1892
Anal. Chem. 1888, 58, 1892-1894
SUMMARY The reaction of primary and secondary amines with 2-dodecen-1-ylsuccinic anhydride provides a simple high-yield route to derivatives that provide useful negative ion SIMS spectra from glycerol solution. Imidization of the amic acid derivative of primary amines provides a method for distinguishing primary and secondary amines. Detection limits for the amic acids lie in the lo4 M range and are improved by the use of cationic surfactants such as hexadecylpyridinium acetate. Addition of a cationic surfactant has the additional advantage that glycerol matrix ions are eliminated from the mass spectrum. ACKNOWLEDGMENT We thank E. A. Williams for assistance with 13C nuclear magnetic resonance spectroscopy. Registry No. A, 102072-09-3;B, 102072-10-6;B (acetimide derivative),102072-16-2;C, 102072-11-7;C (acetimide derivative), 102072-17-3;D, 102072-12-8;E, 102072-13-9; E (acetimide derivative), 102072-18-4; G, 102072-14-0; H, 102072-15-1; hexacytidine amic decylpyridinium acetate, 7439-73-8; cytidine, 65-46-3; acid derivative, 102072-19-5;2-dodecen-1-ylsuccinicanhydride, 19780-11-1; acetic anhydride, 108-24-7. LITERATURE CITED (1) Barber, M.; Bordoli, R. S.; Elliott, G. H.; Sedgewlch. R. D.; Tyler, A. N. Anal. Chem. 1982, 5 4 , 645A.
Busch, K. L.; Unger, S. E.; Vlncze, A.; Cooks, R. G.; Keough, T. J . Am. Chem. SOC. 1982, 104, 1507. KMweii, D. A.; Ross, Mark M.;Chiton, R. J. Blamed. Mass Spectrom 1985, 12, 254. DiDonato, G. C.; Busch, K. L. Biomed. Mass Spectrom. 1985, 12, 364. Clench, M. R.; Garner, G. V.; Gordon, D. B.; Barber, M. Biomed. Mass Spectrom. 1985. 72, 355. L@n. W. V.; Dorn S. B. Int. J . Mass Specworn. Ion Processes 1984. 57, 75. Ligon, W. V. Anal. Chem. 1985, 58, 485-487. Ligon, W. V.; Dorn, S. B. Int. J . Mass Spectrom. Ion Processes 1985, 6 3 , 315. Ligon, W. V.; Dorn, S.B. Anal. Chem. 1985, 5 7 , 1993. Ligon, W. V.; Dorn, S. B. Int. J . Mass Spectrom. Ion Processes 1988, 68, 337. Ligon, W. V.; Dorn, S. B. Int. J . Mass Spectrom. Ion Processes 1904, 67, 113. Busch, K. L.; DMonato, G. C.; Krohe. K. J.; Hittle, L. R. Abstracts, 1985 Pittsburgh Conference & Esposffion on Analytical Chemistry and Applied Spectroscopy, Paper 37 1.
.
Woodfin V. Ligon, Jr.* Steven B. Dorn General Electric Company Corporate Research and Development Schenectady, New York 12301 RECE~VED for review January 21, 1986. Accepted March 17, 1986.
Polar Functional Group Analysis of Mixtures by Silicon-29 Nuclear Magnetic Resonance Sir: Trimethylsilylation is a well-established method for isolation purposes, mass spectrometry, gas chromatography, and numerous synthetic procedures (1). In combination with proton NMR, trimethylsilylation enhances the accuracy of quantitation of polar groups (2-5). Proton chemical shifta of the trimethylsilyl group are, however, not sufficiently sensitive to variations of molecular structure, and so differentiation of various polar functions in polyfunctional compounds or in mixtures is usually not possible by 'H NMR spectroscopy. In total contrast, 29SiNMR chemical shifts in such derivatives are highly sensitive to molecular structure (6) and the conventional 29SiNMR signals are very weak (7). Despite the need for large sample amounts and/or long-time signal accumulation, 29SiNMR spectroscopy has been suggested (6) and used for analysis of polyfunctional compounds (8-12) and recently applied to very complicated mixtures that are encountered in coal extracts and oil fractions (13-16). %i Nh4R has been found to be more convenient than 19F NMR of trifluoroacetyl derivatives for qualitative analysis of such complex mixtures (15). In this communication we wish to report on three improvements of the procedures used in '%i NMR analysis of complex mixtures (13-16). The fist one enhances the NMR signal by application of the INEPT technique; the second increases the reproducibility of chemical shift values and reduces the cost of analysis by replacing the expensive solvent (pyridine-d,) by the much less expensive chloroform-d. Finally, we suggest replacing the volatile reference (tetramethylsilane) with hexamethyldisilane, which is much easier to handle. EXPERIMENTAL SECTION NMR Spectra. The spectra were measured on a Varian XL-200 spectrometer operating at 200 MHz for 'H NMR and at 0003-2700/86/03581892$01.50/0
39.7 MHz for NMR. In a 5-mm broad-band probe, 90° pulses were 8.5 and 49 c long for %i and 'H, respectively. A relaxation period of 40 s was employed for measurements with gated decoupling, unless the relaxation reagent, tris(acety1acetonate)chromium(III), was added to the solution. In the latter case, the delay was only 10 s as in the INEPT measurements. The spectra were measured with 4-kHz sweep width; data were acquired for 1s. FIDs were zero-fied to 16k and exponentially weighted with line broadening of 1.0 Hz. The samples for integral evaluation were approximately 0.5 M solutions of the more abundant component in deuteriochloroform placed into 5-mm NMR tubes. Trimethylsilylation. All work was carried out with dry reagents and under dry nitrogen. Approximately 100 mg of a sample was mixed in a vial with 0.4 mL of bis(trimethylsily1)acetamide and 0 . 4 mL of pyridine, depending on solubility. The heterogenous mixture was shaken for 2 h at 60-80 O C . Then the remaining silylating reagent and pyridine were removed by nitrogen stream, followed by l-h heating (up to 80 "C) under reduced pressure.
RESULTS AND DISCUSSION Conventional ?Si FT NMR spectra (13-16) are measured with gated proton decoupling (to suppress the negative nuclear Overhauser effect), and the measuring time is shortened by a relaxation reagent (7). Much larger time savings can be achieved if the silicon signal is enhanced by polarization transfer from protons. Two general polarization transfer techniques are suitable for this purpose: INEPT and DEPT (for comparison of different variants of these methods and references to the original literature see reviews in ref 17 and 18). For measurement of proton decoupled NMR spectra of trimethylailyl groups, the INEPT technique is slightly superior to DEPT (17). In adopting INEPT pulse sequence (90' [H]-~/2-180~[HI, 180° [Si]-~/2-90' [HI, 90° [Si]-A/2-180° [HI, 180' [Si]@ 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
A/2-decouple, acquire), polarization transfer time, T , and refocusation delay, A, have to be selected for optimum performance of the experiment. In general, the optimum delays T and A can be calculated from the known values of coupling constants and from the formulas derived recently by Schenker and von Philipsborn (19). In trimethylsilyl derivatives we always deal with molecular fragments (CH3)3Si-X-CH, (X being an electronegative element, or group). According to the literature survey, coupling constants in these fragments are fairly constant: 2J(29Si-C-1H) = 7 f 1 Hz, 3J(2BSi-X-C-'H) < 4 Hz,and 5J(1H-C-Si-X-C-1H) < 1 Hz (couplings with protons further removed are even smaller). Setting T = 0.0746 s and A = 0.0161 s (values for maximum enhancement when 2J(mSi-C-1H) = 6.7 Hz and n = 0) yields =Si signal enhancement of about 9.2, which results in a time-saving factor of 80. This factor, combined with a possibly faster pulse repetition rate, results in a time-saving factor of at least 200 when compared with the formerly used gated decoupler technique. The INEPT-increased sensitivity of the measurements speaks again in favor of 29SiNMR for qualitative analysis. For quantitative analysis, however, the use of the INEPT technique means reduced accuracy, as INEPT introduces two possible sources of additional systematic errors. With a fmed setting of the polarization time and refocusation delay, the INEPT enhancement achieved on any silicon line depends on the actual coupling constants of the silicon and protons in the given -CH,-X-Si(CH3)3 fragment. The second source of errors is the relaxation within the duration of the INEPT pulse sequence, which might affect intensities of various 29Si lines to a different extent. In order to estimate the magnitude of errors of the first type, we have calculated the theoretical enhancement factors according to the published formulas (19) for the fixed setting of the delays given above and for all combinations of coupling constant values within the ranges found for these couplings in literature. The calculated enhancement factor varied within the range 8.5-10.0. The smallest enhancement factors were found for >CHl-X-Si(CH3)3 (n = 1) fragments in which maximum values of 2J(29Si-C-1H) = 8 Hz and ,J('H-C-SiX-C-'H) = 1 Hz couplings were combined with 3J(29Si-XC-'H) = 0 Hz. The maximum enhancement occurs if 2J(29Si-C-'H) = 6.4 Hz, 3J(29Si-X-C-'H) = 4 Hz, and ,J('HC-Si-X-C-'H) = 0 Hz in -CH2-X-Si(CHJ3 (n = 2) fragments. Since such combinations of extreme values of coupling constants are unlikely to occur, the calculated range of enhancement factors covers safely all the trimethylsilylated products. The variability of the enhancement factor within this range means that the concentration of an unknown compound can be determined from the INEPT signal intensity with a relative accuracy of only 20%. This accuracy cannot be improved for unknown compounds; for compounds with known structure the accuracy can be increased up to that of conventional NMR measurements (about 5 % ) by suitable calibration. The effect of relaxation on the measured INEPT signal intensity is much more difficult to assess. Since the refocus delay (A) is about 4.5 times shorter than the polarization delay ( T ) , relaxation during the refocus period can be neglected as long as it is not much faster than the relevant relaxation during the polarization. Judging from the line widths in 2gSiNMR and 'H NMR spectra (as approximate measures of relaxation during A and T , respectively), relaxation in the refocusation period is slower than during the polarization delay. With the exception of compounds containing quadrupolar nuclei (14N, 35Cl,37Cl)or samples contaminated with paramagnetic impurities, the line width of trimethylsilyl protons is 1 Hz or less. In such samples, relaxation during the po-
1893
Table I. Molar Ratios of HMDSO and HMDSN to HMDS Determined by Different Methods.a HMDSmethod weight gated decoupler gated + relax INEPT
HMDSO/HMDS 1.000
0.100
1.007
0.108 0.108 0.107
1.008 1.004
0.010 0.017' 0.018' 0.016'
N/ HMDS
error:
1.Ooo
%
0.962 9.919
-7.4% -8.1%
0.803'
-19.7%
Compound abbreviations: HMDSO stands for hexamethyldisiloxane, HMDSN stands for hexamethyldisilazane, and HMDS stands for hexamethyldisilane. Ratio by weight is the true value, relative standard deviation of NMR integration f 2 % . Systematic error for disilazane (see text). 'The involved systematic error is due to circuit nonlinearity. This can be compensated for by calibration. larization period can degrade the signal only by 10% at worst. In practice, however, the line widths of all trimethylsilyl proton lines in a given sample are similar, so the relative error caused by relaxation is much smaller. On the other hand, the silicon concentration determined from signal intensities of compounds containing nitrogen or chlorine or with samples containing a paramagnetic impurity may be too low, by as much as 60% (corresponding to proton line width of 10 Hz). In simple mixtures with resolved proton lines, such fast relaxation of trimethylsilyl protons should be manifested by a considerable line broadening of the corresponding methyl proton line. The experimental results for simple mixtures (Table I) confirm these expectations. In siloxane mixtures, the molar ratios determined with INEPT enhancement agree with those determined by conventional NMR methods. In the silazane, the ratio determined by INEPT has a large systematic error of about -20 % . In qualitative applications it is required that the 93 NMR lines be assigned to the components of the mixture. Since none of the available experimental methods of line assignment (20-24) is applicable to mixtures, one has to resort to chemical shift comparison with model data. For such purposes, however, the shifts must be measured under comparable conditions. This condition cannot be met if one follows the experimental procedure suggested earlier (14-16) in which the spectra of the reaction mixture after silylation with variable amounts of excess reagents and byproducts were measured, using solvents as pyridine-d, (14) or benzene-d6 (16). We have obtained reproducible chemical shifts (with standard deviation of less than 0.05 ppm) only after the excess silylation reagents and solvent (pyridine) were removed in vacuo and the silylated product was dissolved in deuteriochloroform at a low concentration (a referee has pointed out, however, that deuteriochloroform might not dissolve all coal components). In some instances (methoxybenzene derivatives), reproducible shifts were obtained only at concentrations lower than 0.05 M. Measurements at such concentrations are impractical without INEPT enhancement. In quantitative applications, some authors (5-16) used "double referencing" one reference, tetramethylsilane (Me,Si), for chemical shifts and the other, benzoic acid, for integration reference. The two references can be replaced by a single compound, hexamethyldisilane (HMDS), which has several advantages. HMDS is easy to handle and can be weighted for integration referencing. With two silicon atoms in the molecule, HMDS can be used at a lower concentration. The compound is inert, and its chemical shift (6 -19.79 relative to Me4Si in CDC1,) is outside the range of shifts of the silylation products.
Anal. Chem. 1986, 58, 1894-1895
1894
Registry NO.HMDSO, 107-46-0;HMDSN, 999-97-3; HMDS, 1450-14-2;29Si,14304-87-1.
LITERATURE CITED Pierce, Alan E. S//yktbn of Organic Compounds; Pierce Chemical Company: Rockford, IL, 1988. Gebauit, G. M.; Berry, J. M.; Dutton, Guy G. S.; Gibney, K . 6. Anal. Len. 1972, 5 , 413-418. Dutton, Guy G. S.; Funnell, Norman; Glbney, Kelly 6. Can. J . Chem. 1972, 50, 3913-3916. Janssen, A.; Frleling, H.J.; Mellies, R. Z. Anal. Chem. 1977, 266, 239-243. Schwelghardt, F. K.; Retcofsky, H. L.; Friedman, S.; Hough, M. Anal. Chem. 1978, 50, 368-371. Sdvaml, Jan; Pola, Josef; Jancke, Harold; Engelhardt, Wnther; b m g , Mlroslav; Chvalovskjr, Viclav Collect. Czech. Chem. Commun. 1976, 4 7, 360-367. Levy, George C.; Cargioly, Joseph D. In Nuclear MEgneNc Resonance Specfroscopy of Nuclel Other Than Protons; Axenrod. T., Webb, A,, Eds.; Why: New York, 1974; pp 251-274. Gale, D. J.; Haines, Alan H.; Harris, Robin K. Org. M g n . Reson. 1975, 7 , 635-636. Haines, Alan H.; Harris, Robin K.; Rao, R e m C. Org. M a p . Reson. 1977, 9,432-437. Schraml. Jan; Pola, Josef; Chvalovskv, Viciav; Mersmann, Helnrlch C.; BI&ha, Karel Collect. Czech. Chem. Commun. 1977, 42, 1165-1 169. Gale, D. J.; Evans, N. A. Org. Magn. Reson. 1983, 27, 567-569. Schrarnl. Jan; Petrlkovi. Eva; P b r , Otomar; Hlrsch. d n ; Chvalovskjr, Viclav Org. MEgn. Reson. 1983, 2 1 , 666-669. Rose, Kenneth D.; Scouten, C. 0. A I P Conf. R o c . 1980, 70,82-100; Chem. Abstr. 1981, 95, 9677v. Coleman, W. M. 111; Boyd, Alvin R. Anal. Chem. 1982, 5 4 , 133-134.
(15) Dereppe. Jean-Marle; Parbhoc, Bhukandas Anal. Chem. 1984, 56, 2740-2743. (16) Seshadrl. Kakunte S.; Young, Donald C.; Cornauer. Donald C. Fuel 1985, 8 4 , 22-28. (17) Blhka. Thomas A.; Helmer. Bradley J.; West, Robert Adv. Organomet. Chem. 1984, 23, 193-218. (18) Morris. Gareth A. Top. Carbon-73 NMR Spectrosc. 1984, 4 , 179-1 96. (19) Scheneker, K. V.; von Philipsborn, W. J. Magn. Reson. 1985, 67, 294-305. (20) Schraml, Jan J. Magn. Reson. 1984, 59, 515-517. (21) Schraml, Jan; Larln, Michall F.; Pestunovlch, Vadim A. Collect. Czech. Chem. Commun. 1985, 50, 343-347. (22) Schraml. Jan; Petrikovi, Eva; K v b b v i , Magdalena; Chvalovskq, Vaclav J. Carbohydr. Chem. 1985, 4 , 393-403. (23) Past, Jaan; Pwkar, Juri; Schraml, Jan; Llppmaa, Endel Collect. Czech. Chem. Commun. 1985, 50, 2060-2064. (24) Past, Jaan; Puskar, Juri: Alla, Madls; Llppmaa, Endel; Schraml, Jan Magn. Reson. Chem. 1985. 23, 1076-1079.
J a n Schraml* Vratislav Blechta Magdalena KviEalovQ Lubomir Nondek VQclav Chvalovskf Institute of Chemical Process Fundamentals Czechoslovak Academy of Sciences Prague 165 02, Czechoslovakia
REXENEDfor review December 16,1985. Accepted March 11, 1986.
Determination of 1-Nitropyrene in Extracts of Vehicle Particulate Emissions Sir: We have developed a rapid and inexpensive procedure for the routine quantitation of 1-nitropyrene (1-NP), a nitroarene found in vehicle exhaust particulate extracts and other combustion sources. The motive for this work is that nitroarenes are an important class of compounds from the standpoint of mutagenicity and that 1-nitropyrene, as one of the more abundant nitroarenes in vehicle exhaust, is a useful index compound for that class. The procedure uses two disposable liquid chromatography columns to clean up and preconcentrate the vehicle exhaust particulate extract. Isolation of the 1-NP fraction is achieved by using a disposable silica column to remove polar Components and a Florisil column to remove aliphatic components in the extract. The concentrated sample is then analyzed on a capillary column gas chromatograph with electron capture detection (GC/ ECD). The preconcentration process was shown to have a 1-NP recovery of 96.3 f 9.5%. Standard solutions containing 1.25-26 ng of 1-NP in the 0.5-pL GC injection are reproduced with an error of
