Anal. Chem. 2004, 76, 392-398
Application of Nonselective 1D 1H-31P Inverse NMR Spectroscopy to the Screening of Solutions for the Presence of Organophosphorus Compounds Related to the Chemical Weapons Convention Urs C. Meier*
Swiss NBC Defence Establishment, Spiez Laboratory, 3700 Spiez, Switzerland
1D nonselective 1H-31P HSQMBC, HSQC, and 31P decoupled HSQC NMR experiments were applied to the screening of original OPCW proficiency test samples for the presence of organophosphorus (OP) compounds related to the Chemical Weapons Convention. The HSQC and HSQMBC spectra are compared to 1D 1H NMR spectra with WET solvent suppression and 31P{1H} spectra of the same samples. The 1D nonselective HSQC and HSQMBC experiments are shown to be the most sensitive NMR experiments to selectively screen samples for the presence of organophosphorus(OP) compounds. These experiments are at least three times more sensitive than the 31P{1H} NMR experiment and allow the determination of the number of OP compounds present in the sample and their alkyl group bound to the phosphorus atom. Samples spiked at the 5-10 ppm level can be screened within an hour for the presence of OP compounds, whereas for the 31P{1H} experiments, an overnight acquisition is necessary. The sensitivity of the experiments decreases in the order 31P decoupled HSQC, HSQMBC, and HSQC. For the different alkyl groups, the sensitivity of these experiments decreases in the order methyl ∼ isopropyl > ethyl > propyl. The Chemical Weapons Convention (CWC) for the prohibition of the development, production, stockpiling, and use of chemical weapons entered into force in 1997.1 Ever since, the Technical Secretariat of the Organization for the Prohibition of Chemical Weapons (OPCW) has organized interlaboratory proficiency tests in order to designate laboratories for the verification of the CWC.2 Within an OPCW proficiency test, participating laboratories have 15 days to identify and report the substances spiked at trace levels in different matrixes, such as water, soil, paint, etc. Because of its high sensitivity, mass spectroscopy coupled to GC3-5 and LC3,6 is most frequently used for the identification of the spiked * To whom correspondence should be addressed. Phone: +41 33 228 16 92. Fax: +41 33 228 14 02. E-mail.
[email protected]. (1) Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction; Technical Secretariat of the Organisation for the Prohibition of Chemical Weapons: The Hague, 1997. (2) Hulst, A. G.; deJong, A. L.; deReuver, L. P.; van Krimpen, S. H.; van Baar, B. L. M.; Wils, E. R. J.; Kientz, C. E. TRAC 2002, 21, 116-130. (3) Hooijschuur, E. W. J.; Kientz, C. E.; Brinkman, U. A. T. J. Chromatogr., A 2002, 982, 177-200.
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compounds, whereas GC-FTIR7,8 and NMR9,10 spectroscopy are less frequently used. Most chemicals related to the CWC are organophosphorus (OP) compounds. 1H and 31P{1H} NMR spectroscopy are routinely used for the screening of samples for the presence of OP compounds. The samples are usually heavily polluted by chemicals not related to the CWC. Often, the signals from these chemicals partially or completely mask the signals of OP compounds in the 1H NMR spectra. 31P{1H} NMR spectroscopy has the disadvantage of poor information content and sensitivity, as compared to 1H NMR spectroscopy. Therefore, experiments able to selectively detect signals from OP compounds with high sensitivity and eliminating background signals would be ideal for screening samples for the presence of OP compounds. In practice, inverse detected NMR experiments are close to this ideal experiment. Inverse 1H-31P experiments exclusively detect signals from protons coupled to phosphorus nuclei with the sensitivity of 1H NMR. For several years, a variety of 2D inverse experiments have been applied to the structure elucidation of unknown compounds.11,12 These 2D experiments correlate the 1H signals with the signals from heteronuclei. 13C and 15N were almost exclusively used as heteronuclei. Experiments with 31P as heteronuclei are rare, since most OP compounds contain only one phosphorus atom but several carbon or nitrogen atoms.10,13 The construction of a 2D correlation map requires the acquisition of many increments, which is a time-consuming process. Additionally, the resolution of 2D spectra is poor compared to 1D spectra.14 Therefore 2D 1H-31P inverse experiments are not well-suited for (4) Stuff, J. R.; Creasy, W. R.; Rodriguez, A. A.; Dupont Durst, H. J. Microcolumn Sep. 1999, 11, 644-651. (5) Creasy, W. R.; Brickhouse, M. D.; Morrissey, K. M.; Stuff, J. R.; Cheicante, R. L.; Ruth, J.; Mays, J.; Williams, B. R. Environ. Sci. Technol. 1999, 33, 2157-2162. (6) Black, R. M.; Read, R. W. In Encyclopedia of Analytical Chemistry; Mesilaakso, M., Ed.; Wiley: New York, 2000, pp 1007-1025. (7) So¨derstro¨m, M. T.; Bjo¨rk, H.; Ha¨kkinen, V. M. A.; Kostiainen, O.; Kuitunen, M. L.; Rautio, M. J. Chromatogr., A 1996, 742, 191-293. (8) Creasy, W. R.; Stuff, J. R.; Williams, B.; Morrissey, K.; Mays, J.; Duevel, R.; Durst, H. D. J. Chromatogr., A 1997, 774, 253-263. (9) Mesilaakso, M.; Tolppa, E. L. Anal. Chem. 1996, 68, 2313-2318. (10) Albaret, C.; Lœillet, D., Auge´ P.; Fortier, P. L. Anal. Chem. 1997, 69, 26942700. (11) Bodenhausen, G.; Ruben, D. J. Chem. Phys. Lett. 1980, 69, 185-189. (12) Norwood, T. J.; Boyd, J.; Campbell, I. D. FEBS Lett. 1989, 255, 369-371. (13) Morris, G. A.; Freeman, R. J. Magn. Reson. 1978, 29, 433-435. (14) Becker, E. D.; High-Resolution NMR; 3rd ed.; Academic Press: New York, 2000 10.1021/ac0350099 CCC: $27.50
© 2004 American Chemical Society Published on Web 12/09/2003
Figure 1. Pulse sequences used in the NMR experiments, HSQMBC (a) and HSQC (b). 90° and 180° pulses are indicated as gray and black bars.
the screening of samples for the presence of OP compounds. 1D 1H-31P inverse experiments are better suited, since only one increment is required, and the same resolution as in 1D 1H spectra is achieved.15 The only drawback is the loss of the correlation information between the 1H and 31P signals. But this drawback is not severe. Since all OP compounds related to the CWC, with the exception of the pyrophosphates, contain only one phosphorus atom, the number of OP compounds present in a sample can still be deduced from the signals present in the spectra of 1D 1H-31P inverse experiments. Recently, nonselective 1D inverse detected 1H-15N HSQCTOCSY and HSQMBC experiments were published by Parella and Belloc.16 In the present paper, the HSQC and HSQMBC experiments were adapted to 31P and the 1D 1H-31P HSQC, and HSQMBC experiments were applied to original OPCW proficiency test samples and their spectra, as compared to the standard 1D 1H spectra with WET solvent suppression17 and 31P{1H} spectra of the same samples. The 1D nonselective HSQC and HSQMBC experiments are shown to be the most sensitive NMR methods to screen samples for the presence of OP compounds. EXPERIMENTAL SECTION NMR. The HSQC and HSQMBC pulse sequences used in this study are shown in Figure 1. The HSQCTOCSY pulse sequence published by Parella and Belloc16 was slightly modified to optimize the sensitivity of the experiment for the screening of samples for (15) Parella, T. Magn. Reson. Chem. 1996, 34, 329-347. (16) Parella, T.; Belloc, J. Magn. Reson. Chem. 2002, 40, 133-138. (17) Smallcomb, S. H.; Patt, S. L.; Keifer, P. A. J. Magn. Reson. A 1995, 117, 295-303.
the presence of OP compounds. The TOCSY block and the sensitivity enhancement were omitted. The TOCSY reduces the sensitivity by the transfer of magnetization to other protons through scalar couplings and T2 relaxation, and the sensitivity enhancement exclusively doubles the sensitivity of the methine signals, whereas the sensitivity for the methylene and methyl signals is decreased due to T2 relaxation.18 The measurements were carried out in 5 o.d. NMR tubes (Armar UP) at 28 °C on a Varian Inova 500 NMR spectrometer equipped with a broadband probe and 1H and 31P frequencies of 499.83 and 202.34 MHz, respectively. 1H-31P HSQC and HSQMBC spectra were recorded without additional solvent suppression. Pulse widths of 90° for 1H and 31P of 6.4 and 8.4 µs, an acquisition time of 1 s, and a relaxation delay of 2.5 s were used. A total of 13426 complex points were acquired and zero-filled to 64 k, and an exponential filter of 1.1 Hz was applied before Fourier transformation. The gradients G1 and G2 have the same gradient strength but are of opposite sign. The gradient ratio G1/G3 is given by γ1H/2γ31P to inversely detect 31P. A gradient width of 0.750 ms was used for all gradients. The delay ∆ was set to 0.25 JPH. Two- and three-bond JPH coupling constants were usually in the range of 15-22 Hz. A JPH of 18 Hz was used for the HSQC and HSQMBC experiments if not indicated otherwise. 1H NMR spectra were recorded with a 1.7-µs (21°) pulse width, a sweep width of 6712 Hz, and a repetition rate of 4.2s. The WET technique was used for solvent suppression.17 An exponential filter of 1.1 Hz was applied to the FID before Fourier transformation. Water and organic samples were referenced, respectively, to the sodium salt of 3-(trimethylsilyl)-1-propanoic acid-d4 (TSPA, δH ) 0.00 ppm) and tetramethylsilane (TMS, δH ) 0.00 ppm). Proton-decoupled 31P NMR spectra (31P{1H}) were acquired using a 5-µs (45°) pulse width, a sweep width of 30 kHz, and a repetition rate of 1.82s. An exponential filter of 2.0 Hz was applied to the FID before Fourier transformation. The spectra were referenced to external H3PO4 (δP ) 0.00 ppm). Samples. The organic liquid for the first official proficiency test in 1996 contained three OP compounds related to the chemical warfare agent tabun that were spiked at the 50 ppm level: 19 ethyl N,N-dimethylphosphoramidocyanidate (I) or tabun, diethyl N,N-dimethylphosphoramide (II), and (dimethylamino)phosphoryl dichloride (III) (Chart 1). Chlorobenzene and petroleum were added as background chemicals. The sample was stored in a freezer at -18 °C since 1996. No degradation of the three compounds could be detected. A 150-µL portion of C6D6 (Armar 99.95% D) was added to 500 µL of the organic liquid, resulting in a 38.5 ppm spiking level for each compound. The soil sample for the second official proficiency test in 1996 was spiked with three OP compounds: diethyl isopropylphosphonate (IV), ethyl 2-methoxyethyl isopropylphosphonate (V), and isopropyl 2-methoxyethyl methylphosphonate (VI)20 (Chart 1). Diesel oil and CH2Cl2 were added as background chemicals. A (18) Palmer, A. G.; Cavanagh, J.; Wright, P. E.; Rance, M. J. Magn. Reson. 1991, 93, 151-170. (19) Bruce, P.; Laudares, M. Preliminary Evaluation of Results, 1st Official OPCW/ PTS Inter-laboratory Comparison Test: Proficiency Test; Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons, 1996. (20) Evaluation of the Results of the 2nd Official Proficiency Test; Technical Secretariat of the Organisation for the Prohibition of Chemical Weapons, 1997; Vols. 1, 2.
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Chart 1. Spiked OP Compoundsa
a
Protons are indicated by letters.
2.3-g portion of the soil was extracted with two 10-mL portions of CH2Cl2, filtered, concentrated, and dissolved in 650 µL of CDCl3 (Armar 99.95% D). The samples were stored in a refrigerator at 4 °C since 1996. No decomposition of IV, V, and VI was observed. The water sample (W) for the second official proficiency test in 1996 contained isopropyl ethylphosphonate (VII) and ethylphosphonic acid (VIII) (Chart 1) spiked at the 10 ppm level.20 Poly(ethylene glycol) and CH2Cl2, respectively, were added as background chemical and stabilizer to prevent growth of algae. The samples were stored in a refrigerator at 4 °C since 1996. No decomposition of VII and VIII was observed. A 150-µL portion of D2O was added to 500 µL of the water sample, resulting in a spiking level of 7.7 ppm for both compounds. The water (W2) sample for the third official proficiency test in 1997 was spiked with isopropylphosphonic acid (IX) and 2-ethylhexyl methylphosphonate (X) (Chart 1) spiked at the 8.8 and 12 ppm levels, respectively.21 The sample was stored in a refrigerator at 4 °C since 1997. No decomposition of IX and X was observed. A 150µL portion of D2O was added to 500 µL of the water sample, resulting in a spiking level of 6.4 ppm for IX and 9.4 ppm for X. RESULTS Organic Liquid. Figure 2a shows the 1H WET spectrum of the organic liquid sample from the first OPCW proficiency test after 256 scans. Three doublets are observed in the range from 2.35 to 2.59 ppm. The 1H-31P HSQC experiment (Figure 2b), optimized for 3JPH ) 11 Hz, displays three doublets at 2.36, 2.38, and 2.57 ppm with 3JPH of 11.3, 15.8, and 9.8 Hz. This confirms the presence of three OP compounds and that the doublets observed in the 1H WET spectrum between 2.35 and 2.57 ppm are due to the coupling between protons and different phosphorus nuclei. Since the proton signals from alkylester groups coupling with a phosphorus nuclei would exclusively appear in the range from 3.5 to 4.5 ppm and proton signals from alkyl groups directly bonded to a phosphorus nuclei would appear below ∼2 ppm, the (21) Evaluation of the Results of the 3rd Official Proficiency Test; Technical Secretariat of the Organisation for the Prohibition of Chemical Weapons, 1997; Vols. 1 and 2.
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Figure 2. 1H WET (a), HSQC (b), 31P-decoupled HSQC (c), HSQMBC (d), and 31P{1H} (e) spectra from the organic liquid containing ethyl N,N-dimethylphosphoramidocyanidate (I), diethyl N,Ndimethylphosphoramide (II), and (dimethylamino)phosphoryl dichloride (III) spiked at the 37.5 ppm level after 256, 256, 256, 256, and 2048 scans, respectively.
three doublets are assigned to three OP compounds containing a dimethylphosphoramido group. The doublets at 2.36, 2.38, and 2.57 ppm correspond to the H-a signals of I, III and II, respectively, as determined by comparison with spectra of the authentic reference compounds. In agreement with the presence of three OP compounds, the 31P-decoupled 1H-31P HSQC spectrum (Figure 2c) displays three singlets at 2.36, 2.38, and 2.57
ppm. Three antiphase doublets at 2.36, 2.38, and 2.57 ppm with 3J 1 31 PH of 11.3, 15.8, and 9.8 Hz are observed in the H- P HSQMBC spectrum (Figure 2d). Since exclusively signals of protons coupled scalarly to phosphorus nuclei are present in HSQC and HSQMBC spectra, only signals from OP compounds are detected. This avoids the necessity of measuring a blank, followed by a comparison with the sample spectrum to figure out signals from OP compounds, as in the case of 1H NMR spectra. Additionally, because of the selectivity of the HSQC and HSQMBC experiments, the background signals are effectively suppressed. As an example, the background signals due to the petroleum present in the 1H WET spectrum (broad envelope up to 2.25 ppm) are completely eliminated in the inverse experiments (Figure 2b-d). The ability of the HSQC and HSQMBC spectra to suppress background and solvent signals is the same. In both cases, no additional solvent suppression is required, since the residual solvent signal and the signals of the OP compounds are of similar magnitude. The 31P{1H} spectrum (Figure 2e) shows three peaks at -8.94, 11.89, and 18.09 ppm, which are assigned to I, II, and III, respectively. The 31P chemical shift range from -15 to 20 is typical for dialkylphosphoramidates. The signal-to-noise ratios (S/N) of I, II, and III in the 31P{1H} after 2048 scans (62 min) are at least 5 times lower than the S/N of the protons signals H-a of I, II, and III in the HSQC, 31P-decoupled HSQC, and the HSQMBC spectra (Figure 2b-d) after 256 scans (15 min). This clearly demonstrates the sensitivity advantage of the inverse detection of 31P, as compared to the direct observation of 31P. The 31P-decoupled HSQC is the most sensitive experiment, followed by the HSQMBC and HSQC experiments. Soil Sample Extracted with CH2Cl2. Figure 3a shows the 1H WET spectrum from 0.5 to 4.55 ppm of the dichloromethane extract of the soil sample from the second OPCW proficiency test. The background produced by the diesel oil masks all the signals except the methyl doublet of VI at 1.49 ppm with 2JPH ) 17.6 Hz. No signals can be attributed to the isopropylphosphonates IV and V. The 1H-31P HSQC spectrum is shown in Figure 3b. The background signals due to the diesel oil are now completely removed. The HSQC spectrum displays a doublet at 1.49 ppm with 2JPH ) 17.6 Hz, suggesting the presence of a methylphosphonate. In addition, complex multiplets between 1.12 and 1.25 ppm, 1.90 and 2.07, and 4.06 and 4.22 ppm are displayed. On the basis of the intensity ratios of the signals in the ranges from 1.12 to 1.25 ppm and 1.90 to 2.07 ppm and their signal patterns, the presence of an ethyl group bound to the phosphorus atom can be ruled out. In case of an ethyl group, the intensity ratio between the signals of the methyl and methylene groups in HSQC spectra would be ∼2:1, and the patterns in the 1.90-2.05 ppm and 1.121.25 ppm ranges do not resemble, respectively, a doublet of quadruplets for the methylene and a doublet of triplets for the methyl signals of an ethyl substituent (see below). In addition, the range from 1.90 to 2.05 ppm is too much low-field-shifted for the methylene signals of an ethyl group. In case of an n-propyl group, the two methylene signals are well-separated, and three signals would be observed.22 Therefore, the presence of an n-propyl group can be ruled out, too. The methyl signals H-b of (22) The Central OPCW Analytical Database; Technical Secretariat of the Organisation for the Prohibition of Chemical Weapons: The Hague, 2001; Version 4.
Figure 3. 1H WET (a), HSQC (b), 31P-decoupled HSQC (c), HSQMBC (d), and 31P{1H} (e) spectra from the CH2Cl2 extract from the soil sample containing diethyl isopropylphosphonate (IV), isopropyl 2-methoxyethyl isopropylphosphonate (V), and ethyl 2-methoxyethyl methylphosphonate (VI) after 1024, 1024, 1024, 1024, and 2048 scans, respectively.
IV and V are expected to appear as a doublet of doublets. In case of a hindered rotation about the P-Ca bond (Chart 1), the two methyl groups of the isopropyl moiety become nonequivalent, and the number of observable peaks in the spectrum is doubled. Therefore, the observation of 12 peaks in the 1.12-1.25 ppm region of the HSQC spectrum (Figure 3b) suggests the presence of more than one, probably two, isopropylphosphonates. The signals H-a of IV and V appear in the range from 1.90 to 2.05 ppm. The higher multiplicity (doublet of heptuplet) and the smaller number of protons of a methine group, as compared to the methyl groups, result in a considerably lower intensity of the methine H-a signals of IV and V compared to the methyl signals H-b of IV and V. The 31P-decoupled HSQC (Figure 3c) spectrum shows a singlet at 1.49 ppm (H-a of IV), six lines in the 1.121.25 ppm range, and eight lines in the 1.90-2.05 ppm region, in agreement with the presence of one methyl- and two isopropylphosphonates. The 1H-31P HSQMBC (Figure 3d) spectrum displays an antiphase doublet at 1.49 ppm with 2JPH ) 17.6 Hz and complex antiphase doublet patterns in the ranges of 1.121.25 and 1.90-2.05 ppm. The intensity of the signal in the 1.902.05 ppm range in the HSQMBC spectrum is considerably reduced, as compared to the HSQC (Figure 3b and c) spectra, as a result of partial cancellation of the two overlapping antiphase doublets of heptuplets. The intensity of the H-b signals of IV and V in the HSQMBC spectrum are also reduced, as compared to the HSQC spectra, but to a lesser extent than the H-a signals of IV and V. After 2048 scans (62 min), the 31P{1H} spectrum Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
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Figure 4. 1H WET (a), HSQC (b), 31P-decoupled HSQC (c), HSQMBC (d), and 31P{1H} (e) spectra from the water sample W containing isopropyl ethylphosphonate (VII) and ethylphosphonic acid (VIII) spiked at the 7.7 ppm level after 1024, 2048, 2048, 4096, and 30 000 scans, respectively.
(Figure 3e) displays three peaks at 30.21, 35.37, and 35.98, which are assigned to VI, IV, and V, respectively. The chemical shifts suggest that alkylphosphonates are present but do not allow for the identification of the alkyl group bonded to the phosphorus atom. The S/N ratio of the HSQC, 31P-decoupled HSQC, and HSQMBC spectra (Figure 3a-d) after 1024 scans (60 min) are at least three times higher than the S/N of the 31P{1H} experiment after 2048 scans (62 min). This clearly demonstrates the sensitivity advantage of the inverse, proton-based detection of 31P, as compared to the direct detection of 31P by a 31P{1H} experiment, even when using a broadband probe optimized for the detection of heteronuclei. The 31P-decoupled HSQC is again the most sensitive inverse experiment. For H-a of VI, the sensitivity of HSQC and HSQMBC is the same, whereas for H-a and H-b of IV and V, the HSQMBC is less sensitive than the HSQC experiment. Water Sample W. The water sample from the second OPCW proficiency test contains isopropyl ethylphosphonate (VII) and ethylphosphonic acid (VIII) as spiked OP compounds. Figure 4a shows the WET spectrum. Multiplets occur between 0.97 and 1.10, 1.36 and 1.48, and 1.52 and 1.63 ppm, and a doublet occurs at 1.26 ppm with JHH ) 6.1 Hz, which is typical for the methyl signal of an isopropyl ester group. On the basis of the coupling constant of 6.1 Hz, the possibility that the doublet is due to the coupling between methyl protons and a phosphorus atom can be ruled out, since 2JPH would be larger than 15 Hz. The methylene signals H-a of VII and VIII are expected to appear as doublets of quadruplets because of the coupling with a phosphorus atom (2JPH) and a methyl group (JHH, H-b of VII and VIII). The multiplets between 1.36 and 1.48 ppm and 1.52 and 1.63 ppm corresponding to H-a 396 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
of VII and VIII, respectively, display six lines in approximately a 1:3:4:4:3:1 intensity ratio. This intensity pattern corresponds to a doublet of quadruplets with 2JPH ∼ 22JHH. In this case, the two quartets with a 1:3:3:1 intensity distribution will combine to a sextuplet with a 1:3:4:4:3:1 intensity ratio. The multiplet between 1.52 and 1.62 ppm is also a sextuplet, but its intensity pattern is disturbed because of a partial overlap with background signals. Therefore, the two sextuplets centered at 1.42 and 1.58 ppm provide evidence for the presence of two OP compounds with an ethyl group bonded to the phosphorus atom. The complex multiplet from 0.97 to 1.10 ppm is in agreement with the presence of VII and VIII, and its pattern is due to the overlap of two doublets of triplets corresponding to H-b of VII and VIII. Figure 4b shows the HSQC spectrum of the water sample. Two sextuplets centered at 1.42 and 1.58 ppm and a complex multiplet from 0.95 to 1.10 ppm are displayed. The patterns of the multiplets are similar to the patterns in the WET spectrum, further supporting the presence of two ethylphosphonates. The doublet at 1.26 ppm, present in the WET spectrum, disappeared as a result of the absence of a coupling to a phosphorus nucleus. In the 31Pdecoupled HSQC experiment (Figure 4c), the two sextuplets are reduced to two quadruplets at 1.42 and 1.58 ppm with JHH ) 8.1 and 8.0 Hz. The intensity distribution of the lines is close to the expected 1:3:3:1 ratio. This unambiguously confirms the presence of two ethylphosphonates. Two antiphase doublets of quadruplets centered at 1.42 and 1.58 ppm are displayed in the HSQMBC spectrum (Figure 3d). Due to an overlap of the two antiphase doublet of triplets (H-b of VII and VIII), the intensity ratio between the H-b and H-a signals of VII and VIII is considerably lower than in the HSQC spectra. The 31P{1H} spectrum after 30000 scans (Figure 4e) displays two peaks at 25.31 and 29.70 ppm assigned to VIII and VII, respectively. The S/N ratio in the HSQC and 31P decoupled HSQC spectra after 2048 (2 h) scans and the HSQMBC spectrum after 4096 (4 h) scans is at least twice the S/N in the 31P{1H} spectrum after 30 000 (15 h) scans. A comparison of the S/N ratio of VII and VIII in the WET, HSQC, 31P-decoupled HSQC, and HSQMBC spectra (Figure 4a-d) after 1024, 2048, 2048, and 4096 scans, respectively, shows the WET experiment to be the most sensitive experiment, followed by the 31P-decoupled HSQC, the HSQC, and the HSQMBC experiment. Water Sample W2. The water sample from the third official OPCW proficiency test contains isopropylphosphonic acid (IX) and 2-ethylhexyl methylphosphonic acid (X) as spiked OP compounds. The WET spectrum (Figure 5a) shows a doublet of doublets at 1.09 ppm with 3JPH ) 16.4 and JHH ) 7.2 Hz and a partially overlapped doublet at 1.31 ppm. In the 1H-31P HSQC experiment (Figure 5b), the doublet at 1.31 ppm with 3JPH ) 16.2 Hz (H-a of X) and the doublet of doublets at 1.09 ppm with 3JPH ) 16.4 and JHH ) 7.2 Hz (H-b of IX), respectively, prove the presence of a methylphosphonate (X) and isopropylphosphonic acid (IX). In the 31P-decoupled HSQC (Figure 5c) spectrum, a singlet at 1.31 and a doublet at 1.09 with JHH ) 7.2 Hz are observed, which are assigned to H-a of X and H-b of IX, respectively. The signals H-b of IX and H-a of X appear as an antiphase doublet of doublets at 1.09 and a doublet at 1.31 ppm in the HSQMBC spectra (Figure 5d). Two peaks at 26.95 and 29.3 ppm are observed in the 31P{1H} spectrum (Figure 5e), which are assigned to X and IX, respectively. The S/N ratio in the HSQC,
Figure 5. 1H WET (a), HSQC (b), 31P-decoupled HSQC (c), HSQMBC (d), and 31P{1H} (e) spectra from the water sample W2 containing isopropylphosphonic acid (IX) and 2-ethylhexyl methylphosphonate (X) spiked at the 6.4 and 9.4 ppm levels after 2048, 2048, 2048, 2048, and 30 000 scans, respectively.
31P-decoupled
HSQC, and HSQMBC spectra after 2048 (2 h) scans is at least three times higher than in the 31P{1H} spectrum after 30 000 (15 h) scans. A comparison of the S/N ratio of H-b of IX and H-a of X in the WET, HSQC, 31P-decoupled HSQC, and HSQMBC spectra (Figure 5a-d) after 2048 scans shows the WET experiment to be more sensitive than the 31P-decoupled HSQC experiment, followed by the HSQMBC and HSQC experiments.
DISCUSSION The application of 1H NMR spectroscopy to the analysis of environmental samples is often hampered by background signals, which partially or completely mask the signals of interest, limiting the application of 1H NMR spectroscopy in the identification of OP compounds. In 1H-31P inverse NMR experiments (Figure 2-5), the strong background signals are effectively eliminated, and hidden signals become visible as a result of the coherence selection by gradients. The sensitivity of the method is exclusively determined by the amount of spiked OP compound, whereas in 1D 1H NMR, the sensitivity is often limited by the background signals present rather than the amount of OP compounds spiked.9 The sensitivity of the inverse experiments decreases in the order methyl ∼ isopropyl > ethyl > propyl as a result of the higher multiplicity of ethyl (doublet of quartet and doublet of triplet) and propyl (higher order coupling pattern for the two methylene groups and a small 4JPH of ∼2 Hz for the methyl group) signals, as compared to the doublet and the doublet of doublet structures
of the methyl and isopropyl groups, respectively. All the examples presented show the 31P-decoupled HSQC to be the most sensitive experiment, followed by the HSQMBC and the coupled HSQC. However, in the case of an extensive overlap, the coupled HSQC is more sensitive than the HSQMBC. For methyl, ethyl, and isopropyl substituents, all inverse experiments are at least 3 times more sensitive than the 31P{1H} experiment per unit time. For propyl groups, the sensitivity advantage is ∼2. In the case of propyl substituents, optimization of the experiments for 4JPH is better than for 2JPH and 3JPH (see Figure S1 and Table S1 of the Supporting Information). Samples containing OP compounds spiked at the 5-10 ppm level can be screened for their presence within an hour, whereas for the 31P{1H} experiment, an overnight acquisition is required. The use of a probe optimized for the inverse detection of heteronuclei would further increase the sensitivity advantage of the inverse experiments by a factor of 2-3, as compared to the direct detection of 31P with a broadband probe.23 Once the pulse widths, gradients, and the interpulse delays are set up properly, these experiments can be used on a routine basis. Since the coherence selection is achieved by the use of gradients, the application of special solvent suppression techniques, such as presaturation or WET,17 is not required. The measurement of a sample and a blank spectrum, followed by a tedious comparison of both spectra to figure out the signals of OP compounds, as in case of standard 1D 1H spectroscopy,9,10 is not necessary for screening purposes because of the selectivity of the 1H-31P inverse experiments. The signals from the ester groups, with the exception of methyl ester groups, are less suited for screening because 3JPH,ester is ∼10 Hz, as compared to 15-22 Hz for 2JPH and 3JPH for alkyl groups bonded to a phosphorus atom, and consequently, the losses due to relaxation are increased. Furthermore, the signals of ethyl ester or larger alkylester groups often exhibit a complex, non-first-order pattern10,22 that further reduces the sensitivity of the experiment, whereas for alkyl groups bound to a phosphorus atom, only the signals of two methylene groups of the propyl substituent exhibit non-first-order patterns. Ester groups are rather useful for identification purposes by selective and nonselective 1D 1H-31P inverse experiments. In this case, it could be useful to optimize the magnetization transfer for 3JPH,ester, followed by a mixing process (TOCSY, COSY, etc.) to spread the magnetization over the entire alkyl substituent. In addition, the structure of the alkyl substituent at the phosphorus atom can be determined. This is especially important for the distinction of n-propyl and isopropyl substituents, which cannot be differentiated by mass spectrometry using standard electron impact or chemical ionization techniques.24 The information about the exact structure of the alkyl substituent is important, since according to the OPCW criteria for the identification of OP compounds related to the CWC, the identification of the alkyl group bonded to the phosphorus atom is mandatory, whereas for the ester groups, generic reporting is considered sufficient.25 (23) Bruker NMR Accessories and Supplies Catalog, Int. ed.; Part No. 92367, BII4/ 99; p 2. (24) McLafferty, F. W. Tandem Mass Spectrometry; Wiley-Interscience: New York 1983. (25) International Interlaboratory Comparison (Round Robin) Test for Verification of Chemical Disarmament. In Methodology and Instrumentation for Sampling and Analysis in the Verification of Chemical Disarmament; Rautio, M., Ed.; The Ministry for Foreign Affairs of Finland: Helsinki, 1994; Vol. F.
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Owing to the high resolution of the spectra of 1D inverse experiments, small chemical shift differences are sufficient to distinguish between different OP compounds present having the same alkyl substituent (Figure 2). Since the 1D HSQC experiment delivers pure absorption-phase spectra, the signals in HSQC and standard 1D 1H spectra are very similar, allowing for a facile identification of the alkyl substituent in HSQC spectra (Figures 2, 4, and 5). The differences between the standard 1D 1H and HSQC spectra are due to the presence of some antiphase components. The magnitude of these components depends on the mismatch between the JPH present in the OP compounds of the sample and the JPH used for the HSQC experiment. This may prevent 1H-31P HSQC spectra from being phased completely into absorption mode. Despite the pure absorption antiphase signals produced by the HSQMBC experiment, the identification of the alkyl substituents of different OP compounds is straightforward if the various signals are well-separated (Figures 2 and 5). In the case of overlapping signals, the identification of the different OP compounds is more troublesome than in HSQC and 1D 1H spectra, since the antiphase character of the signals in the HSQMBC spectrum leads to partial cancellation of the different signals. This results in more complex multiplet patterns and reduces the sensitivity of the experiment (Figures 3b-d and 4b-d). (26) Serber, Z.; Richter, C.; Moskau, D.; Bohlen, J. M.; Gerfin, J. M.; Marek, D.; Ha¨berli, M.; Baselgia, L.; Laukien, F.; Stern, A. S.; Hoch, J. C.; Dotsch, V. J. Am. Chem. Soc. 2000, 122, 3554-3555. (27) Keun, H. C.; Beckonert, O.; Griffin, J. L.; Richter, C.; Moskau, D.; Lindon, J. C.; Nicholson, J. K. Anal. Chem. 2002, 74, 5488-4593. (28) Spraul, M.; Freund, A. S.; Nast, R. E.; Withers, R. S.; Maas, W. E.; Corcoran, O. Anal. Chem. 2003, 75, 1536-1541.
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The 1D 1H-31P inverse and the 31P{1H} experiments provide complementary information. The 31P chemical shift in 31P{1H} experiments provides information about the type of OP compound, that is, VX, fluoridates, phosphonate, phosphoroamidates, etc., whereas the inverse experiments allow for the determination of the alkyl substituent at the phosphorus atom. Information about the type of OP compound is limited to tabun and its derivatives for inverse detected experiments, since exclusively the alkyl amide signals appear in the 1H chemical shift range from 2.5 to 3.5 ppm. In the future, the use of cryoprobes with their 4-5-fold increased sensitivity, meaning 16-25 times shorter measurement times, as compared to conventional NMR probes,26-28 the screening of samples for the presence of OP compounds spiked at the 10 ppm level will be close to a single shot experiment at 500 MHz or higher frequencies using 1D 1H-31P inverse detected experiments. SUPPORTING INFORMATION AVAILABLE 1H WET, 31P{1H} and 1H-31P HSQC spectra optimized for J PH ) 18 and 2 Hz of a 50 ppm solution of propylphosphonic acid and the corresponding signal-to-noise ratios are available in Figure S1 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review August 28, 2003. Accepted November 5, 2003. AC0350099
