Identification of Crude Oils by Synchronous Excitation Spect rof luorimetry Phillip John* and Ian Soutar' Depariment of Chemistry, Heriot- Watt University, Riccarion, Currie, Edinburgh EH 14 4AS, United Kingdom
The factors influencing the technique of synchronous excitation fluorimetry have been elucidated. Those factors germane to the identification of crude oils-namely, solvent, wavelength increment, concentration, temperature, and frequency bandpass-have been evaluated. The enhanced details in synchronous excitation fluorescence spectra allow firm conclusions to be drawn concerning the identity of an unweathered crude oil sample. Taken In conjunction with the advantages inherent in conventional fluorimetry, this technique shows great promise as a diagnostic tool for the Identification of oil spillages in the marine environment.
ples of Arabian or African origin (Gamba, Hassi Messaoud, Zakum, Brega, Kuwait, and Qatar) and two samples from the North Sea oilfields (Forties and Ecofisk) were examined. Cyclohexane (BDH, fluorescence grade) was used without further purification and was subject to blank experiments to ensure its fluorometric purity. Technique. Fluorescence spectra were obtained on a PerkinElmer MPF-3L instrument. Fluorescence emission was observed at right angles to the incident radiation which impinged upon Samples maintained a t 25 O C in quartz cells of 1-cm path length. Thermostating of the cells was achieved a t higher temperatures by circulation of preheated water from a n external bath.
RESULTS The detection and characterization of crude oil contamination in a variety of environments is currently of particular importance. Fluorescence spectrophotometry is becoming increasingly important for the detection of crude oil especially a t low concentration levels. This technique has been used to study the migration of petroleum in oil fields ( 1 ) and for the detection of crude oil in the marine environment (2-5). While conventional fluorescence spectrophotometry has certain advantages over alternative analytical methods, problems arise in defining crude oil contaminants arising from different locations. Variations in the composition of the complex mixture of hydrocarbons comprising crude oil are insufficient to cause marked changes in fluorescence emission spectra. Published spectra ( 5 , 6) of various crude oils confirm this view and thereby emphasize that conventional fluorescence spectrophotometry should be regarded as only an aid to identification and is of limited use as an exact diagnostic tool. Attempts a t fingerprinting oils have been based mainly on the intensity of fluorescence emission, following a judicious choice of a constant excitation wavelength ( 7 ) , since the fluorescence spectra exhibit minor variations in the band contours. Allied to conventional fluorimetry, the technique of synchronous excitation has been developed by Lloyd (8-10) to identify a number of polynuclear aromatic hydrocarbons and refined petroleum products. In conventional fluorimetry, the fluorescence intensity is recorded as a function of the emission wavelength while the excitation wavelength is held a t a constant value. Excitation fluorimetry involves the reverse situation-namely, variation of the excitation wavelength a t a constant emission wavelength. A third possibility, synchronous excitation fluorimetry, may be envisaged whereby neither wavelength is kept constant with the constraint that the difference between the excitation and emission wavelengths remains constant throughout the spectrum. However, application to crude oil analysis has not yet been forthcoming. In order to rectify this situation, the present study evaluates the synchronous excitation technique as a means of characterizing crude oil samples from a number of different locations.
EXPERIMENTAL Materials. Crude oil samples were kindly donated by the Warren Spring Laboratory, Hertfordshire, United Kingdom. Six sam520
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
The fluorescence emission spectra, a t 25 " C , of three representative crude oil samples are shown in Figure 1 a t a constant excitation wavelength of 260 nm. Previous work ( 7 )has shown that greater distinction between fluorescence emission spectra of refined oil products is possible a t wavelengths less than 290 nm. Despite the use of high energy excitation, the contours in Figure 1 are virtually identical and thus prevent the identification of crude oil samples from differing locations. All the available samples gave similar emission spectra under identical conditions in agreement with previously published data ( 7 ) . The technique of synchronous fluorimetry, described in greater detail by Lloyd (8-10), involves the continuous scan of excitation and emission wavelengths which are separated by a constant increment. The effect on the emission spectra of varying the wavelength difference, AX, a t selected values between 20 nm and 40 nm is illustrated in Figures 2 and 3 for a sample of Ecofisk and Kuwait crude oil, respectively. I t can be observed from these latter figures that the spectral distribution is a function of the wavelength increment and, furthermore, an optimum value for characterization may be chosen for each crude oil. Comparison of the synchronous excitation spectra of each oil sample indicated that a value of AX equal to approximately 20 to 25 nm is suitable for the identification of crude oil samples. A distinct emission profile of each oil sample, using the method of synchronous excitation fluorimetry, is illustrated in Figure 4 in which AX = 20 nm has been employed as an optimum value. A significant feature of the present technique is the sensitivity of the spectral line shapes to the excitation and emission band widths. Two distinct effects are produced on increasing the band widths. First, the fine structure of the band contours is reduced as the resolution is decreased. Second, the relative intensities of the main peaks are less dependent upon the wavelength increment. Furthermore, the distinctive features are still apparent even at large values of the increment. Spectral changes on altering both the band widths from 2 to 5 nm at two values of AX are shown in Figure 5. Fingerprint synchronous excitation spectra of three oil samples, Forties, Kuwait, and Gamba, a t AA = 20 nm and slit widths held a t 5 nm are collated in Figure 6. Compari-
'I
WALCLLNGTH
/
!nm WLVELEFIGTH
Figure 1. Fluorescence emission spectra of A, Ecofisk; B, Zakum; C, Qatar crude oil in cyclohexane solution at 25 OC Excitation wavelength, 260 nm. Concentration of each solution, 100 gI I.-'. Emission and excitation slit widths set at 2 nm. (Abscissa records emission wavelength)
son with the data presented in Figure 4,which employed 2-nm slit widths, indicates that for ease of recognition and subsequent qualitative identification, via a file of reference spectra, larger slit widths are preferable. The spectral profiles of all the samples studied for synchronously excited emission was independent of temperature in the range 20 to 49 "C and, hence, temperature is not a critical factor in deciding the experimental conditions. Comparison of a standard fluorescence spectrum in cyclohexane solution with that of an undiluted sample of crude is illustrated in Figure 7a. A thin film of the undiluted sample was held between two quartz plates and the spectra were measured by frontal illumination. Synchronous excitation spectra, under the same conditions, are shown in Figure 7b and the effect of varying the concentration, over the range lo4 to 10-l 111 l.-I, on the profiles of synchronous spectra are shown in Figure 8. Negligible change in the spectra occurs a t concentrations less than 10-1 ul1.-1.
DISCUSSION The fluorescence emission intensity of a mixture of components is a function of both the excitation and emission wavelengths. However, in conventional fluorimetry, it is normal practice to select a fixed excitation wavelength within the absorption band while scanning the emission frequencies. The efficacy of characterizing a complex mixture by standard fluorimetry using this latter method is reduced on two accounts. First, superimposition of a series of overlapping bands causes a lack of detail in the resulting spectrum particularly if these bands are broad and have closely adjacent maxima. Figure 1 suffices to illustrate this effect. Second, the chosen excitation wavelength will not correspond with the maximum extinction coefficient of each component, thus reducing the contribution to the total fluorescence emission from such species. In addition, migration of electronic excitation energy to species of lower energy in the mixture, causing emission a t longer wavelengths to be enhanced, modifies this latter effect. The con-
/
nm
Flgure 2. Variation of the synchronous excitation fluorescence spectra of Ecofisk crude in cyclohexane solution (100 111 I.-') with wavelength increment, Ax, at 25 C
"
AX = A, 20 nm; 6, 25 nm: C,30 nm; D, 40 nm. Emission and excitation slit widths set at 2 nm. The relative intensities and base lines of individual spectra have been adjusted for clarity
centration dependence of the probability of energy migration has been well documented ( 1 1 ) . Synchronous excitation fluorimetry employs joint variation of both excitation and emission wavelengths. This is achieved by the continuous increase or decrease of the two wavelengths while retaining a constant wavelength differ-
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Figure 3. Variation of the synchronous excitation spectra of Kuwait crude in cyclohexane solution (100 111 I.-') ment, Ax, at 25 "C
with wavelength incre-
AX = A, 20 nm; 6.25 nm; C, 30 nm; D, 40 nm. Emission and excitation slit widths set at 2 nm ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
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b
a
I
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Figure 4, a and b. Synchronous excitation fluorescence spectra of various crude oils in cyclohexane solution (100 PI I.-') at 25 OC Location of source; A, Kuwait; B, Hassi Messaoud; C,Ecofisk, D, Zakum; E, Forties; f, Qatar: G, Gamba; H. Brega. Emission and excitation slit widths set at 2 nm. Wavelength increment, Ai,equal to 20 nm
ence. Thus, the modified technique has the added advantage of both automatically choosing an appropriate excitation wavelength for each component and compressing the frequency range of the resulting spectrum. The consequences of employing this technique are demonstrated in Figures 4 and 6 in which the increase in spectral structure allows delineation of the various crude oils. Spectral differences can now be accounted for by small variations in the relative amounts of aromatic components in each crude. For any component, the maximum fluorescence intensity occurs when the wavelength increment corresponds to that between absorption and emission maxima. Increments which differ from this optimum value will result in a lower contribution from a particular component to the overall
spectrum. Published data (12) of the absorption and fluorescence emission band maxima of the pertinent aromatic molecules indicate that a suitable wavelength increment lies in the range 20 to 25 nm. This is borne out by the data presented in Figures 2 and 3 which demonstrate the spectral changes concomitant with varying the wavelength increment from 20 to 40 nm. Increasing the increment beyond the limits of the absorption and emission envelopes would result in zero contribution from that particular component.
If
,20
Figure 5, a and b. Dependence of the synchronous excitation emission profiles on emission and excitation bandpasses Cyclohexane solution (100 MII,-') of Brega crude at 25 OC. Figure 5a, AA = 20 nm; Figure 5b. AA = 40 nm. Upper curve and lower curves have slit widths set at 5 and 2 nm, respectively
522
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
310
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,
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Figure 6. Synchronous excitation fluorescence spectra of various crudes in cyclohexane solution (100 WII.-') at 25 OC Location of source: A , Forties: B, Kuwait: C, Gamba. Emission and excitation slit widths set at 5 nm. Wavelength increment, 20 nm
a
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900
400
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500
/
.-
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Figure 7a. Comparison of standard fluorescence emission spectrum of neat Ecofisk crude, A, with a spectrum in cyclohexane solution, 8,(100 pl I.-') Neat crude spectrum measured by front surface illumination. Excitation bandwidth 4 nm and emission bandwidth 2 nm for neat crude. Excitation and emission bandwidths for solution both 5 nm
SO"
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Figure 7b. Comparison of synchronous excitation spectrum of neat Ecofisk crude, A , with a cyclohexane solution of the same oil, 8, (100 pi I.-') Excitation and emission bandwidths 5 nm for both neat and solution spectra
Presentation of a range of wavelength differences about the optimum would result in the greatest contribution to the overall fluorescence intensity provided by increments nearer the optimum value. This effect is particularly pronounced since both the amount of absorption and the intensity of fluorescence decrease on altering the increment from the optimum case. Dependence on wavelength increment is thus less marked as the band widths of the emission and excitation wavelength distributions are increased. Figure 5 demonstrates this effect and confirms the latter interpretation. The normal and synchronous excitation spectra both exhibit marked shifts to longer wavelengths as the concentration of the solution is increased. Energy transfer processes become more probable and, hence, emissions from more highly conjugated molecules predominate. At high concentrations, fluorescence from simple aromatics is completely quenched resulting in the observed emission a t longer wavelengths. In addition, self-quenching and self-absorption phenomena distort the spectra a t high concentrations. Figure 8 demonstrates that energy transfer processes, in this system, are reduced a t high dilution and are effectively eliminated a t concentrations less than lo-' p1 1.-'. Despite the constancy of the spectral profile a t low concentrations, it is apparent from Figure 8 that, for practical diagnostic purposes, it is advantageous to observe spectra a t a fixed concentration. In the concentration range 1 to IO3 p1 l.-I, strong emission from both lower and higher molecular weight aromatic components ensures a highly structured spectral profile. Synchronous excitation fluorimetry can also be applied directly to a sample of undiluted crude oil and, as can be ascertained from Figure 7 , greater complexity occurs in synchronous excitation spectra compared to the featureless emission observed using fixed wavelength excitation. However, the characteristics of the synchronous excitation emission spectra of undiluted samples are not sufficiently distinctive to permit their use on a qualitative analytical basis. The effects of self-absorption have been re-
duced to a minimum in Figure 7 for the undiluted sample of Ecofisk crude by the use of frontal illumination. Hence, the absorption maxima occur a t shorter wavelengths than the most concentrated solution in Figure 8. This was confirmed by reduction of the path length of the latter solution. It was observed that the peak maximum of the fixed excitation fluorescence shifted by approximately 40 nm to shorter wavelengths as the cuvette path length was reduced from 10 to 4 mm. The application of derivative techniques to luminescence spectrometry has been demonstrated by Green and O'Hav-
Figure 8. Concentration deDendence of svnchronous excitation spectra of Ecofisk crude oil in' cyclohexane sdlution at 25 O C A , lo-' WII,-': 6, 10 ki I,-'; C, l o 2 MII.-': D, l o 3 1 I,-': E, lo4 1 I.-' ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
523
er (13).We suggest that there is obvious potential in combining synchronous and derivative fluorimetry to enhance minor spectral features and allow a surer identification of oil fingerprint spectra.
ACKNOWLEDGMENT The authors thank Joyce Brown for technical assistance and the Head of the Oil Pollution Division, Warren Spring Laboratory, Hertfordshire for the supply of the crude oil samples.
LITERATURE CITED (1) R. E. Riecker, Bull. Am. Assoc., Petrol. Geolog., 46, 60 (1962). (2) V. Zitko and W. V. Carson, Fish. Res. Board Can. Tech. Rep., No. 217.
(3) E. M. Levy, WaterRes., 5 , 723 (1971). (4) P. D. Keizer and D. C. Gordon, J. Fish. Res. Board Can., 30, 1039 (1973). (5) A . D.Thruston and R. W. Knight, Environ. Sci. Technol., 5 , 64 (1971). (6) M. A. West, lnt. Lab., 41 (1975). (7) J. R. Jadamec, Second Conference on Environmental Quality Sensors. National Environmental Research Center, Las Vegas. Nev.. 1973. (8) J. B. F. Lloyd, J. Forensic Sci. SOC.,2, 83 (1971). (9) J. B. F. Lloyd, J. Forensic Sci. SOC.,2, 153 (1971). (10) J. B. F. Lloyd, J. Forensic Sci. SOC., 2, 235 (1971). (11) J. B. Birks, "Photophysics of Aromatic Molecules", Wiley-Interscience, New York, 1970. (12) I. E. Beriman. "Handbook of Fluorescence Spectra of Aromatic Molecules", Academic Press, New York, 1971. (13) G. L. Green and T. C. O'Haver, Anal. Chem., 46, 2191 (1974).
RECEIVEDfor review September 12, 1975. Accepted November 13,1975.
Fluorescence Measurements of Benzene, Naphthalene, Anthracene, Pyrene, Fluoranthene, and Benzo[ elpyrene in Water Frederick P. Schwarz* and Stanley P. Wasik Institute for Materials Research, National Bureau of Standards, Washington. D.C. 20234
Fluorescence spectra, quantum yields, and concentration dependencies are reported for five representative polycyclic aromatic hydrocarbons (PAH) in water to ascertain the applicability of measuring PAH in aqueous systems by spectrofluorimetry. The fluorescence quantum yields of benzene, naphthalene, anthracene, pyrene, fluoranthene, and benzo[e]pyrene in water are, respectively, 5.3 f 0.5 X lov3, 0.16 f 0.02, 0.25 f 0.02, 0.69 f 0.06, 0.20 f 0.01, and -0.3 f 0.1. With the exception of pyrene, oxygen quenching of the fluorescence in water is at most 30%. The fluorescence concentration dependence was measured by photon counting the fluorescence intensity relative to the excitation light intensity. All the PAH fluorescences exhibited a linear dependency on the concentration. For a fluorescence signal-to-noise ratio of 1, the detection limits are as follows: naphthalene, 0.03 wg/l., anthracene, 0.03 pg/l., pyrene, 0.15 wg/l., fluoranthene, 0.17 pg/I., and benzo[elpyrene, 0.10 pg/I.
Polycyclic aromatic hydrocarbon (PAH) concentrations in fresh water were found by Borneff to range from 0.001 wg/l. in ground water to greater than 0.1 wg/l. in strongly polluted surface water ( I ) . Shabad detected concentrations of benzo[a]pyrene a t the pg/l. level in reservoirs near industrial areas (2). Despite the demonstrated accuracy and simplicity of the measurement of pg/l. pollutant concentrations in air by the fluorescence method ( 3 ) , the application of this technique to the in situ measurement of PAH in water has not been investigated. Naphthalene, anthracene, pyrene, benzo[e]pyrene, and fluoranthene were chosen as five representative PAH ranging in size from 2 to 5 rings. The last three are known to be carcinogenic ( 4 ) .The fluorescence and absorption spectra in water were measured along with their fluorescence concentration dependency. The fluorescence quantum yields in water were determined in order to compare the sensitivi524
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
ty of the fluorescence technique to its sensitivity in hydrocarbon solvents. Similar fluorescence measurements were performed on benzene in water for comparison.
EXPERIMENTAL Most of the fluorescence measurements were performed on t h e apparatus shown in the schematic drawing in Figure 1. T h e fluorescence excitation light was provided by a combination 60-watt deuterium lamp and an f/3.5 monochromator a t a resolution of 3.2 t o 6.4 nm. All the lenses, t h e reflection plate, and sample cell were Suprasil quality quartz. A combination of filters transmitting light from 270 t o 420 n m were used to isolate the naphthalene and benzene fluorescences. A filter transmitting light above 365 nm was used to isolate t h e other PAH fluorescences. T h e two 13-staged photomultipliers, one for measuring the excitation light intensity and the other for fluorescence viewing, possessed S-13 response. T h e fluorescence and excitation light intensities were measured by photon counting over integration times of approximately 30 sec. T h e photon pulses from each photomultiplier were amplified by wide hand pass, low noise amplifiers (51, and transmitted through an updating discriminator into a 20-MHz crystal time based counter-timer. T h e maximum counting rate was -20 000 counts per second. Both the excitation photon pulses from P M 1 in Figure 1 and the fluorescence photon pulses from P M 2 were counted simultaneously by the counter-timer. T h e counter-timer was preset to terminate counting t h e fluorescence photons when the excitation photons attained a preset number of counts. When the counting terminated, the number of fluorescence photon pulses was recorded as t h e fluorescence intensity. Variation of the fluorescence intensity due t o change in the excitation light intensity is thus eliminated. T h e fluorescence-concentration dependence measurements were performed with a modified sample cell. T h e square cell was connected t o t h e base of a 200-ml Pyrex cylinder. T h e sample was diluted over two orders of magnitude by t h e successive addition of known volumes of water. After each dilution, t h e sample cell was shaken t o ensure mixing. The concentration dependence of benzene was not performed since unknown amounts of benzene were lost t o t h e air upon shaking the sample cell. T h e fluorescence quantum yield (ratio of the fluorescence intensity to the absorbed light intensity) was determined by comparing t h e fluorescence photon counts normalized for some pre-set excitation counting rate of the aromatic hydrocarbon in water t o those of
