MlNl REVlEW

Low-level Interferences in Peroxyoxalate Chemiluminescence C. Gooijer? and N. H. Velthorst Free University, Department of General and Analytical Chemistry, De Boelelaan 1083, 1081 H V Amsterdam, The Netherlands

M in peroxyoxalate chemiluminescence is examined based on experimental results available in the literature. Implications for fluorophore and for hydrogen peroxide determinations are discussed. An interpretation in terms of the reaction mechanism is proposed.

The role of interferences at concentrations lower than

INTRODUCTION In 1983 the group of Imai, in a comparative study on the chemiluminescence (CL) efficiencies of TCPO (bis(2,4,6-trichlorophenyl)oxalate)and DNPO (bis(2,4dinitropheny1)oxalate) noted that the use of chloride and especially bromide and iodide in buffers or ionpairing reagents should be avoided in HPLC with peroxyoxalate CL detection (Honda et aL, 1983). Even low concentrations of these ions cause a significant decrease of the CL signal. This aspect has received only minor attention in the literature, although the parameters determining the compatibility of HPLC eluent composition and peroxyoxalate CL reaction conditions have been thoroughly examined. By now it is known, that low-level quenching of peroxyoxalate CL is not limited to halide ions. Other inorganic ions as nitrite and sulfite analogously induce considerable quenching and the same has been shown for a number of organic compounds (Van Zoonen et af., 1986, 1987; Gooijer et af.,1989). Although an extensive screening of possible quenchers has not yet been performed, it may be concluded that, in general, readily oxidizable compounds are efficient quenchers. As already noted in the literature (Honda et af.,1983), this type of low-level quenching can be adequately described by a Stern-Volmer type expression In -=

1

formulated by Rauhut and McCapra (Schuster and Schmidt, 1982), reaction of oxalate and H 2 0 2 under proper conditions leads to the formation of the dioxetanedione, Cz04,followed by a subsequent, crucial electron transfer step from the fluorophore F to C204, producing a radical cation and a radical anion according to C,O,

+ F 2C,O, + F'

[I1

Luminescence is produced in the following reaction sequence C * 0 4 + CO,+ CO,

co,

+ F* + F* + GO,

PI [31

F* + F+luminescence

[41

A readily oxidizable species Q can also react with C z 0 4 via electron transfer so that, parallel to reaction [ 13, the reaction C,04 + Q

I,

+ Q*

~51 leading to consumption of C,O, (without luminescence production) has to be accounted for. Thus the efficiency of reaction [ l ] is reduced to C,O,

where k, and k, are the bimolecular rate constants of reactions [ l ] and [ 5 ] , respectively. As a result the CL signal intensity decreases from loto I , according to

1 + kQ[Q]

wherein Inand I are the CL signal intensities in absence and presence of quenchers, k, is the quenching constant in M - ' and [Q] the concentration (in M ) of quenchers. Efficient quenchers are characterized by a k, on the order of lo5 M-'; for such a species a concentration as low as M causes a CL decrease of 10% while at concentrations higher than lop4 M hardly any signal is observable. At first sight Eqn (1) can be explained readily in view of the CIEEL (chemically induced electron exchange luminescence) mechanism that is most probably operative in peroxyoxalate CL. In its most simple description, t Author to whom correspondence should be addressed

(3)

Equation (3) suggests that k, is determined by both the nature of F (via the rate constant k,) and the concentration of F. If this conclusion were correct, even the presence of low amounts of quenchers would seriously limit the linear dynamic range of fluorophore detection by peroxyoxalate CL. Presently, it is known that the above approach is too simple (Van Zoonen et af., 1986, 1987; Gooijer et al., 1989). Systematic experiments have been performed to investigate the role of low-level interferences. In the subsequent section these experimental results are

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LOW-LEVEL INTERFERENCES IN PEROXYOXALATE C H E M I L U M I N E S C E N C E

assembled and discussed systematically, leading to a possible interpretation. It is emphasized that no detailed kinetic study is presented; only the overall CL intensities have been measured. Of course, a complete mechanistic analysis would require a fully kinetic treatment.

Table 2. TCPO-CL intensities (lo)and quenching constants k , for perylene at different con5 X lo-’ M centrations; quencher: methimazole Perylene

RESULTS AND DISCUSSION Van Zoonen ef al. (1987) have performed a number of experiments on the influence of the nature and the concentration of the fluorophore on k, in a flow injection set-up applying a solid state TCPO reactor and methimazole ( 5 lo-’ ~ M ) as a quencher. Six fluorophores at 4 x M were compared, thus ensuring a wide range of CL. Table 1 presents the results. Although in this table Z,,varies over 4 orders of magnitude, the variation of k , is less than 50%. The influence of the fluorophore concentration on k , was examined using perylene as a model fluorophore and again methimazole ( 5 x lo-’ M ) as a quencher. As is obvious from Table 2, a variation in perylene concentration of over two orders of magnitude only modestly induces a change in k,. From these experiments it is concluded that to a good approximation k , is independent of both the nature and the concentration of the fluorophore. This is a very important conclusion: fluorophore determinations by HPLC peroxyoxalate CL are not adversely affected by the presence of low-level quenchers, provided that the concentrations of quenchers in the detector cell are constant during the elution process. The other parameters possibly influencing k, were studied in experimental set-ups in which the CL detector cell contained 3-aminofluoranthene (3-AF), immobilized on controlled-pore glass beads, as the fluorophore. The influence of the H,O, concentration was investigated for TCPO, in the solid state configuration, again with methimazole as the quencher. For H 2 0 2concentrations between lop2M and lop6M no change in k , was observed. This implies that also in hydrogen peroxide analyses straight calibration lines will be observed even in the presence of quenchers. It is emphasized, however, that the CL efficiencies and thus the slopes of the calibration lines do depend on the quencher concentration. Consequently it is necessary to apply standard addition procedures in order to circumvent systematic errors in the determination of hydrogen peroxide. Of course in fluorophore determinations the H 2 0 2 concentration should be chosen as high as possible since the CL intensity is proportional to the H 2 0 2concentration.

concentration

‘0

(M)

(relative units)

kQ (lo3 M-’)

40 500 10 000 1200

44 48 42

4~10-~ 4x 4 x lo-’

The nature of the oxalate does influence k,. This dependence has already been found by Honda et al. (1983): for DNPO and TCPO the quenching constants of NaCl are 4.6 x lo2 M - ’ and 2.2 x lo3 M - ’ , respectively; for NaI the corresponding values are 4.4 x lo4 M - ’ and 5.5 x 10’ M - I , respectively. Hence, for both halides, the quenching of the TCPO reaction is much more efficient than the quenching of the DNPO reaction. Similar results have been published by Van Zoonen (Van Zoonen ef al., 1987). They compared LC chromatograms of a mixture of anilines by quenched peroxyoxalate CL detection using TCPO and 2-NPO, bis(2-nitropheny1)oxalate. The percentage of quenching induced by a particular aniline is proportional to k,. For 2-NPO the peaks are about a factor of 10 higher than for TCPO, indicating a difference in k, of one order of magnitude. Finally, in Table 3 some detection limits derived from the liquid chromatograms obtained by quenched CL detection using 2-NPO as the oxalate are presented. In this system the analytes are separated on a HPLC column, the effluent is mixed with a reagent flow of 0.05 M H 2 0 2 and 8 mM 2-NPO in acetonitrile and the mixture enters the detector cell containing immobilized 3-AF, placed in front of the photomultiplier. The data collected in Table 3 clearly show that low level quenching of peroxyoxalate CL is not a rare phenomenon. Summarizing, the peroxyoxalate CL mechanism has to account for the following two experimental results. Various readily oxidizable species do induce low-level quenching, described by a Stern-Volmer type relationship, see Eqn (1). The quenching constant in this equation is independent of the nature and the concentration of the fluorophore and of the concentration of

Table 3. HPLC detection limits (amount injected) derived from quenched 2-NPO CL detection Detection limit (ng)

Analyte

Table 1. TCPO-CL intensities (4) and quenching constants (k,) for a number of fluorophores; quencher: 5 X M methimazole; fluorophore concentration 4 X lo-’ M ‘0

Fluorophore

(relative units)

Fluorene Anthracene Fluoranthene Diphenylanthracene Perylene 3-Aminofluoranthene

8 790 3000

5100 40 500 45 000

kQ (103M-’)

28 42 26 44 44 37

Bromide Iodide Sulphite Nitrite p- lsopropylaniline N, N-Dimethylaniline N- Ethyl-m-toluidine N, N-Dipropylaniline Thiourea N - Allylthiourea Ethynyl thiourea Methimazole

1.5 0.3 1.1 0.3

1.4 0.6 1 .o 8.0 1 .o

1.6 2.0 0.4

~

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BIOMEDICAL CHROMATOGRAPHY. VOL 4, NO. 3, 1990 93

C. GOOIJER AND N. H. VELTHORST

hydrogen peroxide, but dependent on the nature of the oxalate. By now, it is known that the peroxyoxalate CL reaction mechanism is much more complicated than the Rauhut-McCapra mechanism denoted in the preceding section. Catherall et al. (1984) have shown that the dioxetanedione is probably not the intermediate participating in electron transfer and Alvarez et al. (1986) in a thorough study have demonstrated that at least three intermediates play a role in a pooled intermediate model. Recently, Gooijer et al. (1989) have been able to interpret the low-level quenching results in terms of the Catherall mechanism. Reaction between oxalate and H 2 0 2 produces a highly energetic intermediate X that subsequently decays, reaction [6], reacts with F, reaction [7], or reacts with Q, reaction [ 8 ] , according to X

[61

non-CL decay

X + F A X 'F"

[71

X + Q k X 'Q"

[81

= 1+ki,rx~[QI

(9)

so that k,, see Eqn ( l ) , can be written as ICllPXF

kQ=

(10)

Here, Q9,"is the efficiency of reaction [9] in absence of quencher and rxF is the lifetime of the radical ion pair in absence of quencher, which is equal to (b+k J ' . Equation (10) does account for the available experimental data on low-level quenching. The quenching constant k , is independent of the concentrations of hydrogen peroxide and fluorophore; also the character of F will hardly influence k, since any fluorophore radical cation will be highly efficiently reduced by an electron-donating species. In contrast, according to Eqn (10) k, will depend on the particular oxalate used in the CL experiment via the lifetime r x F .A reasonable structure for X is shown here and reaction [9] presum0-0

The efficiency of reaction [ 7 ] in presence of Q, given by = k6+

MFI k7[F ] + k 8 [ Q ]

@7,,

is

According to Catherall et al. k6 dominates in the denominator, so that (assuming that the concentrations of F and Q are low enough) Eqn (4)reduces to

Thus, the presence of Q does not reduce the efficiency of the reaction between the intermediate and the fluorophore. In other words, reaction [8] does not account for the phenomenon of low-level quenching. On the other hand Eqn (5) does explain that the CL signal is proportional to [F] and dependent on the oxidation potential of F (via k 7 ) . The most plausible way to interpret the experimental results is to assume that the quencher reduces the efficiency of F* production resulting from the decomposition of XF. Here the following reaction steps must be considered X 'F"

k9

F* +products

5 F+ products

X -.F*.

X

(4)

ably takes place in two consecutive steps (Scheme 1 ) . For a highly efficient quencher, reaction [ l l ] will be diffusion-controlled so that k , , will be about 10" M - ' s-I. For such a compound a lifetime for X-'F*', or more precisely for F", of 1 ps would imply a k , of lo4 M - I , thus explaining low-level quenching. It is speculated that such a relatively long lifetime is not unrealistic in view of the consecutive steps for X-', denoted above.

L

OK

1

~91 [lo1

X ' F * . + Q A F+X-'Q'.+products

[I11

In a steady state approximation for X 'F" the efficiency is cf reaction [93 in presence of Q,

I

,

where k,, k , , and k , are the corresponding rate constants. Hence the redilction in efficiency is given by (7) =l+-

kl1

k9+

k10

[QI

94 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 3, 19%

i2IIB

Scheme 1

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LOW-LEVEL INTERFERENCES IN PEROXYOXALATE CHEMiLUMiNESCENCE

CONCLUSION Low-level quenching of peroxyoxalate CL does not hamper hydrogen peroxide and fluorophore determinations in flow injection analysis and HPLC, provided that

internal standards or standard addition procedures are used. An explanation for the influence of quenchers at concentration levels as low as M can be given in terms of the Catherall mechanism, although a thorough kinetic study might point to the pooled intermediate model of Alvarez et al.

REFERENCES Alvarez. F. J., Parekh, N. J., Matuszewski, B., Givens, R. S..Higuchi, T. and Showen, R. L. (1986). J. Am. Chem. SOC.108. 6435. Catherall, C. L. R., Palmer, T. F. and Cundall, R. B. (1984). J. Chem. SOC.Faraday Trans., 2.80. 823 and 80.837. Gooijer, C., Van Zoonen, P.,Velthorst, N. H. and Frei, R. W. (1989). J. of Bioluminescence and Chemiluminescence, 4,479. Honda, K., Sekino. K. and Imai, K. (1983). Anal. Chem., 55, 940. Schuster. G. B. and Schmidt, S. P. (1982). Adv. Phys. Org. Chem. 18, 187.

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Van Zoonen, P., Kamrninga, D. A., Gooijer, C., Velthorst, N. H. and Frei, R. W. (1986). Anal. Chem., 58, 1245. Van Zoonen. P., Bock, H., Gooijer, C., Velthorst, N. H. and Frei, R. W. (1987). Anal. Chim. Acta., ZOO, 131.

Received 26 July 1989; accepted 21 August 1989.

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