Online Coupling of Gas Chromatography to Nuclear Magnetic

Jun 6, 2008 - Online Coupling of Gas Chromatography to Nuclear Magnetic Resonance Spectroscopy: Method for the Analysis of Volatile Stereoisomers...
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Anal. Chem. 2008, 80, 5481–5486

Online Coupling of Gas Chromatography to Nuclear Magnetic Resonance Spectroscopy: Method for the Analysis of Volatile Stereoisomers Maximilian Kühnle, Diana Kreidler, Karsten Holtin, Harri Czesla, Paul Schuler, Walter Schaal, Volker Schurig, and Klaus Albert* Institute of Organic Chemistry, Chemisches Zentralinstitut, University of Tuebingen, Auf der Morgenstelle 18, D-72076 Tuebingen, Germany The identification of volatile cis/trans-stereoisomers was accomplished by employing a hyphenated GC-NMR system. The chromatographic and spectroscopic conditions were optimized with respect to the 1H NMR detection. A special processing technique was developed to handle the recorded NMR spectra in the gas phase with very low sample amounts. The processed stopped-flow 1H NMR spectra of the investigated chromatographic peaks unequivocally revealed the structure of the corresponding compounds. Nuclear magnetic resonance (NMR) spectroscopy is the most powerful analytical method for the identification of organic compounds and for elucidation of their structure. Naturally organic compounds are not obtainable in the pure state, but they often appear as complex mixtures together with other compounds. The combination of NMR detection and a separation technique avoids the problem of signal overlapping in the NMR spectra and enables the analysis of complex systems. The hyphenation of highperformance liquid chromatography and nuclear magnetic resonance spectroscopy has evolved into a versatile tool in the analysis of complex mixtures. Several applications of this technique are outlined in numerous articles that demonstrate the advantages combining a separation method together with a structure elucidation technique.1–4 However, less evidence is available on the recording of NMR spectra in the gaseous phase.5–8 It would be promising to combine the high separation power and the great variability of stationary phases of GC with the high information content of NMR. * To whom correspondence should be addressed. E-mail: Klaus.albert@ uni-tuebingen.de. Tel.: +49-7071-2975335. Fax : +49-7071-295875. (1) Albert, K. Ed. On-line LC-NMR and Related Techniques; John Wiley & Sons Ltd.: Chichester, U.K., 2002. (2) Albert, K. J. Chromatogr., A 1995, 703, 123. (3) Lindon, J. C.; Nicholson, J. K.; Wilson, I. D. Prog. NMR Spectrosc. 1996, 29, 1. (4) Rehbein, J.; Dietrich, B.; Grynbaum, M.; Hentschel, P.; Holtin, K.; Kuehnle, M.; Schuler, P.; Bayer, M.; Albert, K. J. Sep. Sci. 2007, 30, 2382–2390. (5) Zuschneid, T.; Fischer, H.; Ha¨ndel, H.; Albert, K.; Ha¨felinger, G. Z. Naturforsch. 2004, 59b, 1153–1176. (6) Govil, G. Appl. Spectrosc. Rev. 1973, 7, 47–78. (7) Brame, E. G. Anal. Chem. 1965, 37 (9), 1183–1184. (8) Tsuda, T.; Ojika, Y.; Izuda, M.; Fujishima, I.; Ishii, D. J. Chromatogr. 1972, 69, 194–197. 10.1021/ac8004023 CCC: $40.75  2008 American Chemical Society Published on Web 06/06/2008

To our knowledge, first reports of an online GC-NMR experiment was published from various groups in the 1970s.7–9 Among them, Buddrus and Herzog showed in their application in 1981 that it is possible to obtain acceptable NMR spectra of gas chromatographic fractions with a surprisingly simple equipment. Due to a missing heating system and the simple equipment available to them, Buddrus and Herzog were limited to the analysis of volatile compounds and a continuous recording of 1H NMR spectra was not possible as well. In 1984, Herzog and Buddrus published further experiments.10 Employing a heatable interface, they were able to analyze organic compounds such as monoterpenes with boiling points up to 200 °C. In contrast to previous experiments, the new setup showed an inferior sensitivity so that the injection volume for each substance increased from 3 to 10 µL. The quality of the spectra was notable, but a continuous recording of 1H NMR spectra was still not possible. In recent years, the sensitivity of commercial available NMR instruments improved continuously. This is due to the fact that the magnetic field strength is much higher than 20 years ago. In addition, new developed probes also increase the sensitivity. There are two geometrical types of continuous-flow NMR probes available, which are described in detail in several articles.1 On the one hand, there is the saddle-type coil probe that is used in standard NMR probes. On the other hand, a solenoidal-type microcoil can be used. These microprobes with a solenoidal coil and an active detection volume of ∼1.5 µL outperform the limit of detection of common continuous-flow NMR probes.11–14 Not only the design of the probe shows an influence of the quality of a GC-NMR experiment, also the flow rate has to be carefully chosen to achieve maximum signal intensity and satisfying chromatographic resolution. For a continuous-flow GC-NMR experiment, the optimum flow rate is a compromise between best chromatographic resolution, best spectral resolution, and best sensitivity. As described in the literature, the regular flow rate of (9) Buddrus, J.; Herzog, H. Org. Magn. Reson. 1981, 15, 211–213. (10) Herzog, H.; Buddrus, J. Chromatographia 1984, 18, 31–33. (11) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849–3857. (12) Olson, D. L.; Peck, T. L.; Webb, A. G.; Sweedler, R. L. J. V. Science 1995, 270, 1967–1970. (13) Van Bentum, P. J. M.; Janssen, J. W. G.; Kentgens, A. P. M. Analyst 2004, 129, 793–803. (14) Ehrmann, K.; Saillen, N.; Vincent, F. S.; Tettler, M.; Jordan, M.; Wurm, F. M.; Besse, P.-A.; Popovica, R. Lab. Chip 2007, 7, 373–380.

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Figure 1. Contour plot of a GC-NMR separation of diethyl ether, trans-1,2-dimethylcyclohexane, and cis-1,2-dimethylcyclohexane. Data acquisition parameters: 64 transients with 1.5k time domain points and a spectral width of 5580 Hz were accumulated with a relaxation delay of 10 ms. During the separation, 128 rows with a acquisition time of 13.5 s/row were recorded.

GC compared to an acceptable flow rate for the NMR detection is very fast.10 This leads, due to the higher pulse repetition time, to an increase of the signal-to-noise ratio, but the reduction of the dwell time in the detection cell also causes line broadening. At short dwell times, the line broadening is the decisive effect that makes the recording of the spectra nearly impossible. With the aid of stronger magnets and the newly developed microprobes, the first online GC-NMR spectra could be recorded by Grynbaum et al. The setup of the GC-NMR system is described in detail elsewhere.15 The experiments revealed the high potential of this technique, but it was also shown that the peaks in the GC separation elute up to 10 min, so that the advantage of the high separation performance of GC was lost in the experiment. The required amount for a GC-NMR run was between 1 and 2 mg for each analyte.15 This work reports on improvements in the field of capillaryGC-NMR. These improvements also allow the analysis of stereoisomers, which are usually difficult to separate. In the experiments, it is shown that two different mixtures of volatile stereoisomers were separated by gas chromatography and subsequently detected by a NMR spectrometer. The comparison of the extracted online gas-phase spectra and the stopped-flow gasphase difference spectra with high-resolution reference spectra enables an identification of all compounds that were included in the mixtures. (15) Grynbaum, M. D.; Kreidler, D.; Rehbein, J.; Purea, A.; Schuler, P.; Schaal, W.; Czesla, H.; Webb, A.; Schurig, V.; Albert, K. Anal. Chem. 2007, 79, 2708–2713.

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EXPERIMENTAL SECTION Chemicals. Diethyl ether (Uvasol) was obtained from Merck (Darmstadt, Germany). cis-1,2-Dimethylcyclohexane and trans-1,2dimethylcyclohexane were obtained from Fluka Chemie GmbH (Buchs, Switzerland). Instrumentation. (1) NMR. All NMR spectra were recorded on a Bruker ARX 400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). The outlet of the gas chromatograph was connected to the custom-built, double resonant solenoidal microprobe with an active detection volume of 2 µL by a 2-m fused-silica transfer capillary (250-µm i.d.). For all capillary connections, universal glass connectors were used (Klaus Ziemer GmbH, Langerwehe, Germany). The NMR spectrometer was operated by an O2 workstation (Silicon Graphics) and XWIN-NMR software (Bruker Biospin GmbH, Rheinstetten, Germany). Data analysis was executed with XWIN-NMR 3.5. The 90° 1H flip angle was adjusted to 10 µs (16 dB attenuation/50 W amplifier,). (2) Gas Chromatography. The separations were performed on a Fractocap Series 2350 (Carlo Erba Strumentazione, Milan, Italy) gas chromatograph employing a Chirasil-β-Dex column (25 m, 250-µm i.d., 0.25-µm film thickness.). The carrier gas was dinitrogen 5.0 (Westfalen AG, Muenster, Germany) and was obtained from the centralized supply of the Chemisches Zentralinstitut, University of Tuebingen. A flow rate of 0.22 mL/min and a primary pressure of 0.9 bar were employed. The temperature of the GC column was 60 °C. The synthesis of the gas chromatographic stationary phase Chirasil-β-Dex (monokis-2-octamethylene) [permethyl-β-cyclodex-

Figure 2. Continuous-flow gas phase 1H NMR difference spectra (400 MHz) extracted from the contour data file shown in Figure 1: (a) cis1,2-dimethylcyclohexane; (b) trans-1,2-dimethylcyclohexane.

Figure 3. Comparison of the 1H NMR spectra (a) of the stoppedflow gas phase spectrum of trans-1,2-dimethylcyclohexaneand (b) of the empty GC-NMR probe with 1H NMR background signals.

trin bonded to poly(dimethylsiloxane)] has been described in detail elsewhere.16 Data Processing. All recorded NMR spectra show a significant background signal. In order to obtain more significant information, a special baseline correction procedure was performed. For the correction of the 1D spectra, one 1H NMR spectrum with exactly the same parameters but without any sample was recorded. This spectrum was subtracted from the previously recorded stopped-flow spectrum. For the correction of the rows in the 2D plots, one of the first rows that show no eluted analyte was chosen. This row was subtracted from each other row in the 2 D plot. (16) Jung, M.; Schurig, V. J. Microcolumn Sep. 1993, 5, 11–22.

RESULTS AND DISCUSSION Figure 1 depicts the contour plot of the GC-NMR separation of diethyl ether and of trans-1,2-dimethylcyclohexane and cis-1,2dimethylcyclohexane. For this experiment, a mixture of 0.1 µL of diethyl ether, 0.15 µL of trans-1,2-dimethylcyclohexane, and 0.15 µL of cis-1,2-dimethylcyclohexane was injected into the gas chromatograph, which was connected to the NMR spectrometer. This contour plot shows the retention time (F1 dimension) versus the 1H chemical shift axis (F2 dimension). The flow rate was calculated from the residence time τ of diethyl ether in the detection cell and the known size of the detection volume. The residence time τ in the detection cell can be calculated according to Wflow ) Wstationary + 1 ⁄ τW ) signal half-width

(1)

The calculated residence time in the detection cell is 0.416 s. The very short residence time leads to a line broadening of 2.4 Hz. The corresponding flow rate comes to 0.216 mL/min. As already mentioned in the introduction, the flow rate is a compromise between chromatographic resolution, spectral resolution and a satisfactory signal-to-noise ratio. It does not represent the van Deemter optimum, but higher flow rates would lead to shorter residence times and very broad NMR signals. The van Deemter optimum also depends on the type of carrier gas that is used.17 In order to reduce the negative chromatographic influence of the low flow rate, a carrier gas was chosen (17) Baugh, J. Ed. Gaschromatographie: Vieweg & Sohn Verlagsgesellschaft mbH: Wiesbaden, Germany, 1997.

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Figure 4. Comparison of stopped-flow gas-phase 1H NMR difference spectra of cis-1,2-dimethylcyclohexane, trans-1,2-dimethylcyclohexane, and the high-resolution reference spectra recorded in a 5-mm NMR tube; * impurity. (a) Stopped-flow gas-phase spectrum of trans-1,2dimethylcyclohexane; (b) high-resolution reference spectrum of trans-1,2-dimethylcyclohexane; (c) stopped-flow gas-phase spectrum of cis1,2-dimethylcyclohexane; (d) high-resolution reference spectrum of cis-1,2-dimethylcyclohexane.

Figure 5. Contour plot of a GC-NMR separation cis/trans-2-pentene and cis/trans-2-hexene. Data acquisition parameters: 64 transients with 1.5k time domain points and a spectral width of 5580 Hz were accumulated with a relaxation delay of 10 ms. During the separation, 128 rows with a acquisition time of 13.5 s/row were recorded.

that possesses its van Deemter optimum at a lower flow rate than, for example, helium. For further experiments a carrier gas that shows its van Deemter optimum at lower flow rates than dinitrogen has to be also taken in to account. 5484

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Figure 1 also shows that it is possible to separate the two cisand trans-stereoisomers in the gas phase and perform a continuous-flow NMR detection. In addition, the 2D plot confirms that the analytes do not condense in the flow cell. In this case, the contour plot would show a continuously eluted sample. The total sample amount of each analyte is 0.9 µmol of diethyl ether and 1.1 µmol of cis/trans-1,2-dimethylcyclohexane. This means that the required sample amount with respect to previous experiments is dramatic reduced by the factor of 20.15 The dramatic reduction of the injected sample amount referred to former GC-NMR experiments15 is mainly caused by two effects. First, the main reason for the dramatic reduction is the larger inner diameter of the used transfer capillary as well as the larger inner diameter of the inlet of the detection cell. Experiments under the same conditions employing another capillary with an inner diameter of 50 µm show no NMR signals up to an injection volume of 1 µL for each substance. As a result, the constriction from the outlet of the GC column to the inlet of the detection cell has a restrictive influence of the gas flow, which makes the whole GC-NMR system much more ineffective. The second reason for the advancement is the optimized recording of the NMR spectra. Due to the very short dwell times of the analytes in the detection cell, the data acquisition time referred to former experiments15 was more reduced, which led to a better single-to-noise ratio and an insignificant line broadening. With the new settings, it was possible to record the 4-fold number of transients in half-time. The data acquisition parameters are given in the figure caption of the continuous-flow GC-NMR experiment.

Figure 6. Comparison of the extracted gas-phase 1H NMR difference spectra from the data file shown in Figure 5. (a) High-resolution reference spectrum of cis-2-hexene; (b) continuous-flow gas-phase spectrum of cis-2-hexene; (c) high-resolution reference spectrum of trans-2-hexene; (d) continuous-flow gas-phase spectrum of trans-2-hexene.

For more detailed information, the 1H NMR spectra can be extracted from the contour plot file. This is shown in Figure 2 comparing the continuous-flow 1H NMR spectra of trans-1,2dimethylcyclohexane and cis-1,2-dimethylcyclohexane. Due to the small residence time of 416 ms in the flow cell, the signals are broadened but the differences in the NMR pattern of cis- and trans1,2-dimethylcyclohexane are clearly detectable. Additional information can be obtained by recording stoppedflow NMR spectra. Due to the very low concentration of the analyte in the detection cell, the background signal, which is mainly caused by embedded protons in the glass of the detection cell, is very high and it seems that a great deal of the spectrum can not be used for interpretation (Figure 3a). This problem can be solved by recording a background spectrum without any substance (Figure 3b). This spectrum has to be recorded and processed the same way as the recorded stopped-flow spectrum. After processing, the spectrum can be subtracted from the stopped-flow spectrum of the analyte. This is shown in Figure 4a. With the performed stopped-flow spectra and the high-resolution reference spectra, it is indeed possible to identify these two peaks as cis- and trans-1,2-dimethylcyclohexane. In order to document the preliminary results, further experiments were performed. Figure 5 depicts the contour plot of the capillary-GC-NMR separation of 2-cis/trans-pentene and 2-cis/trans-hexene. For this experiment, 0.2 µL of each compound was injected. The separation

was performed with a high-selectivity perpentyl-γ-cyclodextrin stationary phase. In most cases, the distinction between two cis/trans-stereoisomers can be done by a comparison of the recorded spectra with the reference spectra from an available spectra database. Sometimes a common chemical shift predictor may achieve the correct stereochemical assignment as well. Figure 6 shows that the information content of the extracted spectra of Figure 5 is sufficient to identify these two samples as 2-cis/trans-hexene, but for more detailed information, stopped-flow spectra, which are shown in Figure 7, could be helpful. In the case on the cis/trans-pentene stereoisomers, the separation factor was not enough to perform stopped-flow spectra of the pure substances, but the changing of the chemical shifts of the protons at 2.1 and 5.4 ppm are clearly detectable and enables an assignment by a comparison of the NMR spectra. The preliminary results obtained are very promising for future experiments. All GC-NMR experiments have been performed on an unshielded NMR spectrometer at relatively low field strength of 400 MHz. By increasing the field strength up to 800 MHz, the signal-to-noise ratio would be quadrupled. This implies that also the injected sample amount can be reduced dramatically, which also leads to a better chromatographic resolution because in the majority of cases the GC-capillary columns are overloaded. Also, a shielded magnet would make the GC-NMR system much more effective because this would lead to shorter transfer lines and thus Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

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Figure 7. Comparison of stopped-flow gas-phase 1H NMR difference spectra of cis/trans-2-hexene and the high-resolution reference spectra; * impurity. (a) High-resolution reference spectrum of cis-2-hexene; (b) stopped-flow gas-phase spectrum of cis-2-hexene; (c) high-resolution reference spectrum of trans-2-hexene; (d) stopped-flow gas-phase spectrum of trans-2-hexene.

to lower loss of chromatographic resolution caused by diffusion effects. A major technical challenge in this technique is still the heating of the whole system including gas chromatograph, transfer capillary, and the custom-built probe head. This assembly would support an investigation of samples with boiling points above 80 °C. With the heating system an analysis of a complex mixture of terpenes, for example, the ingredients of a fragrance, could be achieved.

Note Added in Proof: The employed stationary phase Chirasil-Dex16 is chiral as are cis- and trans-1,2-dimethylcyclohexane.18 Whereas the former isomer undergoes fast enantiomerization,16 preventing any enantioresolution at ambient temperature, the latter may be enantiodiscriminated in the course of further refined experiments. Successful enantiomeric differentiation of chiral alkanes and cyclodextrin selectors has meanwhile been performed by hyphenation GC-NMR for the first time in the present laboratories and will be communicated in due course.

CONCLUSION The presented results demonstrate the high potential of the hyphenation of capillary GC to microprobe 1H NMR detection. It is shown that the combination of highly selective GC separation phases and NMR detection with the help of a spectra database can achieve an identification of stereoisomers in a complex mixture. Even stopped-flow measurements of very low sample amounts, which are often essential for analysis of compounds with very low concentrations, can be performed. The sample amount for a successful online detection is ∼100-300 µg at 400 MHz, but instrumental improvements as well as stronger and shielded magnets are anticipated to decrease this amount.

ACKNOWLEDGMENT The authors thank Dr. Klaus Eichele from the Institute of Inorganic Chemistry of Tuebingen University for the critical evaluation of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg “Chemie in Interphasen” Grant 441/3), Bonn-Bad Godesberg, Germany.

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Received for review February 27, 2008. Accepted May 6, 2008. AC8004023 (18) Schurig, V.; Nowotny, H.-P.; Schmalzing, P. Angew. Chem., Int. Ed. Engl. 1989, 28, 736–737.