Anal. Chem. 1997, 69, 1421-1425
Direct On-Line Coupling of Capillary HPLC with 1H NMR Spectroscopy for the Structural Determination of Retinyl Acetate Dimers: 2D NMR Spectroscopy in the Nanoliter Scale Go 1 tz Schlotterbeck,† Li-Hong Tseng,† Heidrun Ha 1 ndel,† Ulrich Braumann,‡ and Klaus Albert*,†
Institut fu¨ r Organische Chemie, Universita¨ t Tu¨ bingen, Auf der Morgenstelle 18, D-72076 Tu¨ bingen, Germany, and Bruker Analytische Messtechnik GmbH, Silberstreifen, D-76287 Rheinstetten, Germany
This paper presents the application of directly coupled capillary high-performance liquid chromatography (capillary HPLC) and proton high-field nuclear magnetic resonance spectroscopy (NMR) for structural elucidation of a so-far unknown kitol isomer. One- and two-dimensional continuous- and stopped-flow NMR spectra were recorded in a 180 µm i.d. capillary, corresponding to a detection volume of only 200 nL. Unequivocal structural assignment on the basis of 1D and 2D stopped-flow capillary HPLC-NMR experiments was performed. The kitol isomer mixture was present in a sample of thermally isomerized retinyl acetate and separated on a capillary column. The coupling of liquid chromatography and NMR spectroscopy offers an effective tool to analyze mixtures of organic compounds in polymer,1,2 pharmaceutical,3,4 and biological5,6 samples. Until now, flow cells with volumes of 60-200 µL and analytical HPLC columns have been used with conventional proton-containing HPLC grade solvents as eluents.7,8 During recent years great efforts have been made to reduce the solvent consumption, either for ecological reasons or due to limitations in the amount of sample available. As more sensitive detectors are now available, miniaturized separation techniques such as capillary HPLC and capillary electrophoresis (CE) are gaining increasing interest. Compared to conventional detecting systems, NMR seems to be less sensitive. New developments such as the now comercially available ultrahigh-field 800 MHz spectrometers or new radio frequency (rf) microcoils have made NMR spectroscopy applicable to previously unreachable analytical areas. In the present study, we show the practical use of directly coupled capillary HPLC with †
Universita¨t Tu ¨ bingen. ‡ Bruker Analytische Messtechnik GmbH. (1) Hatada, K.; Ute, K.; Kitayama, T.; Nishimura, T.; Kashiyama, M.; Fujimoto, N. Polym. Bull. 1990, 23, 549-554. (2) Albert, K.; Braumann, U.; Streck, R.; Spraul, M.; Ecker, R. Fresenius’ J. Anal. Chem. 1995, 352, 521-528. (3) Sidelmann, U. G.; Gavaghan, C.; Carless, H. A. J.; Spraul, M.; Hoffmann, M.; Lindon, J. C.; Wilson, I. D.; Nicolson, J. K. Anal. Chem. 1995, 67, 44414445. (4) Spraul, M.; Hofmann, M.; Dvortsak, P.; Nicholson, J. K.; Wilson, I. D. Anal. Chem. 1993, 65, 327-330. (5) Albert, K.; Schlotterbeck, G.; Braumann, U.; Ha¨ndel, H.; Spraul, M.; Krack, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1014-1016. (6) Pursch, M.; Strohschein, S.; Ha¨ndel, H.; Albert, K. Anal. Chem. 1996, 68, 386-393. (7) Albert, K.; Bayer, E. HPLC Detection Newer Methods; VCH: New York, 1992; pp 197-229. (8) Albert, K. J. Chromatogr. A 1995, 703, 123-147. S0003-2700(96)00902-X CCC: $14.00
© 1997 American Chemical Society
NMR spectroscopy to determine the structure of an unknown kitol isomer (1). 2D NMR spectra were recorded in detection cells of 180 µm i.d. and a volume of 200 nL. The coupling of capillary HPLC and NMR spectroscopy9 offers several practical advantages compared to the conventional hyphenation of HPLC and NMR. Extremely high costs of fully deuterated solvents forbid their use as eluents in conventional LC-NMR coupling, but the small flow rates of capillary chromatography make the use of deuterated eluents economically feasible. Therefore, solvent suppression, which leads to distortion of parts of the NMR spectrum, is no longer necessary. Since even deuterated solvents are not completely deuterated, small proton signals remain detectable in 1H NMR spectroscopy. But suppression of these small signals results only in a distortion of the spectrum and does not yield a further increased NMR receiver gain. Adaptation of the detection cell dimensions to the analytical problem could be easily achieved by using the capillary as a flow cell. Miniaturized flow cells10 with rf microcoils11 show a higher NMR mass sensitivity compared to that of traditional Helmholtz-type rf coils.12,13 With small amounts of sample, higher concentrations of analyte in the detection cell are obtained while column dimensions are reduced in capillary separation techniques. Kitols, natural retinol dimers, were initially found in whale liver oil.14-16 Kitol and its derivatives were regarded as a 1,4 cycloaddition product of the 11,13-diene part of the first retinol molecule with the 13-monoene part of the second retinol molecule, leading to the formation of the central cyclohexene ring17,18 (see Figure 1a). The spectroscopic data for kitols are based mainly on 1D 200 MHz 1H NMR spectra19 and only two structures, 2 and its 9′-cis isomer 2a, have been discussed in the literature. (9) Behnke, B.; Schlotterbeck, G.; Tallarek, U.; Strohschein, S.; Tseng, L. H.; Keller, T.; Albert, K.; Bayer, E. Anal. Chem. 1996, 68, 1110-1115. (10) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849-3857. (11) Peck, T. L.; Magin, R. L.; Lauterbur, P. C. J. Magn. Reson. B 1995, 108, 114-124. (12) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. J. Am. Chem. Soc. 1994, 116, 7929-7930. (13) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1969. (14) Pritchard, H.; Wilkinson, H.; Edisbury, J. R.; Morton, R. A. Biochem. J. 1937, 31, 258-265. (15) Burger, B. V.; Garbers, C. F. J. Chem. Soc., Perkin Trans. 1 1973, 590595. (16) Mousseron-Canet, J. C. Bull. Soc. Chim. Fr. 1966, 3043-3044. (17) Pfoertner, K. H.; Englert, G.; Schoenholzer, P. Tetrahedron 1988, 44, 10391052. (18) Mousseron-Canet, M.; Lerner, D.; Mani, J. C. Bull. Soc. Chim. Fr. 1968, 4639-4645.
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Figure 1. Formation of kitol. (a) Reaction scheme of kitol formation from retinyl acetate. (b) Configuration of the different kitol isomers.
Chromatographic separation of a mixture containing kitol isomers and the monomer, retinyl acetate, show the existence of at least three kitol isomers. Investigations of stopped-flow 1D and 2D 1H NMR spectra recorded in a capillary detection cell of 180 µm i.d. and 200 nL volume lead to the complete structural assignment of a new retinyl acetate dimer (1), including exact coupling constants and chemical shifts. EXPERIMENTAL SECTION Chemicals. Deuterated solvents were purchased from Deutero GmbH (Herresbach FRG) (acetonitrile-d3, 99%) and Merck AG (Darmstadt,FRG) (acetone-d6, 99.8%). Diethyl phthalate was obtained from Aldrich (Steinheim, FRG), and a mixture of thermally isomerized retinyl acetate was obtained from BASF AG (Ludwigshafen, FRG). Capillary Chromatography. Fused silica capillaries of 180 µm i.d./350 µm o.d. were obtained from Polymicro Technology (Phoenix, AZ). The packing of the capillary columns with 3 µm C18 stationary phase over a length of 150 mm and 250 µm i.d. was performed by Grom Co. (Herrenberg, FRG). The HPLC system consisted of a Bischoff pump, a Valco injection device, a stainless steel T-piece, and a resistance capillary. The HPLC pump, T-piece, and resistance capillary were located at a distance of about 3 m from the 14 T cryomagnet. Solvent splitting was accomplished with a stainless steel T-piece and a resistance capillary of 50 µm i.d. and 30 cm length, yielding a split ratio of approximately 1:100. The flow rate was adjusted to about 3 µL/min. The split solvent was recycled. The eluent was filtered on a guard column before splitting to avoid clogging of the capillary columns. An isocratic elution in acetonitrile-d3 was performed to separate the mixture (19) Tsukida, K.; Ito, M. J. Nutr. Sci. Vitaminol. 1980, 26, 319-322.
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of vitamin A derivatives. For the on-line coupled separation, the total amount injected onto the column was about 50 µg. The analytical capillary separation was carried out under the same conditions but with respect to minimized dead volume. The HPLC system additionally consisted of a Shimadzu SPD-10A UVvis detector and a LC Packings U-Z View flow cell of 140 nL volume. The separation was monitored at 280 nm; 100 nL of a 0.001% (m/v) solution of the retinyl acetate reaction mixture was injected. NMR Spectroscopy. NMR spectra were recorded by use of a Bruker AMX 600 spectrometer equipped with a 2.0 mm capillary microprobe matched with an rf coil selective for protons and an “insert” capillary detection cell of 200 nL volume. Shimming was performed prior to measurement on the lock signal of the eluent acetonitrile-d3. As usual in conventional HPLC-NMR, coupling measurements were carried out without spinning. Static NMR Experiments. Static measurements were performed by injecting a 0.1% solution of diethyl phthalate in acetone-d6 directly into the capillary cell. A total of 16 K data points with a spectral width of 5208 Hz, resulting in an acquisition time of 1.57 s, were recorded. The relaxation delay was set to 2 s, and 128 transients were coadded with a total acquisition time of 7 min 43 s. The data were processed without applying any window function. On-Line NMR Experiments. For continuous-flow measurements, a packed capillary column (150 mm × 0.25 mm) was located directly before the capillary NMR detection cell. For online measurements, about 1 µL of a 5% (m/v) solution of retinyl acetate reaction product in acetonitrile-d3 was separated. Twentyfour transients with 4K complex data points and a spectral width of 5345 Hz were recorded. A relaxation delay of 1 s and an acquisition time of 0.38 s/transient were used. The pulse angle
was set to 45°. During the separation, 64 FIDs with an acquisition time of 33.1 s/FID were recorded. Data were treated as a 2D NMR matrix (t1, retention time) and processed with XWINNMR software. A phase-sensitive Fourier transformation was performed in the t2 direction only. Prior to Fourier transformation, a shifted sine bell function (shift 2.0) was applied to the FID in F2 only. Stopped-Flow 1D NMR Experiments. Stopped-flow spectra were recorded when the peak maximum of the chromatographic peak had entered the detection volume. This event could be checked only by simultaneous monitoring of the chromatographic separation by continuous-flow 1H NMR spectroscopy. The stopped-flow 1D NMR spectra were recorded with 16K data points and a spectral width of 5435 Hz, resulting in an acquisition time of 1.5 s. A relaxation delay of 1 s was used. Usually, 128 transients were coadded with a total acquisition time of 320 s. Exponential window function with 0.5 Hz line-broadening was applied before the Fourier transformation step. Stopped-Flow 2D NMR Experiments. A phase-sensitive TOCSY experiment was carried out on kitol 1. A total of 400 t1 increments with 88 transients and 4K complex data points were acquired in simultaneous mode with a spectral width in both dimensions of 4505 Hz. With an acquisition time of 0.45 s and an applied mixing time of the MLEV spin lock of 65 ms, the total acquisition time amounts to 15 h. The data were apodized with a shifted squared sine bell window function in both dimensions and zero-filled in the F1 dimension to 1024 data points. RESULTS AND DISCUSSION Characteristics of the NMR Probe. Compared to previous experiments with a modified 2.5 mm microprobe,9 we used here an improved design with an rf coil of 2.0 mm diameter. This resulted in an increased filling factor and better line shapes. Even in the picomolar range, high-resolution NMR spectra with line widths in the order of 1.5 Hz were recorded.20 A signal-to-noise ratio (S/N) of 18 was calculated for the signal of the methyl group of 900 pmol of diethyl phthalate at δ ) 1.35 ppm. For comparison of NMR capillary detection cells, an approximation of the detection limit (S/N ) 3) was made analogous to ref 13. A limit of detection (LOD) of 150 pmol was obtained for the triplet without any prior apodisation of the FID. These data show that the sensitivity of nanoliter flow cells equipped with rf microcoils is better than that obtainable in conventional HPLC-NMR experiments. To show the capability of the NMR probe for on-line NMR detection of chromatographic separations Figure 2illustrates the on-line 1H NMR spectrum of all-trans-retinyl acetate recorded at the chromatographic peak maximum during a separation. A structural assignment of this compound is easy based on the data recorded in the nanoliter flow cell. The spectrum recorded in a capillary detection cell “on the fly” reveals the same information content as conventionally recorded NMR spectra. All coupling constants, integration ratios, and chemical shifts are visible in the spectrum. The spectrum in Figure 2 clearly shows the power of the insert flow cell design to examine small amounts of sample for structural elucidation. Monitoring of the Separation by Continuous-Flow 1H NMR Spectroscopy. An isocratic capillary HPLC separation was used to separate the kitol isomers from retinyl acetate monomers. The (20) Albert, K.; Schlotterbeck, G.; Tseng, L. H.; Braumann, U. J. Chromatogr. A 1996, 750, 303-309.
Figure 2. Characteristics of the improved capillary NMR probe. Extracted on-line 1H NMR spectrum of all-trans-retinyl acetate.
Figure 3. Capillary HPLC separation of a retinyl acetate reaction mixture.
resulting NMR chromatogram (a pseudo-two-dimensional NMR contour map) is illustrated in Figure 4. In such a representation, the 1H NMR frequency domain is in the horizontal dimension, and the chromatographic separation time is in the vertical dimension. The signals of the first-eluting all-trans-retinyl acetate monomer can easily be assigned. Besides the retinyl acetate, at least two peaks of different dimers of retinyl acetate (kitols) are visible in the contour plot. The chromatogram along the F1 axis was reconstructed by a summation over the 1H NMR signal intensities of the olefinic part of the spectrum. Although such a reconstruction suffers from a small number of data points, a separation of three compounds is still visible. The resolution of the reconstructed chromatogram is in the same order as a UV trace of a separation with the same amount of sample injected. The quality of the separation obviously suffers from overloading the capillary column, but due to the introduction of a second dimension, the NMR chemical shift, this drawback is more than outweighted. In contrast to conventional HPLC-NMR coupling, no additional detection of the separation by UV spectroscopy was performed. For stopped-flow experiments, the chromatographic peak maximum was detected by continuously monitoring the separation progress only by NMR spectroscopy. Therefore, in contrast to conventional capillary HPLC (see Figure 3), a higher amount of sample has to be injected onto the column since NMR spectroscopy is less sensitive than UV-visible spectroscopy. A detailed analysis using 1D and 2D stopped-flow spectra of these compounds shows the existence of a hitherto unknown kitol isomer. Characterization of the New Kitol Isomer. The dimerization of retinyl acetate in a concerted 1,4 cycloaddition leads to kitol Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
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Figure 4. Capillary HPLC-NMR chromatogram of the kitol separation in a 180 µm i.d. column. Peak 1 corresponds to all-trans-retinyl acetate, peak 2 to kitol 1, and peak 3 to a mixture of kitol 2 and kitol 2a.
Figure 5. Stopped-flow 1H NMR spectrum of kitol 1 for the separation in a 180 µm i.d. column, recorded in a capillary detection cell (128 coadded transients).
isomers with different configurations at the newly formed central cyclohexene ring. In addition to the known kitols 2 and 2a, we found in a mixture of thermally isomerized retinyl acetate, the kitol isomer 1 (see Figure 1b), which possesses a different configuration at the central cyclohexene ring. The steric hindrance of the axial trienyl and tetraenyl side chains explains the observed instability of this kitol isomer. We found that it decomposes rapidly, and so attempts of isolation failed. Thereby, the advantages of the closed system, the directly coupled capillary HPLC and NMR spectroscopy, are quite obvious. The structural assignment (the molecular mass was determined by LC/MS) is based on stopped-flow 1D 1H NMR (depicted in Figure 5) and 2D COSY and TOCSY spectra that were recorded in the capillary (TOCSY shown in Figure 6). We briefly discuss the derivation of the structure of kitol 1. Special attention is drawn to the proton chemical shifts and coupling constants of those 1424 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
protons involved in the ring formation, i.e., H-14/H-14′, H-10, H-11, and H-12. The upfield shift of H-11 and H-14/H-14′ compared to the retinyl acetate data indicates the declared reaction site of the cycloaddition: the 11,13-diene part of one retinol molecule reacts with the 13-monoene part of the other retinol molecule, leading to the formation of the central cyclohexene ring. The small coupling constant between protons H-14/H-14′ (J14,14′ < 2 Hz) leads to the conclusion of a quasi axial/equatorial conformation at the cyclohexene ring. We assign the one-proton doublet at 5.4 ppm as H-10 with a coupling constant J10,11 of 10.2 Hz and the oneproton doublet at 5.5 ppm as H-12 with J11,12 of 6.8 Hz with respect to the Carplus equation. The assignment of H-12 arises from the smaller angle between H-11 and H-12 (about 30°), whereas the angle between H-11 and H-10 appears to be 180°. Therefore, the orientation of the trienyl and tetraenyl side chains in the unfavorable quasi axial direction results in the twisted half-chair form of
Table 1.
1H
NMR Spectroscopic Data for Kitol 1, Using the Numbering Scheme Shown in Figure 1 proton olefinic
no. ppm no. ppm
12 * 5.49 12′ 11′ 5.77 6.49
10 5.39 10′ 6.01
methyl 8 6.06 8′ 6.06
7 6.10 7′ 6.17
13 1.74 13′ 1.17
9 1.74 9′ 1.92
5 1.70 5′ 1.70
others 1 1.04 1′ 1.04
15 4.37 15a′ 4.30
14 2.22 15b′ 3.90
11* 2.84 14′ 2.25
4 2.02 4′ 2.02
coupling constants 3 1.64 3′ 1.64
2 1.49 2′ 1.49
OCH3 2.16 OCH3 2.16
J12,11 10.39 J12′,1!′ 10.97
J11,10 6.83 J11′,10′ 15.93
J7,8 14.00 J7′,8′ 16.00
J15,15 not obsd J15′,15′ 4.00
J15,14 2.50 J15′,14′ 10.00
Figure 6. Stopped-flow TOCSY of the capillary HPLC separation. Kitol 1 was measured in a cell volume of 200 nL.
the central cyclohexene ring. The assignment of the methyl groups was derived from the connectivities observed in the TOCSY spectrum. The detailed analysis of chemical shifts, coupling constants, and integration ratios results in the structure assignment shown in Table 1. Figure 7 illustrates the differences in the olefinic region of the 1H NMR spectra of the new kitol isomer 1 in comparison to the spectra of the previously reported kitol isomers 2 and 2a. CONCLUSION Our study of retinyl acetate dimers shows that miniaturized hyphenated NMR techniques are an effective tool to solve analytical problems without any loss of structural information, while gaining more benefits than conventional coupling of liquid chromatography and NMR spectroscopy. A main advantage of coupling capillary HPLC with NMR spectroscopy is the use of fully deuterated solvents, which allows the acquisition of undisturbed spectra. On the basis of stopped-flow 2D NMR spectra, it was possible to identify a so-far unknown kitol isomer from a mixture of thermally isomerized retinyl acetate. An important step
Figure 7. Stopped-flow 1H NMR spectra of the different kitol isomers. (a) Kitol 1 recorded in a 200 nL capillary cell. (b) Kitol 2 recorded in a 120 µL cell. (c) Kitol 2a recorded in a 120 µL cell.
to further minimization of coupled liquid chromatography separation techniques and NMR spectroscopy has been done. Substances susceptible to air and light, such as conjugated polyene systems, can easily be investigated with closed miniaturized systems. ACKNOWLEDGMENT We thank BASF Aktiengesellschaft (Ludwigshafen, Germany) for the supply of kitol samples. We also acknowledge the support of Sabine Strohschein and Matthias Pursch for reading the manuscript and for helpful discussions. Received for review September 10, 1996. January 19, 1997.X
Accepted
AC960902B X
Abstract published in Advance ACS Abstracts, February, 15, 1977.
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