Flow injection system for determination of singlet oxygen quenching efficiencies utilizing on-line dioxetane chemiluminescence detection

201

Analytica Chimica Acta, 290 (1994) 201-214 Elsevier Science B.V., Amsterdam

Flow injection system for determination of singlet oxygen quenching efficiencies utilizing on-line dioxetane chemiluminescence detection H.A.G. Niederhinder,

M.M. de Jong, C. Gooijer and N.H. Velthorst

Department of General and Analytical Chemistry, Free University, De Boelekaan 1083, 1081 HVAmsterdam (Netherlands) (Received 25th May 1993)

Quenchers of singlet oxygen are believed to exhibit a protective action against oxidative stress. Previously, the bimolecular quenching rate constants have been determined by means of elaborate batch experiments. In the present paper a novel analytical technique is presented, i.e. a flow injection system in which photochemically generated singlet oxygen reacts on-line with 1,2-diethoxyethene to form 3,4-diethoxy-1,Zdioxetane. In the detector cell thermal decomposition (70°C) in the presence of a fluorophore (9,10-dibromoanthracene-2-sulfonate) leads to chemiluminescence (CL). Upon injection of a quencher a decrease of the CL intensity is observed and, as outlined theoretically, the associated singlet oxygen quenching rate constant can readily be determined. Furthermore it is shown that effects of interferences can easily be accounted for experimentally. Obtained data are in good agreement with available literature data, provided that solvent influences are considered. The novel method is fast, and quenching rate constants in a wide variety of solvent compositions can readily be obtained. Keywork Chemiluminescence; Flow injection; Dioxetane chemiluminescence detection; Singlet oxygen quenching efficiencies

Reactive oxygen intermediates play an important role in the metabolism of aerobic organisms [l]. Under normal conditions the cells in these organisms are well protected against reactive oxygen intermediates, so that no oxidative damage can occur. The balance between generation and deactivation of these intermediates, however, may be affected by various mechanisms. Some chemicals, such as drugs and pollutants, may diminish cellular antioxidant defenses, or stimulate the formation of reactive oxygen species [2]. Other chemicals may generate reactive oxygen intermediates themselves, for instance under the influCorrespondence to: C. Gooijer, Department of General and Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam (Netherlands).

ence of radiation [3,4]. This will result in oxidative stress, sometimes leading to cell death or cancer. However also some therapeutic uses have been published [3,4]. Mechanisms for generation and deactivation of reactive oxygen intermediates have been extensively studied in for instance pharmacology, toxicology, biochemistry and medicine [5]. In these studies frequently compounds, either synthesized or naturally occurring, are used that exhibit protective action against oxidative stress. Biological data are usually correlated with chemical antioxidant activities obtained through an independent route. If singlet oxygen mediated processes are considered, rate constants for quenching are generally determined by measuring the effect of the quencher on singlet oxygen phosphorescence, in-

OtXl3-2670/94/$07.00 0 1994 - Elsevier Science B.V. All rights reserved SSDI 0003-2670(93)E0607-9

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duced by laser excitation [6,‘7], or generated chemically [1,7], or by following inhibition of singlet oxygen mediated reactions [7-91. These batch experiments are rather elaborate, expensive and time consuming. Furthermore, since long singlet oxygen lifetimes have to be ensured for most of these techniques, expensive deuterated or chlorinated solvents are frequently used, generally differing substantially from the matrices for use of the quenchers in biological systems. In this paper a novel technique for the measurement of singlet oxygen quenching efficiencies is described, based on the competition between the quencher and a singlet oxygen reagent with a known reaction rate constant for addition of singlet oxygen. Unlike other methods our technique is operated in a flow injection (FI) system and there is no need to utilize chlorinated or deuterated solvents. The product of the reaction of singlet oxygen with the reagent is easily monitored through chemiluminescence (CL) detection, so that the effect of an injected singlet oxygen quencher can directly be observed as a decrease of the CL signal. Each measurement takes no more than a few minutes and no time is needed for exchanging the sample or cleaning the detection cell. Below, the theoretical background of the novel method is outlined, together with an evaluation of the mathematical equations needed for calculating the quenching rate constants. Subsequently the experimental details are described. Some of the experimental parameters are optimized and the constants needed for calculating absolute singlet oxygen quenching rate constants are determined. Quenching rate constants for some known singlet oxygen quenchers, measured with our method, are compared to those obtained by conventional methods. Finally the value of the method as a reference system in pharmacological studies is underlined.

per is based on the addition reaction of singlet oxygen with 1,Zdiethoxyethene (DEE), leading to formation of 3,4-diethoxy-1,2-dioxetane (DEDO), a product that will give CL upon decomposition in the presence of a fluorophore (9,10-dibromoanthracene-2-sulfonate, DBAS). This method for detection of singlet oxygen was previously developed in our group with the objective of determining singlet oxygen sensitizers in a liquid chromatography system [lO,lll. In this paper the attention is not focussed on sensitizers, but on quenchers of singlet oxygen. These quenchers can interfere with the production of DEDO, thus affecting the chemiluminescence signal. Ideally only singlet oxygen will be quenched and other reaction pathways are not influenced. In that case the CL signal can directly be used to monitor singlet oxygen quenching. Some quenchers however will also quench triplet excited states, playing a key role in the production of DEDO and the CL signal, or even react with the DEDO that has been formed. We will refer to these processes as interferences, since they will hamper direct monitoring of singlet oxygen quenching. Below, monitoring of singlet oxygen quenching and the effect of interferences will be discussed in more detail.

Monitoring singlet oxygen quenching In our method singlet oxygen is produced by a photosensitizing process. A well known sensitizer for production of singlet oxygen is rose bengal (RB) [12]. RB has a strong absorption band at long wavelengths and a very high quantum yield for intersystem crossing to the triplet state [13]. If a solution containing both oxygen and RI3 is irradiated, singlet oxygen is generated with an efficiency of about 80 percent (Scheme 1) 1131. Once singlet oxygen has been generated it can decay to the ground state through different routes, depending on the compounds present in the soluRB+ hv +lRB*

THEORETICAL

ASPECTS

(1)

O,&RB)

1RB*-3RB*

The technique for measurement of singlet oxygen quenching efficiencies presented in this pa-

3RB* +0,(3Z,)2 Scheme 1.

(2) RB+O,(‘A,)

(3)

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et aL /Anal. Chim. Acta 290 (1994) 201-214

(4) + DEE 2 0,

(

DEDO

203

ciency for production 8

of DEDO is given by Eqn.

(5)

ka,&‘EEl DEDQ-prod =

‘A, +Q&oducts )

k,,,[DEE]

-t

k,[Q]

+ T-~(~O,) (8)

0,(1A,)+Q~0,(3Z,)+Q Scheme 2.

tion (Scheme 2). First of all the solvent itself plays an important role in the deactivation of singlet oxygen (Eqn. 4). The lifetime of singlet oxygen in solution ranges from 2 ps in water to more than 1 ms in chlorofluorocarbons [7]. In general, hydroxylic substituents shorten the lifetime, whereas deuteration, chlorination or fluorination reveals the opposite effect. In the literature singlet oxygen is frequently monitored by means of its ‘Ag +3Z, emission at 1270 run [7]. Since this is a relatively slow process [14] emission can only be clearly observed in solvents that ensure relatively long singlet oxygen lifetimes. In our method singlet oxygen is monitored by its addition reaction with DEE (Eqn. 51, leading to a product (DEDO) that can be used for CL detection. This (second) route for deactivation of singlet oxygen in presence of adequate DEE concentrations is fast (kadd = 4.7 X 10’ 1 mol-’ s-l [15]), so that solvents giving relatively short singlet oxygen lifetimes can also be used. Thirdly, if competitive pathways for singlet oxygen decay become available, the efficiency for production of DEDO will decrease so that the CL signal measured in the detection process will diminish. This occurs for instance if a singlet oxygen quencher is present, acting either chemically (Eqn. 6) or physically (Eqn. 7). We will not distinguish between these two processes. Interaction of singlet oxygen with the quencher will be described by one overall bimolecular rate constant (k,) which is the sum of the rate constant for chemical reaction (Eqn. 6) and the rate constant for physical quenching (Eqn. 7) (k, = k, + ks). If the above three routes for singlet oxygen decay are taken into account the quantum effr-

as can be readily conceived following a steady state approach. Hence the efficiency for production of dioxetane is inversely related to the quencher concentration and the quenching rate constant; evidently the same holds for the CL signal observed. The quotient of the CL signals obtained in absence and presence of a quencher reveals a Stem-Volmer type dependence on the quencher concentration (Eqn. 9). so

-=

s

&EDO-prod 4 DEDO-prod

=1+

kQ

k,,,[DEE]

+ F1fO,)

’ [‘I

(9)

Interferences Apart from singlet oxygen, some other intermediates play a key role in obtaining the dioxetane CL signal in the system concerned. Each of these intermediates might be affected by an added quencher, thus causing a decrease in the effrciency of production of the dioxetane. If such processes occur, the observed decrease of the CL signal will not be exclusively dependent on the interaction of the quencher with singlet oxygen. Effects of the quencher on other intermediates will thus have to be avoided, or should be corrected for. Interferences can be divided in two major classes. First of all triplet excited states, playing a key role in the production of singlet oxygen (Scheme 1, 3RB*), or in the production of the &l!3Md% DEDO -

EF + 3EF *

00)

3EF* +DBASkTS-EF+‘DBAS*

(11)

3EF * + DBAS 5 T-q%*) 3EF*-EF

(12)

EF + ‘DBAS *

(13)

k,(EF)

3EF* +Q -EF+Q Scheme 3.

04)

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singlet excited fluorophore (Scheme 3, 3EF * ), may be quenched. Secondly DEDO may give catalytic decomposition under the influence of the quencher, or even react with it. Below, these classes of interferences will be discussed in more detail. Triplet quenching. Singlet oxygen is generated by energy transfer from the relatively long lived triplet excited state of rose bengal, 3RB*, to ground state molecular oxygen (Scheme 1, Eqn. 3). In principle a quencher might interfere in this process. However, since the reaction rate constant for quenching of a triplet state by oxygen is generally very high (almost diffusion controlled [12,16]) and. solvents, if aerated, contain oxygen concentrations of about 10T3 M [17], 3RB* quenching will only play a significant role for very efficient triplet quenchers that are present at fairly high concentrations [181. A similar type of interference might occur in the detection process (Scheme 3). In the detector DEDO is thermally triggered to decompose (Eqn. 10) into two molecules of ethyl formate (EF), one in the electronic ground state and the other in the triplet excited state with an efficiency of almost unity [19]. Subsequently, energy transfer from 3EF* to DBAS gives singlet excited DBAS (Eqn. 11) [20], leading to CL. Although this spinforbidden transfer process is exceptionally efficient for DBAS [20], spin-allowed energy transfer to DBAS, resulting in triplet excited DBAS (Eqn. 12), and intersystem crossing from 3EF* to the ground state (Eqn. 13), are in competition with this CL productive pathway. Furthermore, a quencher can also cause interference by attacking the triplet excited state of ethyl formate (Eqn. 14). If the various routes for decay of triplet ethyl formate are. taken into account, the quantum efficiency for production of singlet excited DBAS, which is proportional to the CL intensity, is given by Eqn. 15. 4 ‘DBAS*

= {4sEDBASl){h&‘BASl

+k,[DBAS]

+ k,(EF)[Q]

(15)

Thus the CL signal is inversely proportional to the quencher concentration and the reaction rate constant for 3EF* quenching. The Stern-Volmer dependence of the CL signal on the quencher concentration will now reflect both the effect of the quencher on singlet oxygen (Eqn. 9) and the effect of the quencher on triplet EF (Eqn. 16). -= 4 ‘DBAS*

k,( EF) kTs[ D&IS] + k,,[ D&IS] + T-~(~EF*)

*[Ql

(16) The total effect will be the product of these two separate contributions (Eqn. 17). S0

&nno-prod

s=$l

DEDO-prod

4p~~~* .4 ‘DBAS * kQ

k,,,[DEE]

X { 1+

+ +(10,)

* la1

[k,(EF)l[k,s[DBASl

+k,[DBAS]

+ T+(~EF*)]

-‘. [Q]) (17)

;

=

(1 + Cl[Q])(l

+ C2[Q])

=

1 + (Cl + G>[Ql + tWd[Ql*

= 1+ (Cl + C2)[Q]

(18)

As can be seen from Eqn. 18 (a simplified form of

Eqn. 17) there is no linear relationship between S,/S and the quencher concentration. However, at quencher concentrations lower than about 10e3 M, the second order term can generally be neglected, resulting in a linear relationship, where the slope (Cl + C,) is a summation of the StemVolmer constants for both quenching processes. Since spin-forbidden energy transfer from 3EF* to DBAS (k,, (Eqn. 11) is about 10’ 1 mol-’ s- ’ [19,20]) will be slow in comparison to spin-allowed quenching of 3EF *, an added quencher may give significant interference in the detector process (Scheme 3), even if present at

HAG. NieMdr DEDO + Q k

decomposition

products

(19)

Scheme 4.

relatively small concentrations. 3EF * quenching may be reduced by using a high DBAS concentration. If however triplet quenching in Eqn. 14 is fast it has to be corrected for. Dioxetane &composition. A somewhat different type of interference may occur as soon as some DEDO has been produced in the photochemical reactor. Dioxetanes are reactive towards several types of compounds and can be susceptible to catalytic decomposition. Some quenchers might induce DEDO decomposition (Scheme 4, Eqn. 191, resulting in a decrease of the CL signal. Catalytic decomposition cannot easily be prevented, so that it has to be corrected for. This is not a type of process that can be dealt with in terms of efficiencies. Analysis of the kinetics of DEDO decomposition will however give information about the effect of quenchers on the resulting dioxetane concentration entering the detector cell and thus about the effect on the CL signal. The amount of DEDO that will be formed is determined by the rate for production and the rate for decomposition (Eqn. 20). d[ DEDO] dt

205

et al. /Anal. Chim Acta 2W (1994) 201-214

-

k.dQI[DEDOl

Solving this differential 21,

[DEDOlt= [DEElo-

leads to

kad,[‘~*I I

2

-{em kdsf[Qlt

(20)

equation

k,dd[10

_

[DEDO]y= ({ 1 -

+

lQl

k

dec

e-kaddf’021r

I

e-kaddI’021’t})

[DEDOI, kadd[102] add [ lo,]

+

kdec[Ql

(23)

and reflects the relative effect on the CL signal @a/S). This is not a simple inverse proportionality as was obtained for singlet oxygen quenching (Eqn. 8), or for ethyl formate quenching (Eqn. 15). Under certain circumstances however Eqn. 23 will reduce to a simpler form. If, at any time during the irradiation, the production of DEDO is much faster than its decomposition (k,,[Q] < k,,,[‘O,] = k,,,[‘O,]‘) and furthermore the irradiation time t (which is of course equal to the interaction time for DEDO decomposition) is long enough, it is readily seen that Eqn. 23 can be approximated by Eqn. 24. [DEDO]~ [DEDO],

WE1

= k,JO,l

of DEDO produced in absence and in presence of a quencher is given by Eqn. 23

{l-O] = ekdcc[Q]r (24) 1. {e-kc&W _ 0) z

Besides it is noted that the magnitude of k,,[‘O,] will depend on the amount of sensitizer present in the reactor. The total effect of singlet oxygen quenching, triplet EF quenching and dioxetane decomposition on the quotient of the CL signal in absence and in presence of a quencher is the product of the three separate effects on this quotient (Eqn. 25).

which reduces to Eqn. 22

&,

[DEDO]~ = [DEE], - ( 1 - e-k~‘021”}

s=4J

if the concentration of quencher is zero. It should be noted that the singlet oxygen steady state concentrations in Eqns. 21 and 22, denoted as [‘O,] and [‘O,]’ respectively, are not identical. The quencher offers an additional route for deactivation of singlet oxygen, so that [‘O,] is somewhat lower than [‘O,]‘. The ratio of the amount

Singlet oxygen quenching rate constants can be evaluated for most compounds if the effect of the quencher on singlet oxygen can be separated from the effect on other processes playing an important role in generating the CL signal. In our approach the interferences are quantitated by utilizing an alternative arrangement of the FI

&ED0prod

&hws*

[DEW: (25)

DEDO-prod

*-*4

‘DBAS*

[DEW,

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systems. Consecutively the reagents cals used are discussed.

Fig. 1. Original FI set-up for measuring the combined quenching effect of an injected compound on singlet oxygen, DEDO and triplet ethyl formate.

set-up and are subsequently corrected for in the data used for calculating singlet oxygen quenching rate constants.

EXPERIMENTAL

Two configurations of the FI system are used, viz., the original system and the modified system. In the original system (Fig. 1) the effect of the interferences as well as singlet oxygen quenching on the CL signal is measured. In the modified system (Fig. 2) only the interferences are measured, while singlet oxygen quenching plays no role. Both configurations are built from the same components and switching from one configuration to another is easily realized. Below, the components and composite parts, as well as the instrument settings, are described, viz., the photochemical reactor, the CL detector and the solvent, quencher and reagent delivery

Fig. 2. Modified FI set-up for measuring the quenching effect of an injected compound on DEDO and triplet ethyl formate. Singlet oxygen is only present inside the photochemical reactor, so that no contact with the injected quencher is possible.

and chemi-

Apparatus The photochemical reactor part consists of a Model 93110 @O-W, 25 mm arc length) mediumpressure mercury lamp (Philips, Eindhoven), mounted with a laboratory-made cylindrical quartz filter cuvette (9 mm path length) (Free University, Amsterdam). Filter solutions are pumped round, through the filter cuvette and a water cooler, with a Model 1022 centrifugal pump (Eheim, Berlin). The polytetrafluoroethylene (PTFE) reactor tubing (Eriks, Alkmaar), 0.33 mm i.d., 0.73 mm o.d., is knitted around the filter cuvette (5.7 cm diameter), to decrease band broadening. The tubing is 5.0 m in length, providing an irradiation time of 75 s at a flow-rate of 0.34 ml min-‘. In the original system (Fig. 1) DEDO decomposition occurs during its residence time inside the photochemical reactor. In the modified system (Fig. 2) the second reactor (reactor B) consists of 5.0 m of identical knitted PTFE tubing, ensuring exactly the same interaction time of DEDO with the quencher as in the original system. The CL detector part consists of a Model 980 fluorescence detector (Applied Biosystems, Ramsey, NJ) with the lamp turned off and with a 25-~1 detector cell installed. The stainless-steel bar containing the cell compartment is adapted with a thermocouple and a thermocoaxial electrical heater. The temperature is controlled with a laboratory-made electronic temperature programmer (Free University) at 70.0 f O.l”C. Boiling and creation of bubbles inside the detector cell are avoided by operating the detector at about 8 bar back-pressure. DEE and RB are added with a Model 140A dual syringe solvent delivery system (Applied Biosystems) at a flow-rate of 0.09 ml min-’ in the original system and 0.22 ml min-’ in the modified system. This flow-rate results from the two syringes delivering equal amounts of solution, one syringe delivering a solution of DEE in mobile phase and one syringe delivering a solution of RB in mobile phase.

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The quencher, solved in mobile phase, is introduced into a flow of pure mobile phase by means of a laboratory-made six-port injection valve (Free University) equipped with a 200-~1 injection loop. This flow is delivered by a Model 300 solvent pump (Gynkotek, Munich), at a flow-rate of 0.25 ml min-’ in the original system and 0.12 ml min-’ in the modified system. At these flow-rates the interaction times in the photochemical reactor (Fig. 1) and reactor B (Fig. 21, in the original and the modified system, are identical (reactor flow is 0.34 ml min-’ in both situations). DBAS solved in methanol is added with a laboratory-made syringe pump (Free University) at a flow-rate of 0.2 ml min-‘.

hydrogenphosphate and sodium dihydrogenphosphate in Milli-Q water. The various quenchers were supplied by Aldrich. They were all at least 95% pure. Stock standard solutions (about lo-’ Ml were prepared (every 1 or 2 weeks) in mobile phase and stored at 4°C in the dark. Filter solutions were made in deionized water. They will be referred to as follows: A 2 260 nm is 1.2% (w/v) potassium iodide in 0.5 M sodium hydroxide, A 2 330 nm is 2 M sodium nitrate, A r 395 nm is 10% (w/v) sodium nitrite and A r 495 nm is 0.5% (w/v) potassium dichromate.

RESULnTSANDDISCUSSION

Chemicals and reagents 1,2_Diethoxyethene (DEE) was prepared as described by Baganz et al. [21] and purified by double distillation prior to use. Rose Bengal (RB) was supplied by Aldrich (Brussels) and used as obtained. Fresh solutions in mobile phase were made every two or three days. The concentrations of DEE were 2.0 X lo-’ M in the original system and 5.6 X 10V3 M in the modified system. For RB the concentrations were 1.7 x 10m7 M in the original system and 4.1 x lo-’ M in the modified system. DBAS was prepared as described by Catalani et al. [201 and purified by recrystallisation from water. A fresh solution (2.0 x 10m3 M) in methanol was made every one or two months. The concentrations mentioned are the pump concentrations. Dilution factors are 0.13 for DEE and RB in the original system, giving the photochemical reactor concentrations, and 0.37 for DBAS in the original and the modified system, giving the detector concentration. Acetonitrile was purchased from Westburg (Leusden) and methanol from Baker (Deventer), both LC grade. “Milli-Q water” was prepared by ultrafiltration using a Mill&Q system (Millipore, Bedford, MA). Disodium hydrogenphosphate, sodium dihydrogenphosphate, potassium dichromate, sodium nitrite, sodium nitrate and potassium iodide were supplied by Baker. The mobile phase was either pure acetonitrile, or acetonitrile-buffer (70 + 30, v/v). The buffer solution, at pH 6.6, was prepared from disodium

The photochemical reactor process and the CL detector process are determined by a number of variables, such as the concentrations of reagent (DEE) and fluorophore (DBAS), the irradiation time, the irradiation wavelength window, the detector temperature and the solvent composition. Most of these variables have been optimized in previous investigations [lO,ll]. In the system under consideration only few of these variables have to be reevaluated. The irradiation wavelength window will be optimized and the effect of the concentration of the singlet oxygen sensitizer will be studied. Furthermore singlet oxygen lifetimes, in connection to the solvents used, have to be determined (if not known from literature). Consecutively correction methods for dioxetane decomposition and triplet quenching are stated, leading to reliable procedures for calculating singlet oxygen quenching rate constants. Finally some singlet oxygen quenching rate constants obtained will be compared to literature values. Irradiation window If processes, other than dioxetane production through singlet oxygenation induced by RB, take place in the photochemical reactor the effect of a singlet oxygen quencher may be obscured, or at least become extra complicated. The most important disturbances of a simple singlet oxygenation mechanism occur because of absorption of radiation by compounds other than the sensitizer, such

208

as the quencher itself, the reagent for addition of singlet oxygen (DEE), the dioxetane produced (DEDO) and impurities present in solvents and reagents. If radiation is absorbed by the quencher itself, this may lead to production of singlet oxygen, assuming that the excited state of the quencher can operate as a singlet oxygen sensitizer. The quencher will have a dual effect in this situation, both generating and quenching singlet oxygen. It would therefore be impossible to calculate a quenching rate constant from the results. Similar consequences can evolve from absorption of radiation by DEE or impurities. A more complicated situation is encountered for absorption of radiation by DEDO. Dioxetanes, such as DEDO, are sensitive to photolytic decomposition, yielding excited decomposition products [22,23]. Absorption by the dioxetane will therefore lead to a decrease in the concentration of the dioxetane directly proportional to the amount of dioxetane produced. The effect will be more pronounced for a bad singlet oxygen quencher than for a good one. Decomposition may moreover lead to triplet excited decomposition products, giving rise to production of singlet oxygen, which will strongly complicate the overall kinetics. All these effects can be avoided by irradiating at long wavelengths, so that DEDO, DEE and quenchers cannot absorb the light and only the sensitizer is excited. In Fig. 3 the CL intensity is plotted as a function of the cut-off wavelength of the excitation filter used. The background CL signal is measured in a system where only DEE is present and reflects the amount of dioxetane obtained through oxygenation sensitized by for instance excited impurities, or DEE. The sensitized CL signal observed on top of the background, in a system where both DEE and RB are present, reflects the amount of dioxetane obtained by singlet oxygenation sensitized by RB. In both situations the signal is subject to photolytic dioxetane decomposition. It is clear that the background signal reduces, as expected, with increasing cut-off wavelength. Besides, the sensitized CL signal increases strongly as the wavelength changes from a cut-off wavelength of 250

HAG. Niederliinder et aL /Ad

Chim. Acta 290 (1994) 201-214

Fii. 3. Background CL signal (dot-dashed lie) and the CL signal sensitized by 1.7 X 10m9 M rose bengal (dashed line) as a function of the cut-off wavelength of the excitation filter, the right hand axis. Molar absorptivity of DEDO (solid line), the left hand axis.

mn to 500 nm. Figure 3 also shows part of the absorption spectrum of DEDO [19]. It is clear that there is an inverse relationship between absorption by DEDO and the sensitized CL signal. It can therefore be concluded that at wavelengths longer than about 450 mn DEDO is exclusively generated by RB-sensitized singlet oxygenation and photolytic decomposition of DEDO can be excluded. In practice an excitation filter with a cut-off wavelength at 495 mn was used. Sensitizer concentration The concentration of RB determines the absolute sensitized CL signal intensity whereas the relative CL signal intensities in presence and absence of a singlet oxygen quencher, as given in Eqn. 9, is independent of the RB concentration, provided that it is high enough to justify the simplification of Eqn. 23 to Eqn. 24. The concentration of the sensitizer therefore only has to be optimized with respect to the signal to noise ratio obtained in an unquenched situation. In practice the signal to noise ratio remained nearly unchanged on varying the sensitizer concentration, indicating that mainly correlated noise is dealt with. A sensitizer concentration of 2 X 10-s M in the photochemical reactor was chosen to give an easily detectable signal. This concentration is low enough to prevent any possible interference of the sensitizer in other processes.

HAG. Nieahkier

et at!/Ad

Singlet oxygen lifetimes

The Stem-Volmer constant for singlet oxygen quenching (Eqn. 9) does not directly give absolute information about the rate constant of singlet oxygen quenching for the quencher under consideration. It can only be calculated if the reaction rate constant for addition of singlet oxygen to DEE and the lifetime of singlet oxygen in the (reactor) solvent used are known. The reaction rate constant for addition of singlet oxygen to DEE is 4.7 X 10’ 1 mol-’ s-l in acetone [U] and has been shown to be essentially independent of solvent [15,24]. As a contrast the lifetime of singlet oxygen changes strongly with solvent composition. This lifetime is known for various solvents and solvent combinations [7]; but, unfortunately, in other cases it has to be determined. Measuring the effect of a singlet oxygen quencher for two different concentrations of DEE will lead to two different Stem-Volmer constants (Eqns. 26 and 27).

ksv(2)=

kQ

(27)

k,,[DEElz ++(h)

Since the reaction rate constants remain unchanged, substitution of Eqn. 26 into Eqn. 27, cancelling out the rate constant for quenching of singlet oxygen, will lead to Eqn. 28.

T-*(*0*) =

(b4l)[DEEl1-

k,(2)[DEElz}

.k add

W&)-

209

Chh. Acta 290 (1994) 201-214

(%)

~sv(l)l

Substitution of the two Stem-Vohner constants, the two concentrations of DEE in the photochemical reactor and the reaction rate constant for addition of singlet oxygen to DEE reveals the lifetime of singlet oxygen in the solvent or solvent composition under consideration. In many of the investigations the solvent was a composition of acetonitrile and water (70 + 30, v/v), containing 1.5 mM of phosphate buffer at a pH of 6.6. For this solvent composition the lifetime of singlet oxygen was calculated based on

the Stem-Vohner quenching constants obtained for trolox at two different DEE concentrations. The compound trolox was used for this purpose, because no interferences are experienced for this quencher. The singlet oxygen lifetime in the solvent of the above composition is about 24 ps. Correctionfor inte#erences In the modified arrangement of the FI system, where the quencher under consideration is added directly after the photochemical reactor, singlet oxygen quenching is excluded. In this situation the remaining possible negative influences are (catalytic) decomposition of DEDO and quenching of the triplet excited state of ethyl formate. This modified set-up can therefore be used to quantify the effects of these interferences on the CL signal in the original FI set-up, provided that they are equal in both systems. Dioxetane &composition. Catalytic decomposition and reactions of DEDO are generally not very fast 122,231, allowing the simplification of Eqn. 23 to Eqn. 24. To account for these effects it is necessary to insert a second (not irradiated) reactor after the point where the quencher is added to the modified system. The rate of DEDO decomposition in this second reactor is described by Eqn. 29; d[DEDO] dt

= k,,[Q][DEDO]

solving this differential [DED~]:

equation yields Eqn. 30.

= [DEDOlo e+=lQIt

(30)

The ratio of the DEDO concentration in absence and presence of a quencher, and therefore the partial effect of induced dioxetane decomposition on the total CL signal, is now given by Eqn. 31,

[IDEDO];~ [DEDO];

[DEDOlo

= [DEDO]oe-kd=tQlt = ekds[Qy = Eqn. 24

(31) which is equivalent to Eqn. 24. The effect of the quencher in the modified system is therefore equal to the effect of the quencher in the original system, where interaction with DEDO takes place inside the photochemical reactor itself (length

HAG. Niederlkder et al. /Anal. Chim. Acta 290 (1994) 201-214

210

and flow-rates for both reactors were made identical). Trip&t ethyl formate quenching. The effect of the quencher on the CL process through quenching of the triplet excited state of ethyl formate (see Eqns. 15 and 161, in the original and the modified set-up, can easily be made identical. The reactor flow-rate and the fluorophore addition flow-rate should be identical in both systems. If these conditions are met all terms in Eqn. 16 are equal for both the original and the modified system. Correction for dioxetane decomposition and triplet ethyl formate quenching is now simple. A

Stern-Vohner plot of the data obtained in the original system will meet Eqn. 25 and a SternVolmer plot of the data obtained in the modified system will meet Eqn. 32. S; --. 4&,,~ F - #loBAS*

[DEDOI? [DEDO],

(32)

Multiplying the expression for S,/S with the expression for S’/Si will lead to Eqn. 33,

(33)

TABLE I Data obtained in pure acetonitrile; Stern-Volmer constants for quenching obtained with the original system and the modified system and experimental rate constants for singlet oxygen quenching calculated from these Stem-Volmer constants Quencher

Sulfur conlpounds Dimethyl sulfide Diethyl sulfide Di-isopropyl sulfide Di-tert. -butyl sulfide Lipoic acid d

Stern-Volmer constants (1 mol-t X lo-*)

Quenching rate constants (I mol-’ s-l X lo-‘)

Original system

Modified system

Experimental

10.0 a (* 1) 3.75 a ( f 0.07) 0.9 ( f 0.05) < 0.8 ’ 26.0 a (k5)

5.8 (kO.2) 1.8 (kO.03) no quenching ’ no quenching ’ 16.2 (kO.9)

5.2 ( f 2.6 ( f 1.2 ( f < 1.0 13.0 ( f

Amines 112,2,6,6-Pentamethylpiperidine 2,2,6,6-Tetramethylpiperidine 4-Hydroxy-2,2,6,6tetramethylpiperidine 1,4-Diazabicyclo[2,2,2loctane N, N, N ‘,N ‘-Tetramethylp-phenylenediamine

490.0 ( f 20)

Phenols 2,6-Di-tert.-butyl-4methylphenol Trolox d

< 0.8 b 20.0 = (*4)

19.5 (*0.2)

no quenching ’

1.3) 0.13) 0.07) 7.8)

25.0(*0.3)

Literature

1.7 b 0.25 b 0.02 b 13.8 e

9.2 f

< 0.8 ’

no quenching ’

< 1.0

< 0.02 f

< 0.8 ’

no quenching ’

< 1.0

< 0.02 g

72.0 ( f 5)

3.3 ( f 0.2)

89.7 ( f 6.5)

5.2 g

no quenching ’

637.0 ( f 26)

100.0 h 330.0 i

no quenching ’ 2.5 (It 1)

< 1.0 23.4 ( f 6.5)

0.6 i 47.0 k

The literature rate constants for singlet oxygen quenching are from different references and were determined in various solvents. a Best results were obtained from fitting a second order polynomal. The linear part of this polynomial is presented, being a summation of Stern-Volmer constants. b From Ref. 29, solvent is methanol. ’ No observable quenching was obtained for quencher acid, trolox is 6-hydroxy-2,5,7,8-tetramethylchromanconcentrations as high as 10e3 M. d Lipoic acid is 1,2-dithiolane-3pentanoic 2-carboxylic acid. e From Ref. 1, solvent is D,O. f Expected based on Ref. 8,32, solvent is chloroform. g From Ref. 8, solvent is chloroform. h From Ref. 30, solvent is methanol. i From Ref. 31, solvent is H,O. i From Ref. 9, solvent is methanol. k From Ref. 1, solvent is D,O-ethanol (1: 1). ’ No interferences of the quencher were observed for quencher concentrations as high as 10v3 M.

HAG. Niederlliinderet al./Anal.

211

Chim. Acta 290 (1994) 201-214

which is identical with the Stem-Vohner dependence of Eqn. 9, from which the rate constant for singlet oxygen quenching is easily obtained. As emphasized previously (Eqn. 181, the StemVolmer constant for singlet oxygen quenching can generally be obtained by simply subtracting the slope for the interfering processes (as a function of quencher concentration) from the slope for the total process. If this is not allowed the data have to be fitted to a nonlinear function and corrected numerically for these processes. It should be noted that, for the same quencher concentration injected, its concentration in the reactor and in the detector are not the same for the original and the modified system. For fitting functions to the obtained data the real (local) quencher concentrations in the system under consideration should be used.

Determination of singlet oxygen quenching rate consfanfs Numerous singlet oxygen quenching rate constants have been determined in various kinetic studies in literature [1,6,8,9,25-291. We have studied some of these quenchers in our system, using two solvent compositions [pure acetonitrile and acetonitrile-water (70 + 30, v/v)], and calculated the rate constants for singlet oxygen quenching. CL signals were measured in both the original set-up and the modified set-up. The associated Stem-Volmer constants are presented in Tables 1 and 2, for data obtained in acetonitrile and in acetonitrile-water respectively. The quality of the fits is sufficient to give relatively small standard errors in the Stem-Volmer constants (regression coefficients > 0.998). Quenching rate constants for singlet oxygen quenching are ob-

TABLE 2 Data obtained in acetonitrile-water (70 + 30, v/v); Stem-Volmer constants for quenching obtained with the original system and the modified system and experimental rate constants for singlet oxygen quenching calculated from these Stern-Volmer constants Quencher

Stern-Volmer constants (1 mol-’ X 10-‘) Original system

Amines 1,4-Diaxabicyclo[2,2,2Joctane 1,2,2,6,6-Pentamethylpiperidine N,N,N’,N’-tetramethylp-phenylenediamine Nicohe Miscellaneous Trolox Ascorbic acid Histidine Lipoic acid

2.9 ( f 0.3)

Quenching rate constants (1 mol-l s-l X lo-‘) Modified system

0.6 ( f 0.04)

Experimental

3.9 ( + 0.6)

Literature

2.2 a

< 0.8 b

no quenching ’

< 1.4

313.0 ( f 3)

12.0 ( f 1)

515.0 ( f 6.8)

330.0 d loo.0 e

10.2 ( f 0.5)

no quenching ’

17.4 ( f 0.9)

5.9 f

22.3 ( f 2) 20.2 ( f 0.9)
no quenching ’ 4.7 ( f 0.9) no quenching ’ nonlinear fit j

38.0 (It 3.4) 26.4tk3.1) < 1.4 20.9 ( f 12)

47.0 s 0.83 h 10.0 i 13.8 k

The literature rate constants for singlet oxygen quenching are from different references and were determined in various solvents, mainly H,O, or D,O, or solvent compositions containing H,O or DaO. a From Ref. 8, solvent is methanol. b No observable quenching was obtained for quencher concentrations as high as 10m3 M. ’ No interferences of the quencher were observed for quencher concentrations as high as 10e3 M. d From Ref. 31, solvent is HaO. ’ From Ref. 30, solvent is methanol. f From Ref. 33, solvent is acetonitrile-Da0 (4: 1). g From Ref. 1, solvent is ethanol-D,0 (1: 1). h From Ref. 27, solvent is H,O. i From Ref. 25, solvent is D,O. j Roth the measurements for the original system and the modified system could only be fitted with a nonlinear function (clearly indicating catalysed DEDO decomposition). The quenching rate constant is obtained by numerical corrections. IrFrom Ref. 1, solvent is D,O.

212

tamed by subtraction of the Stem-Volmer constant for the modified set-up from the StemVolmer constant for the original set-up and multiplying with the numerical value of the denominator [k,,,[DEE] + ~-t(~0~)] in Eqn. 9. Acetonitrik In this solvent singlet oxygen has a lifetime of about 60 ps [7]. The reaction rate constant for addition of singlet oxygen to DEE is assumed to be fixed at 4.7 x lo7 1 mol-’ s-i [151 and the reactor concentration of DEE was 2.73 X 10m3 mol 1-r. Given these values the denominator [k,,,[DEE] + r-1(1O2)] of the theoretical Stem-Vohner constant is 1.3 x 10’ s-l, which is dominated by the term for addition of singlet oxygen to DEE. It is seen from Table 1 that the experimental rate constants for sulfides show more or less the same trend as was previously outlined in literature [29], wherein decreasing singlet oxygen quenching efficiencies for dialkyl sulfides are ascribed to the steric effects of the alkyl substituents. In our measurements the rate constants decrease on going from dimethyl sulfide to ditert. -butyl sulfide, underlining this interpretation. For diethyl sulfide the absolute value of the rate constant is in good agreement with the literature value. For the two less efficient quenchers, di-isopropyl sulfide and di-tert. -butyl sulfide, hardly any quenching was observed, leading to large standard deviations in the experimental rate constants. The only sulfur compound which does not belong to this series, lipoic acid, resulted in a singlet oxygen quenching rate constant that is almost identical to the value obtained in the literature [l]. Larger deviations from literature rate constants were obtained for the amines measured. However for these compounds the quenching rate constants are strongly solvent dependent Dl. In solvents were the amine is hydrogen-bonded the rate constant is relatively small. Indeed if acetonitrile-water (70 + 30, v/v> is used as the solvent (vide supra) considerably lower values are found (Table 2). The rate constants obtained in acetonitrile are even higher than those measured in chloroform (literature data, denoted with f and g, in Table l), so that it might be concluded that the

HAG. Niederlinder et al. /AnaL Chim. Acta 290 (1994) 201-214

amine is even less solvated in acetonitrile than in chloroform, Only for the arylamine N,N,N’,N’tetramethyl-p-phenylenediamine is the rate constant obtained relatively close to the literature values observed in hydroxylic solvents [30,31]. TWO phenolic quenchers were measured. It is obvious that for trolox the obtained rate constant differs only a factor of two from the literature value, where the solvent was a mixture of D,O and ethanol. 2,6-Di-tert. -butyl-4-methylphenol did not give a measurable effect in our system, which is in line with the literature. Acetonitde-water (70 + 30, u/u). In this solvent composition singlet oxygen has a lifetime of about 24 ks, as calculated above. Hence, the denominator of the theoretical Stem-Vohner constant is equal to 1.7 X lo5 s-t, which is again dominated by the term for addition of singlet oxygen to DEE. The associated Stem-Volmer constants and quenching rate constants are presented in Table 2. It is obvious that most of the presently determined rate constants are in reasonable agreement with the literature data, which were all measured in hydroxylic, or even aqueous solvents, which permits a more direct comparison than in Table 1. For the amines the quenching rate constants have decreased considerably with respect to those obtained in pure acetonitrile; in fact they are in close proximity to those obtained in water or methanol. For 1,2,2,6,6+entamethylpiperidine the reaction rate constant is now too small to be measured with our method. For this compound no literature quenching rate constant in a hydroxylic solvents is available. However, taking into account that quenching rate constants for amines decrease with a factor of about 4.5 on going from chloroform to methanol [8], a quenching rate constant of about 2 X lo7 1 mol-’ s-l would be expected in methanol, starting from the literature value for the 4-hydroxy compound in chloroform (9.2 x lo7 1 mol-’ s-r) [8,32]. This value is close to the limiting rate constant that can still be determined with our system, in agreement with our measurements for 1,2,2,6,6_pentamethylpiperidine. The quenching rate constant for histidine is also beyond detection with our method. At first

HAG. Nie&rhder

et al. /AnaL Chim. Acta 290 (1994) 201-214

sight, this is in disagreement with the literature value presented 1251(Table 2). It should be realized however that the literature value refers to D,O at a pD of 8.4 while our system was buffered at a pH of 6.6. In the same article [25] it is shown that the quenching rate constant of histidine is strongly pD dependent, falling to a value below 2 x 10’ 1 mol-’ s-l at a pD lower than about 6.5, which brings the rate constant in close agreement with our result. The only compound that reveals a large difference between the rate constant obtained with our system and the literature value [27], that cannot be easily explained, is ascorbic acid. The obtained results indicate that the presented method offers a fast and reliable route for determining singlet oxygen quenching rate constants in a wide variety of solvents and solvent compositions. Since a flow system is used the conditions for each measurement are identical and well defined. A large number of quenchers, over a wide concentration range, can be measured in a relatively short time (up-to 15 measurements per hour). Moreover the system can be easily automated. Furthermore the reaction product (DEDO) is monitored on-line, unambiguously and with a simple detection system. The method can therefore be valuable as a standard reference for interpreting pharmacological data. In a current project at the Pharmacology Department of our University [34], singlet oxygen quenching rate constants obtained (with our method) for some 21-amino-steroids 1351showed an interesting correlation with some pharmacological properties of these compounds. We would like to thank Dr. J. de Jong and Prof. Dr. A. Bast (Department of Farmacochemistry, Free University, Amsterdam) and Drs. S.A.B.E. van Acker and Prof. Dr. W.J.F. van der Vijgh (Department of Oncology, Free University Hospital, Amsterdam) for their cooperation and for stimulating discussions. REFERENCES 1 P. di Mascio, M.E. Murphy and H. Sies, Am. J. Clin. Nutr., 53 (1991) 194s.

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