Total Luminescence of Coumarin Derivatives Isolated from Expressed Lime Oil Howard W. Latz and Brooks C. Madsen Department of Chemistry, Ohio Uriioersity, Athens, Ohio 45701
The crystalline components responsible for the room temperature fluorescence, low temperature phosphorescence, and low temperature fluorescence of expressed lime oil have been isolated and identified. Two of the eleven components have not previously been isolated from expressed lime oil. Luminescence characteristics for the individual components are compared with those for samples of expressed lime oil of different geographical origin. The coumarin and psoralen derivatives responsible for the various emission characteristics of the whole oil are identified.
AN EXTENSIVE total luminescence (fluorescence, phosphorescence, and low temperature luminescence) study of the substituted coumarin derivatives which occur naturally in expressed lime oil is presented. These crystalline components were separated chromatographically, purified, and examined individually after their identity had been confirmed by R, values, melting points, mass spectra, and ultraviolet absorption spectral data. Nine previously reported components and two additional components, one tentatively identified, were isolated. These coumarin derivatives are responsible for the fluorescence and phosphorescence exhibited by ethanol solutions of expressed lime oils. Similar coumarin derivatives probably are responsible for the fluorescence of other expressed citrus oils. Luminescence spectrophotometry can enjoy certain advantages over absorption methods of analysis. Luminescence techniques are generally much more sensitive than absorption methods and sometimes are more specific for certain compounds. Utilization of appropriate excitation and emission wavelengths can resolve a mixture of two or three luminescent species in the presence of other nonluminescent species. Disadvantages of luminescence spectroscopy include interferences which arise from impure solvents and the limitation of using only solvents which form clear uncracked glasses at liquid nitrogen temperature for the investigation of phosphorescence and low temperature fluorescence. The application of luminescence measurements to the study of complex mixtures in combination with thin-layer chromatography or gas chromatography has demonstrated the usefulness of luminescence spectroscopy as a qualitative and quantitative tool. Drushel and Sommers ( J ) employed fluorimetry and phosphorimetry in combination with gas chromatography for analysis of petroleum fractions. Sawicki, Stanley, and Johnson (2) employed fluorimetry and thin-layer chromatography using direct measurement of the emission from the thin-layer plate and solution fluorescence to study aromatic compounds of interest in air pollution studies. Hood and Winefordner (3) measured the low temperature fluorescence and phosphorescence for a series of (1) H. V. Drushel and A. L. Sommers, ANAL.CHEM., 38, 10 (1966). (2) E. Sawicki, T. W. Stanley, and H. Johnson, Microchemical J., 8, 257 (1964). (3) L. V. S. Hood and J. D. Winefoldner, Anal. Chim. Acta, 42, 199 (1968). 1180
ANALYTICAL CHEMISTRY
carcinogens using thin-layer chromatography to separate the compounds. The fluorescence of certain compounds present in expressed citrus oils has been utilized to locate these compounds on thin-layer chromatograms (4-7) which subsequently are examined spectrophotometrically (8). Vannier and Stanley (9) developed a method which employs fluorimetry in the analysis of mixtures of grapefruit oil in lemon oil. Fisher and Nordby (6, 10, I J ) reported partial fluorescence data for substituted coumarin derivatives isolated from Florida grapefruit peel oil. D’Amore and Corigliano (12) used fluorimetry for the characterization of expressed mandarin, lemon, and bergamot oils. No information describing phosphorescence or low-temperature luminescence properties of citrus oils has been reported in the literature nor have extensive investigations been conducted to study the fluorescence and phosphorescence characteristics of the individual naturally-occurring components present in citrus oils. This paper demonstrates an application which can be made of luminescence spectroscopy in the study of complex natural product mixtures. EXPERIMENTAL Apparatus. An Aminco Bowman spectrophotofluorometer (SPF) with a high pressure Xenon arc source lamp, RCA 1P21 phototube, and X-Y recorder (American Instrument Co., Silver Spring, Md.) was used to obtain all luminescence spectra and analytical data. An Aminco-Keirs phosphorescence attachment was used with the above instrument when phosphorescence was measured. Phosphorescence decay curves were traced on the X-Y recorder by manually blocking the excitation radiation with a blank shutter placed in the excitation monochromator slit holder while simultaneously initiating the recorder sweep at two inches per second. LOW temperature luminescence (low temperature fluorescence and phosphorescence) was measured by removing the phosphoroscope from the light path. Fluorescence data were obtained in 1,000-cm quartz cells; phosphorescence and low-temperature fluorescence data were obtained in quartz tubes (approximately 1.5 mm i.d.) at liquid nitrogen temperature. Relative luminescence intensities were obtained from the photomultiplier microphotometer supplied with the SPF by multiplying the meter multiplier setting times the scale reading in per cent transmittance. ___
(4) W. L. Stanley and S. H. Vannier, J. Amer. Chem. SOC.,79, 3488 (1957). ( 5 ) W. L. Stanley and S. H. Vannier, Phyrochem., 6, 585 (1967). (6) J. F. Fisher and H. E. Nordby, J . FoodSci., 30, 869 (1965). (7) W. L. Stanley, A. C. Weiss, R. E. Lundin, and S. H. Vannier, Tetrahedron, 21, 89 (1965). (8) W. L. Stanley and S. H. Vannier, J. Ass. Ofic. Agr. Chemists, 40, 582 (1957). (9) S. H. Vannier and W. L. Stanley, ibid., 41, 432 (1958). (10) J. F. Fisher and H. E. Nordby, Tetrahedron, 22, 1489 (1966). (11) J. F. Fisher, H. E. Nordby, A. C. Weiss, Jr., and W. L. Stanley, ibid., 23, 2523 (1967). (12) G. D’Amore and F. Corigliano, A m . Fac. Econ. Commer., Unio. Studi Messim, 4 (2), 413 (1966); Chem. Abstr., 68, 16063N, (1968).
A
*c VJ
z w
c I w
2 !-
U -I w K
400
500
400
500
600
WAVELENGTH, nm
Figure 1. Fluorescence (-), phosphorescence (- -), and low temperature luminescence (- -) excitation and emission spectra for expressed lime oil. Emission spectra appear at longer wavelength than excitation spectra. Correlation of relative intensities from spectrum to spectrum is not intended. Quantitative evaluation of emission intensities and specific excitation and emission wavelength maxima are presented in Table I1
-
A Cary Model 14 recording spectrophotometer with 1.000cm quartz cells was used to obtain all absorption spectra. A Hitachi Perkin-Elmer RMU-6E mass spectrometer with source operated at 70 eV was used for mass spectra measurements. Developed thin-layer chromatograms were examined under 254 nm and 365 nm ultraviolet light with a Mineralight (Ultra-violet Products, Inc., San Gabriel, Calif.). E. Merck precoated analytical layer chromatoplates, silica gel F-254 (Brinkmann Instruments Inc., Westbury, N. Y . ) were cut to 3 X 10 cm size for use in the examination of fractions isolated by column chromatography. A Brinkmann sandwich developing chamber for TLC was used to develop 20 cm2 plates. Melting points were determined on a Buchi melting point apparatus and are uncorrected. Materials. All absorption and luminescence spectrophotometry studies were carried out in absolute ethanol purified according to the method of Winefordner and Tin (13). Luminescence Characteristics. Fluorescence, phosphorescence, and low-temperature luminescence spectral characteristics were measured for several samples of expressed lime oil, for the crystalline components isolated from expressed lime oil and for samples of expressed lemon, grapefruit, and orange oils. Analytical curves for fluorescence and phosphorescence were obtained from solutions prepared by successive dilution of an ethanol stock solution of each component and lime oil sample. Fluorescence excitation and emission spectra and analytical curves for the various lime oil samples and for coumarin derivatives I1 and IX (Table I) were measured with both slit program 3-2-3-3-2-3-2 (slit arrangement 3) and slit program 5-4-5-5-4-5-5 (slit arrangement 5). For the remaining coumarin derivatives slit arrangement 5 was used. Phosphorescence excitation and emission spectra were obtained with slit program 3-1-1-3-2. Analytical curves for phosphorescence were obtained with slit program 4-4-4-4-4. The small slit numbers give the highest resolution. Phosphorescence decay curves were obtained from solutions containing 1.0 pg/ml of the individual coumarin derivatives and 10 pg/ml of the expressed lime oils. Low-temperature luminescence ~
(13) J. D. Winefordner and M. Tin, Anal. Chin?. Acta, 31, 239 (1964).
Figure 2. Emission spectra for ( A ) expressed grapefruit oil, fluorescence spectrum (-) with excitation at 332 nm and phosphorescence spectrum (--) with excitation at 320 nm and ( B ) expressed orange oil, fluorescence spectra (-) with excitation at 335 and 354 nm for samples 1 and 2, respectively, and phosphorescence spectrum (- -) for both samples with excitation at 338 nm. Correlation of relative intensities from spectrum to spectrum is not intended excitation and emission spectra and analytical curves were obtained with slit program 3-1-1-3-2. Instrument Calibration. The sensitivity of the SPF for fluorescence was adjusted daily to a predetermined value using a 0.8 pg/ml solution of quinine sulfate in 0.1N sulfuric acid. For phosphorescence and low temperature luminescence the SPF was standardized using the above quinine sulfate solution at room temperature with the instrument modified to measure low temperature luminescence. Separation of Coumarin Derivatives. Chromatographic separation of coumarin derivatives present in expressed lime oil was achieved using a procedure similar to that of Stanley and Vannier ( 5 ) using silicic acid with redistilled ligroine (bp 63-75 "C),ethyl acetate, and ethanol. Table I lists the various coumarin derivatives isolated. R, values reported in Table I were obtained by spotting appropriate amounts of each component 2 cm from the bottom of a 20 cm2 precoated thin-layer plate which was then developed to 17 cm with 1 :3 (v/v) ethyl acetate-hexane. Coumarin derivatives show intense blue fluorescence under 254 nm and 365 nm ultraviolet light. Psoralen derivatives show yellow or brown fluorescence of moderate to low intensity under 365 nm ultraviolet light and therefore are more easily located as dark spots on a yellow fluorescent background under 254 nm ultraviolet light. The coumarin derivatives isolated were purified by recrystallization and thin-layer chromatography. The purity of components used in the luminescence study was established by thin-layer chromatography. RESULTS AND DISCUSSION
General Characteristics of Whole Oils. A preliminary luminescence examination revealed variations in the solution luminescence of various expressed citrus oils. Lime oil (Figure 1) and lemon oil exhibit identical spectral characteristics. Fluorescence emission spectra for grapefruit and orange oils (Figure 2) differ from those obtained for lemon and lime oils while the phosphorescence spectrum for orange oil (Figure 2) is different from the identical phosphorescence spectra obtained for lemon, lime, and grapefruit oils (Figures 1 and 2). Solution fluorescence of each oil is radically different from the fluorescence shown by whole oils. The VOL. 41, NO. 10, AUGUST 1969
1181
differences between whole oil and solution fluorescence can be attributed to the inner filter effect. Successive dilution of the lime oil samples with ethanol results in a gradual shift of excitation and emission spectra obtained from the whole oil until the spectra correspond to those obtained in dilute solution. It is doubtful that the excitation and emission spectra of the whole oil can be used in analysis although these spectra vary for samples of lime oil of different origin. A wide variation in luminescence intensities from ethanol solutions of the four oils is observed. The fluorescence
Fractions combined 50-144a 200-230 251-269 291-340 363-387 426-438 476-489 476-489 490-565 636-794 954-962
Table I. Crystalline Components Isolated from Expressed Lime Oil % Ethyl acetate Compound in eluate 2.5 I 5-geranoxypsoralen I1 5-geranoxy-7-methoxycoumarin 7.5 5-geranoxy-8-methoxypsoralen 10.0 111 10.0 IV 8-geranoxy psoralen 10.0 V 5-isopenteneoxy-8-methoxypsoralenb 15.0 VI 8-i~openteneoxypsoralen~ VI1 15.0 VI11 5-methoxy-8-isopenteneoxy psoralenb 15.0 IX 5,7-dimethoxycoumarin 15.0 X 5,8-dimethoxypsoralen 15.0 XI 5-dihydroxyisopentanoxypsoralen 10.0"
One liter of ligroine and 4.0 liters of 2.5 Tentative identification. Ten per cent ethanol, 90% ligroine.
I I1 111 IV V
VI VI1
VI11 IX X XI
intensity for lime oil is approximately one order of magnitude greater than for lemon and grapefruit oils and two orders of magnitude greater than for orange oil. The variation in phosphorescence emission intensity for the oils is not nearly as great. Lime oil has a phosphorescence intensity approximately one order of magnitude greater than that of lemon and orange oils with the phosphorescence intensity of grapefruit oil of intermediate intensity. Identification of Coumarin Derivatives. Compounds I, 11, 111, IV, IX, X, and XI have been previously identified in
Rf 0.73 0.67 0.63 0.43 0.52 0.34 0.15 0.34 0.42 0.24 0.02
ethyl acetate-ligroine in fractions 1-49.
Table 11. Luminescence Characteristics of Coumarin Derivatives" Room temperature Low temperature luminescence' fluorescinceb-Low temperature phosphorescenced ex (nm) em (nm) R.I.# ex (nm) em (nm) R.1.c ex (nm) em (nm) R.I." 7 (sec) 0.11 314 430 314 474 (474) 0.60 314 474 0.41 474 0.42 508 (508) 0.54 1.8 508 0.35 335 480 (472) 0.28 335 385 3.3 335 420 20.4 472 0.28 510 (508) 0.27 1 .o 508 0.18 318 485 0.11 318 517 318 - (500s) 0.01 508 (518) 0.06 0.8 308 435 0.03 312 460 308 472 (462) 0.34 0.01 466 0.12 498 (494) 0.38 0.8 498 0.12 312 484 0.29 312 502 (502) 0.13 0.8 314 513 0.02 5243 (518s) 0.12 308 435 0.03 308 472 (462) 0.38 0.02 312 460 462 0.14 500 (494) 0.44 0.8 494 0.I5 346 430 2 340 488 (482) 0.15 346 460 4 515 (518) 0.12 1.2 316 470s 0.19 316 500 (496) 0.17 1.o 0.02 317 506 492 0.20 5259 (520s) 0.14 335 380 4.0 335 478 (472) 0.33 1 .o 335 420 28.8 472 0.57 508 (508) 0.31 508 0.35 314 485 0.28 314 505 (500) 0.12 318 514 0.02 5243 (524) 0.12 0.9 316 430 0.10 316 481 (474) 0.98 314 474 0.68 476 0.49 510 (508) 0.90 1.2 510 0.41
Intensities for the three types of emission are not comparable because of the different slit programs and instrument modifications used. Slit Arrangement 5, uncorrected for instrumental response. Relative fluorescence intensity, 0.1 fig/ml. Slit program 4-4-4-4-4, uncorrected for instrumental response; maxima in parentheses were obtained with slit program 3-1-1-3-2 and are preferred for characterization. e Relative phosphorescence intensity, 1 .O pglml. f Slit program 3-1-1-3-2, uncorrected for instrumental response. Relative low temperature fluorescence and phosphorescence intensity, concentration 1.0 pg/ml. 0
1182
ANALYTICAL CHEMISTRY
217
50
IO0
150
232
200
i
250
m/e
Figure 3. Partial mass spectrum of VI1 showing major peaks expressed lime oil (5). Verification of the presence of these compounds was established by comparison of R, values, melting points, ultraviolet absorption spectra, and mass spectra. The mass spectra of 11, 111, IV, and XI have not been reported. However, the fragmentation patterns for these compounds parallel those for other coumarin derivatives (6, 14). Compounds V, VI, VII, and VI11 could not conclusively be identified from the small amount of material isolated. However, the ultraviolet absorption spectra and the luminescence spectral characteristics found for all except VI1 are specific enough to identify V and VI11 as 5,8-dialkoxypsoralens and VI as an 8-alkoxypsoralen, Compounds V, VI, and VI11 have been tentatively identified as indicated in Table I. Compound VI1 was not identified. A melting point of 151-152.5 OC, mass spectra (Figure 3), ultraviolet absorption maxima at 255 (shoulder), 263 and 324 nm, and R, value (Table I) cannot be correlated to establish identity of this compound as being any of the coumarin derivatives previously reported in expressed citrus oils (4-7,1 0 , I I ) . The mass spectra with prominent mJe ions at 232 and 217 may correspond to a 5,8-alkoxy-methoxypsoralen.The ultraviolet absorption spectrum does not correspond to a 5-alkoxy, 8-alkoxy, or 5,8dialkoxypsoralen. The fluorescence excitation and emission maxima are very similar to those reported for 5-[(3,6-dimethyl6-formyl-2-heptenyl)oxy]-psoralen (10). It riust be concluded that this component has not been found by previous investigators. Initial investigation of the fluorescence characteristics for 111, V, VI, and VI11 revealed fluorescence emission which resembled that of I1 and IX although the ultraviolet absorption spectra for these compounds did not indicate the presence of impurities. It can be concluded that for compound I11 a very small amount of I1 was isolated with I11 and that VI11 was isolated with a trace of IX present. This emphasizes the extreme sensitivity of fluorescence measurements in particular situations where other methods are not as selective or sensitive for analysis. Compounds I11 and VI11 are very weakly fluorescent while I1 and IX exhibit intense fluorescence. Luminescence Characteristics. The luminescence characteristics measured for the coumarin derivatives are summarized in Table I1 and Figure 4. The trends which occur in the room-temperature fluorescence emission spectra (Figure 4 4 ~
(14) C. S. Brown and J. L. Occolowitz, Aust. J. Cliem., 17, 975 ( 1964).
C 4
300
400
600 300 WAVELENGTH, nm
500
400
600
SO0
Figure 4. Luminescence excitation and emission spectra of coumarin derivatives from expressed lime oil A . Fluorescence excitation and emission spectra B. Phosphorescence excitation and emission spectra C . Low-temperature luminescence excitation and emission spectra I . 5-Alkoxypsoralens 2. 8-Alkoxypsoralens 3. 5,7-Dialkoxycoumarins 4. 5,8-Dialkoxypsoralens 5. Component VI1 Emission spectra appear at longer wavelength than excitation spectra. Wavelength maxima for excitation and emission spectra are presented in Table 11. Correlation of relative intensities from spectrum to spectrum is not intended. Quantitative evaluation of emission intensities is presented in Table I1 for the similarly substituted coumarin derivatives are analagous to the variations previously observed in the ultraviolet absorption spectra (5) for these compounds. The large variation in emission intensity observed for the coumarin derivatives is in striking contrast to the similar molar absorptivities (5)shown by these compounds. The variations in phosphorescence spectra (Figure 4B) are not as pronounced as the variations in fluorescence spectra. The large variation in emission intensity observed for fluorescence is not present for phosphorescence. In addition a phosphorescence emission at approximately 435 nm with excitation maximum at 275 nm is observed for the eleven components isolated from lime oil and for all samples of expressed lemon, lime, grapefruit, and orange oils. This phosphorescence emission is present only at very dilute concentrations. Nonexponential phosphorescence decay is observed for this emission. Typical low-temperature luminescence spectra are shown in Figure 4C. Seven components 11, 111, V, VII, VIII, IX, and X exhibit low-temperature fluorescence which is more intense than phosphorescence. Only low-temperature fluorescence is observed for 111, V, VIII, and X. The fluorescence intensity is much greater than the phosphorescence intensity. Therefore the phosphorescence emission spectrum cannot be observed superimposed on the low-temperature fluorescence VOL. 41,NO. 10, AUGUST 1969
0
1183
Table 111. Luminescence Characteristics of Expressed Lime Oil"
Florida Extra
Room temperature fluorescence* ex (nm) em (nm) R.Lc
ex (nm)
420
318
335
Mexican 335 Extra
420
Persian
420
335
West Indian 335 Select
420
335 Origin Unknown
420
2.0 1 1 .o
7.7 14.1 7.3
318
314 318
318
Low temperature phosphorescenced em (nm) R.I.E r (sec)
Low temperature luminescencey ex (nm) em (nm) R.I.0
475 (474) 508 (508)
0.14 0.13
1.2
335 318
480 (475) 508 (510)
0.32 0.30
1.3
335 318
480 (476) 509 (510)
0.34 0.32
1.2
480 (472) 508 (508)
0.42 0.39
1.2
335 320
480 (475) 508 (510)
0.24 0.23
1.1
335 318
380 478 510 380 475 510
385 472 510 385 475 510
0.2 0.06 0.05
0.6 0.14
0.10
1.5 0.24 0.18 0.5 0.10
0.07
Intensities for the three types of emission are not comparable because of the different slit programs and instrument modifications used. Slit Arrangement 5 , uncorrected for instrumental response. Relative fluorescence intensity, concentration 1 .O pg/ml. d Slit program 4-4-4-4-4, uncorrected for instrumental response, maxima in parentheses were obtained with slit program 3-1-1-3-2 and are preferred for characterization. e Relative phosphorescence intensity, concentration 10.0 Fg/ml. Slit program 3-1-1-3-2, uncorrected for instrumental response. Relative low temperature fluorescence and phosphorescence intensity, concentration 10.0 pg/ml. 5
f
spectrum. In each case the low temperature fluorescence shows a blue shift compared to the room temperature emission. All luminescence spectra are characteristic for the type of substituted coumarin derivative responsible for the emissionLe., 5,7-dialkoxycoumarin, 5-alkoxypsoralen, 8-alkoxypsoralen, or 5,8-dialkoxypsoralen. Although the roomtemperature and low-temperature fluorescence emission spectral characteristics of VI1 are similar to those for 8alkoxypsoralens, the fluorescence emission intensity is much higher than that observed for other 8-alkoxypsoralens. The phosphorescence and low-temperature luminescence spectra and excitation spectra of VI1 do not correspond with those of the previously mentioned coumarin derivatives. Fluorescence analytical curves are linear from 0.0001 to 0.1 pg/ml for I1 and IX and 0,001 to 0.1 pg/ml for I and XI. Emission intensity from 111, IV, V, VI, VIII, and X is so low that fluorescence is not useful for determination of these compounds. Analytical curves for VI1 were not determined because of its unknown nature. Phosphorescence analytical curves are linear from 0.01 to 1.0 pg/ml for the compounds investigated (phosphorescence and low-temperature luminescence analytical curves were not obtained for 111, VII, and VIII). Low-temperature luminescence analytical curves obtained by measuring the long wavelength emission (low temperature fluorescence for V and X and phosphorescence for I, 11, IV, VI, IX, and XI) were linear over the same concentration range as were the phosphorescence curves. Characteristic downward curvature due to the inner-filter effect occurs for all the above described curves once the indicated concentration range is exceeded. The luminescence characteristics for samples of expressed lime oil in ethanol (Table 111, Figure l), when compared with the characteristics of the coumarin derivatives indicate that compounds I1 and IX are responsible for the intense blue fluorescence exhibited by the oil. While I, 11, 111, IX, and X make up essentially all of the total coumarin derivative concentration in expressed lime oil (5), the fluorescence intensity from I, 111, and X is much lower than from I1 and IX. Phos1184
ANALYTICAL CHEMISTRY
phorescence of the oil in ethanol is characteristic of emission from I, 11, IX, and XI. The relatively small concentration of XI in expressed lime oil makes its contribution to the phosphorescence of the oil very small. The phosphorescence efficiency for I11 and X is lower than for I, 11, and IX and therefore does not exert appreciable effect on the whole oil spectrum. Low temperature luminescence from ethanol solutions of the whole oil is comprised of low temperature fluorescence due to I1 and IX and phosphorescence which can be attributed to I, 11, and IX. Fluorescence, phosphorescence, and low-temperature luminescence analytical curves for expressed lime oil samples of different geographic origin showed linear behavior from 0.01 to 1.0 pg/ml, 1.0 to 100 pg/ml, and 1.0 to 100 pg/ml, respectively. All luminescence emission intensities for the oils showed wide variation apparently depending on origin of the sample. Ultraviolet absorption by the oils at 317 nm shows that same wide variation. Because only one sample of each oil was investigated a definite correlation between sample origin and emission intensity and therefore coumarin derivative content cannot be established. The method of Sale (15)used in the detection of adulteration of expressed citrus oils is based on the ultraviolet absorption of the oil. In lime oil the presence of the coumarin derivatives I, 11, 111, IX, and X is responsible for this absorption. The other coumarin derivatives identified in lime oil (5) are present in such small amounts that their contribution to the total absorbance is insignificant. This investigation has shown that two coumarin derivatives I1 and IX are exclusively responsible for the room temperature fluorescence of ethanol solutions of expressed lime oil. Use of the 420-nm emission in place of the 317-nm absorbance to establish authenticity of lime oil samples could therefore be based on the presence of two instead of five components. Phosphorescence emission or low temperature luminescence essentially due to three components could be used as well.
(15) J. W. Sale, J. Ass. Ofic. Agr. Chemists,36, 112 (1953).
However, because of the increased difficulty inherent in making phosphorescence and low-temperature luminescence measurements. fluorescence is favored. Luminescent species provide characteristic excitation spectra. Utilization of the excitation and emission spectra, to detect the presence of compounds such as chalcones and menthyl salicylates which may be added to adultrated expressed oils to bring the absorption of the oil up to levels prescribed by the method of Sale, is a definite possibility. Expressed lemon oil exhibits the same fluorescence characteristics in ethanol as expressed lime oil. This blue emission can be attributed to 5-geranoxy-7-methoxycoumarin,5,7dimethoxycoumarin, and 5-isopenteneoxy-7-methoxycoumarin which are present in lemon oil (5). Expressed grapefruit oil shows maximum emission from ethanol solution of the oil below 400 nm. This can be attributed to the presence of 7-geranoxycoumarin(6) which apparently is present
as the major coumarin derivative. Further examination of the total luminescence from expressed lemon oil and grapefruit oil should result in observations similar to those made for expressed lime oil. The major crystalline components in expressed orange oil are flavones with smaller amounts of coumarin derivatives. Orange oil shows emission and excitation maxima at higher wavelength than other citrus oils which may be due to the flavones. ACKNOWLEDGMENT
Samples of expressed citrus oils were provided by Fritsche Brothers, Inc., Dodge and Olcott, Inc., and the Florida Home Juice Company. RECEIVED for review March 5, 1969. Accepted May 19, 1969. Research supported in part by the Esso Foundation.
Novel Impedance Measurements on Ion-Selective Liquid-Membrane Electrodes M. J. D. Brand and G. A. Rechnitz’ Chemistry Department, State University of New York, Buffalo, N . Y. 14214
-
-
Ion selective, liquid membrane electrodes were studied using a novel impedance measurement technique. Evaluation of several commercially available membrane electrodes was carried out in detail as a function of frequency and of solution variables. From these studies, it was possible to identify and characterize some of the processes-e.g., electromigration and ion exchange-occurring within the membrane and at its interfaces. The data obtained suggest a possible means for the quantitative evaluation of fundamental electrode parameters, not accessible to potentiometric studies, and may lead to an experimental test of the current theories of ion-selective electrode operation.
A CONSIDERABLE NUMBER of ion selective electrodes have now been developed to the stage of analytical utility ( I ) , and it seems certain that many others will be introduced in the future. Among the various membrane types used in these electrodes, liquid ion-exchange resins are now firmly established as offering a wide range of possibilities. While liquid membrane electrodes have been known for many years, it was the introduction of the calcium-selective electrode by Ross (2) which led to recent developments. Subsequently, commercially available electrodes have been evaluated for measurement of the activity of not only calcium ( 3 ) but also cupric (4), nitrate (5), perchlorate (6), fluoroborate (7), and chloride (8) ions. Alfred P. Sloan Fellow; to whom reprint requests should be addressed. ( 1 ) G. A. Rechnitz, Chem. Eng. News, 43,(25), 146 (1967).
(2) J. W. Ross, Jr., Science, 155, 1378 (1967). (3) G. A. Rechnitz and Z . F. Lin, ANAL.CHEM.,40, 696 (1968). (4) G. A. Rechnitz and 2. F. Lin, Anal. Letters, 1, 23 (1967). (5) S. Potterton and W. D. Shults, ibid., p 11. (6) T. M. Hseu and G. A. Rechnitz, ibid., p 629 (1968). (7) R. M. Carlson and J. L. Paul, ANAL.CHEM.,40, 1292 (1968). (8) T. G. Lee, ibid., 41, 391 (1969).
A general theory of liquid membrane electrodes based on ion-exchange properties has been presented by Eisenman et al. (9-11) and by Sandblom (12, 13). Potentiometric response to a given counter ion depends not only on the activity of the ion in solution and in the membrane but also on the equilibrium constant of the ion-exchange process and on the mobility of the ion in the membrane. Ionic migration has been assumed to be the only process responsible for the passage of electricity through the membrane. This is equivalent to treating the membrane as a pure resistance (IO, 13) for which the measured conductivity is independent of the frequency of the applied ac signal. Measurements of the conductivity of solid ion-exchange membranes have indicated only small variations with frequency (14, 15). The mechanism of ion transport through membranes and across the membrane-solution interface is not well understood ; kinetic data on the processes involved are not available although it is thought that the ion-exchange reaction is not rate determining (16). It is apparent that such information is not obtainable from steady state electrode potential measurements and a different experimental approach is required. One possible approach involves the study of the power spectrum of noise generated by passage of a relatively high density current through a solid ion-exchange membrane ( I 7). Buck has discussed the impedance of glass electrodes (18) and (9) J. Sandblom, G. Eisenman, and J. L. Walker, J. Phys. Chem., 71, 3862 (1967). (IO) Ibid., p 3871. (11) G. Eisenman, ANAL.CHEM.,40, 310 (1968). (12) J. Sandblom, J. Phys. Chem., 73,249 (1969). (13) Zbid., p 257. (14) J. H. B. George and R. A. Courant, ibid., 71, 246 (1967). ( 1 5 ) V. Subrahmanyan and N. Lakshminarayanaiah, ibid., 72, 4314 (1968). (16) F. Helfferich in “Ion Exchange,” J. Marinsky, Ed., Marcel Dekker, New York, Volume 1, 1966, p 65. (17) M. E. Green and M. Yafuso, J. Phys. Chem., 72, 4072 (1968). (18) R. P. Buck,J. Electroanal. Chem., 18,381 (1968). VOL. 41,NO. 10,AUGUST 1969
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