Anal. Chem. 1995,67,930-935
OnmLine Monitoring of the Supercritical Fluid Extraction Process with Proton Nuclear Magnetic Resonance Spectroscopy Ulrich Braumann, Heidtun Handel, and Klaus Albert* lnstitut f i r Organische Chemie, Universifat Tubingen, Auf der Morgenstelle 18, 0-72076lllbingen, Germany Rainer Ecker Hewlett Packard GmbH, Ermlis-Allee, 0-76337Waldbronn, Germany Manfred Spraul Bruker Analytische Messtechnik, Silberstreifen, 0-76287Rheinstetten, Germany
Supercritical fluid extraction (SFE) is performed with carbon dioxide as eluent, and the extraction process is monitored with ‘HNMR spectroscopy as the detection technique. It is shown that the extraction process can be easily controlled with the information provided by the NMR spectra. Normal conditions for the SFE concerning pressure and flow rate can be applied. Spectra are recorded in the continuous-flowand stopped-flowmode; 2D spectra can be acquired. The possibilities are demonstrated by monitoring the extraction of roasted coffee and of black pepper. Extraction with supercritical fluids (SFE) was introduced as a technical process several years ago.’Z2 Now it becomes, similar to the related technique of supercritical fluid chromatography (SFC), more and more i m p ~ r t a n t . ~The - ~ most commonly used eluent is COZdue to its chemical inertness and easy to handle critical parameters. The SFE with COZprovides a rapid, mild technique of extraction of low or medium polar substances. The solvent strength can be easily and infinitely variable-adjusted by altering the pressure and thereby the density. For more polar extractands, modfiers such as methanol may be added to increase the solvating power. Once the sample is extracted, the solvent can be easily removed by decompressing the solution into an adequate container. This might be a trapping column or a vessel containing a low-boiling solute l i e dichloromethane. A direct observation of the extraction process with FT-IR was reported by Kirschner and Taylor? but the information provided by conventional SFC detectors is not very specitic. They are only suited to observe the extraction in a very coarse manner. In fact, the extraction process in most cases is not monitored directly. The extracted samples are analyzed discontinuously by chromatc(1) Stahl, E.; Schilz, W. Chem. Ing. Tech. 1976,48,773-778. (2) Stahl, E.; Schilz,W. 2. Anal. Chem. 1976,280, 99-104. (3) King, J. W. J Chromatogr. Sci. 1989,27, 355-364. (4)Chester, T. L.; Pinkston, J. D.; Raynle, D. E.Anal. Chem. 1994,66,106R130R. (5) Lee, M. L.; Markides, K E. Analytical Supercn’tical Fluid Chromatography and Extraction; Chromatography Conferences, Inc.: Provo, UT, 1990. (6) Kirschner, C. H.; Taylor, L. T. Anal. Chem. 1993,65, 78-83.
930 Analytical Chemistry, Vol. 67,No. 5,March 7, 7995
graphic methods which allow identification and quantification of known compounds when standards are available. Nevertheless, an identification of unknowns requires spectroscopic methods. As shown already, the detection of ‘H NMR spectra in the supercritical state is possible and can be used in SFC.7-9 The limiting factor for use of NMR as a detector with chromatographic techniques is the low sensitivity, a problem that is difficult to overcome in any chromatographic technique. As during the extraction with SFE the components of the sample are already separated by their solubility, it can be considered as a kind of chromatographic technique. The big advantage is that there is virtually no limitation in the amount of the sample used. Whereas in most analytical-scale chromatographic techniques and also in SFC the on-column loadings are limited to the lower milligram or upper microgram region, the sample in SFE can be easily scaled up containing extractables in the upper milligram region. The only limitation factor for sample concentration is the solubility in the mobile phase. EXPERIMENTAL SECTION Apparatus. The apparatus is shown in Figure 1. The instrument was a Hewlett Packard supercritical fluid chromatograph G1205A which was modified for use as SFE equipment. The NMR flow cell was connected between the column outlet and the back-pressure regulator. To apply the pressure and modifier gradients, the “no inject” run modus of the chromatography software was used. The extraction cell was a 125 x 4.6 mm analytical HPLC column from Bischoff (Leonberg, Germany). The sample was packed in the column, and it was closed on both sides with the conventional 5 pm metal frits to keep the sample in the column. The column and the NMR flow cell were connected to the system by two Valco valves. They allowed the disconnection of (7) Albert, K; Braumann, U.; Tseng, L.-H.; Nicholson, G.; Bayer, E.; Spraul,
M.; Hofmann, M.; Dowle, C.; Chippendale, M.Anal. Chem. 1994,66,30423046. (8) Braumann, U.; Tseng, L.-H.; Albert, K; Spraul, M. GIT Fachz. Lab. 1994, 38, 77-79.
(9) Albert, K; Braumann U. In Frontiers in Analytical Spectroscopy; Andrews, D. L., Davies, A. M., Eds.; The Royal Society of Chemistry: Cambridge, U.K, in press.
0003-2700/95/0367-0930$9.00/0 0 1995 American Chemical Society
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the extraction vessel as well as the detection cell from the system. This was necessary to exchange the flow cell or to isolate the extraction cell for carrying out stopped-fow experiments without disturbing the system. SFE Parameters. COZ (99.9995%purity) was supplied from Messer-Griesheim. The pepper and coffee were normal available products which were bought already ground. No compression was used for packing of the sample. For the pepper extraction -1000 mg of black pepper was used. A C02 flow of 0.5 mL/min and an oven temperature of 42 "C was applied. The eluent was pure CO2 without modifler. The density was increased in 0.05 g/mL steps from 0.2 (71 bar) to 0.9 g/mL (294 bar). The density was increased after the signals of the extracted sample vanished or if no change in the NMR spectrum occurred. For the coffee extraction, a linear pressure gradient from 85 to 220 bar within 55 min and a COZ flow of 1mL/min was applied. The oven temperature was also 42 "C. NMR Parameters. The NMR spectrometer was a Bruker ARX 400. It was equipped with a specially designed, pressureproof probe head with a cell volume of 120pL, resisting pressures up to 400 bar and temperatures to 100°C. The probe is shown in Figure 2. For a more detailed description see ref 7. Heating of the probe head was performed using the temperature control instrument BVT 2000. A constant temperature of -315 f 0.2 K was kept for all experiments to keep supercritical conditions. It was not adjusted to the temperature of the extraction cell. The NMR data were collected on a ASPECT I station, using the 2D software of the Bruker UXNMR system. The recorded data were stored as 2D files on disk. Trpical memory requirements were 2-5 MB. This is in the order of the size of conventional 2D NMR experiments,but far less than the amounts of data that have to be collected and processed for modern 3D NMR experiments. Therefore data storage and processing is not a problem. The stored data allow extractions of data for both dimensions, chemical shift and time dependence. For a distinct state of the extraction, the corresponding NMR spectrum can be extracted from the 2D file. Information about the time dependence of the
Figure 2. SFC probe: (a, a') heated air, (b, b') eluent in/out, (c) double-tuned proton/lock coil, and (d) sapphire flow cell.
extraction process can be generated by summing up the NMR intensities within a selected chemical shift area. Those projections represent the concentration vs time of a compound that shows a NMR signal in this selected chemical shift range. As the relaxation times are prolonged due to the supercritical conditions, a flip angle of -50" was used to give maximum intensity of the NMR signals. Prolonged relaxation delays were used during the stopped-flow measurements. During the pepper extraction, the NMR spectra were recorded with a sweep width of 4425 Hz, which with 4K data points yielded an acquisition time of 0.463 s. Together with a relaxation delay of 0.5 s and 32 transitions per spectrum, a time resolution of 0.514 min/row was reached. A total of 339 spectra were recorded during 2 h 54 min 19 s to monitor the extraction. The parameters for the stopped-flow 1D spectrum were as follows: 4425 Hz sweep width, 8K data points, 90" pulse, 0.926 s acquisition time, 8 s relaxation delay, and 64 transitions. The resulting experiment time was 9 min 31 s. The parameters for the COSY45 were for the f2 domain: 3289 Hz sweep width, 1K data points, 0.156 s acquisition time, 4 s relaxation delay, and four transitions for each row. For f l , 128 rows were recorded in 38 min. The parameters for the coffee extraction were as follows: sweep width 4808 Hz, 8K data points, acquisition time 0.852 s, relaxation delay 0.4 s, and 16 transitions per spectrum. A time resolution of 0.336 min/row was reached. A total of 256 rows were acquired over a period of 1 h 26 min. As already mentioned, the chemical shift of the signals is strongly dependent upon the conditions of the solution, which made the calibration relatively arbitrary. A highfield-shifted impurity of the C02 was therefore used as internal standard and set to 0 ppm. Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
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Figure 3. Extraction of roasted coffee using an linear pressure gradient from 85 to 220 bar in 55 min. Projection reconstructed from intensities of NMR signals, 3.4-2.9 ppm for caffeine, 4.5-5.3 ppm for the triglycerides. m
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RESULTS Two systems were used to demonstrate the feasibility of the technique. The most popular and one of the earliest application of SFE is of course the decaffeination of coffee with C02.loJ1 In our case, in contrast to the conventional technique, roasted coffee was treated. Roasted Coffee.12-14 The contour plot of a extraction of 820 mg of coffee is shown in Figure 3. One can clearly see the caffeine,which is extracted at 105bar as indicated in the spectrum. What also is conspicuous is the upfield shift of the signals caused by the increasing density. (10)Zosel, K Deutsche Patentamt, Berlin: Offenlegungsschrift 2.005.293, 1971. (11)Zosel, K Angew. Chem. 1978,90, 748-755. (12) Horman, I.; Viani, R. Colloq. Int. Chim.Cafes Verts, Torrefies h u m Den'u., IC.R.1 1973,5, 102-107. (13) Baltes, W. Colloq. Sci. Int. Cafe, IC.R.1 1977,8, 85-96. (14)Murgia, E.; Richards, P.; Walton, H. F.J. Chromatogr. 1973,87,523-533.
932 Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
The projections are generated by summing up the intensities of the NMR signals of the indicated compound and therefore represent an extraction profile. The spectrum that is drawn from the 2D spectrum is shown in Figure 4 and allows the identification of the caffeine. Other components are extracted over a very wide range of higher pressures. This is most probably a complex mixture of triglycerides and other ingredients of the coffee, which are not separated by their solubility. The spectrum correspondingto the maximum of the extraction profile at 200 bar is shown in Figure 5. The enlarged section shows the pattern corresponding to the glycerin. Examination of the extraction process shows that the composition of the sample extracted between mainly 110 and 220 bar is uniform. It reaches its maximum at -200 bar. This extraction profile can be explained by different, chemically similar substances
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region, which occur during the whole extraction and may belong to lipids and related compounds, one can recognize that at least at three different densities additional NMR signals appear. They are indicated in the stackplot at 0.25 (v), 0.7 (A), and 0.9 g/mL (090).
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Figure 6. Extraction of black pepper using a manually adjusted density gradient from 0.2 (71 bar) to 0.9 g/mL (294 bar). Spectra collected at different densities. The spectra are shifted relative to each other to compensate the density-induced upfield shift. The main differences between the spectra are indicated.
which show no substantial difference in the NMR spectrum. Also, extraction of the same compound from different sites in the matrix may lead to a broader extraction profile. Black P e ~ p e r . ' ~ -The ' ~ extraction of black pepper is shown in Figure 6. Apart from the nonresolved signals in the aliphatic
The quality of the spectra collected at lower density is slightly inferior to the later ones. This can be explained by two different kinds of influences. At first the concentration of the sample during the lower density is lower. Also in the contour plot of the coffee it is obvious that the pressure dependence of the signal is stronger in COz with high density. This also means that instabilities of the system have a more marked effect during the first part of the extraction and may not affect the quality of the spectra during the later part, At a pressure of 294 bar (0.9 g/mL) two compounds are extracted: the first one vanishes rapidly and the second appears earlier and remains visible longer. In a second extraction, the sample was stopped at 294 bar after the first component (0) vanished. The stopped-flow proton spectrum is shown in Figure 7, and the compound can be identified as piperine. Also, a COSY45 was recorded for 38 min (shown in Figure 8). The connectivities are indicated. In this special case, with the background information available for the treated sample, the extracted compound could be already identified by the proton NMR spectrum. In other experiments where this might not be the case, only with the additional information provided by 2D assignment techniques would an identification be possible. The pressure of 294 bar was kept stable overnight, so that longer lasting 2D experiments should be possible if the SFC probe was equipped with an external lock or the extraction was carried out with deuterated m o d ~ e r . ~~
(15) Freist, W.; Chemie Unserer Zeit 1 9 9 1 , 2 5 , 135-142. (16) Grewe, R; Freist, W.; Neumann, H.; Kersten, S . Chem. Ber. 1 9 7 0 , 103, 3752 -3782. (17) Cleyn, De R; Verzele, D. Chromatograjhia 1 9 7 5 , 8, 342-344.
Analytical Chemistry, Vol. 67, No. 5,March
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DISCUSSION Direct monitoring provides all the advantages inherent in a continuous analytical method. The direct coupling of the extraction technique and the spectroscopic method minimized the need of sample treatment which may lead to loss, dilution, contamination, or decomposition of the sample. NMR spectroscopy provides the most structural information of all spectroscopic methods. Additionally, it provides linearity over the whole detectable range and is compatible with all proton-containing samples. On-LineMeasurements. The acquisition of on-line spectra most times means that NMR spectra are continuously acquired with identical parameters. The comparison of the spectra allows control of the extraction proceedings. Those spectra are normally collected as pseudo-2D files, which allow comfortable data treatment. The x-axis in this case represents the NMR axis in ppm, whereas the y-axis is the time, respectively, the pressure/density axis. There are principally two possibilities for varying the pressure or the density of the solvent respectively: a somewhat linear 934 Analytical Chemistry, Vol. 67, No. 5, March 7, 7995
variation or a step gradient. Both have advantages and disadvantages in respect to the acquisition of the NMR spectra which shall be discussed in the following. From the viewpoint of extraction a linear gradient seems to be more convenient. Principally, the changing density of the COZ causes an upfield shift of all NMR signals. If the density-induced shift is strong enough (Le., larger than the line width of the NMR signals) during the acquisition of one spectrum, it destroys the NMR resolution. Therefore, care has to be taken not to acquire too many transitions for each spectrum. Several spectra may be recorded instead, which can afterwards be shifted manually and summed up to achieve an increased signal-to-noise ratio (S/N). The advantage of a linear gradient is the smoothly changing eluting power, which means that two substances are separated even if they differ slightly in their solubility. A linear pressure gradient causes a relatively large change of the density, and thereby of the chemical shift, in the proximity of the critical point. This means that the shift is relatively large during the beginning of the extraction and decays to nearly zero at the end, when the increasing pressure causes only a small increase of the density. The use of a linear density gradient leads to a more uniform change during the whole experiment. In fact, the relation between density and signal shift is also not linear, especially near the critical point, so that periods with large signal shifts occur. Thus, both kinds of gradients can be used without distinct advantages for NMR detection. If a step gradient is used principally, the quality of the NMR spectra is better and they can be easily coadded for S/N improvement during the isobaric periods. F'roblems appear during the increase of pressure, which actually lasts a finite time. During those very steep pressure gradients, the NMR resolution is completely lost. If the pressure values are not carefully adjusted, during the first one the solvent strength may lie slightly under the solubility of a substance, whereas in the second it is clearly above. In this case, the sample may elute completely during the pressure step. Another disadvantage is that a group of substances is extracted without daerentiation. The normal approach is to apply a linear gradient to screen the substance for the appearing substances with the corresponding
pressures. This can naturally be done be W detection. The required pressures values can be estimated by observing the extraction profile. During a step gradient, the duration of the extraction for a distinct density can be manually adapted, to elute the substance completely and to avoid a cany-over to the next fraction. Increasing the density leads to an increase of the NMR intensity to a constant higher level. The adequate extractingdensity is reached when the intensity rapidly passes a maximum and drops to nearly zero. Use of Modifer. The addition of a modifier, such as methanol, principally introduces a relatively intense signal into the NMR spectrum. The advantage of the CO2 as proton-free eluent is partly lost. This may be overcome by using the corresponding deuterated form of the modifiers. A relatively low amount (-1-5%) leads to a negligible increase of cost but reduces the background signal dramatically. Another advantage is that the deuterated solvent provides an internal lock for the spectrometer. This eliminates most of the pressure-dependent shift of the NMR signals as both sample and background (modifier) show the same effect. The acquisition and processing of the NMR data is much easier. A severe problem is introduced by the commonly used MeOH, which shows two NMR signals. The chemical shift of the hydroxyl proton strongly depends on pressure, temperature, and composition of the mobile phase. Especially during the beginning of the extraction, when the sample is not moistened by methanol and all the modifier is adsorbed in the extraction cell, a very unstable NMR signal of the modifier is observed, which means that it cannot be used as internal lock. All the discussed problems of stability, especially with the use of modifier, may be overcome if the spectra are acquired in stopped-flow rather than in on-line mode. But even if the quality of the on-line spectra may be inferior to those recorded in stopped flow, this normally only means that the information about coupling constants is lost. An identification and quantification of known ingredients is still possible. Stopped-FiowMeasurements. If a sample shall be observed in more detail or with higher sensitivity, stopped-flow measurements are possible. Two switching valves allow one to fuc the sample in the NMR flow cell and simultaneouslydisconnect the extraction cell from the pump. The system normally slowly loosens the pressure over the back-pressure nozzle. By the second valve, the extraction cell is
isolated from the system and the pressure can be kept. This means that a few minutes after the end of the NMR experiment, when the system has reached the pressure, the extraction can be continued without introducing disturbances in the sample. It has been proved that it is possible to keep even a pressure of 290 bar stable for many hours. which is the precondition to perform all kinds of 2D NMR experiments which are required if the technique is used for structural elucidation of unknown compounds. During earlier experiments, pressure was controlled by an additionally connected sensor. Now the pressure is controlled by the NMR signal itself. Also, the temperature has to be very stable because instabilities introduce fluctuation in the chemical shifts. The only limitation factor now was the lack of an external lock to stabilize the NMR spectrometer. Nevertheless, we were able to record a COSY45 for 38 min, and with our spectrometer, -3 h seemed to be the limit after which the drift of the spectrometer destroys the spectrum. The advantage of stopped-flowmeasurements over preparative SFE is that there is no danger of contamination by the solvent which is used to collect the sample or to elute it from the trapping column. Also, on-line detection avoids loss of substance due to inadequate sample trapping techniques. CONCLUSION It was shown that it is possible to monitor the extraction process. This allows a rapid tuning off of the extraction conditions. In principle, NMR spectroscopy is not a very good method for the analysis of mixtures. In contrast to, for example, W absorption bands, the high dispersion of the NMR signals allows the specific detection of a known compound in relative independence from the presence of one ore more chemically related compounds. As NMR detection is a noninvasive method it may also be used to fractionate the extraction in a proper way for subsequent chromatographic and spectroscopic investigations. Received for review October 7, 1994. Accepted December 19, 1994.@ A69409896 Abstract published in Advance ACS Abstracts, February 1, 1995.
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