W31-X655/7X/l IOI-MhSS02.0010 Phorochnrrbrr! und Phorohiologi,. Vol. 28. pp. 465-46% 8 Pcrpamon Press Ltd.. 197X Printed in Great Britain

A PRELIMINARY INVESTIGATION OF THE USE OF LIMONENE FOR DETECTING SINGLET OXYGEN

AS A COMPONENT OF POLLUTED AIR

H. RALPH RAWLS* and FRANCE L. EsTEst

Gulf South Research Institute P.O. Box 26518. New Orleans, LA 70186, USA.

(Received I March 1978; accepted 9 March 1978)

Abstract-Exploratory experiments using simulated conditions indicate that the terpene. ( + )-limonene can serve to detect 02( 'Ag) in polluted atmospheres. '0, attack on limonene results in the formation of specific oxidation products in a specific distribution; quantitative identification of these products is a highly specific test for '0,

INTRODUCTION

Singlet oxygen is postulated to be a reactant in pol- luted air (Khan et a/.. 1967; Pitts et a/., 1969; Kummler and Bortner, 1970). It is a metastable, hard- todetect species and is difficult to identify in the open atmosphere (Pitts, 1969). However, a number of studies have provided evidence that '02 is involved in photochemical air pollution (Trozzolo, 1970; Schaap, 1976). In an attempt to bring additional evi- dence to light and mainly to demonstrate a potential method for detecting small quantities of '02 in the open atmosphere, we have utilized the reaction between ( + )-limonene and 'OZ,

When attacked by lo2, limonene forms six different isomeric hydroperoxides which can immediately be converted to the corresponding alcohols by in situ reduction (Gollnick and Schneck, 1964; Foote el al., 1965; Wexler, 1966: Gollnick and Schade, 1973). Although four of these alcohols are also formed under free-radical oxidation conditions, two of them are uniquely the result of '02 attack. Moreover, the product distribution among the several alcohols for '0, oxidation is entirely different from that obtained with free-radical oxidation such as might occur with ground-state molecular oxygen. In addition, '02 oxi- dation produces optically active products, while the same products are racemic when formed by free-radi- cal oxidation of (+)-limonene (Foote et a/., 1965; Wexler, 1966). Thus, the quantitative identification of the optically active limonene oxidation products formed as a result of exposure to a given polluted atmosphere should determine the presence of '02. The identification of the other oxidation products could be used to determine the presence of any species capable of initiating free-radical oxidations. Such observations would help to explain the diversity of biological and chemical responses which are observed to take place in polluted air.

Present addresses: *LSU School of Dentistry, Department of Biomaterials, 1100 Florida Avenue, New Orleans, Loui- siana 70119, USA. tBox 33, Mendon, Michigan 49072

465 P A P . 2 8 A / % D

MATERIALS AND METHODS

Among the probable sources of '02 are the direct exci- tation of ground state 302. photolysis or O3 (especially in the presence of NO2). energy transfer from light-absorb- ing pollutants (including olefin-02 charge-transfer com- plexes), and decomposition of peroxyacetylnitrate (Pitts. 1969). The first three of these potential sources were simu- lated in our laboratories by exposing various gaseous mix- tures to a Hanovia 450-W, medium pressure. Hg lamp enclosed in a Pyrex absorption sleeve (300nm cut-off). Mixtures of I-butene (representative of the olefins found in polluted air), NO, and air were used to simulate smog- forming polluted air. Reactant concentrations somewhat higher than those in typical polluted air were used in order to obtain observable reaction products in reasonable time periods. GLC retention time was used to identify reaction products. The product work-up and GLC identification were based on that described by Footc er ul. (1965) and Wexler (1966).

Limonene samples were exposed to air alone from a gas cylinder, to irradiated air, to irradiated air containing 0.86 ppm NO, and to various irradiated butene/NO,/air mixtures. In addition to simulated photochemical-air-pol- lution mixtures, limonene samples were also exposed to a chemical source of '0, ( H 2 0 2 + NaOCI) and to air which had not been irradiated.

Limonene was purified by preparative gas chroma- tography. The purified material was free of carvone and gave a single major chromatographic peak. A few percent of impurities remained but none interfered with the oxi- dation products of interest. The treated samples were reduced with NaBH,, extracted with pentane, and then concentrated, according to procedures described by Wexler (1966). Likewise, the chemical oxidations with NaOCl and H 2 0 2 were carried out as described by Wexler (1966) except that no free-radical scavenger was used.

In the photochemical experiments 2.8 m/ of limonene were dissolved in a 1 : I mixture of butanol and methanol and placed in an ice bath. This served as a trap for '0,. The inlet tube to this trap was connected to the gas irradia- tion chamber through a light trap. The desired concen- trations of NO, and/or butene in air were obtained by serial dilution using a device which has been described by Estes and Frenczy (1968). After exposure to the gas stream, the limonene solution was reduced, extracted. dried and concentrated by evaporation.

The limonene products were analyzed on a Barber-Cole- man Gas Chromatograph with a hydrogen flame detector. A 10 ft. 1/4 in (3.2 m x 0.63 an) packed column containing 15% diethylene glycol succinate on 80-100 mesh Gas-' Chrom P at 150°C with a carrier gas flow of 65m//min

466 H. RALPH RAWLS and FRANCES L. ESTES

Table 1. Distribution of oxidation products (?A in mixture)

Alcohol No NaOCl Air Air Air Olefin-N02 photo-oxidation produced treatment + only + + I (Butene/NO, ratio)

from H202 hv hv limonene + 300 100 10 5 oxidation NO2

I

I I

1v

V

111

VI

Products produced by both free radical and singlet 0, oxidation - 10 69 - 31 - 61 57 100

31 -

43 - - 13 16 trace

Singlet-0,-specific oxidation products

57 16 33 26 27 - - 31 -

- - 37 25 trace trace - -

Experimental Exposure (hr) 6 12 6 6 6 6 6 conditions Flow (mlimin) 120 120 120 60 120 I20 I20

NO2 (ppm) 0.86 7.3 0.5 5 6.6 Butene (ppm) 2000 50 50 33

- = No GLC peak observed. Trace = Insufficient amounts observed for quantification.

was used. Peak areas were measured by an attached Info- tronics Co. chromatography readout system. The retention times of the products of the NaOCl + H202 'reaction cor- related well with those reported by Wexler (1966). Both limonene and carvone served as internal references to establish the correct product identities.

RESULTS AND DISCUSSION

Table 1 summarizes the results. These results show that GLC peaks attributable to '02-characteristic alcohols are only observed under conditions which are capable of producing singlet oxygen. The control "air only" experiment in which there was no irradia- tion, produced neither of the '02 -characteristic alco- hols.

The oxidation of lirnonene with the hypochlorite/ H z 0 2 solution is expected to produce all six alcohols (Foote et al., 1965; Wexler 1966). However, free-radi- cals are formed by the rapid breakdown of the initial-

ly-formed hydroperoxides and these promote autoxi- dation of both libonene and the initial oxidation products. The addition of 2,6-di(t-butyl) phenol as a free radical scavenger was found to obscure the chro- matographic analysis. The lack of such inhibition is probably the major cause for the non-appearance of some of the expected products. However, the appear- ance of alcohol 111 served to establish that the reten- tion time of this '0,-specific product was the same as that observed by Wexler (1966).

When irradiated just prior to entering the reaction flask, air alone was found to produce a singlet oxy- gen-specific product (alcohol 111). This is rather sur- prising in light of the extremely low transition prob- ability for direct, excitation to the singlet state (Kearns, 1971). Singlet-oxygen-producing photosensit- izing contaminants together with light leaks, may account for this unexpected formation of 111. The par-

Singlet O2 in polluted air 467

ticularly long exposure time used in this experiment (12 h vs 6 h in the other experiments) makes it es- pecially vulnerable to light-leak problems. No products were observed in this experiment after only 6 h irradiation.

The inclusion of NOz is expected to greatly in- crease the chance of singlet-oxygen formation via NOz photolysis to form singlet-oxygen-producing intermediates (Pitts, 1969). The results shown under "air + hv + NO; in Table 1 bear this out. Both sing- let oxygen-specific alcohols are observed and the product distribution is similar to that reported for '02 and photooxidations (Foote et a/., 1965; Schenck et a/ . , 1964).

Olefinic compounds, of which 1-butene is represen- tative, are also expected to enter into the photo- chemical reactions in polluted air and to influence production of loz (Khan et a/., 1967; Pitts et al., 1969). The results reflect this in that the distribution of alcohols is altered when varying amounts of I-butene are included in the gas mixture at various ratios of 1-butene to NO2 (Table I ) . Changing butene from 2000 to 50 to 33ppm with little change in NO2 concentration results in a reduction in 'OZ-specific product formation from 58 to 27:/, to zero. On the other hand, when butene is kept at 50 ppm no change in product distribution is observed when NOz con- centration is changed ten-fold. The ratio of olefin to NO2 is the important factor. NOz at 0.86ppm in air produces both products 111 and IV when irradiated. They also appear at a ratio of 300:l. As the ratio is reduced first VI and then 111 drops out.

When butene and NO2 are irradiated in air. NO, atomic oxygen, ozone and '02 are produced. NO2 is regenerated and aldehydes and other organic reac- tion products are also produced (Pitts, 1969). A very complex set of reactions take place, some of which

produce lo2, and some of which consume it. NOz is generally an oxidant, but can also serve as a radical scavanger. The interaction between butene and NOz vary with their relative concentrations. Because of these complexities, we prefer not to speculate further concerning the implications that the present results have for reaction mechanisms is polluted atmos- pheres. The point was to determine if 'Oz forms in polluted atmosphere-like circumstances and i f so, under what conditions. From these experiments it would appear that '02 does form and is strongly dependent upon the relative concentrations of such pollutants as olefins and NOz.

The technique of using limonene as a chemical trap which produces '02 specific products, has been demonstrated. The present identifications are depen- dent upon GLC retention times and must be verified by independent means. However, if appropriately refined, it should be possible to establish this tech- nique as a method for identifying '0, in trace steady- state concentrations in actual polluted environments.

CONCLUSIONS

Although these experiments are incomplete, the observations are encouraging and support the conten- tion that singlet oxygen may be an important air pol- lution component. Further, the potential usefulness of (+)-limonene for identifying the presence of singlet oxygen in low concentrations in the gas phase and in complex mixtures has been demonstrated.

Acknowledgement-This work was supported through an NIH general research support grant (5-FOI-RR-05672) and an NIH Research grant (AP-1153). John L. Guagliardo performed most of the experimental procedures at Gulf South Research Institute in Baton Rouge. LA 70808.

REFERENCES

Estes, F. L. and K. 0. M. Frenczy (1968) Foote. C. S.. S. Wexler and W. Andro (1965) Gollnick K. and G. 0. Schenck Gollnick. K. and G. Schade (1973) Tetrahedron Lett . 857-860. Kearns. D. R. (1971) Chem. Revs. 71 (4) 39S427. Khan, A. U., J. N. Pitts, Jr. and E. 9. Smith (1967) Kummler. R. H. and M. H. Bortner (1970) J . Geophys. Res. 75, 3115-3122. Pitts, J. N.. Jr. (1969) Pitts, J. N., Jr., A. U. Khan, E. 9. Smith and R. P. Wayne (1969) Schaap. A. P.. (Ed). (1976) Singlet Molecular Oxygen. Wiley, Stroudsberg, PA. Schenck, G . 0.. K. Gollnick, G. Buchwald, S. Schroeter and G. Ohloff (1964)

Trozzolo. A. M. (Ed) (1970) Wexler, S. (1966) Dissertation. University of Calif., Los Angeles.

ACS preprint, Dic. of Water, Air. and Waste 8, 86. Tetrahedron Lett . 41 11-4118.

(1964) Pure Appl. Chem. 9, 507-525.

Enciron. Sci. Techno/. 1, 656657.

Adc. Enuiron. Sci. Techno/. I, 309-337. Enuiron. Sci. Techno/. 3, 241-247.

Ann. Chem. 674, 93-117.

Ann. N .Y . Acud. Sci. 171, 1-302.

DISCUSSION 3. Were individual products stable in the system such that allylic rearrangements did not occur '?

H. R. RAW= 1. No. Although it would be interesting to explore the

contribution of NO, alone, because of the nature of NO2 it would require an extended series of experiments. I t is, doubtful if any single NO2 experiment could be defined as a control.

Chairpersons: R. SANTUS and E. BEN-HUR

L. L. SMITH

that is, without O2 or butene?

hydride reduction of the reaction products?

1. Was a control reaction with NO2 (irradiated) run-

2. Were hydroperoxides actually formed prior to boro-