Anal. Chem. 1997, 69, 3832-3837
Application of On-Line HPLC-1H NMR to Environmental Samples: Analysis of Groundwater near Former Ammunition Plants Markus Godejohann and Alfred Preiss*
Fraunhofer-Institut fu¨ r Toxikologie und Aerosolforschung, Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany Clemens Mu 1 gge
Humboldt-Universita¨ t zu Berlin, Institut fu¨ r Chemie, Hessische Strasse 1-2, D-10115 Berlin, Germany Gerold Wu 1 nsch
Universita¨ t Hannover, Institut fu¨ r Anorganische Chemie, Lehrgebiet Analytische Chemie, Callinstrasse 9, D-30167 Hannover, Germany
Coupling of HPLC to NMR was applied for the first time to the analysis of environmental samples, i.e., water samples from an ammunition hazardous waste site. Using the continuous flow mode at very low flow rates (e0.017 mL/min) and large volume injection (400 µL), the confirmation of many nitroaromatic compounds could be achieved down to the microgram-per-liter level after solid phase extraction of a groundwater sample from a former ammunition production site. At a flow rate of 0.006 mL/ min, it is possible to detect less than 29 nmol (5 µg) of 1,3-dinitrobenzene injected on a 75 mm × 4 mm reversed phase C-18 column (particle size, 5 µm). The results obtained by HPLC-NMR are compared to those obtained by HPLC-PDA (photodiode array) of the same sample, demonstrating that many more compounds can be identified by the former compared to the latter method as a result of coelution of major and minor components in the HPLC chromatogram. HPLC-NMR is a relatively new analytical method1,2 (LC probe heads have been commercially available since the early 1990s) which generally is used to separate a target compound from a mixture to allow the unequivocal interpretation of the NMR spectrum of the pure compound. In the past, this method was applied in particular to the determination of drug metabolites in biofluids like urine or blood plasma.3 Other applications deal with the analysis of wine concentrate, polymeric mixtures, and fuels or with the photoisomerization of azadirachtin, a natural insecticide.4 This method has not yet been applied to environmental problems. Such an application is presented here for the first time. The NMR detector offers unique information for the identification of unknown compounds or the confirmation of already known (1) Watanabe, N.; Niki, E. Proc. Jpn. Acad. Ser. B 1978, 54, 194. (2) Bayer, E.; Albert, K.; Nieder, M.; Grom, E.; Keller, T. J. Chromatogr. 1979, 186, 497-507. (3) Lindon, J. C.; Nicholson, J. K.; Wilson, I. D. Advances in Chromatography; Marcel Dekker: New York, 1996; Vol. 36, pp 315-382. (4) Korhammer, S. A.; Bernreuther, A. Fresenius J. Anal. Chem. 1996, 354, 131-135.
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compounds. Unfortunately, NMR spectroscopy is a “low-sensitivity” method and is also time-consuming compared with other methods like mass or UV spectroscopy. This is the main drawback for the determination of ultratrace amounts (nanogramper-liter range) of compounds in environmental samples using this method. However, there are a variety of environmental problems, such as the analytical characterization of hazardous waste sites, where pollution levels are sufficiently high for HPLC-NMR analysis. It has previously been shown that, as a result of the very high information content of NMR, this method can be applied directly to the characterization of environmental samples present as mixtures.5,6 However, if these mixtures are very complex, coupling of HPLC to NMR is desirable to enhance the resolution of information even further. In this case, it is not necessary to achieve optimum separation by HPLC. Incompletely resolved chromatograms are sufficient, and thus short columns for chromatographic separations can be used. Coupling of HPLC to NMR can be achieved in three ways: (a) In the “stop flow” mode, the flow will be stopped after a defined transfer time, after which a part of the chromatographic peak will be located in the NMR measuring cell. If necessary, one- and two-dimensional experiments can be performed for several hours. While the chromatographic run is stopped, peak broadening on the column in isocratic separations may occur. (b) To overcome this problem, a part of the chromatographic peak can be transferred into loops without interruption of the HPLC run (“sampling mode”). Samples stored in the loops are subsequently transferred to the NMR cell for measurement. (c) To get an overview of all proton-carrying compounds eluting from the column, the “continuous flow” mode can be used. In this case, the chromatographic run will not be affected in any way. While the mobile phase with the analytes is flowing through the NMR cell, the spectrometer records 1D spectra. These spectra are stored as rows in serial files and may be subsequently (5) Preiss, A.; Levsen, K.; Humpfer, E.; Spraul, M. Fresenius J. Anal. Chem. 1996, 356, 445-451. (6) Preiss, A.; Lewin, U.; Wennrich, L.; Findeisen, M.; Efer, J. Fresenius J. Anal. Chem. 1997, 357, 676-683. S0003-2700(97)00142-X CCC: $14.00
© 1997 American Chemical Society
Table 1. List of Standard Compounds Used peak assignment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
compound 1,3,5,7-tetranitro-1,3,5,7tetrazacyclooctane 2,4-dinitrobenzoic acid 2-nitrobenzoic acid 1,3,5-trinitrohexahydro1,3,5-triazine 1,3,5-trinitrobenzene picric acid 4-nitrophenol 3-nitrophenol 1,3-dinitrobenzene 4-nitrobenzoic acid 3-nitrobenzoic acid 2,4,6-trinitrotoluene 2,6-dinitro-p-cresol 3,5-dinitrophenol 2,4-dinitrotoluene 2,6-dinitrotoluene 3,4-dinitrotoluene 2-amino-4,6-dinitrotoluene 4-amino-2,6-dinitrotoluene 4,6-dinitro-o-cresol 2-nitrotoluene 4-nitrotoluene 3-nitrotoluene
purity (%)
origin
>99
Promochema
96 96 96
Promochem Aldrichb Promochem
>99 99.8 >99 >99 >99 99 99 99 98 >98 99 99 99 99 99 99 99 99 99
Promochem Merckc Flukad Fluka Fluka Aldrich Aldrich Promochem Aldrich custom-made (11) Riedel-de-Hae¨ne Riedel-de-Hae¨n Promochem Promochem Promochem Promochem Promochem Promochem Promochem
a Promochem, Wesel, Germany. b Aldrich, Steinheim, Germany. Merck, Darmstadt, Germany. d Fluka, Buchs, Switzerland. e Riedelde-Hae¨n, Seelze, Germany.
c
presented as a function of the (chromatographic) time analogous to HPLC (PDA) and GC/HPLC (MS), where UV or mass spectra are recorded at fixed intervals and again presented as a function of time. Methods a and b are usually preferred, as they provide ample time for an accumulation of NMR scans and thus optimum sensitivity. In contrast, in the continuous flow mode, there is a time limit for accumulation of scans because the chromatographic peak is flowing through the measuring cell. The optimum time resolution for best sensitivity in continuous flow measurements is given elsewhere.7 However, in this mode, lower detection limits can also be achieved by decreasing the flow of the mobile phase, as shown below. In this report, HPLC-NMR is applied in the on-flow mode to the analysis of groundwater samples near a former ammunition plant. It was previously demonstrated that this groundwater is polluted by high concentrations of explosives and their byproducts as well as by compounds formed by bio- and photodegradation therefrom, which form a very complex mixture.8-10 Many of these compounds are thermolabile or nonvolatile and thus cannot be analyzed by gas chromatography coupled to mass spectrometry (GC/MS), while HPLC, as a result of both its limited separation efficiency and the low specificity of the UV/PDA detector usually employed, will allow the identification and quantitation of only the main compounds. To detect compounds in the microgram-per-liter range in groundwater samples by coupled HPLC-NMR, special consid(7) Griffiths, L. Anal. Chem. 1995, 67, 4091-4095. (8) Mussmann, P.; Eisert, R.; Levsen, K.; Wu ¨nsch, G. Acta Hydrochim. Hydrobiol. 1995, 23, 13-19. (9) Steuckart, C.; Berger-Preiss, E.; Levsen, K. Anal. Chem. 1994, 66, 25702577. (10) Levsen, K.; Mussmann, P.; Berger-Preiss, E.; Preiss, A.; Volmer, D.; Wu¨nsch, G. Acta Hydrochim. hydrobiol. 1993, 21, 153-166.
Figure 1. Comparison of HPLC chromatograms at 210 nm obtained by injecting the standard solution of compounds known to be present in the groundwater of the ammunition hazardous waste site Elsnig (a) with the chromatograms of the original water sample (b) and the diluted extract after sample enrichment (c). For peak assignments, refer to Table 1.
erations are required. Based on the known limit of detection of the HPLC-NMR method, it is necessary to preconcentrate the compounds from the water sample, which in this investigation was achieved by solid phase extraction. Furthermore, the largest possible injection volume (500 µL of extract diluted in the mobile phase is the maximum injection volume for the system used) should be selected. EXPERIMENTAL SECTION Instruments. (i) HPLC-PDA. Routine measurements were carried out using an analytical system consisting of a Waters 501 HPLC pump and a Waters 996 photodiode array detector (Waters, Milford, MA). Chromatograms were extracted from the PDA data at a wavelength of 210 nm. For chromatographic separation, a Analytical Chemistry, Vol. 69, No. 18, September 15, 1997
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Figure 3. Pseudo-two-dimensional NMR chromatogram of a 5 µg injection of typical nitroaromatic compounds and nitramines at a flow rate of 0.006 mL/min and 144 scans per row, leading to a time resolution of 27 min/row. For peak assignments, refer to Table 1.
phase that consisted of a methanol-D2O mixture (45%/55% v/v) buffered to a pH value of 2.3, as mentioned above. Chromatographic separation was carried out on a Merck LiChrospher 60 RP-Select B C-18 column (75 mm length, 4 mm i.d., 5 µm particle size) at a flow rate of 0.017 mL/min and, for highest sensitivity, at a flow rate of 0.006 mL/min. The injection volume of the groundwater extract was 400 µL. The chromatographic system consisted of a LC gradient mixer from Bischoff, Model 1155, a Bischoff HPLC pump, Model 2250, equipped with micropump heads, and a Bischoff UV detector, Lambda 1000, operated at wavelengths mentioned in the UV chromatograms. Pseudo-2D spectra were recorded on a Bruker AMX 600 spectrometer at 600.13 MHz with a 1H-13C inverse flow probe (4 mm o.d. of measuring cell, with a detection volume of 120 µL). Solvent suppression was done by application of a 1D version of the NOESY pulse sequence
(RD-90°-t1-90°-tm-90°-FID)
Figure 2. Effect of flow rate reduction (a-c) on the HPLC separation efficiency (d). For peak assignments, refer to Table 1.
Merck LiChrospher 60 RP-Select B C-18 column from Merck (Darmstadt, Germany), 250 mm in length and 4 mm i.d., with a particle size of 5 µm, was used. Isocratic chromatographic conditions with a composition of the mobile phase of 55% (v/v) water and 45% (v/v) methanol, adjusted to a pH value of 2.3 with a dihydrogen phosphate-phosphoric acid buffer, were used. The flow was 0.5 mL/min at room temperature. (ii) Continuous Flow HPLC-NMR. The extract of the groundwater sample was freeze-dried and diluted with the mobile 3834
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with presaturation during relaxation delay (RD) and mixing time (tm) on two frequencies simultaneously (Bruker, Rheinstetten, Germany). The t1 value is not incremented and is set to 3 µs. The pseudo-2D spectrum has been recorded within 64 rows, each row consisting of 128 (144) free induction decays (FIDs) (sweep width, 14 706 Hz), collected into 32K data points, with a relaxation delay of 10 s, a mixing time of 80 ms, and a flip angle of 90°. This leads to a time resolution of 24 (27) min per row. Data were multiplied with an exponential function in f2 (LB ) 1 Hz) and processed with UXNMR software. 1H NMR chemical shift values are referenced to the methanol resonance line, which was set to 3.30 ppm. Reagents. The origin and purity of the compounds are listed in Table 1. Deuterium oxide (99.9%) was from Promochem (Wesel, Germany).
Figure 5. Coaddition of rows 6-12 (144-288 min) of NMR chromatogram of the upper level extract (Figure 4a). For peak assignments, refer to Table 1.
a stream of nitrogen, the analytes were eluted three times with 3 mL of a methanol-acetonitrile mixture (50/50 v/v). For HPLCNMR investigations, 3 L of groundwater sample was enriched on three SPE cartridges. The extracts were combined, freeze-dried, and diluted in 1 mL of the mobile phase. Acidic compounds were dissolved by sonification of this mixture for 2 min. It has been previously demonstrated that this extraction method leads to high recoveries for explosives and related compounds.12
Figure 4. Confirmation of compounds in a groundwater sample (a) by direct comparison with the pseudo-two-dimensional NMR chromatogram (only 1000 min is shown) of the standard solution (b) obtained under the same chromatographic and spectroscopic conditions (flow rate, 0.017 mL/min; 128 scans/row). For peak assignments, refer to Table 1.
Standard Solution. The standard solution contains compounds known to be present in the groundwater sample described below.5,6 Safety Considerations. Explosives and related nitroaromatic compounds are toxic [RDX (1,3,5-trinitrohexahydro-1,3,5triazine), HMX (1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane), 2,4,6trinitrotoluene, dinitrotoluenes, picric acid, 2,4-dinitrophenol, 4,6dinitro-o-cresol], highly toxic (1,3,5-trinitrotoluene, 1,3-dinitrobenzene), carcinogenic or suspectedly carcinogenic (2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,4,6-trinitrotoluene), and mutagenic (dinitrotoluenes, 2,4,6-trinitrotoluene, 4,6-dinitro-o-cresol, 2-nitrotoluene) and should, therefore, be handled with special care. Samples. Groundwater samples were obtained from the former ammunition site at Elsnig, Saxony, Germany. Procedure. Enrichment of the compounds from the aqueous phase was performed by solid phase extraction (SPE) on LiChrolut EN cartridges from Merck (Darmstadt, Germany) by adding sodium chloride (5 g/L) and adjusting 1 L of aqueous sample to pH 1.0 with hydrochloric acid. After the cartridge was dried with
RESULTS AND DISCUSSION HPLC-PDA. For comparison reasons, HPLC-PDA chromatograms of the standard solution (Figure 1a), the directly injected groundwater sample (Figure 1b), and the diluted SPE extract (Figure 1c) were recorded. All compounds named in the chromatograms of the groundwater sample were confirmed by PDA library search. Because of coelution of several minor compounds under these chromatographic conditions, only the main contaminants were confirmed by PDA library search. Continuous Flow HPLC-NMR. (i) Reduction of the Flow Rate. To increase the sensitivity of continuous flow HPLC-NMR measurements, the flow rate of the HPLC eluent was substantially reduced. At a flow rate of 0.8 mL/min, a chromatographic peak had a width at half-height ranging from 0.2 to 0.5 min for the isocratic separation shown in Figure 2a. Upon reducing the flow to 0.05 mL/min (cf. Figure 2c), the widths increased by factors of 10 for the early-eluting compound and 25 for the last-eluting compound (the peak volumes remain almost constant), without decreasing the separation efficiency, even on conventional columns (4 mm i.d., 5 µm particle size) for the early-eluting compounds, as shown in Figure 2d, which shows the variation of the number of theoretical plates with flow rate. Therefore, the number of scans for each row of the NMR chromatogram can be increased by at least 10 times without deteriorating the time resolution. As the S/N ratio increases with the square root of the number of scans, (11) Hantzsch, A. Ber. Deutsch. Chem. Ges. 1907, 40, 330. (12) Kruppa, J.; Preiss, A.; Levsen, K.; Kabus, H. P. Acta Hydrochim. Hydrobiol. 1996, 24, 226-231. (13) Godejohann, M.; Preiss, A.; Levsen, K.; Wu ¨ nsch, G. Chromatographia 1996, 43, 612-618.
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Table 2. Confirmation of Analytes Known To Be Present in Groundwater Samples from Former Ammunition Sites by PDA Library Search and Continuous Flow HPLC-NMRa sample 1 compound 1 2 3 4 5 6 7 2,4-DNP 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
sample 2
HPLC-PDA
HPLC-NMR
+ +
+ + + + (+) + + + + + + + + + + + + + + /
(+) (+) + +
+ +
HPLC-PDA
+
+ +
+ +
sample 3
HPLC-NMR + + + + + + + + + + + + + -
HPLC-PDA
+ +
+
+ + +
HPLC-NMR + + + + + + + + + + + + + + + +
a For compound assignments, see Table 1. 2,4-DNP, 2,4-dinitrophenol. Sample 1, 5-7 m below ground; sample 2, 15-17 m below ground; sample 3, 25-27 m below ground. Key to symbols: -, below detection limit if present; (+), coelution or overlapping of resonance lines; /, at the detection limit (lack of weak resonance lines).
an improvement in sensitivity by a factor of 3 is achieved for the early-eluting compounds. However, for the later-eluting compounds, an isocratic separation will lead to broader HPLC peaks, thus reducing the sensitivity. Retention times, however, increase with decreasing flow rates almost linearly until a flow rate of 0.2 mL/min is reached (Figure 2b). Below this flow rate, retention times increase faster compared to the decrease of the flow rate. This can be explained as due to a more intense mass transfer of the analyte molecules between the particles of the stationary phase and the mobile phase at very low linear velocities, according to the van Deemter theory. (ii) Limit of Detection. Figure 3 shows the NMR chromatogram of an injection of 5 µg of typical nitroaromatic compounds on the short HPLC column at a flow rate of 0.006 mL/min. Under isocratic conditions, the resonance lines of 1,3-dinitrobenzene (5 µg or 29 nmol injected onto the column) can be observed with 144 scans per row, with a good S/N ratio, without optimization of the chromatographic and spectroscopic conditions. For the methyl groups of the toluenes, the detection limit can be estimated to be less than 1 µg injected onto the column. (iii) Continuous Flow Measurements of Groundwater Extracts. The injection of 400 µl of the groundwater extract diluted in the mobile phase is shown in Figure 4a. To confirm the presence of selected compounds in this extract, the NMR chromatogram of the standard solution (12.5 µg per compound injected onto the column), obtained under the same experimental conditions, is shown in Figure 4b. Simply by comparing retention times and chemical shift values, the presence of analytes can be clearly confirmed down to the detection limit of the analytes, as shown in Figure 5, which is a projection of the coadded rows marked in Figure 4a by an asterisk. 3836
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It is also clearly apparent from Figure 4a that many still unknown compounds are present in the upper level extract. The chemical shift values of these compounds suggest the presence of di- or trisubstituted nitroaromatic compounds with low retention on RP columns. Comparison of HPLC-PDA and HPLC-NMR. In Table 2, confirmation results of three different groundwater levels, obtained by HPLC-PDA and HPLC-NMR, are given. Confirmation was achieved by comparing the extracted UV or NMR spectra of the groundwater extract to those obtained from pure reference compounds measured under exactly the same chromatographic and spectroscopic conditions. With the routine HPLC-PDA measurement, only major compounds in the upper microgram-per-liter range can be confirmed in these samples due to their complexity. By injecting the original water sample, a fast screening can be performed with sufficient sensitivity. The coelution of minor compounds makes it impossible to detect compounds in the lower microgram-perliter range without very time-consuming cleanup of the sample. CONCLUSIONS The results demonstrate that the HPLC-NMR method allows the detection of nitroaromatic compounds and other explosives down to the microgram-per-liter range without special optimization of the HPLC method. It is likely that the method can be applied with equal success to other environmental pollutants. Mixture analysis by NMR (without chromatographic separation) may be used for a first overview of the contaminants present in samples from hazardous waste sites. However, if these mixtures are very complex, continuous flow HPLC-NMR should be used in a second step. This method allows the identification
and quantitation of contaminants down to the microgram-per-liter range in a single run, justifying the long measuring times for onflow HPLC-NMR chromatograms (quantitation of pollutants by HPLC-NMR will be reported in a later paper). Even the determination of as-yet unknown compounds could be achieved without a second injection of the sample by structure elucidation of the desired pollutant. Once the pollutants have been identified, less sophisticated methods such as GC or HPLC (also coupled to MS) may be developed and employed for routine monitoring of such a hazardous waste site.
ACKNOWLEDGMENT Financial support from the Federal Department of Research and Technology (Contract No. 146 1063), Bonn, Germany, is acknowledged. Thanks are due to Manfred Spraul of Bruker Spectropin for his helpful discussions. Received for review February 5, 1997. Accepted May 29, 1997.X AC970142K X
Abstract published in Advance ACS Abstracts, July, 1, 1997.
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