Application of high-performance liquid chromatography coupled to nuclear magnetic resonance and high-performance liquid chromatography coupled to mass spectrometry to complex environmental samples

27

trends in analytical chemistry, vol. 19, no. 1, 2000

Application of high-performance liquid chromatography coupled to nuclear magnetic resonance and high-performance liquid chromatography coupled to mass spectrometry to complex environmental samples Karsten Levsen*, Alfred Preiss

Fraunhofer-Institut fuër Toxikologie und Aerosolforschung, Nikolai-Fuchs-Str. 1, D-30625 Hannover, Germany

Markus Godejohann

Bruker Analytik, Silberstreifen, D-76287 Rheinstetten, Germany HPLC^1H NMR in combination with HPLC^MS is particularly suited to the identi¢cation of unknown polar, non-volatile compounds in complex environmental samples, i.e. to non-target analysis of such samples, if pollutants are present in the ppb range. Both methods give complementary structural information. Optimum information on unknowns is gained if both techniques are coupled on-line. This review discusses general aspects of the application of HPLC^NMR and HPLC^MS to environmental samples and compares the advantages and disadvantages of these methods. The potential of the combined application of these hyphenated techniques to environmental samples is illustrated with contaminated ground water from a former ammunition plant, leachate from industrial waste disposal sites and waste water from a textile company as examples. z2000 Elsevier Science B.V. All rights reserved. Keywords: High-performance liquid chromatography^ mass spectrometry; High-performance liquid chromatography^nuclear magnetic resonance; Industrial waste water; Ammunition hazardous waste site; Industrial land ¢lls

*Corresponding author. Tel.: +49 (511) 5350 218; Fax: +49 (511) 5350 155. E-mail: [email protected]

1. Introduction The pollution of the environment by a large variety of chemicals represents a particular challenge to the analytical chemist. This is especially the case for hazardous waste sites where both soil and ground water may be contaminated by complex mixtures of organic chemicals, comprising compounds with very different polarities present in a wide range of concentrations. Industrial waste water or leachates from industrial waste disposal sites are other examples where pollutants are present as complex mixtures. Identi¢cation of the constituents is dif¢cult as not only the original compounds released into the environment are present in these samples. Chemical, photochemical and microbiological transformation processes may have led to additional and unexpected products. Monitoring of such environmental samples is usually restricted to known or suspected compounds or compound classes (`target analysis') for which speci¢c methods have been developed and optimized. Identi¢cation of unknown compounds (`non-target analysis') is much more dif¢cult. Here, sample extraction and separation must take into account the very different ( and a priori unknown ) physical and chemical properties of the individual organic compounds, while the instrumental method must have high separation ef¢ciency and provide optimum structural information. For compounds of high to medium volatility which are thermally stable, gas chromatography coupled to mass

0165-9936/00/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 9 ) 0 0 1 7 8 - 8

ß 2000 Elsevier Science B.V. All rights reserved.

TRAC 2593 20-12-99

28

trends in analytical chemistry, vol. 19, no. 1, 2000

spectrometry ( GC^MS ) under electron impact conditions is the method of choice, as this method combines the high separation ef¢ciency of the GC with the structural speci¢city of MS. Large MS libraries are available for compound identi¢cation and the fragmentation mechanisms of the odd electron molecular ions formed under electron impact conditions are well documented. Moreover, quanti¢cation is readily possible, in particular if isotopically labeled internal standards are used, which compensate both for reduced recovery during extraction and for variation in the instrumental response. Polar, non-volatile or thermally labile compounds in environmental samples are more dif¢cult to identify. For GC or GC^MS analysis, these compounds can be derivated to enhance their volatility. This approach, although time-consuming, is frequently used for target analysis, but can hardly be used if the compound to be analyzed is completely unknown. Although high performance liquid chromatography ( HPLC ) is well suited to the separation of polar or thermally labile compounds, this method is hardly suited to the structure elucidation of unknowns, even if a photodiode array detector is used. Moreover, the separation ef¢ciency of HPLC is signi¢cantly poorer than that of GC. Finally, detection by photodiode array is only possible if a chromophoric group is present in the molecule. More structural information is available if HPLC is coupled to MS, which is now widely used for the analysis of polar, non-volatile compounds in environmental samples [ 1^3 ]. While this technique is ideally suited to the analysis of known or suspected compounds ( even if a chromophoric group is lacking ), the structural elucidation of unknowns by this approach remains dif¢cult, as discussed below. Although nuclear magnetic resonance (NMR ) has been known as a powerful technique for structural elucidation for many decades, this technique was generally considered to be too insensitive for the analysis of organic compounds in environmental samples. However, with the advance of modern high-¢eld NMR instruments, the sensitivity of this method was signi¢cantly improved. Moreover, as a result of the very narrow NMR signals the `resolution of information' provided by this method is good [ 4 ] and was continuously increased by developing instruments of higher magnetic ¢eld strength ( see below ).

Recently it was shown by us that NMR ( without any chromatographic separation ) can be applied to a direct mixture analysis of environmental samples [ 4^6 ]. However, the resolution of analytical information may be further enhanced if the NMR spectrometer is on-line coupled to chromatographic systems, for instance to HPLC [ 7,8 ]. Since the commercial introduction of HPLC^NMR systems, this hyphenated technique has been mainly applied to the structural elucidation of metabolites in drug development studies [ 9 ] and the identi¢cation of natural compounds in plant extracts [ 10 ]. We have recently demonstrated for the ¢rst time the application of this technique to the analysis of environmental samples [ 11^14 ]. However, the optimum structural information is obtained if both HPLC^MS and HPLC^NMR are used together to identify unknowns in complex mixtures. This is demonstrated in this review using the analysis of contaminated ground water in the vicinity of former ammunition production sites, leachates from industrial waste disposal sites and industrial waste water as examples. In the following, ¢rst the HPLC^NMR and the HPLC^MS techniques are presented and their advantages and disadvantages discussed before the application of these techniques to environmental samples is reported in Section 5.

2. HPLC^NMR There are two basic requirements for the coupling of HPLC to NMR [ 9 ]: 1.

TRAC 2593 20-12-99

The conventional probe tubes have to be replaced by a £ow through cell. Today this is usually a U-shaped glass with an internal diameter of 2^4 mm inserted vertically into the magnet with resulting detection volumes of 40^120 Wl ( the detection volume should not be larger than the HPLC peak volume, which can be approximated by 2Upeak width at half-height times the £ow rate ). The RF transmitting and receiving coils are directly ¢xed to the outside of the glass cell to obtain an optimum `¢lling factor' ( this is the ratio of the sample volume to the volume inside the detector coil ) for maximum sensitivity. The £ow cell should be designed in such a way that a laminar £ow of the HPLC eluent is achieved to maintain the chromatographic resolution.

29

trends in analytical chemistry, vol. 19, no. 1, 2000

2.

With such an arrangement, a spectral resolution of 2^5 Hz can be achieved. When HPLC is coupled to NMR using protoncarrying solvents, the resonance signals of the mobile phase are by far the most intense signals in this spectrum. With the spectrometer systems currently available, a detection of weak analyte signals in the presence of strong solvent signals is not possible due to the limited dynamic range of the digitizer and the receiver. In principle, this problem could be overcome by using fully deuterated solvents, but this is generally too expensive. Today, very ef¢cient methods for suppression of the solvent signals exist, which are based on conventional presaturation techniques or newer pulse sequences such as WET ( water suppression enhanced through T1 effects ). However, multiple solvent suppression is easier if D2 O is used in the mobile phase instead of H2 O, where the cost of D2 O is relatively modest.

2.1. Coupling modes Coupling of HPLC to NMR can be achieved in three modes [ 9,15 ]: 1.

2.

3.

In the `stopped-£ow' mode, the entire chromatographic process is halted after a de¢ned transfer time, after which the center of the HPLC peak will be located in the NMR measuring cell. Now one- or two-dimensional experiments can be performed for several hours. If the £ow is halted for less than 2 h, no peak broadening due to diffusion is observed for the subsequent HPLC peaks after this time, while at longer times peak broadening may occur, especially with isocratic separation. To overcome this problem and to save HPLC operation time, various fractions of the chromatogram or parts of the chromatographic peaks can successively be transferred into loops without interruption of the HPLC run. Peaks stored in the loops are subsequently transferred to the NMR cell for measurement. In the `on-£ow' mode, the eluent is continuously transferred to the NMR measuring cell. While the mobile phase with analytes £ows through the cell, the spectrometer records 1D spectra. These spectra are stored and may be subsequently presented as a function

of the ( chromatographic ) time analogous to HPLC^DAD, GC^MS or HPLC^MS, where UV or mass spectra are continuously acquired and stored and a UV spectrum or a mass spectrum may be represented as a function of time at ¢xed intervals. Alternatively, the total HPLC^ NMR experiment can be represented as a pseudo-two-dimensional NMR chromatogram or contour plot, where the chemical shift values are plotted on one axis, the retention times on the second axis of the graph and the intensities of the signals are represented by contour lines ( see below ).

2.2. Structure elucidation With HPLC^1 H NMR experiments, chemical shift values, peak multiplicities and coupling constants and retention times can be used for structural elucidation. Complete chromatographic separation, which is often dif¢cult with complex environmental samples, is not a prerequisite for structural elucidation. As a result of the very narrow resonance signals, in many cases several distinct compounds within one NMR spectrum can be identi¢ed simultaneously. In particular, coelution of two compounds generally does not present a problem. For sensitivity reasons, HPLC^NMR measurements will often be restricted to 1 H NMR. In particular, direct HPLC^13 C NMR will probably not be realized in the near future, but several correlation spectra such as heteronuclear multiple quantum coherence ( HMQC ), heteronuclear single quantum coherence ( HSQC ), 2D total correlation spectroscopy (TOCSY ) and 2D nuclear Overhauser effect spectroscopy (NOESY ) have been successfully acquired in the past to obtain additional structural information [ 10 ].

2.3. Quanti¢cation It is often overlooked that NMR is well-suited to quanti¢cation, which is particularly important in environmental analysis. Quanti¢cation of a mixture is based on the following equation: Ca ˆ

cs ns Fa WMA Fs WMs na

where c stands for the concentration and the subscript s and a stand for the internal standard and analyte, respectively. n represents the number of

TRAC 2593 20-12-99

30

trends in analytical chemistry, vol. 19, no. 1, 2000

protons generating the NMR signal selected for quanti¢cation, F the area of the NMR signal and M the molecular weight of the compound. For quanti¢cation an internal standard has to be added to the sample. It is of advantage that quanti¢cation is possible without acquiring calibration data. Quanti¢cation is even possible when no reference compounds are available. In HPLC^NMR quanti¢cation is carried out in the on-£ow mode as described previously [ 16 ].

2.4. Validation In previous publications, we reported on ¢rst validation data of the NMR and the HPLC^NMR method [ 6,16 ]. As expected, the precision depends on the signal-to-noise (S /N ) ratio of the integrated resonance line and decreases with decreasing S /N, as illustrated in Fig. 1 for several resonance signals of ¢ve compounds measured by 1 H NMR without prior chromatography. While for an S /N ratio of 12 a precision of only 7% RSD was observed, this precision increases to 1% for S /N s 100 [ 6 ]. For on£ow measurements of several explosives and related compounds, a precision of 6 5 RSD was found for S /N s 100 whereas at lower S /N values ( e.g. 910 ) the precision is signi¢cantly poorer. For the same compounds spiked into an aqueous solution, quanti¢cation was possible with an accuracy ranging from 1 to 10% (S /N s 20 ). In the on-£ow mode under optimized conditions ( low £ow rate, large volume injection ), absolute detection limits of 3 Wg 2,4,6-trinitrotoluene and 5 Wg 1,3-dinitrotoluene ( injected onto the column ) were determined [ 16 ]. Detection limits in the upper nanogram range are achieved in the stopped-£ow mode. It is expected that the absolute detection limit in HPLC^NMR will reach values 910 ng in the near future, when cryoprobes and new capillary £ow through cells are brought onto the market [ 17 ].

2.5. Advantages and disadvantages of HPLC^NMR in environmental analysis HPLC^NMR uses the high structural information of the NMR technique which is of particular advantage in non-target analysis. Due to the low analyte concentration in environmental samples and the limited sensitivity of NMR discussed above, HPLC^NMR is often restricted to 1 H NMR and the full potential of two-dimensional NMR techniques,

Fig. 1. Dependence of the precision of the procedure (RSD: relative standard deviation ) on the signal-to-noise ( sino ) ratio of the integrated resonance line.

and 13 C NMR is generally not available. Thus, identi¢cation of unknowns will not be possible in every case. The technique is particularly powerful for the differentiation of isomers, while the differentiation between functional groups leading to similar chemical shift values of neighboring protons ( e.g. COOH versus NO2 group ) is not always easy. Of particular importance is the high `resolution of information' available for small molecules. This `resolution of information' has been de¢ned for NMR as the ratio of the expected chemical shift range to the width of the resonance signals ( in Hz [ 4 ]) and may be as large as 3000. (Note that the presence of several resonance signals even for a small molecule reduces this number. ) If, for chromatographic methods, such a `resolution of information' is de¢ned as the ratio of the maximum retention time to the peak width, it becomes apparent that e.g. for HPLC this `resolution of information' is much poorer than for NMR ( e.g. 100 ). As a result of the coupling of the two independent analytical methods, the chromatographic resolution of all analytes in a complex environmental sample is not a necessary prerequisite for their identi¢cation by HPLC^NMR. This is demonstrated in Fig. 2 which shows the results of an HPLC^NMR experiment of an arti¢cial mixture of aromatic acids ( which were identi¢ed or suspected in the leachate of an industrial waste disposal site ). The ¢gure represents the 1 H NMR spectra as a function of the retention time and shows that the high `resolution of information' of the NMR methods allows the identi¢cation of several analytes in one NMR spectrum, while all analytes are only resolved on

TRAC 2593 20-12-99

31

trends in analytical chemistry, vol. 19, no. 1, 2000

Fig. 2. Dispersion of the NMR information by the chromatographic process.

the chemical shift axis if NMR is coupled to HPLC ( even with modest resolution ). Compounds with a wide range of polarities may be analyzed by HPLC^NMR, including thermally labile and non-volatile compounds which are not amenable to GC^MS. The identi¢cation of this class of non-volatile compounds, in environmental samples in particular, has been dif¢cult up to now. In contrast to GC^MS and HPLC^MS, HPLC^NMR is a non-destructive method. Thus, the sample can be retrieved after analysis and then analyzed by other methods.

It is a further advantage that in 1 H NMR the detection response of an analyte directly re£ects its concentration ( i.e. for a given molecular mass the signal intensity expressed as peak area only depends on the concentration of the compound and the number of equivalent protons ), while with HPLC^ MS, the detection response depends on the ionization ef¢ciency, i.e. the chemical properties of the analytes. Finally, as mentioned above, quanti¢cation of analytes is possible in the on-£ow mode without reference compounds and without calibration.

TRAC 2593 20-12-99

32

trends in analytical chemistry, vol. 19, no. 1, 2000

A main disadvantage of HPLC^NMR is its modest sensitivity, which in general is three orders of magnitude lower than that of HPLC^MS. Thus, ultratrace analysis in environmental samples will not be possible. Moreover, if analytes are present at low concentrations in the sample ( i.e. in the upper ppt range ) the technique is time-consuming as several 1000 FIDs have to be accumulated in an overnight run to obtain an acceptable S /N ratio. It is a further disadvantage that solvent impurities are often of the order of the analyte signals and that the solvent suppression often in£uences ( reduces ) the intensity of signals in the neighborhood of the solvent signals. Furthermore, analyte signals which are superimposed with solvent signals are suppressed. Finally, the equipment required is expensive. Thus, in general, HPLC^NMR will not be used for routine analysis of environmental samples. Rather, the method gives a ¢rst complete survey of the pollutants present in an environmental sample ( in the ppb range ) and permits the identi¢cation of unknowns. On the basis of these HPLC^NMR results, simpler and less expensive methods may be developed in a second step.

3. High performance liquid chromatography coupled to mass spectrometry ( HPLC^MS ) A large variety of interfaces for the coupling of HPLC to MS have been developed over three decades [ 1 ]. While interfacing these two techniques, two major problems had to be overcome: õ

õ

the vacuum system of the MS had to handle the large amounts of solvents eluting from the HPLC; new methods for the ionization of non-volatile, polar compounds (present in solution) had to be developed.

The vacuum problem was solved on the one hand by introducing the mobile phase of the HPLC as a spray into the MS ion source where only a small fraction of this solvent enters the MS analyzers, and on the other hand by the development of sophisticated differential pumping systems which permit the handling of pressure differences of eight orders of magnitude. Moreover, new ionization methods have been developed which differ

in the mechanism for ion formation, but are all based again on the introduction of the HPLC eluent into the MS ion source as a spray of ¢ne droplets. With the introduction of ion sources operating at atmospheric pressure and the invention of atmospheric pressure chemical ionization ( APCI ) [ 18,19 ] and electrospray ionization ( ESI ) [ 20,21 ], robust and dedicated HPLC^MS instruments came onto the market which are now used routinely in the bioanalytical area of many laboratories ( instead of conventional HPLC ). The potential of this technique in environmental analysis has been demonstrated for many years and is well documented [ 3 ], while the application of this technique in this ¢eld is not growing as quickly as in the bioanalytical area. In the following section, the ionization methods and mechanisms and the instrumental details of HPLC^MS are not discussed. Rather, the general approach of applying HPLC^MS to non-target analysis in complex environmental samples as well as the advantages and disadvantages of this technique ( in particular as compared to HPLC^NMR ) will be discussed.

3.1. Target analysis by HPLC^MS If compounds are too polar to pass a GC ( for GC^ MS determination ), HPLC^MS is the method of choice for target analysis or identi¢cation of suspected compounds ( for instance, metabolites formed by biotic or abiotic degradation can be considered examples of suspected compounds, as the common routes for such degradation reactions are known at least for some compound classes ). HPLC^MS is by far more selective and in most instances also more sensitive than HPLC (UV ). In most cases, the ( quasi-)molecular ion and the retention time are used for compound con¢rmation. For complex environmental samples this approach may not be suf¢ciently speci¢c. The speci¢city can be enhanced by optimized clean-up procedures and, in particular, by the use of collision-induced fragmentation. Although already in single-stage mass spectrometers fragmentation may be induced by applying suitable voltages to a skimmer or a lens, tandem mass spectrometers with a neutral collision gas in a collision cell are far superior in generating these fragments. This approach, termed mass spectrometry^mass spectrometry (MS^MS ) [ 22 ], not only leads to the formation of ( structure-speci¢c ) fragments, but reduces interference from coeluting components

TRAC 2593 20-12-99

33

trends in analytical chemistry, vol. 19, no. 1, 2000

and the matrix. Speci¢c scanning procedures, such as the neutral loss scan, allow the identi¢cation of common functional groups within a class of compounds. Tandem mass spectrometry can be realized not only in space ( i.e. by two consecutive quadrupoles ) but also in time ( i.e. by inducing fragmentation in an ion trap ) [ 22 ]. While originally HPLC^MS was mainly applied to compound con¢rmation in target analysis, the precision of this method is suf¢ciently high to allow the quanti¢cation of analytes in environmental samples by external or internal calibration.

3.2. Non-target analysis by HPLC^MS The identi¢cation of unknown compounds ( non-target analysis ) by HPLC^MS in complex environmental samples is far more dif¢cult and often not successful if based on mass spectrometry alone. Compared to capillary GC^MS, the chromatographic resolution in HPLC ( and thus HPLC^MS ) is signi¢cantly poorer. Thus, in complex environmental matrices, coelution is a common problem. In APCI and ESI many neutral analytes yield both positive and negative quasi-molecular ions ( e.g [ M+H ]‡ and [ M3H ]3 or [ M ]3 ions ). Thus, one of

the ¢rst steps in identifying an unknown compound in a complex mixture is the search for these complementary positive and negative ions. If e.g. only negative ions are formed, this may be considered a ¢rst indication of the presence of an acidic compound. Isotope peaks can also be used to gain structural information, where not only halogen isotopes, but also 34 S and 13 C give valuable structural information, in particular in the negative ion mode ( i.e. on the approximate number of carbon atoms ). ESI spectra are often devoid of any fragment ions. Here MS^MS techniques are particularly valuable. However, with these methods too, the structural elucidation of unknowns still remains dif¢cult. MS libraries of the even-electron ions formed by APCI or ESI ( comparable to those of the odd-electron ions formed by electron impact ( EI )) are not yet commonly available. Moreover, while the fragmentation of the main compound classes after electron impact is well documented ( as mentioned above ), our knowledge of the fragmentation of the evenelectron ions is far poorer. Non-speci¢c fragmentation ( e.g. the loss of water ) and rearrangement reactions complicate the interpretation of the mass spectra of unknowns. However, in general,

Fig. 3. Coupling of HPLC^NMR^MS ( schematic setup for on-£ow and stopped-£ow measurements ).

TRAC 2593 20-12-99

34

trends in analytical chemistry, vol. 19, no. 1, 2000

TRAC 2593 20-12-99

35

trends in analytical chemistry, vol. 19, no. 1, 2000

Fig. 4. Comparison of a ground water sample contaminated by explosives with a mixture of 23 reference compounds ( explosives and transformation products ). Upper part: HPLC chromatograms; lower part: pseudo-two-dimensional NMR chromatograms ( contour plots ).

6

functional groups are readily detected by MS^MS using speci¢c fragments. Thus, in the negative ion mode the loss of a neutral of 44 amu is indicative of a carboxylic group and formation of an ion at m / z = 80 indicative of a sulfonic acid. Isomers differing in their substitution pattern at the aromatic ring are dif¢cult to differentiate by mass spectrometry ( ortho-substituted compounds which show a pronounced ortho effect represent an exception to this rule ). Finally, in contrast to EI-GC^MS and in particular HPLC^NMR, the peak area of an unknown compound in the chromatogram of an environmental sample provides no direct information on the concentration of the compound in the sample since, under APCI and ESI conditions, ionization ef¢ciencies of compounds may differ substantially. In the case of APCI, the proton af¢nities of the individual compounds determine both the type of ( quasi- )molecular ion formed and its abundance. Depending on the mobile phase and the presence of e.g. buffers, not only the above-mentioned ( quasi- )molecular ions, but also other cluster ions are formed which further complicate the structure elucidation of unknowns. If compounds of different proton af¢nities coelute, the ionization of one component may be largely suppressed. Thus, structural identi¢cation of unknowns by HPLC^MS is more dif¢cult than by GC^MS and will not always be successful if additional analytical information is not available. Such additional analytical information may be gained from HPLC^NMR, as discussed above.

particularly the case for aromatic compounds. The fact that the peak area of an NMR signal at a given concentration directly re£ects the number of equivalent protons ( as mentioned above ) is an advantage in structural elucidation. NMR spectra also provide straightforward information on the substitution pattern of the aromatic ring. It is apparent that HPLC^NMR and HPLC^MS often provide complementary information on the structure of unknowns. Thus, if both techniques are used together, an optimum level of structural information is available. HPLC^MS and HPLC^NMR have been used suc-

3.3. Comparison of the potential of HPLC^NMR and HPLC^MS for the structural elucidation of unknowns in environmental samples Mass spectrometry allows the determination of the molecular weight and ( in the case of high resolution ) the elemental composition, which is not possible by nuclear magnetic resonance spectroscopy. Moreover, functional groups ( e.g. halogen, COOH, NO2 , SO3 H ) are readily detected by MS. This is not possible by 1 H NMR. On the other hand, 1 H NMR spectra in general give more detailed structural information than mass spectra. This is

Fig. 5. HPLC (UV ) chromatogram of a leachate sample from an industrial waste disposal site ( for peak assignment see Table 2 ).

TRAC 2593 20-12-99

36

trends in analytical chemistry, vol. 19, no. 1, 2000

Table 1 Aromatic carboxylic and sulfonic acids identi¢ed in a leachate sample by HPLC^MS with thermospray ionization No. Compound

Mr

tR ( min )

Relative abundance ( l ) Negative ions 100%

1 2 3 4 5 6 7 8 9 10 11 a

p-Chlorobenzenesulfonic acid Phthalic acid Terephthalic acid Isophthalic acid Phenylacetic acid Benzoic acid K-Chlorobenzoic acid K-Hydroxybenzoic acid 3-Phenylpropionic acid m-Chlorobenzoic acid p-Chlorobenzoic acid

192

5.8

166 166 166 136 122 156 138 150 156 156

8.7 12.5 14.0 17.3 20.3 21.6 25.0 30.7 41.0 41.5

pKa Positive ions

20% 6 M 6 100%

100%

20% 6 M 6 100%

156 [ M3Cl3H ]3 191 [ M3H ]3 50%

0.7a

165 [ M3H ]3 165 [ M3H ]3 211 [ M+COOH ]3 181 [ M+COOH ]3 167 [ M+COOH ]3 201 [ M+COOH ]3 183 [ M+COOH ]3 195 [ M+COOH ]3 201 [ M+COOH ]3 201 [ M+COOH ]3

2.89 3.51 3.54 4.28 4.19 2.92 2.97 4.17 3.82 3.98

148 [ M3H2 O ]3 20% 184 [ M+NH4 ]‡ 167 [ M+H ]‡ 80% 211 [ M+COOH ]3 80% 184 [ M+NH4 ]‡ 165 [ M3H ]3 30% 184 [ M+NH4 ]‡ 154 [ M+NH4 ]‡ 140 [ M+NH4 ]‡ 155 [ M3H ]3 30% 3

155 [ M3H ] 30% 155 [ M3H ]3 30%

168 [ M+NH4 ]‡

pKa of benzenesulfonic acid=0.7.

cessfully by us to analyze complex environmental samples, as illustrated below. In these studies, separate HPLC^MS and HPLC^NMR systems were used. However, this off-line approach has some disadvantages: if complex environmental samples with many peaks in the HPLC chromatogram are analyzed by both techniques, it is often dif¢cult to correlate the MS and NMR data of a given unknown compound unambiguously as the retention times in the two experiments may differ. Slight differences in retention times are, for instance, encountered when D2 O is used in HPLC^NMR and H2 O in HPLC^MS experiments. Such differences in retention times are also observed if different HPLC equipment is used with both hyphenated techniques. Further uncertainties in the interpretation of the data may result from the suppression of the ionization of some analytes under APCI or ESI conditions.

4. On-line HPLC^NMR^MS Recently, a simultaneous coupling of HPLC to NMR and MS ( HPLC^NMR^MS ) was reported [ 23,24 ] and a ¢rst commercial version is now available in which the HPLC^NMR and MS units are controlled by common software. The schematics of this coupling is shown in Fig. 3. The HPLC eluent ( possibly after passing the peak sampling unit, BPSU ) is split. As the mass spectrometer ( an ion trap operating under electrospray conditions ) is far more sen-

sitive than the NMR instrument, 5% of the eluent is transferred to the MS and the remaining 95% to the NMR instrument. To improve the ionization conditions of the MS, an additional buffer may be added to the mobile phase before entering the MS via a Tpiece and a syringe pump. A loop is also incorporated to compensate for the different transfer times of the eluent from the split to the NMR and MS instruments. It is not necessary for these transfer times to be exactly equal, i.e. the mass spectrum and the NMR spectrum recorded at the same time: if the difference in transfer times is determined accurately, this constant difference allows an unambiguous assignment of a peak. If the HPLC eluent enters the MS before entering the NMR, the mass spectrum can be used to trigger the NMR instrument in the same way that the UV signal of the HPLC is used to trigger the NMR instrument in the stopped-£ow experiment. This approach is particularly valuable if compounds without a chromophoric group are investigated.

5. Application of HPLC^NMR in combination with HPLC^MS to environmental samples Three examples will be selected to illustrate the potential of the combined application of HPLC^ NMR and HPLC^MS to the non-target analysis of environmental samples:

TRAC 2593 20-12-99

37

trends in analytical chemistry, vol. 19, no. 1, 2000

Fig. 6. Time slices of the NMR chromatogram of the leachate sample shown in Fig. 5 ( for peak assignment and NMR chemical shift values see Table 2 ).

1. 2. 3.

contaminated ground water from a former ammunition plant; leachate water from an industrial waste disposal site; waste water from a textile company.

The extraction of the aqueous sample will not be discussed in detail. In general, extraction was performed by solid phase extraction (SPE ) using special polystyrene^divinylbenzene copolymers. If acidic organic compounds are to be extracted, the

sample must be acidi¢ed. In several instances, SPE extracts were lyophilized prior to analysis. Mass spectrometric detection was achieved using thermospray ionization (TSP ), APCI or ESI in both the positive and negative ion mode. A quadrupole or an ion trap was used as mass analyzer. The ion trap allowed multiple MS^MS experiments (MSn ) to be performed, which proved particularly valuable for structural elucidation. HPLC^NMR measurements were carried out in the on-£ow or stopped-£ow mode on a 600 MHz NMR spectrometer.

TRAC 2593 20-12-99

38

trends in analytical chemistry, vol. 19, no. 1, 2000

Table 2 1 H NMR chemical shift valuesa of reference compounds No. Compound

H2

H3

H4

1 2 3 4 5 6 7 8 9 10 11

7.45 ( pd )b

7.72 ( pd ) 7.74 ( m ) 8.06 ( s )

7.61 ( m )

p-Chlorobenzenesulfonic acid Phthalic acid Terephthalic acid Isophthalic acid Phenylacetic acid Benzoic acid K-Chlorobenzoic acid K-Hydroxybenzoic acid 3-Phenylpropionic acid m-Chlorobenzoic acid p-Chlorobenzoic acid

8.06 8.54 7.22 7.97

(s) (d) ( pd ) ( pd )

7.22 ( d ) 7.95 ( s ) 7.97 ( pd )

7.27 7.47 7.52 6.94 7.27

( pt ) ( pt ) (d) ( dd ) (t)

7.50 ( pd )

8.19 7.20 7.60 7.41 7.50 7.20 7.60

( dd ) ( pt ) ( pt ) ( t )c ( dt ) (t) (d)

H5

H6

7.72 ( pd ) 7.61 ( m ) 8.06 ( s ) 8.60 ( t ) 7.27 ( pt ) 7.47 ( pt ) 7.40 ( t )c 6.92 ( dt ) 7.27 ( t ) 7.46 ( t ) 7.50 ( pd )

7.45 ( pd ) 7.74 ( m ) 8.06 ( s ) 8.19 ( dd ) 7.22 ( pd ) 7.98 ( pd ) 7.82 ( d ) 7.84 ( dd ) 7.22 ( d ) 7.90 ( d ) 7.97 ( pd )

-CH2 -

3.60 ( s )

2.60 ( t ), 2.87 ( t )

a

Referenced to the solvent peak of acetonitrile (=2.00 ppm ). Multiplicities of the signals: s, singlet; d, doublet; t, triplet; dd, double doublet; m, multiplet; pd, pseudo-doublet; pt, pseudo-triplet. c Assignment may be interchanged. b

5.1. Ammunition hazardous waste sites As a result of extensive production of ammunition before and during World War II, a large number of hazardous waste sites exist in Germany, where both soil and water are polluted by explosives and their transformation products. In Germany as many as 3400 potentially contaminated sites have been identi¢ed [ 25 ]. To carry out a risk assessment of these sites, a large number of soil and water samples were analyzed, where the analysis was usually restricted to target compounds such as explosives expected or found on the site, but also to several byproducts and neutral transformation products formed by photo- or biodegradation of these precursors. Recent studies by us and others [ 4^ 6,11,12,26 ] have revealed that aqueous samples from such ammunition hazardous waste sites may also contain a variety of acidic compounds which were overlooked in previous investigations during target analysis. Contaminated ground water was analyzed by us using direct NMR and HPLC^NMR and the tentative structures elucidated by these techniques con¢rmed by HPLC^MS [ 6,11,12 ]. Fig. 4 compares part of the pseudo-two-dimensional NMR chromatogram of a contaminated ground water sample with that of a mixture of 23 reference compounds. In the upper part of this ¢gure, the HPLC chromatogram of the sample and the mixture of reference compounds is presented. Chromatography was achieved with a short column ( 75U4.5 mm; 5 Wm particles ) at a very low £ow rate ( e.g. 0.017 ml / min ). This £ow rate allows the accumulation of a suf¢ciently large number of FIDs per

row ( here 128 scans / row ). HPLC conditions were not optimized and poor resolution was accepted as the coupling of HPLC to NMR leads to such an

Fig. 7. Partial HPLC (UV ) chromatogram of a waste water sample from a textile company ( for peak assignment see Table 3 ).

TRAC 2593 20-12-99

39

trends in analytical chemistry, vol. 19, no. 1, 2000

Fig. 8. HPLC^NMR analysis of a waste water sample from a textile company. Selected NMR spectra of some HPLC peaks acquired under stopped-£ow conditions ( for peak assignment see Table 3 ).

TRAC 2593 20-12-99

40

trends in analytical chemistry, vol. 19, no. 1, 2000

Table 3 Summary of the NMR and MS data of the waste water sample from a textile company, peak assignment and structure of the identi¢ed compounds

TRAC 2593 20-12-99

41

trends in analytical chemistry, vol. 19, no. 1, 2000

Table 3 ( continued )

a†

s: singlet, d: doublet, pd: pseudo-doublet, t: triplet, m: multiplet

TRAC 2593 20-12-99

42

trends in analytical chemistry, vol. 19, no. 1, 2000

increase in selectivity that compound identi¢cation is possible even with a minimum of chromatographic separation. Both the resonance signals of the aromatic protons ( middle part of Fig. 4 ) and the methyl groups ( lower part of Fig. 4 ) can be used for identi¢cation. Comparison of the retention times and the chemical shift values of the signals allowed not only the con¢rmation of many explosives and related compounds known from common target analysis, but also the identi¢cation of some acidic compounds such as 3,5-dinitrophenol, 1,3,5-trinitrophenol ( picric acid ), mono- and dinitrobenzoic acids [ 11 ]. At the same time it became evident that this sample contained many more hitherto unidenti¢ed compounds, parts of which were elucidated in later studies where structural identi¢cation was corroborated by TSP^HPLC^MS [ 12 ]. Thus, 2,4,6trinitrobenzoic acid ( a photooxidation product of 2,4,6-trinitrotoluene ) was identi¢ed in the pseudotwo-dimensional NMR chromatogram at low ¢eld as a singlet and as a very early eluting compound. The thermospray MS of this compound is characterized by loss of CO2 and NO, which is consistent with the proposed structure. When investigating a larger number of contaminated ground water samples from ammunition hazardous waste sites in Lower Saxony ( Germany ) an unknown peak appeared in the NMR spectrum and HPLC chromatogram [ 6 ]. This peak was assumed to result from 2-amino-4,6-dinitrobenzoic acid. The HPLC^MS spectrum supported this assignment which was ¢nally con¢rmed when the NMR spectrum became available to us…1† . Once identi¢ed, this compound could be quanti¢ed in several ground water samples without a reference compound ( which was not commercially available ) demonstrating the power of NMR in quantitative studies.

5.2. Leachate from industrial land¢lls Leachates from industrial waste disposal sites may frequently contain a broad mixture of different chemicals. These compounds may be the ¢nal products, precursors or intermediates of the process, or else impurities and by-products obtained in a way that is often dif¢cult to predict and, as a consequence, dif¢cult to control. The leachate may

…1†

We are grateful to Dr. Steinbach (Marburg ) for providing the NMR spectrum of this compound.

contain pollutants originally contained in the waste; furthermore, transformation occurs during the aging of the waste and, as a consequence, the leachate may collect contaminants produced inside the land¢ll. Transformation products are often more polar than precursors. Leachate water from two industrial land¢lls was characterized by us using HPLC^NMR and thermospray HPLC^MS where particular attention was paid to acidic compounds [ 13 ]. The more volatile compounds were analyzed by GC^MS, as reported in the same publication [ 13 ]. To remove the excess neutral and basic analytes, the water sample was ¢rst made basic and preextracted with methylene chloride. After acidi¢cation, the aqueous phase was then extracted by SPE. The sample was ¢rst analyzed by HPLC with diode array detection (PDA ) as shown in Fig. 5 where two chromatograms at different detection wavelengths are shown. From these chromatograms it becomes obvious that the separation ef¢ciency of the HPLC method was insuf¢cient for this type of application. Compound identi¢cation by the UV spectrum alone was not possible. Furthermore, UV-inactive compounds such as aliphatic carboxylic acids or sulfates cannot be detected. Therefore, the LC^PDA method is not suitable for the analysis of such complex mixtures. In the next step the sample was analyzed by thermospray HPLC^MS in both the negative and positive ion mode. Using this approach, 11 organic acids could be identi¢ed in the sample as summarized in Table 1. In this table, the main ions found in the negative and positive ion spectra are also shown. The identi¢cation was corroborated by comparison with reference compounds and by HPLC^NMR ( see below ). As expected, the TSP mass spectra show abundant quasi-molecular ions and cluster ions in both ionization modes. Under negative ion conditions, [ M3H ]3 and cluster ions formed by the buffer ammonium formate, i.e. [ M+HCOO ]3 , are predominantly formed. The weak acids phenylacetic acid and phenylpropionic acid show more abundant positive than negative ions under TSP conditions, where the [ M+NH4 ]‡ ion dominates the spectrum. These ions are also intense in the spectra of the isomeric phthalic acids and benzoic acid, although with these compounds more negative than positive ions are formed. Finally, HPLC^NMR experiments were carried out. Fig. 6 shows various time slices of the NMR

TRAC 2593 20-12-99

43

trends in analytical chemistry, vol. 19, no. 1, 2000

Fig. 9. HPLC^MS analysis of a waste water sample from a textile company: a = APCI spectrum ( positive ions ) of peak 7 in Fig. 7; b^e = MS2 ^MS5 spectra, ( o ) = odd electron ions; ( e ) = even electron ions.

TRAC 2593 20-12-99

44

trends in analytical chemistry, vol. 19, no. 1, 2000

Fig. 10. HPLC^NMR^MS analysis of a commercial dye sample. A: HPLC (UV ) spectrum at 254 nm. B: Photodiode array contour plot. C: Total ion chromatogram under negative ion electrospray conditions. D: Density plot of the negative ion electrospray mass spectra. E: 1 H NMR spectra of peaks 1, 3 and 5 of the HPLC chromatogram in A.

chromatogram. The spectra are rather simple and can be analyzed without use of further techniques. Eight organic acids could be identi¢ed in the NMR chromatogram on the basis of their retention times and by comparison with the chemical shift values of the reference compounds, as summarized in Table 2. Note again that the NMR spectra shown in Fig. 6

consist of several compounds. HPLC chromatography simpli¢es the NMR spectra to such an extent that the NMR spectra of the individual compounds do not show interference from other components. However, with this technique too, several early and late eluting compounds could not be identi¢ed ( see for instance rows 14^17 ).

TRAC 2593 20-12-99

45

trends in analytical chemistry, vol. 19, no. 1, 2000

Fig. 10 ( continued ).

5.3. Ef£uent from a textile company Industrial waste water is an important potential source of pollution of the aquatic environment. It is assumed that usually only V20% of the organic constituents in the waste water of a chemical plant are known, where the unknown components are considered to be mainly polar, non-volatile compounds. For the identi¢cation of these polar compounds, the combined use of HPLC^NMR and

HPLC^MS is particularly promising, as illustrated by us with the analysis of untreated waste water from a textile company as an example [ 14 ]. Again, the more volatile compounds of the sample were identi¢ed by GC^MS, as reported by Benfenatti et al. [ 27 ]. The HPLC (UV ) chromatogram in Fig. 7 gives an overview of the early eluting components of this waste water sample ( which was extracted by methylene chloride ).

TRAC 2593 20-12-99

46

trends in analytical chemistry, vol. 19, no. 1, 2000

Fig. 10 ( continued ).

TRAC 2593 20-12-99

47

trends in analytical chemistry, vol. 19, no. 1, 2000

The NMR spectra of the various peaks were acquired using the stopped-£ow method. Some NMR spectra are shown in Fig. 8 while the complete NMR data and the structure assignments are summarized in Table 3. The interpretation of the NMR spectra has been reported in detail previously [ 14 ]. Here, only the NMR spectrum of peak 7 in Fig. 8 will be discussed as an example. The 1 H NMR spectrum of this peak shows in the chemical shift region of the aromatic protons, two multiplets ( two protons each ) centered at 7.77 and 8.27 ppm typical for the ring A protons ( H 5,8 and H 6,7, respectively ) of an anthraquinone skeleton ( see Scheme 1 ). The doublets of the AB subspectrum at 7.47 and 7.51 ppm, respectively, correspond to the protons of the C ring of the anthraquinone skeleton. They indicate that the substituents at positions 1 and 4 of the ring differ. Furthermore, a singlet in the aliphatic region at 3.11 ppm is within the expected shift range of the N-bonded methyl group, while the two triplets at lower ¢elds ( 3.59 and 3.80 ppm ) are in the expected shift range of N- and O-bonded methylene groups, respectively. Thus, the compound corresponding to peak 7 was tentatively identi¢ed as disperse blue 3. This assignment was con¢rmed by mass spectrometry where HPLC^MS was performed using atmospheric pressure chemical ionization both in the positive and negative ion mode. The mass spectral data of this and other compounds in the sample are also summarized in Table 3, while Fig. 9 represents the positive ion APCI spectrum and the MS2 ^ MS5 spectra of the collision-induced fragments as recorded in an ion trap. Not only the quasi-molecular ions [ M+H ]‡ at m / z 297 and [ M ]3 at m / z 296, but also the fragments are consistent with the proposed structure ( see scheme in Fig. 9 ). The ¢nal con¢rmation of the structure assignment was achieved by comparing the NMR and MS spectra of peak 7 with those of the reference compounds. The fragmentation of this compound highlights the above statement that the fragmentation rules of odd-electron ions as known from electron impact mass spectrometry do not apply to the even-electron [ M+H ]‡ ions formed by APCI^HPLC^MS. While ionized amines ( such as disperse blue 3 ) should show an abundant cleavage of the K-C-C bond, cleavage of the N-C bond is observed in the MS2 spectrum of the [ M+H ]‡ ion. This cleavage leads to an odd-electron ion which, in the following fragmentation step (MS3 ), shows the well-known K-cleavage.

As summarized in Table 3, several anthraquinone-type dyes and their by-products, a £uorescent brightener, a by-product from polyester production and auxiliaries such as anionic and non-ionic surfactants and their degradation products were also identi¢ed in this waste water sample. With the exception of nonylphenol and nonylphenolmonoethoxylates, the compounds listed in Table 3 could not be identi¢ed by GC^MS. Some additional ionic compounds could be identi¢ed by ion-pair chromatography coupled to HPLC^MS, as reported by Castillo et al. [ 28 ].

5.4. HPLC^NMR^MS analysis of a dye sample As a ¢rst example of an application of directly coupled HPLC^NMR^MS, the analysis of a commercial dye sample similar to that found in the waste water of a textile company ( as discussed in Section 5.3 ) is reported. Such commercial dyes usually contain several components. The results of the HPLC ( DAD )^NMR^MS analysis are summarized in Fig. 10. Fig. 10A represents the HPLC (UV ) chromatogram at 254 nm while the corresponding PDA contour plot is shown in Fig. 10B. Six main components are found in this sample. The PDA contour plot reveals that these six main and two minor components are dyes since they exhibit a strong absorption at wavelengths s 450 nm. Fig. 10C shows the total ion chromatogram under negative ion electrospray, Fig. 10D the negative ESI spectra of the ¢ve main components as density plot. Peak 1 ( disperse blue 14 ) is characterized by an [ M ]3 ion at m / z 326, peak 3 ( disperse blue 3 ) by an [ M ]3 at m / z = 296 and peak 5 ( disperse blue 23 ) by an [ M ]3 at m / z = 266 ( see also Table 3 ). The 1 H NMR spectra obtained for peaks 1, 3 and 5 under stopped-£ow conditions are shown in Fig. 10E. The corresponding compounds are identi¢ed as disperse blue 14, 3 and 23. The simultaneous acquisition of NMR and

Scheme 1.

TRAC 2593 20-12-99

48

trends in analytical chemistry, vol. 19, no. 1, 2000

MS spectra after HPLC chromatography facilitates the structural identi¢cation signi¢cantly.

Acknowledgements Financial support from the Ministry for Science, Education, Research and Development ( Grant 146 10 63 ) and the European Commission ( Grant ENV CT95-0021 ) is kindly acknowledged.

References [ 1 ] W.M.A. Niessen and J. van der Greef, Liquid Chromatography^Mass Spectrometry, Marcel Dekker, New York, 1992. [ 2 ] R. Willoughby, E. Sheehan and S. Mitrovich, A Global View of LC /MS, H.D. Science, Toton, 1998. [ 3 ] D. Barceloè ( Editor ), Application of LC-MS in Environmental Chemistry, Elsevier, Amsterdam, 1996. [ 4 ] A. Preiss, K. Levsen, E. Humpfer, M. Spraul, Fresenius' J. Anal. Chem. 356 ( 1996 ) 445. [ 5 ] A. Preiss, U. Lewin, L. Wennrich, M. Findeisen, J. Efer, Fresenius' J. Anal. Chem. 357 ( 1997 ) 676. [ 6 ] M. Godejohann, A. Preiss, K. Levsen, K.M. Wollin, C. Muëgge, Acta Hydrochim. Hydrobiol. 26 ( 1998 ) 330. [ 7 ] E. Bayer, K. Albert, M. Nieder, E. Grom, T. Keller, J. Chromatogr. 186 ( 1979 ) 497. [ 8 ] N. Watanabe, E. Niki, Proc. Jpn. Acad. Ser. B 54 ( 1978 ) 194. [ 9 ] J.C. Lindon, J.K. Nicholson and I.D. Wilson, Advances in Chromatography, Marcel Dekker, New York, 1996, Vol. 36, p. 315. [ 10 ] J.L. Wolfender, K. Ndjoko, K. Hostettmann, Curr. Org. Chem. 2 ( 1998 ) 575. [ 11 ] M. Godejohann, A. Preiss, C. Muëgge, G. Wuënsch, Anal. Chem. 69 ( 1997 ) 3832. [ 12 ] M. Godejohann, M. Astratov, A. Preiss, K. Levsen, C. Muëgge, Anal. Chem. 70 ( 1998 ) 4104. [ 13 ] E. Benfenati, P. Pierucci, R. Fanelli, A. Preiss, M. Godejohann, M. Astratov, K. Levsen, D. Barcelo, J. Chromatogr. A 831 ( 1999 ) 243. [ 14 ] A. Preiss, U. Saënger, N. Kar¢ch, K. Levsen and C. Muëgge, Anal. Chem. ( submitted ). [ 15 ] M. Spraul, M. Hoffman, J.C. Lindon, I.D. Wilson, Anal. Proc. 30 ( 1993 ) 390. [ 16 ] M. Godejohann, A. Preiss, C. Muëgge, Anal. Chem. 70 ( 1998 ) 590. [ 17 ] M. Spraul, Bruker Company, personal communication. [ 18 ] D.I. Caroll, I. Dzidic, R.N. Stillwell, K.D. Haegele, E.C. Horning, Anal. Chem. 47 ( 1975 ) 2369. [ 19 ] T.R. Covey, E.D. Lee, J.D. Henion, Anal. Chem. 58 ( 1986 ) 2453.

[ 20 ] C.M. Whitehouse, R.N. Dreyer, M. Yamashita, J.B. Fenn, Anal. Chem. 57 ( 1985 ) 675. [ 21 ] A.P. Bruins, T.R. Covey, J.D. Henion, Anal. Chem. 59 ( 1987 ) 2642. [ 22 ] K.L. Bush, G.L. Glish and S.A. McLuckey, Mass Spectrometry /Mass spectrometry: Techniques and Application of Tandem Mass Spectrometry, VCH, New York, 1988. [ 23 ] F.S. Pullen, A.G. Swanson, M.J. Newman, D.S. Richards, Commun. Mass Spectrom. 9 ( 1995 ) 1003. [ 24 ] G.B. Scarfe, I.D. Wilson, M. Spraul, M. Hofmann, U. Braumann, J.C. Lindon, J.K. Nicholson, Anal. Commun. 34 ( 1997 ) 37. [ 25 ] J. Thieme ( Editor ), Suspected sites of ammunition hazardous waste sites in Germany ( in German ), Report 103 40 102, German Federal Agency of the Environment, Berlin, 1993. [ 26 ] T.C. Schmidt, M. Petersmann, L. Kaminsky, E.v. Loëw, G. Storck, Fresenius' J. Anal. Chem. 357 ( 1997 ) 121. [ 27 ] E. Benfenati et al., Trends Anal. Chem. ( 1999 ) [ 28 ] Castillo et al., Trends Anal. Chem. ( 1999 ) Prof. Levsen studied chemistry at the University of Bonn ( Germany ). He received his PhD degree in 1971 in Physical Chemistry and the habilitation degree in 1975 and was appointed Professor of Physical Chemistry in 1979. Since 1985 he has in addition been head of the Department of Bio- and Environmental Analysis at the Fraunhofer Institute of Toxicology and Aerosol Research in Hannover ( Germany ). In the area of environmental analytical chemistry he has developed and applied new methods and instruments for sampling and analysis of contaminants in water, air ( with emphasis on indoor air ) and hazardous waste using hyphenated techniques and on-line coupling of sample preparation, separation and instrumental analysis. In bioanalytical chemistry emphasis is on the study of adsorption, distribution, metabolism and excretion ( ADME ) of industrial chemicals, pharmaceuticals and pesticides and the development of methods for human biomonitoring. Dr. A. Preiss studied chemistry at the University of Leipzig ( Germany ). He received his PhD degree in 1970 in organic chemistry. From 1970 to 1979 he was head of the NMR laboratory at a chemical plant in Bitterfeld, from 1980 to 1987 he worked as a research scientist at the Institute of Plant Biochemistry in Halle in the ¢eld of structural elucidation of natural products. From 1988 to 1990 he was responsible for the chemical quality control in a pharmaceutical company in Switzerland. Since 1990 he has been deputy head of the department of Bio- and Environmental Analytical Chemistry at the Fraunhofer Institute in Hannover. The main ¢elds of research are environmental analyses, pharmaceutical analyses and natural product chemistry. For the last two years he has been engaged in the development of methods for the analysis of complex mixtures by hyphenated techniques, in particular HPLC^NMR and HPLC^MS. Dr. M. Godejohann studied chemistry at the University of Hannover and received his PhD in analytical chemistry in 1998. Since 1999 he has been working with the Bruker company in the area of HPLC^NMR and HPLC^NMR^MS.

TRAC 2593 20-12-99