Anal. Chem. 1994,66, 3042-3046
On-Line Coupling of Supercritical Fluid Chromatography and Proton High-Field Nuclear Magnetic Resonance Spectroscopy Klaus Albert, Ulrlch Braumann, Ll-Hong Tseng, Graeme Nicholson, and Ernst Bayer' Institut fur Organische Chemie, Auf der Morgenstelle 18, D- 72076 Tubingen, Germany Manfred Spraul and Martin Hofmann Bruker Anal'ische Messtechnik GmbH, Silberstreifen, 0-76287 Rheinstetten, Germany Chris Dowle I C I Materials Research Centre, Wilton, Middlesbrough, TS 90 85E, UK Margaret Chippendale ZENECA Specialties, Blackley, Manchester, M9 3 DA, UK
The direct on-line coupling of supercritical fluid chromatography and proton high-field nuclear magnetic resonance spectroscopy is demonstrated. For this purpose, a specially designed pressure-proof continuous-flow probe head has been developed, from which a signal line width of 1.5 Hz can be obtained in the supercritical state. A separation of phthalates is carried out under supercriticalconditionswith carbon dioxide as eluent and is monitored on-line with IH NMR on a 400MHz NMR instrument. All the components can be identified from their NMR spectra. The advantage of carbon dioxide as eluent is that the whole spectral range of the 'H NMR spectra can be observed and no solvent suppression techniques are necessary to obtain the NMR spectra. Continuous-flow 'H NMR spectra acquired under supercritical conditions are of the same quality as those recorded in liquids. The on-line coupling of high-performance liquid chromatography (HPLC) with high-field lH NMR spectroscopy is becoming an established hyphenated technique. Numerous applicationsl-10 have been reported in the literature, revealing a wealth of structural information which cannot be obtained with any other detector. Despite all the improvements in the technique of on-line HPLC-NMR coupling, the use of protoncarrying solvents still leaves unobservable windows at the solvent signal positions. (1) Bayer, E.; Albert, K.; Nieder, M.; Grom, E.; Keller, T. J . Chromatogr. 1979,
186, 497. (2) Bayer, E.; Albert, K.; Nieder, M.; Grom, E.; Wolff, G.; Rindlisbacher, M. Anal. Chem. 1982,54, 1747. (3) Dorn, H. C. Anal. Chem. 1984, 56, 741. (4) Laude, D. A., Jr.; Wilkins, C. L. Trends Anal. 1986, 9, 231. (5) Albert, K.; Bayer, E. Trends Anal. Chem. 1988, 7, 288. (6) Albert, K. Habilitationsschrift, TCIbingen University, 1988. (7) Albert, K.; Kunst, M.; Bayer, E.; Spraul, M.; Bermel, W. J. Chromatogr. 1989, 463, 355. (8) Albert, K.; Kunst, M.; Bayer, E.; De Jong, H. J.; Genissel, P.; Spraul, M.; Bermel, W. Anal. Chem. 1989, 61, 115. (9) Albert, K.; Bayer, E. In HPLC Detections Newer Methods; Patonay, G., Ed.; VCH Publishers: New York, 1992; p 197. (10) Spraul, M.; Hofmann, M.; Dvortsak, P.; Nicholson, J. K.; Wilson, I. D. Anal. Chem. 1993, 65, 327.
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In fact, the first attempt to couple HPLC and 'H NMR avoided those background signals by using carbon tetrachloride or relatively inexpensive fully deuterated solvents such as deuteriochloroform, thus restricting the separation technique to adsorption chromatography. In addition, gel permeation chromatography with pure deuterium oxide as eluent can be performed.' I In reversed-phase chromatography, the commonly used eluent mixture is acetonitrile/water. While water may be replaced by the relatively cheap deuterium oxide, strong signals from the remaining acetonitrile and contaminant HDO are much more intense than the sample signals. Even if these remaining signals can be well suppressed with elaboratesolvent suppression techniques, they cover parts of the spectra and reduce the informationcontent drastically. From theviewpoint of NMR spectroscopy the reestablished supercritical fluid chromatography (SFC)12 with supercritical C02 as mobile phase should be an ideal chromatographic method for direct N M R on-line coupling, as it provides no 'H NMR signals. Even SFC with a modifier seems to be feasible because the added amounts are normally in a concentration range which allows the use of deuterated solvents. The first attempts to acquire NMR spectra in the supercritical state mainly dealt with measurements of relaxation times of quadrupolar n~c1ei.l~ Dorn14recorded proton spectra and also used the method for on-line coupling with SFC, but no detailed description of the quality of the spectra is reported. Further, the separation was carried out isocratically and isobarically; thus, the chromatographic conditions were not suitable for real separation problems. Therefore it was our goal to monitor a separation carried out under realistic chromatographic conditions with respect to pressure programming, temperature, and, in particular, flow rate and samde concentration. (1 1) Hofmann, M.; Spraul, M.; Streck, R.;Wilson, I. D.; Rapp. A. Laborpraxis 1993, 10, 36. (12) Lee, M. L.; Markides, K. E. Analytical Supercritical Fluid Chromatography and Extraction; Chromatography Conferences, Inc.: Provo UT, 1990. (13) Lamb, D. M.; Vander Velde, D. G.; Jonas, J. J. Magn. Reson. 1987.73.345. (14) Allen, L. A.; Glass, T. E.; Dorn, H. C. Anal. Chem. 1988, 60, 390.
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Figure 1. SFC-NMR equipment. Key: (a) Bischoff HPLC pump for specialexperiments, (b) Bruker LC 22 pump, (c) cryostatfor precooling of COPand pump heads, (d) C02 cylinder with dip tube, (e) GC oven with mixing chamber and steel capillary, (f) Valco C6W injection valve, (9) Hewlett-PackardHP1050UV detector, (h) air supply for probe-head heating, (i) Bruker AMX 400 with SFC probe, (k) Jasco back-pressure regulator, and (I) PC.
EXPERIMENTAL SECTION Apparatus. The equipment used for the SFC-NMR coupling is shown in Figure 1. The chromatographic system was assembled from commercially available GC and HPLC components. The C02 pump was a Bruker LC 22. To meet the demands of SFC, specially manufactured piston seals and extra check valves were used. The pump heads were cooled to approximately 3 “C with a cryostat. C02 was supplied from a gas cylinder with diptube and was cooled through 2 m of steel capillary (i.d. 0.5 mm), which was positioned in the cryostat used to cool the pump heads. The modifier pump, a Bischoff HPLC 2200, was connected with a T-piece to the system after the C02 pump. As the separations were carried out without modifier, the pump was only used for continuously adding sample during special setup operations(e.g., shimming.) Heating of the column was performed with an Dani 3900 GC oven. To facilitate sufficientmixing of the added modifier and thermal equilibration, a mixing chamber and 2 m of steel capillary (i.d. 0.5 mm) were placed in sequence in the GC oven. This sequence was completed by attaching the steel capillary to a Valco C6W injection valve, which was positioned outside the oven. The injection valve was equipped with a 10-pL external sample loop. Detection was carried out with a HP 1050VariableLwavelength detector, which was equipped with a Jasco high-pressure cell. Using a Jasco 880-8 1 backpressure regulator, it was possible to adjust pressure and flow rate independently. The program LC 22 (Bruker) was used to control the LC 22 pump. The program Chromstar (Bruker) was used to control the back-pressure regulator via the analog interface LC 234 M (Bruker) and to record both the signal of the UV detector and the actual back pressure. Both programs were run under WINDOWS on an Olivetti 80286 PC.
Figure 2. SFC-NMR probe head. Key: (a) container with external lock (b) radio frequency coils for 2H lock, (c) ’H ‘coil, (d) flow cell, (e) hot air for heating of probe head, and (f) capillaries carrying eluent.
To avoid disturbance of the magnetic field by the chromatographic system, the column effluent was connected via PEEK capillaries (i.d. 0.25 mm, total length 3 m) to the NMR probe. In the first experiments, electrically heated steel capillaries were used for the connection between the probe head and chromatographic system. Later experiments showed that this heating was not necessary, and the steel capillaries were replaced by PEEK capillaries. SFCProbe Head. For NMR detection, a new probe (shown in Figure 2) was developed. The flow cell used is a 5-mm sapphire tube (i.d. 3 mm) giving a NMR-active cell volume of 120 pL. During the first experiments, the SFC probe contained a double-walled cylinder concentric to the flow cell containing deuterioacetonitrile as external 2H lock. As it was outside the homogeneous magnetic field, any impurity produced a very broad signal, and additionally, the deuterium signal could not be used for shimming. On the contrary, it made shimming more difficult as it interfered with the internal deuterium signal. Therefore the container was removed. The probe was temperature-controlled by means of an airflow of 6 L/min. This air was electrically heated using the Bruker variable-temperature unit BVT 2000. To maintain supercritical conditions in the flow cell, the temperature was set to 320 K and showed a deviation of A0.2 K. The NMR spectrometer was a Bruker AMX 400 operating at 9.4 T. It was controlled by a Bruker X32 computer system. The NMR data acquisition was performed using the 2D routines of the data aquisition system UXNMR. The resulting pseudo “2D” files were only transformed in the f2 dimension while the fi dimension represents the time/chromatographic axis. During the separations, the spectra were recorded without fieldfrequency stabilization. NMR Parameters. The acquisition parameters for the SFC-NMR coupling were 8 or 16 scans/spectrum. A total of 4K data points, using quadrature detection, were acquired. AnalyticalChemistry, Vol. 66, No. 19, October 1, 1994
3043
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Figure 4. 'H NMR spectrum (400 MHz) of (a) 0.1 % ethylbenzene in iiqulddeuterloacetone and (b) 0.1 % ethylbenzene in 90% supercritical Conwith 9.9% deuteriochloroform at 165 bar and 321 K; flip angle ~ 1 2relaxation , delay 601120 s, 16K data points, and SW 3521 Hz. Solutlons were not degassed. Table 1. lH Spin-Lattlce Relaxatlon Tlmos of Butyl Benzyl Phthalate Recorded In the Llquld and the Supercrnlcal States (250 bar). T?"( ~ l
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With a sweep width (sw) X of 4000 Hz, an acquisition time of 0.508 s resulted. The delay between the scans amounted to 30 ms to allow data storage, no additional relaxation/delay was added, thereby a time resolution of 4.3 (8 scans) and 8.7 s (16 scans) was achieved. Typically 128 spectra (=rows) were recorded to observe a complete chromatogram. The flip angle of 40° (90° flip angle, 5.2 ps) was optimized to give maximal intensity of the NMR signals. An exponential window function of 0.5-Hz line broadening was the only data manipulation applied. The spectra were calibrated on the dichloromethane signal (5.3 ppm), which was used as solvent for the samples. A total of 8 scans/spectrum were acquired during the HPLC-NMR coupling experiments. A total of eight K data 3044
Analyfical Chemistty, Vol. 66,No. 19, October 1. 1994
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points were acquired, using quadrature detection. A sweep width of 5050 Hz was used, resulting in an acquisition time of 0.8 11 s. The solvent suppression was implemented by use of a 1331-pulse train with water on resonance. A total of 190 spectra were recorded to produce the complete chromatogram. A polynomial baseline correction and a shifted sine bell curve were applied to the FID, to reduce the remaining water signal. The HPLC coupling experiments were carried out on the Bruker AMX400 using an inverse lH(l3C]probe head with a cell volume of 120 pL. During the separation, the spectrometer was locked on the 2% D2O in the eluent. SFC Parameters. For all separations, the stationaryphase was LiChrosphere RP Select B 60 A ( 5 pm) in a Hibar 250 X 4.6 mm column (Merck, Darmstadt, Germany). The separations were carried out with a flow rate of 2.0 mL/ min, a linear pressure gradient from 115 to 180 bar in 20 min, and a temperature of 80 OC. The C02 was of 99.9995% purity (Messer-Griesheim). The solvents were of Uvasol quality (Merck, Darmstadt). For shimming under supercritical conditions, 0.2 mL of a solution of 10% (v/v) chloroform in deuterioacetone was added to 1 mL of COz with the second pump, yielding a total concentration of 1.8% chloroform. This mixture was pumped into the cell and then the flow was stopped by switching the injection valve into an intermediate position and closing the back-pressure regulator. HPLC Parameters. The HPLC separation was carried out with a Bischoff HPLC pump, using a premixed eluent. UV detection and data were acquired using the same equipment and column as described for the SFC experiments. The acetonitrile was of HPLC gradient grade quality (Merck). The HPLC separations were carried out under isocratic conditions at room temperature and with a flow rate of 0.75 mL/min. Sample. The sample was a mixture of diallyl, di-n-propyl, di-n-butyl, butyl n-benzyl, and diphenyl phthalate in approximately the same molar amount. Solutions of the sample in dichloromethane, ranging from 2 to 1096, were used for the SFC coupling experiments, whereas a solution of 10% sample dissolved in eluent was used for the HPLC separation. The phthalates were purchased from Aldrich and had a purity of >98%.
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RESULTS AND DISCUSSION An important feature of lH NMR probes is the signal line width at the height of the l3C satellites. This hump test is usually performed with a sample of chloroform in deuterioacetone. To obtain a basic shim set in liquid phase, a solution of 3% chloroform in deuterioacetone was injected into the flow cell. After changing to COz as solvent, the shimming had to be repeated due to the different susceptibilities of the liquid and supercritical solvent. In order to guarantee absolutely stable conditions, the final shimming was carried out in stopped-flow mode. Hump tests measured with the SFC probe in the liquid and supercritical state are shown in Figure 3. There is no change in line width in the lH NMR spectrum of chloroform recorded in the liquid and the supercritical sample. This is confirmed by the 'H NMR spectra of ethylbenzene, shown in Figure 4, recorded in the stopped-flow mode under supercritical and conventional conditions. Whereas lH T2 values do not seem to be severely
affected by applying supercritical conditions, the work of DornI4 suggests that there must be a strong influence on T1 due to the differing viscosities of the supercritical versus the liquid state. As it contains different kinds of protons, butyl benzyl phthalate was used as sample for determination of TI. Because T1 values are of interest only under realistic experimental conditions during the coupling of chromatography and NMR, the samples were not specially prepared, Le. not degassed. The results are shown in Table 1 and Figure 5 . In the liquid state, increasing temperature causes an increase in T Ivalues due to the decrease in correlation times. The same effect is responsible for the longer TIvalues observed in the supercritical state. The lH NMR chromatogram (contour plot) of a separation of the 10% phthalate mixture of five phthalates is shown in Figure 6. As the separation was performed without modifier and finally without lock container, the spectra were acquired without field-frequency stabilization. One can clearly see all AnalyticalChemistry, Vol. 66,No. 19, October 1, 1994
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Figure 8. IH NMR chromatogram (contour plot, 400 MHr) of a HPLC separatlon of five plastifiers: (a) diethyl, (b) dl-+propyl, (c) diphenyl, (d) benzyl +butyl, and (e) dl-+butyl phthalate.
five components in the N M R spectrum. Except for a small peak at 1.2 ppm, which arises from an impurity in the COz, the spectrum is free from interferences, and therefore, the whole spectral range isobservable. In our case, this is especially necessary to detect the ester protons, which may be obscured by thewater signal of an acetonitrile/water mixtureof a HPLC separation. Without any further data manipulation, such as extensive baseline correction, it is possible to observe and identify all five phthalates. If we extract a spectrum, e.g., that of the diallyl phthalate in Figure 7, we can clearly see that the NMR resolution is excellent. Even the small long-range coupling of the terminal protons (at 5.25 and 5.1 ppm) to the ester protons and the geminal coupling, both on the order of 1.4 Hz, are resolved without any further resolution enhancement. From the impurity peak at 1.2 ppm, which appears through the whole chromatographic run, it is clear that the signals shift upfield with increasing pressure. The relationship is not linear and can be described as approximately inversely proportional to the pressure. This correlates to the density of the CO1. As all the signals show the same trend, the cause can only be the changing density of COZ and no chemical process should be involved. The drifting signals limit the number of scans which can be coadded to one spectrum. The dependency of the signal on density is maximal at the beginning of the pressure gradient, where the change is approximately 1.7 Hz/bar. If one assumes that the drift should be less than 1 Hz,not more than 16 scans (over a period of 9.1 s) should be acquired for one row in this case. The separation was repeated with different sample concentrations. Even with concentrations as low as 2%, which corresponds to an amount of 60 pg/component, it was still possible to detect all five phthalates.
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Analytical Chemistry, Vol. 66, No. 19, October 1, 1994
For comparison, the contour plot of a HPLC separation of five phthalates monitored on-line with IH N M R is shown in Figure 8. In the region between 3.9-3.3 and 1.9-1.7 ppm, the spectrum is completely obscured by the solvent signals.
CONCLUSION From our experiments, we have shown that coupling of SFC and NMR can be used as a powerful tool for the analyst. It is clear that the quality of the NMR spectra obtained from SFC-NMR is not inferior to those obtained from HPLCN M R coupling. The disadvantage of longer 'H TI values in the supercritical state is compensated by the major advantage arising from COz as eluent, which depicts the whole * HNMR spectrum without any solvent window. No solvent-suppression techniques are required; therefore, 2D experiments in stoppedflow mode should be much more easily implemented than with eluents that produce strong background signals. Even if the expense of the required equipment seems to be higher than that necessary for HPLC-NMR coupling, the additionally gained information can make this worthwhile, as the NMR is no longer restricted by the conditions dictated by the chromatographic separation. ACKNOWLEDGMENT The authors express their appreciation to Ken P. Evans (ZENECA Specialties, Blackley, Manchester, M9 3DA, UK) for providing conditions for the phthalate HPLC separation. Received for revlew January 3, 1994. Accepted June 2, 1994." e Abstract
published in Aduance ACS Abstracts, July 15, 1994.
