Anal. Chem. lQ82, 5 4 , 2131-2133
Table I. Ratio of Double Hydrogen Migration to Total Olefinic Cleavage (A/(A + B)) in the Mass Spectra of CH,PO(-YR)(-XR')' 100 x A/(A t B) for Y-R for X-R' no. YR XR' cleavage cleavage S-ethyl 64 1 1 0-n-propyl 2 0-n-butyl S-ethyl 90 b S-n-propyl 80 b 3 0-n-butyl S-n-propyl b 7 4 0-ethyl S-n-butyl b 26 5 0-ethyl S-n -pent yl b 37 6 0-ethyl S-n-hexyl b 38 7 0-ethyl 0-ethyl 98 98 8 0-ethyl 0-n-propyl b 89 9 0-ethyl 10 0-ethyl S-methyl 11 11 S-n-pentyl S-n-pentyl 31 31 ' Abundance values of fragment A were corrected for Very low isotopic contribution from fragment B. abundance values for both A and B. nation of an alkenyl iradical takes place (Scheme I, X = 0, route a) in decisive predominance over the simple olefinic cleavage (the McLafferty-type rearrangement, route b) in which only one hydrogen migrates. When an alkyl cha:in (longer than CH3) is bound directly to the phosphoryl group (Scheme I, 8; = CHJ, the carbon atom adjacent to the ]phosphorus cannot be protonated, and route b, therefore, is found exclusively (2). We found that when a thioalkyl moiety is attached to the P=O group, the sulfur atom behaves in a way which is intermediate between that of an oxygen and that of a carbon; in cases where the sulfur bears a short alkyl chain, the group suffers an almost exclulsive single hydrogen migration (Scheme I, X = S, route b), whereas heavier chains cause an increase in the relative abundance of route a in a predictable way (7). Route b is, however, always dominant in the olefinic cleavage
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of unbranched aliphatic alkylthio moieties. The ratios of the abundance of double hydrogen migration (fragment A) to the total olefinic cleavage (A + B) for some dialkyl esters of methanephosphonic acid are tabulated in Table I. From the table it is clear that while A/(A + B) for 0-R cleavage is usually over 80% I the value for S-R cleavage is lower than 40% even for the longer aliphatic chains. Thus, by measuring the ratio of single to double hydrogen migration of an olefinic cleavage, it is easy to determine whether the olefin (or the olefinic radical) originated from the alkoxy or alkylthio part of the molecule. It is interesting that the presence of sulfur reduces to some extent the tendency of the molecule as a whole to suffer a double hydrogen migration. Thus, the 64% value for the 0-propyl cleavage of the mixed 0,sdiester 1 (see Table I) is considerably lower than the 89% found for the same cleavage of 9 which lacks sulfur. The effect is pronounced for an 0-ethyl cleavage and is evident when the high proportion of double hydrogen migration found for the 0-ethyl cleavage of 8 is compared to the low value found for the same cleavage of the mixed 0,s diester 10. Thus, a low value of A/(A B) for an X-ethyl cleavage is not necessarily indicative of X = S. Nevertheless, the assignment of the alkyl groups by the proposed method is still possible using the A/(A B) value of the other alkyl moiety (compare compounds 4-7).
+
+
LITERATURE CITED (1) Mclafferty, F. W. Anal. Chem. 1956, 28, 306-316. (2) Budzlklewlcz, H.; Fenselau, C.; Djerassl, C. Tetrahedron 1966, 22, 1383-1398. (3) Occolowltz, J. L.; White, G. L. Anal. Chem. 1963, 35, 1179-1182. (4) Occolowitz, J. L.; Swan, J. M. Aust. J . Chem. 1966, 79, 1187-1195. (5) Whelan, D. L.; Johanssen, J. C. Aust. J . Chem. 1971, 2 4 . 887-890. (6) Nishiwaky, T. Tetrahedron 1967, 23, 2181-2187. (7) Tashma, 2 . ; Katzhendler, J.; Deutch, J. Org. Mass Spectrom. 1973, 7, 955-961.
RECEIVED for review March 3,1982. Accepted June 8,1982.
Photoreduction-,Fluorescence Detection of Aliphatic Alcohols, Aldehydes, and Ethers in Liquid Chromatlography Mltchell S. Gandelnian and John Wr. Birks" Department of Chemistry and Cooperatlve Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, Colorado 80309
The application of high-performance liiquid chromatography (HPLC) to the determination of trace organic compounds is presently limited by the availability of sensitive and selective detectors. Compounds that only weakly absorb light in the visible or near-ultraviolet spectral regions pose particularly difficult analysis problems. Recently, several investigators have applied postcolumn photochemical reactors to improve the UV-visible or fluorescence detection properties of selected groups of compounds (2-5). We have investigated the use of a sensitized, photwhernical reaction to detect compounds that do not absorb W-visible radiation at all (6). In that detection scheme an anthraquinone was added to the HPLC solvent reservoir and the HPLC mobile phase saturated with O2 The sensitized photooxygenation reaction produces hydrogen peroxide which is subsequently detected by its chemiluminescence reaction with luminol. However, we have dis-
covered that when oxygen is excluded from the HPLC solvent the anthraquinone is reduced to the highly fluorescent hydroquinone. The combination of this photochemical reaction with a conventional fluorescence detector provides a much simpler detection scheme with about 3 orders of magnitude improvement in detection limits. We describe here the application of the new photoreduction-fluorescence (PRF)detector to the determination of aliphatic alcohols, aldehydes, and ethers. Figure 1shows the reaction scheme for the photoreduction of anthraquinone by 2-propanol (7). The photochemical reaction begins with the abstraction of a hydrogen atom by the triplet-excited anthraquinone (8). A subsequent thermal disproportionation reaction leads to the highly fluorescent hydroquinone (9,lO-dihydroxyanthracence). Those compounds having C-H bond strengths less than about 95 kcal
0003-2700/82/0354-2131$01.25/0 0 1982 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982 AP
+
hv--AP*
(singlet,S)
AQ*(S) -+ AQ* (triplet,T) AP'(T)
+
(CH,),COH ZAPH*-AQ
-
(CH,),CHOH
AQH,+ (CH3)2COH
+ AQ --AQH-
+
(CH,),
C.0
+APH,
Flgure 1. Probable mechanism for photoreduction of anthraquinone by 2-propanol: AQ is anthraquinone; AQH. is the semiquinone radical; AQH, is 9, IOdihydroxyanthracene. HPLC
PHOTOCHEMICAL
Table I. Detection Limits for Compounds Tested detection compound limit, ng
REACTOR
A. Alcohols ethanol 2-propanol (isopropyl alcohol) 1-butanol 2-met h yl-1-propan01 ( is0but yl alcohol ) 1-hexanol 1-octanol
4 4 5 5 5 7
B. Ethers FLUORESENT LAMP
HPLC MOBILE PHASE 80% CH CN 485 x l b 4 M AQDS
PYREX SLEEVE
/
Figure 2. Schematic diagram of PRF detector. AQDS is anthraqulnone-2,6disulfonate.
1,3-diethylenedioxide (dioxane) diethylene oxide (tetrahydrofuran) diethyl ether
12 3
C. Aldehydes 1,5-pentanedial (glutaraldehyde) propanal ( propionaldehyde) 3-methylbutanal (isovaleraldehyde)
31
7
4
7
mol-l can reduce anthraquinone in this reaction and thus be detected. Such compounds have been termed "hydrogenatom-donating substrates" (9). Additional classes of compounds other than those listed in the title of this paper that are likely to respond to the PRF detector are amines and those compounds having either allylic or benzylic hydrogens.
EXPERIMENTAL SECTION Reagents. Anthraquinone-2,6-disulfonatedisodium salt (Aldrich, Milwaukee, WI) was used without further purification. HPLC grade acetonitrile (Fisher, Denver, CO) was used in the mobile phase of the HPLC. Chromatographic Apparatus. The chromatographic equipment consists of an Altex llOA high-pressure pump, a Rheodyne Model 7120 injector with a 20-pL sample loop, and an Altex CIS column (250 mm X 4.6 mm) with 10 pm diameter particles. Detector. The PRF detector can be subdivided into two separate functional units-photochemical reactor and fluorometer-which are connected in series. Figure 2 is a scheM matic diagram of the PRF detection system. A 4.85 X solution of anthraquinone-2,6-disulfonatein 80% acetonitrile is used as the HPLC mobile phase and pumped throughout the entire system-column, photochemical reactor, and fluorometer. The postcolumn photochemical reactor is a 1-m length of 0.38 mm i.d. transparent PTFE tubing (Bel Arts Products, Pequannock, NJ). Scholten, Welling, Brinkman, and Frei (5) have previously described the use of PTFE tubing in postcolumn photochemicalreactors. Connectionsto the photochemicalreactor were made with 1.5-mmAltex tube end fittings. The dead volume (220 pL) of the photochemical reactor was measured by weighing the amount of water displaced as a bubble of air traversed the length of tubing. The PTFE tubing has a 50% transmittance at the center of the emission envelope of the UV source (365 nm). In order to avoid excessive heating of the HPLC solvent, we coiled a 1-m length of tubing around a 26 cm long X 2 cm 0.d. Pyrex cylinder which slides over the fluorescent tube of the UV lamp. The fluorescent lamp (Sylvania Model E 8TS 1BLB) was purchased from a local hardware store for approximately $20. Finally, the entire photochemical reactor was covered with aluminum foil to increase the photon flux by reflection. The fluorometer used here is a Schoeffel Instrument Corp. Model FS 970. The excitation wavelength was set at 365 nm, and a 470-nm cutoff filter was used for the emission. RESULTS AND DISCUSSION Since we wanted the photoreduction to occur in aqueousacetonitrile solvents, an anthraquinone soluble in polar solvents was required. I t has been shown that many of the sulfonate salts of anthraquinone have quantum yields near unity in this sensitized photoreduction (9). We have not encountered any problems by adding the anthraquinone-
1
O
l
2
3 4 mP
5
E
Flgure 3. Chromatogram of aliphatic alcohols (flow rate, 0.5 mL/min): (1) 15.8 pg of ethanol; (2) 16.2 Mg of 1-butanol; (3) 16.3 pg of 1-hexanol; (4) 16.5 pg of 1-octanol.
2,6-disulfonate to the mobile phase of the HPLC; however, we have observed an enhancement of the resolution during the separation of small aliphatic alcohols, aldehydes, and ethers when the sensitizer is present (6). Table I lists the detection limits for various oxygen-containing compounds. The detection limit was determined from the equation 3rs
mL = (S/mi) where 3u is half the peak-to-peak noise, S is the signal peak height obtained from an injection of a 0.1% solution of the analyte in 80% acetonitrile, and mi is the mass of the analyte in that sample. No special pretreatment of the samples (deoxygenation) was required. Figure 3 demonstrates the separation and detection of four aliphatic alcohols using a CI8 column. In conclusion, we believe the PRF detector is both the least complex and most sensitive detector available for the determination of these aliphatic oxygen-containing compounds. In the past the refractive index (RI) detector was employed to quantitate these analytes; however, the PRF detector offers two major advantages over the RI detector. First, the P R F
Anal. Chem. 1982, 5 4 , 2133-2134
detector is at least 2 orders of magnitude more sensitive than the R1 detector, and second, it responds to a much narrower class of compounds. The latter is a large advantage when the interpretation of a complex chromatogram is required. All of the compoundri listed in Table I are volatile and thus may also be determined by gas chromatography. Recent work has concentrated on nonvolatile compounds that can only be determined by liquid chromatography. In collaboration with Frei and Brinkman we have achieved detection limits of approximately 10 ng for a variety of digitalis glycosides separated by reversed-phase HPLC. Applications of the PRF detection scheme to these and other nonvolatile compounds such as saccharides will be demibed in future articles.
ACKlVO WLEDGMEIVT We are especially thankful to Kenneth Sigvardson and Tad Koch for helpful discussions.
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LITERATURE CITED (1) Iwaoka, W.; Tannenbaum, S. R. IARC Scl. Pub/. 1979, 14, 51. (2) Twltchett, P. J.; Willlams, P. L.; Moffat, A. C. J. Chromatogr. 1978, 149, 683. (3) Scholten, A. H. M. T.; Frei, R. W. J. Chromatogr. 1979, 176, 349. (4) Scholten, A. H. M. T.; Brinkman, U. A. Th.; Frel, R. W. Anal. Chim. Acta 1980, 114, 137. (5) Schoiten, A. H. M. T.; Welling, P. L. M.; Brinkman, U. A. Th.; Frei, R. W. J. Chromatogr. 1980, 199, 239. (6) Gandelman, M. S.; Blrks, J. W. J. Chromatogr. 1982, 242, 21. (7) Wllklnson, F. J. Phys. Chem. 1962, 66, 2569. (8) Bridge, N. K. Trans. Faraday Soc. 1980, 56, 1001. (9) Turro, N. J. "Modern Molecular Photochemistry"; Benjamin-Cummlngs: Menlo Park, CA, 1978; pp 362-380.
RECEIVED for review February 2,1982. Accepted July 9,1982. The authors gratefully acknowledge the support of the National Science Foundation (Grant No. CHE-7915801). J.W.B. thanks the Alfred P. Sloan Foundation for a Research Fellowship.
Noise Reduction in Liquid Phase Photoacoustic Spectroscopy M. R. Fisher Department of Chemistty, University of Nebraska, Lincoln, Nebraska 68588
N. S. Nogar" Group CNC-2, MS G738, /-os Alamos National Laboratory, Los Alamos, New Mexico 87545
Cell geometry has been shown in the past to have a significant affect on both signal and noise levels in photoacoustic spectroscopy, PAS (1). A number of different cell designs have been used in the past for piezoelectric detection of liquid phase PAS signals. These include placing a scirew-mounted transducer in direct contact with the analyte solution (2, 3 ) , fabricating the entire sample cell from a piezoelectric material (4,5),and placing a transducer in contact with a quartz vessel which contains or is attached to the sample of interest (6-8). This latter arrangement has a number of advantages over other designs. It allows detection without contact between sample and transducer, a serious consideration when dealing with incompatible materials. In addition, sample change is expedited, and the prolblem of formation of microscopic air bubbles on the transducer face is eliminated. However, poor signal reproducibility inay be a probleim (8) with this arrangement. It is this latter problem that we address.
EXPERIMENTAL SECTION The excitation source used was a nitrogen llaser (NRG 0.7-5-200) capable of delivering l-mJ, 6-11s pulses at repetition rates up to 60 Hz. Two spherical Suprasil quartz lenses, 1000 and 250 mm focal lengths, were used to focus the beani into the cell. The detector employed was a piezoelectric pressure transducer (Celesco LG-65, Canoga Park, CA) which was screw mounted into the side of an aluminum C-clamp. This transducer exhibits a 60-kHz mechanical resonance. A spring mounted on the opposite side of the C-clamp applied pressure to the sample cell and ensured good acoustic contact of the cell with the transducer using mineral oil as the coupling fluid. A standard Suprasil quartz fluorescence cuvette, 5 cm in height, was used in addition to a modified cell. The modified cell was made by reducing the height of a standard cell to 2 cm. Signal processing has bisen described previously (9,lO) and will only be outlined here. Tlhe signal was passed through an impedance matched pre-amp, Celesco LG-1344 (40 dB, 1000 mil, 20 Hz-100 kHz) and then through a Tektronix 7A18 plug-in, used
as an amplifier. Recording electronics consisted of a transient digitizer (Biomation 805) interfaced to an &bit microcomputer (North Star Horizon) (IO),where signal averaging could be used to compensate for shot-to-shot fluctuations of the nitrogen laser. The data acquisition cycle is triggered by a photodiode monitoring the laser pulse. Hard copies of the data were obtained by outputting to an X-Y recorder (Omnigraph 2000). Doubly glass distilled water was used as the sample for these experiments.
RESULTS AND DISCUSSION It has been our experience (8)that the poor signal reproducibility often seen with the above apparatus is due to the presence of low-frequency acoustical noise which is asynchronous with the laser pulse. Alternation of the cell geometry offers a simple way of controlling this problem, without electronic filtering. Figure 1 shows the acoustic signal generated when the N2 laser is directed through a standard cuvette containing doubly distilled water. The relatively large low-frequency excursions seen in a nominally nonabsorbing sample are due to detection of room acoustic noise. While this noise is random in phase relative to the laser generated signal, and can thus be reduced by signal averaging, there are a number of difficulties associated with this procedure. First, the acoustic noise is often comparable to, or greater than, the sample generated signal, thus making averaging very timeconsuming. Second, the finite dynamic range of the detection system @bit storage) limits the extent to which signal can be retrieved from noise (11-13). Figure 2 shows the result of preforming the same experiment with a cuvette reduced in height to 2 cm. A dramatic decrease in low-frequency noise is obvious. In a parallel series of experiments using Pyrex glass tubing in place of the cuvette, the acoustic noise was seen to scale directly with length of the tube over the range 1-10 cm. Actual PAS signals were found to be independent of cell height. Further, the noise level for a given cell height was found to depend only weakly on the
0003-2700/82/0354-2133$01.25/0 @ 1982 American Chemical Society
