) increases the toxicity and mutagenicity of these mixtures to other species. For example, fish mortality dramatically increases on irradiation of water taken from the area of a fuel oil spill (8). Oxidation will increase the extractability of the organics into water as well. The utility of on-line, postcolumn reactions in enhancing the selectivity and/or sensitivity of high performance liquid chromatography (HPLC) detection has been demonstrated in many articles. Photochemical reactions have been applied with considerable success ( 1 0 , l l ) . Quinones exhibit unique photochemical characteristics that can be exploited to enhance both their detection and the detection of other analytes. Gandelman and Birks have reported two systems based on the photochemistry of anthraquinone derivatives for the detection of HAD analytes. A unique feature of both systems is the detection of analytes that do not adsorb light themselves. In the first of these systems, photooxygenation chemiluminescence (POCL), H202is produced from HAD compounds via their reaction with photoexcited anthraquinone 2,6-disulfonate (AQDS). H202 produced in the photoreactor is detected by luminol chemiluminescence (12). Unfortunately, the utility of the aerobic reaction is limited by large photochemical backgrounds. In the second of their detection schemes, an anthraquinone derivative is photoreduced by HAD analytes to the corresponding dihydroxyanthracene. Fluorescence of the dihydroxyanthracene is observed when HAD analytes elute from the high-performance liquid chromatography (HPLC) column (13). While the aerobic photooxidation reaction is of little use in detecting HAD species, it can be reversed to detect quinones. In this mode, the mobile phase is hydrogen atom donating (e.g., methanol), and H202 production indicates the presence of “type 1 photooxygenation” sensitizers. Type 1 sensitizers have triplet states capable of abstracting hydrogen atoms from compounds with weak carbon-hydrogen bonds (14). Because no light-absorbing compounds are added to the mobile phase, the photooxidation background is much lower. H202serves as a surrogate molecule in this scheme and is quantified by peroxyoxalate chemiluminescence. Peroxyoxalate chemiluminescence (15) has been used in several analytical detection schemes (16,17). In addition to selectively determining H202in flow injection analysis (FIA) (18),extremely sensitive detection of fluorescent molecules

0003-2700/90/0362-1242%02.50/0 ~-.. . 0 1990 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990

in both HPLC and FIA has been demonstrated (19,20). Since peroxyoxalate chemiluminescence is very selective toward Hz02,a separation is not necessary for its determination. The introduction of postcolumn reactions that generate H202,in an on-line mode from specific classes of analytes, allows peroxyoxalate peroxide detection to be applied to HPLC. In earlier work, solid-phase reagent addition methods for peroxyoxalate Hz02 detection were investigated by using the photocatalytic chemiluminescence detector (PCCL) as a model system for postcolumn generation of H202(21, 22). In the present work, a two-pump, postcolumn system is employed to deliver TCPO, rubrene, and a tris(hydroxymethy1)aminomethane buffer (TRIS) in solution. While peroxyoxalate chemiluminescence is not discussed in depth, its compatibility with PCCL should be emphasized. The high polarity modifier concentrations, necessary for both the chromatography and photochemistry, are in the optimal range for a sensitive peroxyoxalate response (18, 21-23), Under typical chromatographic conditions, a linear range in excess of 4 orders of magnitude is observed for peroxyoxalate detection of H202with few interferences (24). For most analytes, the linear range of the PCCL is limited by the photooxidation reaction rather than the peroxyoxalate response. Because of the combined selectivity of the photochemical and peroxyoxalate reactions, PCCL exhibits little response to compounds other than ’type 1photooxygenation” sensitizers. Quinones have high molar absorptivities, so their detection by UV absorption is quite sensitive; however, the selectivity often is insufficient for application to complex mixtures. Because PCCL exhibits such a high degree of selectivity, it may be possible to preconcentrate environmental samples to adequate levels without insurmountable interference problems. In this paper, the detection of anthraquinone in a cardboard extract demonstrates the enhanced selectivity of PCCL relative to UV absorption. Optimizations of PCCL in terms of solvent composition, reaction time, reagent flow rates and reagent concentrations are described. Additionally, the photochemistry of quinones and some “interfering” compounds is discussed. EXPERIMENTAL SECTION Chemicals. Quinone samples, compounds used in determining selectivity ratios, and standards for the photooxidation product study were obtained from Aldrich Chemicals (including the “Alfred Bader Library of Research Chemicals”), Sigma, Fisher, Eastman, or Pfaltz and Bauer. After the samples and standards were screened for impurities by HPLC with UV detection, these were used as received. TCPO was synthesized by the method of Mohan and Turro (2.5) with additional recrystallizations from spectrograde benzene (Fisher)and ethyl acetate (Burdick and Jackson) followed by a wash with spectrograde hexanes (Fisher). Particulate matter was removed by a hot vacuum filtration through a sintered glass filter funnel (medium frit, 10-15 pm) during the benzene recrystallization. Fresh solutions of TCPO and rubrene (Aldrich) were prepared daily in Spectrograde acetone (Fisher). HPLC Apparatus. Figure 1 is a schematic diagram of the PCCL system. The HPLC system consists of a Kratos Spectroflow-400 pump, a Rheodyne 7125 injector (20-pL loop), a Du Pont Zorbax ODS column (25 cm X 4.6 mm), and a Shimadzu CR-3A integrating recorder. The chromatographic eluents were mixtures of methanol and water (Burdick and Jackson, HPLC grade), 80-100% methanol by volume. For vitamin K1 detection, a mobile phase consisting of 60% methanol and 40% 2-propanol (IPA, EM Science Omni-Solv) was used. The solvent reservoir was equipped with a gas dispersion tube to facilitate purging with oxygen (UHP grade, Air Products) or nitrogen (USP grade, General Air). W detection utilized a Schoeffel SF-770 or Kratos SF-773 variable wavelength detector. Photochemical Reactors. Reactors were prepared from 30gauge PTFE tubing (0.30-mm i.d. with a 0.15 0.05-mm wall thickness) purchased from Small Parts, Inc. They were plumbed and crocheted by the method of Poulsen et al. (26).By variation

*

1243

TCPOIRubrene

A

Mobile Phase

Photochemical Reactor

4

TRIS Butler

Figure 1. Schematic diagram of the PCCL system.

of the length from 2.7 to 29 m, reaction volumes from 0.2 to 2.4 mL were generated. During use the reactors were wrapped in a reflective foil and cooled with a muffin fan. Light Sources. A fluorescent poster lamp (Sylvania F8T5/ BLB “black light” bulb), purchased at a local hardware store for about $30, was the most useful photoreactor source. The emission spectrum of this lamp is centered at 366 nm. Three ‘pencil lamps” also were tested. These low-pressure mercury lamps emit most of their radiation at 254 nm (Pen-Ray, Model 11 SC-1, UV Products, Inc., San Gabriel, CA) with phosphor coatings available to change the emission maximum. Lamps with specified emission maxima of 366 nm (UV Products Pen-Ray, Model 11SC-1L) and 351 nm (Analamp, Model 80-1055-01,BHK, Inc., Monrovia, CA) were tested. Post-Column Reagent Delivery. Detection of the hydrogen peroxide was accomplished by pumping a TCPO (0.75-1.0 g/L) and rubrene (20-50 mg/L) solution into a four-way, Valco ZDV mixing tee connected to the photochemical reactor. The TCPO and rubrene were dissolved in Spectrograde acetone and delivered by an ISCO Model 314 syringe pump at a flow rate of 0.33 mL/min. TRIS (Sigma,reagent grade), buffered to a pH of 7.8-8.2 with reagent grade HNOB(Fisher), was used as a catalyst in most experiments. TRIS (0.5-10.0 mM) in water-methanol-acetone, 11316 by volume, was delivered by a Sage Model 341 syringe driver at 0.15 or 0.092 mL/min. The TRIS concentration of the mixed solution entering the chemiluminescence detector cell was ~ 0 . 5 mM. Chemiluminescence Detection. Chemiluminescence was detected with a Kratos Model FS-970 fluorometer operated with the excitation source turned off and emission filter removed. Photomultiplier tube (PMT) voltages ranged from 900 to 1460 V. Paper Product Extractions. To prepare the cardboard extracts, samples (10-15 g) were cut up and placed in a Soxhlet extraction thimble (Whatman No. 2800 339, 33 mm X 94 mm, cellulose). An empty Soxhlet thimble was extracted, and no antraquinone residue was detectable. A 90-mL portion of methanol was kept boiling through 12-15 Soxhlet cycles. The boiling extract was allowed to evaporate under nitrogen to *35 mL and filtered through a Millex LS 5-pm syringe filter (Millipore Corp., Bedford, MA) to remove particles. About 15 mL of methanol was used to rinse the filter, and then the combined fitrate was diluted to volume with water such that the final solvent composition was 4 0 % methanol and 30% water. A portion of the extract, which had turned cloudy after the water was added, was filtered through a 0.45-pm Millex HV filter unit, followed by another filtration through a 0.22-pm Millex GV, syringe filter. An eluent consisting of 85% methanol and 15% water was delivered at a flow rate of 1.20 mL/min for UV-251 nm detection and 0.80 mL/min for PCCL. For PCCL, a 27 m long photoreactor (2.1 mL, tRm = 152 s) was inserted between the column and reagent mixing tee. TCPO (0.84 g/L) and rubrene (23 mg/L) were delivered in acetone at 0.33 mL/min, and TRIS (pH = 8.1,6 mM) was delivered at 0.15 mL/min in 58/38/4 acetone-methanolwater, by volume. The PMT was biased at 1300 V with a 2.5-s time constant. CHEMISTRY OF PCCL Aerobic Quinone Photochemistry. Under the conditions for HPLC detection of quinones, sensitized photooxidation by quinones can be described in terms of a simple hydrogen

1244


abstraction mechanism (27). Some of the productive reaction pathways possible when methanol is the hydrogen atom donor are outlined below (27, 28)

Q + hv

+

-

'Q*

(1)

(2)

'Q* -+ 3Q*

3Q* + CHBOH 'QH 'CHZOH 2'QOOH 2'00CHzOH

+ O2 +02

-

+

2'00CHzOH 'QOOH

'QOOH 'OOCHZOH HOO'

+

'OOCH2OH

(44

'OOCH20H

(4b)

2

(54

HzOz + 02 + 2CH20

(5b)

H202 + 2HCOOH

(54

---c

-+

+ HOO'

H2Oz + 0 2 + CH2O + Q (5d)

Q + HOO' CH2O + HOO'

(6b)

H202 + 0 2

(7)

-- + + +

+ 'CHzOH 'QOOH + 'QH

'QOOH

(3)

'QOOH

2Q + H2Oz + 0

-+

+ '00CH20H

'CHzOH + 'QH

+ 'CHZOH

-

Q

(6a)

CH20 + Hz02 (8a) 2Q H2Op (ab)

-

+ H202 Q + CHzO + HzOz 2CH20

'OOCHZOH + 'QH Q = quinone analyte 'QH = semiquinone radical

(8~) (8d)

Quinone sensitizers serve as catalysts for the reduction of oxygen and the oxidation of HAD substrates. H202and oxidation products derived form the substrates, such as aldehydes, ketones, and carboxylic acids, are produced by this reaction (27-31 1. Lower energy T , A * and /or n,r* absorption bands of the quinone must be used to initiate this reaction in order to avoid photolyzing the HzOP These absorptions are much weaker than the shorter wavelength r,r* transitions used for UV detection of quinones. The excited states produced by light absorption rapidly decay to the lowest excited singlet state of the analyte through internal conversion and subsequently undergo intersystem crossing to the triplet manifold. Regardless of the excitation wavelength, it is still the lowest triplet state that is involved in the photochemical reaction (32). The lowest triplet state of the quinone abstracts a hydrogen atom from a compound which has a weak carbon-hydrogen bond (HAD)-methanol as shown-to produce the semiquinone radical and an a-hydroxy alkyl radical (Reaction 3). HAD compounds typically contain nitrogen or oxygen and have at least one hydrogen bound to the carbon a to the heteroatom (33,34). Triplet deactivation mechanisms other than hydrogen abstraction are thought to be unimportant during aerobic detection of sensitizers, because methanol, the hydrogen atom donor, is present at such a high concentration. Quenching by hydrogen abstraction is extremely efficient under these conditions (32). Quenching by oxygen (reaction 9) is inefficient, because the HAD substrate concentration is -lo5 times greater than that of oxygen (35).

-

3Q* + 30z 'Q

+ IOz*

(9)

quenching by oxygen The semiquinone and a-hydroxyalkyl radicals react rapidly with oxygen (reaction 4) (30,35). Given the level of oxygen

in solution (10+10-3 M)(36)and the reactivity of the primary radicals toward it, the peroxy radicals will control the secondary chemistry of the system (30).Radicals produced from weak sensitizers by pulse radiolysis have similar reactivities to those produced from strong sensitizers (35). Hence, the differences in sensitizer activity stem from the nature of the reactive excited state rather than the secondary free radical chemistry (35). In terms of sensitizer detection, the most important feature of the aerobic photooxidation scheme is the fact that the quinone is regenerated and can act as a catalyst by going through the reaction cycle many times. The limiting quantum yield for HzOz production by strong sensitizers in alcoholic solutions in unity (27, 28, 31). Hence, photooxidation by quinones is a cyclic reaction sequence rather than a free radical chain reaction initiated by the sensitizer (35, 37). Peroxyoxalate Chemiluminescenca. Chemiluminescence occurs when a highly exergonic (-AG) reaction leaves one or more products in excited electronic states. Just as excited states produced by light absorption may decay by fluorescence, emission of light provides a pathway for excited states resulting from chemiexcitation to return to the ground state. Light emission from the excited state products is indistinguishable from their fluorescence emission spectra. Analytical applications typically utilize chemiluminescent reactions that have very little emission in the absence of the analyte. Hence, the signal is measured against a near-zero background. Peroxyoxalate chemiluminescence is one of the most efficient chemiexcitation reactions known to occur in solution (38) and one of the most widely used of the chemiluminscent detection schemes for HPLC (16,17). Peroxyoxalate chemiluminescence is useful in detecting either H20z or certain fluorophores. When an oxalate ester such as bis(2,4,6-trichlorophenyl) oxalate (TCPO), H202, and a fluorophore are mixed together, chemiluminescence is observed. In most cases, a buffer, formed from triethylamine, imidazole, sodium salicylate, or tris(hydroxymethy1)aminomethane (TRIS) and a strong acid, is added to enhance the rate of light emission. Excitation is thought to occur by an electron transfer mechanism in which annihilation of a radical ion pair leaves a fluorescent molecule in an excited state (39). Linearity over 4 to 5 orders of magnitude between the analyte concentration and the chemiluminescenceintensity is observed in most case8 (24). The sensitivity and selectivity of peroxyoxalate-H202 detection are unequaled in both dynamic flow and static systems. Organic peroxides are much less efficient at initiating the peroxyoxalate reaction than Hz02(40). When fluorophores are the analytes, H202and the oxalate ester are mixed with the sample in the presence of a catalyst. Chemiluminescence indicates that a fluorophore capable of accepting the chemiexcitation energy is present. In fluorophore detection, the structure of the acceptor has a large effect on sensitivity (20, 4 1 ) . The nature of the fluorophore has a much weaker effect on the sensitivity when H202 is the analyte (18,21,22). Here, the fluorophore is in large excess relative to the intermediate available to excite it. Typically, rubrene and perylene are used as fluorophores in this mode. Factors such as reagent solubility frequently play a role in the selection of the fluorophore for liquid-phase peroxyoxalate-HzOz detection systems. Rubrene is more soluble in polar solvents, while perylene has a slightly higher chemiexcitation efficiency (42). Peroxyoxalate chemiluminescence is compatible with reversed-phase HPLC in terms of reaction kinetics, reagent solubility, sensitivity, and low background emission from the reagents themselves. For these reasons, it was hypothesized that the combination of photochemical generation of H202 with peroxyoxalate chemiluminescence detection would be a


1245

3

highly selective and sensitive way to detect quinones.

RESULTS AND DISCUSSION Photochemistry. Compounds that are good sensitizers have a lowest triplet state with predominantly n,?r* character (14). These bands exhibit hypsochromic shifts as the solvent polarity increases. Raising the energy of the n,?r* triplet state causes the amount of T,T* character mixed into the lowest triplet state to increase. In extreme cases, the lowest triplet may become T,A* in character. This implies that the yield of HzOzwill be highest at high polarity modifier concentrations (least polar solvents). Such behavior is observed in PCCL; however, the solvent dependence of the peroxyoxalate reaction is a more significant contributor to the decline in sensitivity with increasing polarity. Furthermore, the strongest sensitizers are affected the least by the change in solvent conditions. These compounds also are the best PCCL analytes. Three possible quenching reactions have the potential to be important in PCCL: chemical reaction with a HAD molecule (reaction 3), quenching by oxygen (reaction 91,and energy transfer (reaction 10). Reactions 3 and 9 are the most

3Q* + A

-

‘Q

+ A*

(10)

energy transfer to a n acceptor, A probable routes to the ground state, because the concentrations of HAD species (methanol and its oxidation products) and oxygen are high. Oxygen is present in air-equilibrated methanol at 2.1 mM and in water at 0.27 mM (36). However, the hydrogen abstraction reaction proceeds with a quantum efficiency near unity, even in the presence of oxygen (30,32, 35). Energy transfer to compounds other than oxygen (reaction 10) is unlikely, because the concentrations of possible acceptors (impurities) are very low in HPLC grade solvents. The peroxide yield is slightly lower when the oxygen concentration is lowered by flushing the mobile phase with nitrogen. PTFE tubing is permeable to oxygen, so the secondary oxidation reactions still are possible; however, the oxygen concentration is limited by the rate a t which it can diffuse into the photoreactor and possibly by its consumption. At high levels of oxygen saturation, the signal-to-noise ratio was found to be relatively insensitive to the oxygen content of the mobile phase, so most experiments were carried out using mobile phases equilibrated with air. This eliminates the need for a flow of gas through the mobile-phase reservoir. Since a carbon-hydrogen bond is broken during the abstraction reaction, its strength will influence the efficiency of the reaction. This is borne out to the extent that the signal intensities of both vitamin K1and anthraquinone are increased by adding isopropyl alcohol (IPA) to the mobile phase. However, this is of little practical utility due to increases in the base-line noise caused by reagent decomposition and background photochemistry. The reason for the increased photochemical background is not known. For PCCL, IPA is a useful mobile-phase additive only for compounds that are retained so strongly that 100% methanol is impractical in terms of analysis time. In fact, the photochemical background noise is one of the most important considerations in determining whether or not a given solvent will be compatible with PCCL. To fully realize the advantages of chemiluminescence detection-the ability to amplify small photocurrents to a much greater extent than would be possible in a fluorescence detector where stray light causes a large background photocurrent-signals produced in the absence of an analyte must be minimized. While the background signals caused by the photochemical reaction and the chemiluminescent reagents themselves are quite small when methanol is the polarity modifier, there is a small increase in the noise relative to the P M T dark current (Figure 2). The ultimate sensitivity of PCCL is limited by these

2

-s

0 Figure 2. Noise contributions in PCCL detection. Conditions were as follows: The HPLC mobile phase, 85/15 methanol-water, was delivered at 0.80 mL/min (fh = 152 s). TCPO (0.84 g/L) and Nbrene (23 mg/L) in acetone were delivered at 0.33 mL/min. TRIS buffer (pH = 8.2, 6.3 mM) in 4/38/58 water-methanol-acetone was delivered at 0.15 mL/min. The PMT was set at 1300 V. The magnitudes of the background signals are as follows: (0) fluorometer and recorder set to “zero”;(1) W dark current; (2) PMT dark current peroxyoxalate reagent background; (3) PMT dark current peroxyoxalate reagent background photochemical background. The change between 0 and 3 corresponds to =4 nA.

+

+

+

Table I. Catalytic Factors for Quinone Detection by PCCL

compound

87.5% methanol/ 12.5% water short long reactor reactor (0.87 mL) (2.3 mL)

anthraquinone anthraquinone-2-sulfonate

20 20

45 55

anthraquinone-2,6-disulfonate

28

69

25

62 59 59

2-methylanthraquinone 2-ethylanthraquinone 2-tert-butylanthraquinone 9,lO-phenanthrenequinone

19 22

2.5

1,2-naphthoquinone 1,4-naphthoquinone

10

P-methyl-1,4-naphthoquinone 2,3-dimethyl-l,4-naphthoquinone 2,7-dimethyl-1,4-naphthoquinone 2,6-dimethyl-l,4-naphthoquinone

15 0.2 17 11

0.5

8.9

21

27 0.5

29 19

background reactions. If these sources of background chemiluminescence could be eliminated, then operation of the PMT in the “photon counting mode” would increase the sensitivity. An interesting feature of PCCL is the catalytic nature of the photooxidation reaction. Since a quinone analyte will cycle through the photooxidation reaction sequence many times during its residence in the photoreactor, each cycle producing H202, the analyte’s signal is “chemically amplified”. Table I contains “catalytic factors” calculated for several of the sensitizers. The catalytic factor is defined as the number of moles of HzOzproduced per mole of quinone analyte. These factors were calculated from area-based linear regressions of HzOZcalibration plots obtained in the “plug injection” mode (lamp off), while the sensitizers were injected on column to prevent possible interferences from impurities. The magnitude of the catalytic factor depends strongly on the functional groups attached to the quinone moiety and the reaction time. Solvent composition (as long as a HAD substrate is present) and oxygen content play a smaller role in determining the HzOZyield. Acetonitrile and water mixtures are not suitable

1246


Table 11. Selectivity of PCCL against Other Comounds" amt

1

injected, selectivity compound

9-nitroanthracene anthrone benzophenone apdibromoacetophenone 1-nitronaphthalene 9,lO-dibromoanthracene a-acetonylbenzyl-4-hydroxy-

coumarin 2,3-butanedione anthracene 2-methylanthracene 9,lO-diphenylanthracene 2-chloroanthracene 9-anthracenecarboxylicacid naphthalene 2-phenyl-l,34ndandione phenol ethyl benzoate fructose glucuronic acid glucuronic acid lactone glucuronamide propanal chloroform 2-butanone 2-butanol

ratio

VR,bmL

mol

5.1 6.1 26 71 240

6.48

0.050

5.98 5.31 5.07 5.38

0.069

500 560

22.3 3.75

2 100 2 400 2 400 3 500 >4 300 >6 400 >44 000

4.36 7.92

quencher >84 000 >58000 >270000 >400 000 >250000 >100000 >120000 >100OOO >93 000 >91000

c!

Xl0-B

9.75 15.2

-10.0

9.66 6.01 3.44 4.43

5.30

0.26

0.15 7.62

3

3.89 4.26 3.33 1.15 1.03

1.53 10.6

5.04 20.1

13.9 64.8 94.8

4.2

ti

1.43

5.06

60.4 25.3 27.7 24.8

22.3 21.8

a HPLC mobile phase, 95/5 methanol-water, delivered at a flow rate of 0.80 mL/min (tRm = 112 9). TCPO (0.90 g/L) and rubrene (38 mg/L) delivered in acetone at a flow rate of 0.33 mL/min. TRIS buffer (pH = 7.9,6 mM) delivered at 0.15 mL/min. V , = uncorrected retention volume.

mobile phases for PCCL detection. Since neither is a HAD substrate, no significant production of HzOz will occur on a time scale appropriate for reaction detection in HPLC. The photohydroxylation reaction of quinones with water is too inefficient for this purpose. As one would expect for a catalytic reaction, the dependence on reaction time is the most significant. Stability of the quinone analyte is another important consideration. The large differences in sensitizer activity between differently substituted quinones result primarily from triplet reactivity changes. The inherent activity as a sensitizer is best evaluated a t short reaction times, since photodegradation becomes more significant as the irradiation time increases. In some cases, particularly quinones with dialkyl substituents that are both on the same ring and adjacent to the oxygens of the quinone moiety (as is the case with 2,3-dimethylnaphthoquinone and 1,4-dimethylanthraquinone), decomposition causes the H202 yield to be low a t long reaction times. The decomposition of 1,4-dimethylanthraquinoneis so severe that it is barely detected by PCCL and exhibits virtually no increase in sensitivity between reaction times of -20 and -180 s. A drawback in using a broad-band light source is the difficulty in separating the excitation efficiency of the sensitizer from its reactivity. In this case, the catalytic factor is a combination of the analyte absorptivity over the entire range of the lamp output, the intersystem crossing efficiency, the reactivity of its triplet state toward hydrogen abstraction, the speed and efficiency of the subsequent steps in the reaction cycle, the relative importance of deactivation through nonreactive channels, and the possibility for photodegradation or reaction with radicals produced in the reactor. Each factor probably is solvent dependent, which further complicates interpretation. Because of these complications, our optimi-

0 4

Figure 3. Photooxidation of anthracene. When this reaction occurs on particulate matter the yields of the endoperoxide (I), anthraquime (2),banthrone (3),and l-hydroxyanthraquinone(4) are reported to be 2-7%, 12-19%, 8-13%, and 2-5%, respectively (ref 5).

zation of the photochemistry for the PCCL detection scheme is empirical. Selectivity of PCCL. An advantage of the PCCL technique for the detection of quinones is its high degree of selectivity. Table 11includes measured selectivity ratios (SR), relative to anthraquinone (AQ), for some compounds that are not quinones. A few of these exhibit a weak response to PCCL. Here, the selectivity ratio is defined by

I

1I

moles of compound injected moles of AQ injected

I

area of AQ area of compound SR (11) Acetophenone and benzophenone derivatives are well-known as sensitizers of hydrogen abstraction reactions, so the low selectivity against these compounds is not surprising. Most of the other compounds in the table were detected poorly, if at all. This result certainly is expected for those compounds that absorb poorly in the wavelength region of lamp irradiation. For undetected compounds, the selectivity ratio is reported as moles of compound injected detection limit of AQ in moles under the same reaction conditions. Of particular interest is the possibility that singlet-oxygen sensitizers could produce a response to PCCL. Under the aerobic conditions in the photoreactor, most compounds with chromophores in the wavelength range of the source will generate singlet oxygen. A response through this mechanism would severely limit the selectivity of PCCL and increase the potential for these compounds to interfere with quinone detection. Formation of organic peroxides from singlet oxygen is well-known. However, the peroxyoxalate reaction responds to organic peroxides with much lower sensitivity relative to HzOz (40). Another possibility is the conversion of singlet-oxygen sensitizing compounds to "type 1" sensitizers through a photochemical oxidation. For example, anthracene can undergo a type 2 photooxidation to anthraquinone (Figure 3) (5). To differentiate between these two response mechanisms, products generated in the photoreactor from several PAH derivatives were investigated. As shown in Table 11, there is a wide variation in the selectivity toward two- and three-ring PAH compounds, which depends on their substituents. Although PAHs are fluores-


cent, they still sensitize the production of singlet oxygen. Furthermore, PAHs can react with singlet oxygen to form endoperoxides. Some PAH compounds, because of their oxidizability via this endoperoxide mechanism, are able to respond to PCCL through quinones formed during their photooxidation. A third possibility, direct detection through chemiexcitation of the fluorescent PAHs (as an increase in the peroxyoxalate background) was eliminated, because no peak was observed when the photoreactor lamp was turned off. To determine the reason for PAH response, heart cuts of the photoreactor products, generated from a concentrated solution of the parent PAH, were collected with the postcolumn reagents shut off. These fractions were characterized by their fluorescence, 251-nm UV absorbance, PCCL, and anaerobic PRFQ chromatograms (1). While the conversion efficiencies were not rigorously quantified, there are large differences in the yield of quinone products. Furthermore, the response of the PAH compounds to PCCL correlates very well with the extent of conversion to the corresponding quinone and the amount of HzOzsubsequently produced by the quinone in the photoreactor. By use of several different detectors, the confirmation of the quinone photoproduct identities can be made with reasonable certainty. Many other unidentified photoproducts were observed in addition to the quinones. Virtually all of these unidentified products were more polar than the parent PAH. This indicates that oxidation reactions are much more efficient than other reactions, such as dimerization, under the conditions in the photoreactor. In a more general sense, this HPLC-photoreactor system, in combination with heart cutting, makes it possible to investigate photochemical reaction products without interference from reagent impurities. Phenanthrene and anthracene were compared with respect to their response and photoproducts. Anthracene photooxidation produces small but readily detectable amounts of anthraquinone and other early eluting (probably hydroxysubstituted or endoperoxide) photoproducts. The response of anthracene to PCCL is about a factor of 4 greater than phenanthrene. Phenanthrene is barely detected at a level of 1.4 pg injected on-column. Phenanthrene produces only a small amount of phenanthrenequinone in the photoreactor. Photooxidation by singlet oxygen, the reaction which forms anthraquinone, occurs through an endoperoxide intermediate that bridges the 9 and 10 positions of anthracene (5). On the other hand, the reactive "9,lO" carbons of phenanthrene are adjacent and the endoperoxide may be less capable of forming at this position. In fact, phenanthrene has been reported to be very unreactive toward singlet oxygen (43). The spectral properties and reactivity of phenanthrene are described most accurately when it is considered to be a Ybenz"naphthalene as opposed to an anthracene derivative (43). While anthracene and phenanthrene are expected to have similar quantum yields for singlet oxygen production, the interpretation of the much larger response of anthracene to PCCL than phenanthrene is complicated by phenanthrene's lower absorptivity in the wavelength region emitted by the photoreactor source. Still, the formation of anthraquinone and subsequent H202 production in the photoreactor, rather than a direct response to singlet oxygen or organic peroxides formed from singlet oxygen, is the most likely cause of PCCL response to PAHs. Naphthalene is undetected by PCCL, even when 1.4 pg is injected on-column. Nitro-PAH compounds are known to abstract hydrogen atoms in reactions analogous to those of benzophenone and quinone derivatives. Of particular interest is 9-nitroanthracene, which has been reported to form anthraquinone and bianthrone as photoproducts (Figure 4) (44). 1-Nitro-

1247

Figure 4. Photooxidation of 9-nboanthracene ( 4 4 ) .

naphthalene is a poor hydrogen abstraction sensitizer in polar solvents. This has been attributed to 1-nitronaphthalene having a B,R* triplet state (45). In any case, both compounds would be expected to produce significant amounts of singlet oxygen in the photoreactor. In Table I1 the response of the 9-nitroanthracene is shown to be much larger than that of the 1-nitronaphthalene. Three possible sources of this difference are (1) the large difference in absorptivity, (2) a lower efficiency in the photoconversion to the corresponding quinone, and (3) the lower reactivity of naphthoquinone as a sensitizer relative to anthraquinone. A study of the 9-nitroanthracene photoproducts revealed large conversions to anthraquinone. 1-Nitronaphthalene produced 1,4-naphthoquinone, but in much smaller quantities. Even though anthraquinone is a stronger photooxidation sensitizer than naphthoquinone, given the magnitude of the difference in responsiveness it primarily is caused by some combination of the first two factors. 9,lO-Diphenylanthracene (DPA) also is detected poorly by PCCL. Very little anthraquinone is formed in the photoreactor from DPA. The phenyl groups decrease the ability of the 9,lO-endoperoxide to oxidize to anthraquinone. In fact, when the 9,10-endoperoxide of DPA is heated, DPA is regenerated and singlet oxygen is released (46, 47). While a 1,4-endoperoxidewould be capable of forming the 1,4-quinone, this product probably would have a lowest triplet state with B,B* character and, as such, be less reactive toward HzOz formation through hydrogen abstraction. Indeed, compounds such as 6,13-pentacenequinone and benz[a] anthracene-7,12dione, which have fused rings undisturbed by the quinone moiety are very poor photooxidation sensitizers. 6,13-Pentacenequinone was not detected by PCCL when a saturated methanolic solution was injected, while benz[a]anthracene7,12-dione had a detection limit of ~ 2 8 ng. 0 DPA is known to be a much better singlet oxygen trap than anthracene; hence, it is likely that this opening of the endoperoxide is the limiting step of the reaction. Singlet oxygen reacts with DPA at rates that are nearly an order of magnitude greater than it reacts with anthracene (46). 9,lO-Dibromoanthracene (DBA) also produces anthraquinone in the photoreactor. This observation correlates well with the enhanced response of DBA relative to anthracene. Photodehdogenation reactions of aryl halides are well-known. In the case of O,lO-DBA,the dehalogenation could occur either before or after the reaction with singlet oxygen forms the endoperoxide. The higher intersystem crossing rate of DBA increases singlet oxygen production in the photoreactor. Singlet oxygen sensitizers do not appear to respond signifi-

1248


Table 111. Detection of Vitamin K,by PCCL detection limit, pmol ( S I N = 3)" PCCLb 27-m PCCLb PCCLb reactor 27-m 10.4-m UV 100% reactor reactor 247 nm CH30Hc 60/4OC 6O/4Oc 60/4OC phylloquinone (vitamin K,) V,, mL

89

78

29.2

10.6

120

1.8

10.6

10.6

On-column detection limits under the following conditions: HPLC mobile phase, either 100% methanol or 60/40 methanol-%-propanol, delivered at 0.78 mL/min (PCCL) or 1.30 mL/min (UV).bPCCL t b = 158 s with the 27-m reactor, tb = 67 s with the 10.4-m reactor. TCPO (0.95 g/L) and rubrene (32 mg/L) in acetone delivered at 0.33 mL/min; TRIS buffer (pH = 8.1, 0.5 mM) delivered at 0.15 mL/min. Mobile phase, methanol/water. a

cantly to PCCL unless they are able to react to form "type 1" sensitizers (e.g., anthracene forming anthraquinone). Unfortunately, many quinone compounds of biological interest do not respond well to PCCL. These quinones, substituted with hydroxy, amino, and/or carboxyl groups are poor photooxidation sensitizers, because their lowest triplet state is charge transfer or T,T* in character. In the case of hydroxyquinones, photoenolization and deprotonation of the excited state decreases its electrophilicity and, thereby, decreases its reactivity. Quinones of this type are found in many of the electron transport systems of plants and animals (38, 48).

Another group of bioactive quinones, the "K" vitamins, also respond poorly to PCCL (Table 111). The poor detection limits of the K vitamins probably are caused by the phytyl chain photocyclizing via the intramolecular abstraction of an allylic hydrogen (49). The photocyclization product(s) are fairly stable and lower the intermolecular hydrogen abstraction efficiency. The chemiluminescence signal increases on adding IPA to the mobile phase. P A has a weaker a C-H bond than methanol; hence, it is a better hydrogen atom donor. While the improvement in detection limit is not commensurate with the increase in signal intensity, the retention time of this analyte is decreased. Photodecomposition is not the cause of the poor detection limits for vitamin K1, because there is a continued gain in signal intensity with increasing reaction time. Although the detection limit for vitamin K1 only improves by a factor of 1.5, the peak height increases by a factor of 2.6 between the short and long reactor. When the methanol-IPA mobile phase is used, anthraquinone exhibits changes in its detection limit and peak height that are similar in magnitude, 1.8 and 3.5, respectively. Vitamin K3 (menadione), which has no phytyl chain, produces a strong response to PCCL (see Table IV).

Comparison of Light Sources. The key considerations in choosing a source for the photoreactor are its intensity and spectral distribution, absorptivity of the analytes over the range of the lamp output, and minimization of the photodegradation of the desired product(s). In PCCL, there is a small photochemical background that increases at shorter irradiation wavelengths. This background varies from solvent to solvent. Quinones have strong absorptions in the region of 254 nm, indicating that a low-pressure mercury lamp would be an efficient source for their excitation. However, the H202 produced in the photoreactor will photolyze at wavelengths shorter than ~ 3 1 nm 0 (reaction 13) (50). With the 254-nm Pen-Ray lamp, very high backgrounds were observed with almost no signal from quinone analytes or plug injections of H2Oz. HZOZ hv 20" (13)

+

-+

While use of the fluorescent poster lamp (emission centered at 366 nm) avoids the problem of peroxide decomposition, the broad-band emission of this lamp overlaps with much weaker absorption bands of the analytes. Lower absorptivity and lamp intensity translate into longer reaction times for an equivalent gain in sensitivity. Therefore, in the interest of finding a stronger radiation source with emission in this wavelength region, two phosphor-coated pencil lamps were tested. The first had a phosphor with a spectrum centered a t 366 nm, similar to that of the poster lamp. This lamp was slightly less effective than the fluorescent poster lamp, mainly because its geometry was poorly matched to the large photoreactors used with PCCL. The second lamp, which had a specified emission maximum of 351 nm, generated a very high background which was decreased by placing a Pyrex sleeve between the lamp and the reactor. It is likely that the phosphor passes some 254-nm radiation, which causes this background. Sensitivity with the Pyrex sleeve in place was comparable to the poster lamp. Since the poster lamp generates less heat, is longer (a better fit to the photoreactor), and is less expensive, it is the preferred source. Detection Limits. As the variation in peroxide yield with changes in substituents suggests, the detection limits for quinones are dependent on these functional groups and their positions. In Table IV, the detection limits for several quinones are presented. By examination of the detection limits under various solvent conditions and reaction times, the sensitivity and selectivity of PCCL can be optimized for specific applications. Since solvent conditions often are limited by the separation, the choice of reaction time affords the best means of adjusting the selectivity of PCCL to a target analyte. As previously mentioned, decomposition causes some analytes to be detected most sensitively a t short reaction times. Di-

Table IV. Detection Limit Comparisons

t,, min compound

9,10-anthraquinone-2-sulfonate menadione (vitamin K3) 2,7-dimethyl-1,4-naphthoquinone 9,lO-anthraquinone 2-methyl-9,lO-anthraquinone 2-tert-butyl-9,lO-anthraquinone

(UV)

PCCLb 27-m reactor

2.07 5.34 6.31 7.92 10.06 11.83

0.057 0.16 0.17 0.086 0.085 0.087

detection limit. Dmol (SIN = 3)" PRFQE PRFQE 2.6-m 2.6-m reactor reactor A,, = 243.5 nm A,, = 257.5 nm 0.26 0.13 0.083 0.067 0.084 0.11

0.045 0.97 0.51 0.018 0.018 0.021

uv

251 nm 0.10 0.40 0.34 0.25 0.25 0.36

a On-column detection limits under the following conditions: HPLC mobile phase, 89/ 11 methanol-water, delivered at 0.80 mL/min. bPCCL: tRxn = 156 s; TCPO (0.86 g/L) and rubrene (29 mg/L), dissolved in acetone, delivered at 0.33 mL/min; TRIS buffer (pH = 8.1, 6 mM) delivered at 0.15 mL/min. 'Photoreduction fluorescence detection of quinones (ref 1): t~~ = 16.5 s, A,, > 389 nm.


0

I

I

I

10

16

MINUTES

(b)

I

10

MINUTES

3

5

Log (moles x 1013injected)

Flgure 6 . 9,lO-Anthraquinone calibration curve by PCCL. The amount injected covers the range from 86 pg to 345 ng, n = 3 per point, n, = 27. Conditions were as follows: HPLC mobile phase, 85/15 methanol-water, delivered at 0.80 mL/min (fRxn = 152 s); TCPO (0.85 g/L) and rubrene (28 mglL) in acetone delivered at 0.33 mL/min; TRIS buffer (pH = 8.2, 6.3 mM) in 4/36/60 water-methanol-acetone delivered at 0.15 mL/min; PMT biased at 1300 V. Slope = 0.982, uap. = 0.0024, intercept = 0.030, uhwt = 0.0062, r = 0.99993. Because rubrene is a long wavelength emitter, selfabsorption by the analyte is not observed.

~~-251nm

I

1

1246

I

16

Flgure 5. Separation of a quinone mixture. PCCL detection: HPLC mobile phase, 89/11 methanol-water, delivered at 0.80 mL/min (tRm = 155 s); TCPO (0.84 g/L) and rubrene (31 mg/L) in acetone delivered at 0.33 mL/min; TRIS (pH = 8.1, 6.3 mM) delivered at 0.15 mL/min. UV detection (A = 251 nm): HPLC mobile phase, 89/11 methanolwater, delivered at 0.80 mLlmin. Peaks were as follows: (1) 9,lOanthraquinone-2-sulfonate (0.59 pmol), (2) 1,Cnaphthoquinone (2.4 pmol), (3) 2-methyl-1 ,Cnaphthoquinone (vitamin K3) (1.6 pmoi), (4) 2,7dlmethyl-1,Cnaphthoquinone (1.1 pmol), (5) 9,lO-anthraquinone (0.74 pmol), (6) 1,5dichloro-9, lo-anthraquinone (1.1 pmol), (7) 2methyl-9, lhnthraquinone (0.50 pmol), (8) 2-ethyC9,lO-anthraqulnone (0.42 pmol), (9) 2-tert-Butyl-9,lO-anthraquinone (0.55 pmol).

alkylquinones, when both alkyl groups are attached to the same ring, are the best examples of this situation. Short reactors also provide additional selectivity against compounds that can be transformed photochemically into “type 1”sensitizers (e.g., “interfering” PAH compounds). The selectivity toward type 1 sensitizers is a t its highest when the reaction time is short. In Figure 5 a separation of a mixture of nine quinones is presented to facilitate the comparison of PCCL to UV detection. The chromatograms demonstrate an improvement in sensitivity without significant degradation of the chromatographic resolution. Increasing the reaction time causes only small decreases in the resolution and analyte concentration, because the photoreactors are designed to minimize peak broadening (26).The primary limitations on reactor length are practical in nature. Reactor back pressure presents no problem to the pumping system (e.g., -7 bar for a 2.4-mL reactor with a 0.76 mL/min flow of 95/5 methanol-water), but it does limit the reactor length and the flow rates it will accommodate. The 0.30mm-i.d. (30-gauge) tubing sold by Small Parts,Inc., is available in lengths up to 100 ft (volume ~ 2 . mL). 5 We have used

crocheted reactors up to this length at flow rates of 1.0 mL/min without causing the reactor to burst. However, when reactors of this length are necessary, slightly lower flow rates are utilized to increase the residence time and protect the reactor against excessive back pressure. PCCL detection has a wide linear range, as demonstrated by the calibration curve for 9,lO-anthraquinone (Figure 6).

Optimization of the Chemiluminescence Response, The sensitivity of the peroxyoxalate reaction toward H202 depends on the solvent composition, reagent concentrations, pH, and the residence time in the flow cell. In the PCCL system, choice of the chromatographic eluent is limited by the requirement that the mobile phase be “hydrogen atom donating” and still allow separation of the analytes. Fortunately, the quinone analytes are separated efficiently at rather high polarity modifier concentrations. These are optimal for both the photochemistry and peroxyoxalate response. On the other hand, the oxalate esters (TCPO in this system) tend to decompose in the presence of hydroxylic solvents. This necessitates the addition of the ester in a solvent different from the mobile phase, as well as the minimization of the delay time between mixing the reagent and chromatographic streams and measuring the intensity of the resulting chemiluminescence. Under these conditions, efficient mixing is an important consideration in the choice of the reagent addition solvent(s). Since sensitivity is enhanced by increased reagent concentration, another factor in the choice of the addition solvent is the solubility of the reagents. Only one high-pressure syringe pump was available for postcolumn use, so the TCPO and fluorophore (rubrene) were added in a mixed solution. This configuration was more stable and reproducible than delivering one of the reagents via an additional low-pressure syringe driver (Sage Model 341). Therefore, both reagents had to be sufficiently soluble in the addition solvent. Acetone provides the best combination of reagent solubility, stability, and miscibility with the mobile phase. Rubrene has too low a solubility in acetonitrile, while ethyl acetate causes a high background due to mixing noise. Concentrations of both TCPO and rubrene must be lowered as the water content of the mobile phase is increased, or the reagents will precipitate in the flow cell and mixing tee.

1250

ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990 200 T

l

I

I

a

a

li

t?

100

AQ

1 1

L

zi,

L:

8

16

24

e

16

24

0

0

IO

20

33

40

50

0 0

10

20

30

40

RETENTION VOLUME (mL)

50

FLOW RATE (mL/hr)

Flgwe 7. TCPO flow rate dependence of PCCL response: (a) signal-to-noise ratio vs reagent flow rate; (b) peak height vs reagent flow rate. Conditions were as follows: HPLC mobile phase, 9515 methanol-water, delivered at 0.80 mL/min (fhn = 112 s); TCPO (0.93 g/L) and rubrene (37 mg/L) in acetone W i e d at 0.33 mL/min; TRIS (pH = 8.1, 6.3 mM) delivered at 0.15 mL/min in the catalyzed curves; PMT biased at 1460 V. Injections of 4.1 ng of 9,lO-anthraquinone (n = 4 per point).

A low-pressure syringe driver was used to deliver a TRIS buffer. By use of a buffer at a high enough concentration to allow delivery at a low flow rate and in a delivery solvent that minimizes the swelling or shrinkage of the plastic syringe's rubber piston, the performance of these pumps is adequate. Addition of the "catalyst" (buffer) affords a small improvement in detection limits and, perhaps more significantly, a protection from interferences caused by acidic or basic matrix constituents. Once again, matching the composition of the buffer's addition solvent to the chromatographic eluent and reagent addition solvent(s) to promote efficient mixing and prevent reagent precipitation is of critical importance. For TRIS addition, a mixture of methanol, acetone, and water worked well. Pure acetone causes the plastic syringe to jam, because the piston swells. On the other hand, pure methanol causes it to leak due to shrinkage of the syringe piston. Once the solvents for reagent addition were selected, the effect of reagent flow rate on response was explored. Of the factors causing the dependence of the signal on the reagent flow rates-a combination of the detector residence time, delay time after mixing, final solvent composition, reagent concentration, reagent decomposition, and analyte dilution-the effect of reagent concentration appears to dominate in PCCL detection. In the flow-rate-dependence curve (Figure 7), the HPLC solvent composition and flow rate were selected with regard to the chromatography and photoreactor residence time. The buffer flow rate is kept low to minimize dilution of the analyte and the frequency with which the syringe must

Chromatograms of a cardboard extract: (a) PCCL chromatogram; (b) UV absorbance chromatogram at 251 nm. The anthraquinone (AQ) peak is indicated in the PCCL chromatogram and undetectable in the UV chromatogram (the retention volume axis of the PCCL chromatogram is offset by the volume of the photoreactor). See the Experimental Section for chromatographic conditions. Figure 8.

be refilled, while its maximum concentration is limited by the solubility of TCPO in the mixed solution. At very low reagent flow rates, the TCPO/fluorophore concentration is the limiting factor on the response. As the flow rate is increased, the response levels out. Since the background noise also increases with the increase in flow rate, gains in terms of detection limits are slightly smaller than the increase in absolute chemiluminescence intensity. We selected a flow of 20 mL/h, which is on the plateau of the flow-rate-dependence curve, to maximize the sensitivity while conserving the reagents. The optimum reagent flow also was determined with a lower concentration of anthraquinone. No change was observed in the relative shapes of the signal intensity and signal-to-noise ratio curves or in the positions of their optima as the detection limit for anthraquinoine was approached. Application of PCCL to a Cardboard Extract. With the PCCL detection scheme, it is possible to detect anthraquinone residues in finished Ubrownpaper" products, such as cardboard and shopping bags, after direct injection of a filtered extract. Neither prefractionation nor preconcentration is required. As one can see from the chromatograms of a cardboard extract, W absorption detection is unable to detect anthraquinone in this matrix (Figure 8). The sensitivity of the UV detector had to be attenuated to bring the chromatograms on scale; hence, no signal for this low amount of anthraquinone would be observed. Once again, the importance of selective detection is demonstrated, as quantification of anthraquinone in this extract would be impossible with UV detection. Presumably, a large part of the absorbing/fluorescing compounds present in the extract are phenolics.


Electrochemical detection, while very sensitive toward quinones, would probably exhibit similar interference problems, since the phenolic compounds would be electroactive. Another reaction-detection scheme for quinones in this type of matrix is reported to be =4 orders of magnitude less sensitive than PCCL (51). This method is based on the formation of a long wavelength chromophore (detection a t 500 nm) through a postcolumn reaction. The efficiency of the extraction of anthraquinone from cardboard was not investigated. Assuming that 100% of the analyte is removed by the Soxhlet extraction and that none is lost during the filtration step, the level of anthraquinone in the extract corresponds to 4 0 pbb in the cardboard sample, as determined by standard additions. Obviously, the sensitivity and selectivity of PCCL detection result in considerable savings in the analysis time. NO anthraquinone residues were detected in extracts of “white” paper products (e.g., office stationary, toilet paper, paper towels, and newsprint). These extractions were performed in the same manner as the cardboard extracts.

ACKNOWLEDGMENT The authors wish to thank Professor F’ and Mitchell Geckman for the loan of HPLC apparatus during the stages Of this project* The late Professor w’ Frei assisted in all phases of this project. LITERATURE CITED Poulsen, J. R.; Birks, J. W. Anal. Chem. 1989,61, 2267. Holton, H. H.; Chapman, F. L. Tappi 1977,60(11), 121. Haggln, J. Chem. Eng. News 1984,62(42), 20. Chem. Eng. News 1966,64 (21), 19. Fox, M. A.; Olhre, S. Science 1979,205. 582. Rontanl, J. F.; Rambeloarisoa, E.; Bertrand, J. C.; Giusti, G. Chemosphere 1985, 74, 1909. Hendbwk of Environmental Data on Organic Chemicak; Verschueren, K., Ed.; Van Nostrand Reinhold, Co., Inc.: New York, 1983; pp 208-213. Scheler, A.; Gominger, D. Bull. €nviron. Contam. Toxicol. 1976, 16, 595. PMs, J. N., Jr.; Harger, W.; Lokensgard, D. M.; Fitz, D. R.; Scorziefl, G. M.; Mejia, V. Mufat. Res. 1982, 704. 35. Poulsen, J. R.; Birks, J. W. I n Chemilumlnescenceand photochemical ReacHon DerecHon in Chfomatoyaphy; Birks, J. W., Ed.; VCH Publishers, Inc.: New York, 1989; pp 149-230. Krull, 1. S. I n Reaction Detection in Li9uM Chromatography, Chromatographic Science Serbs; Krull, I.s., Ed.; Marcel Dekker, Inc.: New York, 1986; Vol. 34, pp 303-352. Oandelman, M. S.; Birks. J. W. J. Chromatogr. 1982,242, 21. Gandelman, M. S.; Birks, J. W.; Brinkman, U. A. Th.; Frei, R. W.J. Chromatcgr. 1963,282,193. Wells, C. H. J. Introduction to Molecular photochemistry; Chapman and Hall, Ltd.: London, England, 1972; pp 105-107.

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Rauhut, M. M. Acc. Chem. Res. 1969,2 , 80. Sei&, W. R. CRC Crit. Rev. Anal. Chem. 1981, 13, 1. Imai, K.; Weinberger, R. TrAC, Trends Anal. Chem. 1985,4 . 170. Van Zoonen, P.; Kammlnga, D. A.; Gooijer, C.; Velthorst, N. H.; Frei, R. W.; Gubh, 0. Anal. Chim. Acta 1985, 774, 151. (19) Kobayashi, SA.; Imai, K. Anal. Chem. 1960,52, 424. (20) Sigvardson, K. W.;Kennish, J. M.; Birks, J. W. Anal. Chem. 1984,56, 1096. (21) Pouisen. J. R.; Birks, J. W.; Gubtz, G.; van Zoonen, P.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. J. Chromatogr. 1986,360, 371. (22) Poulsen. J. R.; Birks, J. W.; van Zoonen, P.; Gooijer, C.; Velthorst, N. H.; Frei. R. W. Chromatographla 1968. 21, 587. (23) Weinberger, R. J. Chromatogr. 1984,314, 155. (24) Sherman, P. A.; Holzbecher, J.; Ryan, D. E. Anal. Chim. Acta 1978, 97, 21. (25) Mohan, A. G.; Turro, N. J. J. Chem. Educ. 1974,57, 528. (26) Pouisen, J. R.; Birks, K. S.; Gandelman, M. S.; Birks, J. W. ChromatoOraDhkI 1986.22. 231. (27) boknd, J. L.; Cooper, H. R. Proc. R . SOC. London, Ser. A 1954, 225, 405. (28) Wells, c, F, Trans, farahy lgsl, 57, 1703, 1719. (29) Wilkinson, F. J. Phys. Chem. 1962,66, 2589. (30) Carison. S. A.; Hercules. D. M. photoahem. photobid. 1973, 17, 123. (31) Cooper, H. R.; Talbot, B. M. Trans. faraday Soc. 1965, 6 1 , 506. (32) Carlson, S. A.; Hercules, D. M. J. Am. Chem. Soc. 1971,93, 5611. (33) Bruce, J. M. I n The Chemistry of Quinoid Compounds, Part 1 ; Patai, S.. Ed.; Wiley-Interscience: 1974; pp 494-507. (34) Turro, N. J. Modern Molecular Photochemistry; W. A. Benjamin: New York, 1981; pp 362-392. (35) Hulme, B. E.; Land, E. J.; Phillips, G. 0. J. Chem. Soc., faraday Trans. 11972,68, 1992, 2003. (36) Hendbook of Photochemistry; Murov, S. L., Ed.; Marcel Dekker, Inc.: New York, 1973. (37) Cooper, H. R. Trans. faraday SOC. 1966,62, 2865. (38) Tseng, SA.;Mohan. A. G.; Haines, L. G.; Vlzcarra, L. S.; Rauhut, M. M. J . e o . Chem. 1979. 44. 4113. - (39) McCapra, F. Prog. 0rg:’Chem. 1871,8 , 231. (40) Rauhut, M. M.; Bollyky, L. J.; Roberts, B. 0.; Loy, M.; Whltman. R. H.; Iannona, A. V.; Semsel, A. M.; Clarke, R. A. J. Am. Chem. SOC. 1967,89, 6515. (41) Sigvardson, K. W.; Birks, J. W. Anal. Chem. 1983,55, 432. (42) Honda. K.; Miyaguchi, K.; Imai, K. Anal. Chlm. Acta 1985, 777, 111. (43) Kearns, D. R., Khan, A. U. Photochem. Photobiol. 1969, 10, 193. (44) Chapman, 0. L.; Heckert, D. C.; Reasoner, J. W.; Thackaberry, S. P. J. Am. Chem. SOC. 1966,8 8 , 5550. (45) Hurley, R.; Testa, A. C. J. Am. Chem. SOC.1968,90, 1949. (46) Monroe, B. M. J. Phys. Chem. 1978,8 2 , 15. (47) Wasserman, H. H.; Scheffer, J. R.; Cooper, J. L. J. Am. Chem. SOC. 1972. 94, 4991. (48) function of Quinones in Energy Conserving Systems; Trumpower, B. L., Ed.; Academic Press: New York, 1982. (49) Nakata. T.; Tsuchda, E. Methods fnzymol. 1980,67, 148. (50) Wilkinson, F.; Brummer, J. G. J. Phys. Chem. Ref. Data 1981, 70, 809. (51) Kiba, N.; Takamatsu, M.; Furusawa, M. J. Chromatogr. 1985, 328, 309. (15) (16) (17) (18)

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RECEIVED for review August 14, 1989. Revised manuscript received January 22,1990. Accepted January 25,1990. This work was supported by a grant from the U.S.Environmental Protection Agency (No. R-810717-01-0).

Photocatalytic chemiluminescence detection of quinones in high

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