Table 111. PMR Data for Selected Carbamates Carbamate of PPm (6) 0.85 s, 3; 0.89 s, 3; 0.94 s, 3; 1.00 t o 2.60 (-)-Borneol b, m , 7 ; 4.71 d, 1 ; 5.29 b, s, 1. Cedrol 0 . 8 2 d , 3 ; 0 . 9 9 s , 3 ; 1 . 1 9 s , 3 ; 1.5Os,6; l . l t o 2 . 1 b , m , 7 ; 2.07s,1; 2.40d,2; 5.06, b, s, 2. Citronellol 0.90d,3; l.Oto2.0b,m,S; 2.06s,6; 3.97t,2; S.Olb,t,l; 5.52b,s,2. 0.83-0.86 2s, 6; 0.97 s, 3; 1 . 0 to 1.90 b, m, (+)-Isoborneol 7; 4.50 t, 1 ; 5.07 b, m, 2. (-)-Isopulegol 0.93 d, 3; 1 . 1 to 2 . 3 b, rn, 8 ; 1.63 s, 3; 4.3-4.6 b, rn,1; 4.67 s, 2; 5.23 b, 2. Linalool 1.48s,3; l.S7s,3; 1.64s,3; 1.70t02.10 b , m , 6 ; 4 . 8 8 r n , l ; 4.80-5.30b,2; 5.91 m, 1. Linalooloxide 1 . 2 6 s , 3 ; 1.4Os,3; 1 . 4 3 s , 3 ; 1.50-1.90 (cis,H:CH3) b , m , S ; 4 . 0 1 b , t , l ; 4 . 8 8 m , l ; 5.19 b, m, 2; 5.77 m, 1. 4-Terpinenol 0.91 d , 6 ; 1.66 s, 3; 1.60 to 3.00 b, rn, 7; 5.10 b, 3(NH2 and C = CH). a-Terpineol 1.43 s,6; 1.65 s, 3; 1 . 7 0 t o 2 . 4 0 b , m, 7; 4.67 b, 2; 5.30 b, 1. n-Pentanol 0.90m.3; l.lOto1.90b,m,6; 3.94t,2; 5.42 b, 2. n-Hexanol 0.90m3; l.lOto1.90b,m,8; 3.93t,2; 5.35 b, 2. n-Heptanol 0.92 m, 3; 1.10 to 1.90 b, m, 10; 3.92 t, 2; 5.25 b, 2. n-Nonanol 0.92m.3; l.lOtoi.9Ob,rn,14; 3.92t,2; 5.20 b, 2. n-Decanol 0 . 9 2 m , 3 ; l . l O t o 1 . 9 0 b , r n , 16; 3 . 9 2 t , 2 ; 4.97 b, 2.
rates many carbamate pairs. The R, values obtained with benzene are similar to those obtained with 2-chloropropane (3). GLC Kovats values (4) for the carbamates were recorded from short Apiezon L and DEGS columns. Degradation was observed for the tertiary carbamates on the Apiezon (4) E. sz. Koviits, Fresenius’Z. Anal. Chem., 181,351 (1961).
L column; linalool carbamate was not successfully chromatographed. Extraneous peaks which could be attributed to degradation were not observed during chromatography on the DEGA column. Approximation of the carbamate partial KovBts index (61,) (5) can be determined for the Apiezon L column which is essentially free of polar effects. For n-pentanol, ponderal (mass) effect (5) contributions of the hydrocarbon and hydroxyl moieties are 500 and 114. By difference, the 61, for the carbamate is 1070 less 614 = 456. Zk values for the higher n-alkyl alcohol carbamates support this calculation. The IR spectra (CC1,) of all the selected carbamates listed in Table I1 showed five a5sorption bands between 3100 and 3600 cm-’; these characteristic bands can be seen in Figure 1 which shows the spectrum for (-)-isopulegol carbamate. The PMR data for CCll solutions of the carbamates are given in Table 111. All carbamates showed a typical unsplit, broad, amide proton resonance at ppm (6) 4.5-5.5 (two protons). Comparison of each carbamate with the related alcohol showed a downfield shift of ppm (6) 0.5-1.0 for the protons attached to the carbon bonded to oxygen. Deshielding .was also observed for protons in close proximity to the carbonyl group.
RECEIVED for review April 12, 1972. Accepted September 8, 1972. A portion of the M.S. thesis of F. Y. Hutto, Department of Chemistry, Mississippi State University, State College, Miss. 39762; in cooperation with the Mississippi Agricultural Experiment Station, State College, Miss. Mention of a proprietary product in this paper does not constitute an endorsement of this product by the U.S. Department of Agriculture. ( 5 ) P. A. Hedin, J. P. Minyard, Jr., and A. C. Thompson, J . Chro-
matogr., 30,43 (1967).
Scanning Fluorescence Spectrometry Combined with Ultraviolet Detection of High Pressure Liquid Chromatographic Effluents E. D. Pellizzari and C. M. Sparacino Chemistry and Life Sciences Division, Research Triangle Institute, Post Ofice Box 12194, Research Triangle Park, N.C. 27709 FLUORESCENCE SPECTROMETRY, with its inherent high sensitivity, seems well suited as a spectrometric detection system for characterizing chromatographic effluents (1-3). Since emission and excitation spectra are obtainable from a fluorescing molecule, two qualitative aids are available for the identification or verification of purity of liquid chromatographic peaks. Furthermore, when the effluent is monitored with an ultraviolet detector set in tandem with the fluorescent system, it is (1) M. C. Bowman and M. Beroza, ANAL.CHEM., 40, 535 (1968). (2) H. P. Burchfield, R. J. Wheeler, and J. B. Bernos, ibid., 43, 1976 (1971). (3) D. J. Freed and L. R. Faulkner, !hid., 44,1194 (1972). 378
0
possible to simultaneously quantitate each component in a mixture as well as measure retention data and examine peak shape characteristics. Thus, complementary information can be acquired on the nature of each component in a complex mixture. The principle of operation of such an analytical system is based upon a molecule possessing strong absorption and fluorescence characteristics. Polynuclear aromatic hydrocarbons (PaH) present in the combustion products of fuels, tobacco smoke, and smoked foods are naturally suited for this type of analysis. This report describes the interfacing, operation, and potential application of a high pressure liquid
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
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NANOMETERS
WAVELENGTH
Figure 1. Excitation and emission spectra for benzo[3,4]pyrene in microflow cell in static mode
- NANOMETERS
Figure 3. Repetitive emission scans (excitation 285 nm) during elution of benzo[3,4]pyrene See Figure 2A for chromatographic parameters.
Concentration was 38 ng/pl in hexane. Excitation and emission were recorded at 425 and 285 nm, respectively. MM = 1.0
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Figure 4. Repetitive emission scans (excitation 350 nm) during elution of 3-methylcoronene See Figure 2B. MM = 0.001
--
. . TIME
0
2
4
6
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(MINI
Figure 2. Chromatograms from UV photometric detector response for polynuclear arenes . . . ' indicates emission scan periods taken with fluorimeter, see Figures 3-5. Column was Corasil 11. Sensitivity was 0.04 absorbance unit, full scale. Chromatogram A represents 76 ng benzo[3,4]pyrene, B is 192 ng 3-methyl coronene, and Cis 128 ng perylene; D is a representative chromatogram for an injected mixture of these three PaH's at 19, 24, and 32 ng, respectively
.
chromatograph equipped with an ultraviolet detector and a fluorimeter in series for simultaneous monitoring and characterization of PaH in column effluents. EXPERIMENTAL Apparatus. A Varian Model 4010-01 high pressure liquid chromatograph (HPLC, Varian Instrument Co., Walnut
Creek, Calif.) and an Aminco-Bowman spectrofluorimeter (SPF, American Instrument Co., Silver Spring, Md.) were interfaced in series. Polynuclear arenes were chromatographed on a 0.24- x 100-cm stainless-steel column packed with Corasil I1 (Waters Associates, Framingham, Mass.). The packing was supported by a '/*-in. union containing a 0.5-pm stainless-steel frit,and the column was housed in a watercooled jacket which maintained the temperature at =t1 "Cof ambient. Column effluent was monitored in a UV photometric cell (254 nm) having an internal diameter of 1 mm and a path length of 10 mm (dead volume 8 11). The detector signal which was recorded with an A-25 Varian strip chart recorder, was linear in absorbance units (0.02 to 0.64, full scale) and therefore, directly proportional to concentration for solutes that obey Beer's law. After passing through the UV photometric cell, the column effluent was then transferred via a 22-gauge Teflon (Du Pont) line into an Aminco-Bowman microflow cell (3-mm i.d. x 5-mm 0.d. x 38 mm, 50-pl volume) where the polynuclear aromatics were characterized by fluorimetry. The distance between the two detectors was approximately 25 cm. The SPF was equipped with a MO-watt, 7.5-ampere, 17-23 volt dc Hanovia 901C1 Xenon lamp and a IP21 photomulti-
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
379
rates (10 nmjs) offered with the standard Aminco-Bowman fluorimeter. Emission spectra were also obtained when a mixture of the three PaH’s were injected onto a column which was unable to resolve the mixture.
loo[ 90
70
RESULTS AND DISCUSSION
WAVELENGTH - N A N O M E T E R S
Figure 5. Repetitive emission scans (excitation 250 nm) during elution of perylene See Figure 2C. MM
=
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I
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200
250
300
350 400 450 500 WAVELENGTH NANOMETERS
-
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Figure 6. Emission spectra recorded at exci350 nm tation wavelengths of 285 nm (-), of a chro(. ), and 250 nm (-*--) matographic peak containing a mixture of PaH
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See Figure 2 0
plier tube. The photomultiplier microphotometer was operated at full sensitivity and the meter multiplier (MM) settings varied from 0.3 to 0.001. The bandpasses were fixed at 2 mm. An Aminco Model 814 x-y recorder (American Instrument Co., Silver Spring, Md.) was used to display continuous spectral scans. Procedure. Hexane was degassed in cacuo and used as the mobile phase. A head pressure of 150 psi was used to deliver the mobile phase from a large reservoir through the chromatographic column at 0.7 ml/min. Solutions of benzo[3,4]pyrene (BeP), 3-methyl coronene (MeCO), and perylene (PER) at concentrations of 38, 48, and 64 ng/pl, respectively, were injected on-column with a 10-pl Hamilton syringe equipped with a Chaney adapter. Emission and excitation spectra for each polynuclear aromatic were recorded in the fluorimeter microflow cell in the static mode by filling the cell with a standard solution. An excitation wavelength was selected for each PaH and this wavelength was used to obtain continuous emission scans during elution of a chromatographic peak detected by the UV photometer. Continuous emission scans were obtained on single component injections for BeP, MeCO, and PER at maximum scan 380
Excitation and emission spectra for benzo[3,4]pyrene are depicted in Figure 1. The resolution is lower during rapid scanning in a microflow cell; however, the “fingerprint” characteristics can still be utilized for verification of identity or purity of a fluorescent compound. From the spectra of each PaH generated in the static mode, excitation wavelengths of 285, 355, and 250 for BeP, MeCo, and PER, respectively, were selected for acquiring emission spectra during scanning of chromatographic peaks. The series of chromatographic peaks observed by UV detection are given in Figure 2A-C and the corresponding emission spectra for each PaH are depicted in Figures 3-5. The emission intensities for a series of spectra obtained for an individual PaH varied and these differences correlated to the dynamic change in the concentration of solute during its elution from the chromatographic column. However, the characteristics of a spectrum were similar regardless of whether scans were made during an increase or decrease in the solute concentration in the microflow cell and the emission spectra obtained by repetitive scanning of an HPLC peak agreed with those obtained in the static mode. Thus qualitative identification of each compound can easily be made. A lag period of approximately 10 sec was observed between the maximum UV photometric and fluorescence detector responses. The peak widths in UV chromatograms were measured in minutes (at *i2 peak height) and fluorescence chromatograms were also constructed by plotting the maximum emission intensity for each scan cs. time. Comparison of the peak widths from UV and fluorescence revealed that some band spreading did occur in the fluorescence microflow cell but this was only 6-10 seconds greater than in the UV photometric cell. This phenomenon is important especially in those cases when solutes are only partially resolved by a column (as indicated by UV monitoring), since some remixing may occur in the second microflow cell. The technique can also demonstrate that an apparently homogeneous chromatographic peak is composed of a mixture of substances. A mixture of the three PaH’s was injected onto the Corasil I1 column which was unable to resolve the components. Emission spectra were recorded at excitation wavelengths of 285,355, and 250 nm, respectively, during maximum UV detector responses. Although Figure 2 0 reveals that the peak is apparently homogenous to UV monitoring, it is seen in Figure 6 that the chromatographic peak is multicomponent when emission spectra are taken at several different excitation wavelengths. Furthermore, if emission spectra are recorded at several excitation wavelengths during the elution of a peak, then homogeneity can be established without comparison to reference spectra for the pure solute. These results suggest that both identity and purity of a substance giving the peak can be established utilizing the scanning fluorescence technique. Furthermore, it is possible to dynamically acquire alternate excitation and emission spectra for peak characterization. The sensitivity limits for characterization by this means is dependent upon many factors governed by the conditions employed for effecting chromatography-e.g., flow rates, mobile phase as well as rate of fluorescencescanning, fluores-
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
cence cell dimensions, and obviously by the fluorescent yield of the solute under study. The potential utility of this analytical method can be fully appreciated when applied to analyses of complex sample extracts such as those obtained from air particulate, where it is not only desirable to quantitate each compound, but also where additional information (besides retention index) is necessary to assure the analyst that each quantified peak is homogeneous.
ACKNOWLEDGMENT We thank M. E. Wall and C. E. Cook for their helpful suggestions. RECEIVED for review July 25, 1972. Accepted October 3, 1972. This work was supported by Contract PH-43-65-1057 from the National Institutes of General Medical Sciences, National Institutes of Health.
Pulsed Source, Time Resolved Phosphorimetry Determination of Phosphorescence Lifetimes K. F. Harbaugh, C. M. O’Donnell,’ and J. D. Winefordner2 Department of Chemistry, University of Florida, Gainesville, Fla. 32601 PULSEDSOURCE, TIME RESOLVED PHOSPHORIMETRY was introduced as a possible analytical method by O’Haver and Winefordner ( I , 2 ) . Recently, Fisher and Winefordner (3) have described a laboratory-constructed pulsed source phosphorimeter, have compared the pulsed source system to the conventional CW systems, and have applied their pulsed phosphorimetric system to the time resolution of several synthetic binary and one ternary mixture of spectrally-similar millisecond decaying phosphors. Also O’Donnell, Harbaugh, Fisher, and Winefordner ( 4 ) have time resolved and quantitatively measured several halo-biphenyl mixtures with a modified version of the pulsed source phosphorimeter described by Fisher and Winefordner (3). Time resolved phosphorimetry should have considerable potential for the quantitative analysis of structurally-similar organic molecules, particularly those of biological interest. However, only recently has the quantitative measurement of the phosphorescence of biologically important molecules become feasible ; prior to the development of the rotating capillary cell ( 5 , 6), it was not possible to measure reliably phosphorescence from predominately aqueous solutions at liquid nitrogen temperatures. However, with the rotating capillary cell, it is possible to measure sub-nanogram quantities of many organic molecules in microliter quantities of predominantly aqueous solutions at 77 OK. The rotating capillary cell in combination with the pulsed source phosphorimeter (3, 4 ) allows even smaller quantities of organic molecules to be measured and even resolved from other structurally-similar species as well as from phosphorescence imOn leave. Department of Chemistry. Colorado State University, Fort Collins, Colo. 80521. Author to whom reprint requests should be sent. (1) T. C. O’Haver and J. D. Winefordner, ANAL. CHEM.,38, 602 (1366). ( 2 ) J. D. Winefordner, Accounfs Cl7em. Res., 2,361 (1969). (3) R. P. Fisher and J. D. Winefordner, ANAL. CHEM.,44, 948 (1972). (4) C. M. O’Donneli. K. F. Harbaugh, R. P. Fisher, and J. D. Winefordner, ibid., in press. ( 5 ) R . J. Lukasiewicz, P. A. Rozynes, L. B. Sanders, and J. D. Winefordner. ibid., 44,237 (1972). (6) R. J. Lukasiexicz, J. J . Mousa, and J. D. Winefordner, ibid., p 1339.
EMISSION
WAVELENGTH
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Figure 1. Phosphorescence emission spectra of 4’4rifluoromethylacetophenone (- - - ) and 3’-trifluoromethyl acetophenone (-)
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purities present in the solvents, reagents, and quartz cells used in the phosphorimetric studies. In the present study, pulsed source phosphorimetry is utilized for the measurement of phosphorescence lifetimes of a number of structurally- and spectrally-similar organic molecules. The influence of changing the environment on the phosphorescence lifetimes of several molecules is shown, and the great potential of time resolved, pulsed source phosphorimetry is clearly demonstrated. EXPERIMENTAL Apparatus. The pulsed source phosphorimeter was identical to the one described previously (3, 4). All phosphorescence lifetime measurements were performed with a stationary sample cell (5-mm 0.d. x 3-mm i.d.). Reagents. All chemicals with the exception of 4-hydroxyacetophenone, which was recrystallized, were used without further purification. Acetophenone, 2‘-hydroxyacetophenone, 4’-hydroxyacetophenone, 3-(tri-fluoromethyl)acetophenone, 4’-(trifluoromethyl)acetophenone, 4,4’-bis-(dimethyl-
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