Recent Progress in LC-NMR

Transcription

1 Recent Progress in LC-NMR Sumitomo Chemical Co., Ltd. Organic Synthesis Research Laboratory Takashi TOKUNAGA Masahiko OKAMOTO LC-NMR has been noted as a practical method for mixture analysis in recent years. Technical backgrounds of high performance LC-NMR are discussed from the point of view of NMR, chromatography and related technologies. Constituent profiling and LC-2D NMR are introduced as practical applications. Further hyphenated techniques such as LC-NMR/MS and chiral LC-CD-NMR are also described. Hyphenated technique 1) HPLCNMR LC-NMR Fig ) 3), 4) LC-MS 2000 HPLC Pump Detector NMR Flow-cell NMR Solvent (Deuterated) Column Drain Storage Loop Workstation LC-NMR system Fig. 1 Schematic view of conventional LC-NMR system 2010-

2 NMR LC-NMR LC- NMR Fig. 2 LC-NMR LC-NMR NMR 1 LC-NMR LC- NMR Flow-cell Fig. 1 HPLC NMR Fig. 3(A) 5) HPLC 30 µl 120 µl 4.6 mm 1 ml/min 120 µl ) Fig. 3(B) 800 MHz magnet (A) HPLC Drain SPE (Solid phase extraction) unit HPLC UV detector MS 8 sec (120 μl) Flow-cell (120 μl) 100% Column Storage loop (B) HPLC Drain Photo at National Institute of Biomedical Innovation Fig. 2 High performance LC-NMR system (800 MHz LC-SPE-NMR/MS equipped with cryogenic probe) 40 sec 8 sec (120 μl) 60% Fig. 3 Peak width and sample concentration in a flow-cell LC-NMR NMR LC-NMR 2 mm LC-NMR 1/5 LC-NMR 2010-

3 0.2 ml/min 120 µl 40 LC-NMR HPLC SPE Solid phase extraction LC-NMR SPE Fig : Organizer 2: HPD LC-SPE-NMR Dispenser 3: ACE LC-SPE-NMR Interface 4: Dilution pump SPE cartridge (Spark Hyspher Resin GP10, 96 cartridges, volume 30 μl) SPE unit (96 catridges 2) Photo at National Institute of Biomedical Innovation Spark PROSPEKT 2 TM SPE system LC-NMR 7) SparkSPE Fig. 4 LC-NMR SPE µl LC-NMR NMR NMR DQF-COSY HMBC 8) LC-NMR 9) SPE 2 NMR NMR LC-NMR NMR 3/2 10) LC-NMR Fig MHz 11) MHz 50 NMR 12)

4 MHz NMR 100 LC-NMR Fig. 5 Magnetic field strength in NMR and LC-NMR LC-NMR 800 MHz LC-NMR Fig µg 1 H NMR 500 MHz 500 MHz scans, 20 h year ppm Bruker AVANCE TM II (800 MHz) 512 scans, 30 min MHz NMR LC-NMR LC-NMR ODS HPLC LC-NMR 1990 PFG (Pulsed Field Gradient) WET 13) LC-NMR ODS LC-NMR SPE Fig ppm 1 H NMR spectrum of a 0.1% component with an 800 MHz LC-NMR equipped with cryogenic probe

5 Fig. 1 1 LC-NMR LC-NMR HPLC 1 H NMR H NMR Fig % NMR LC-NMR 2 LC-2D NMR LC-NMRNMR 1 H- 13 C NMR HSQC HMBCHSQC - 1 JCH HMBC 2 JCH 3 JCH HMBC Intens mau loop1 loop2 loop3 loop4 loop5 loop6 loop7 loop8 loop9 loop10 loop11 loop12 loop13 loop14 loop15 loop1 : 1.38% loop2 : 1.45% loop3 : 0.94% loop4 : 0.85% loop5 : 0.28% loop6 : 82.08% loop7 : 1.43% loop8 : 0.68% loop9 : 1.56% loop10 : 0.85% loop11 : 0.47% loop12 : 0.11% loop13 : 0.61% loop14 : 0.63% loop15 : 1.53% Fig. 7 Constituent profiling by 1 H LC-NMR (aromatic region) 2010-

6 1 H NMR LC-NMR LC-NMR SPE 300 µg HMBC Fig. 8SPE 3 SPE HMBC SPE 13 C HSQC HMBC Table Fig HMBC spectrum with LC-SPE-NMR (800 MHz, cryogenic probe) 0.0 LC-NMR LC-NMR NMR LC-NMR MS LC-NMR/MS LC-CD-NMR NMR MS LC-NMR/MS1990 Pfizer 14) LC-NMR/MS LC NMR Exarchou SPE LC- SPE-NMR/MS Fig. 9 15) SPE NMR NMR Table 1 HMBC and HSQC experiments by LC-NMR Sample Frequency (MHz) Cryogenic probe Plant extract Plant extract Plant exudate Plant extract Plant extract NOESY, HSQC, HMBC HMQC, HMBC COSY, TOCSY, HSQC, HMBC COSY, HSQC, HMBC COSY, TOCSY, HMBC Synthetic peptides Model mixture Drug degradation products Minor component Minor component Capillary LC-NMR Semi-preparative LC-SPE-NMR Column trapping COSY, HSQC, HMBC TOCSY, HMBC COSY, HMBC a) E. Garo, J. Wolfender, K. Hostettrnann, W. Hiller, S. Antus and S. Mavi, Helv. Chim. Acta, 81, 754 (1998). b) V. Exarchou, M. Godejohann, T. A. van Beek, I. P. Gerothanassis and J. Vervoort, Anal. Chem., 75, 6288 (2003). c) G. Karagianis, A. Viljoen and P. G. Waterman, Phytochem. Anal., 14, 275 (2003). d) C. Clarkson, D. Stark, S. H. Hansen and J. W. Jaroszewski, Anal. Chem., 77, 3547 (2005). e) A. Pukalskas, T. A. van Beek and P. de Waard, J. Chromatgr. A, 1074,81 (2005). f) P. Hentschel, M. Krucker, M. D. Grynbaum, K. Putzbach, R. Bischoff and K. Albert, Magn. Reson. Chem., 43, 747 (2005). g) F. Xu and A. J. Alexander, Magn. Reson. Chem., 43, 776 (2005). h) T. Murakami, N. Fukutsu, J. Kondo, T. Kawasaki and F. Kusu, J. Chromatgr. A, 1181, 67 (2008). SPE Other techniques Experiment Reference a) b) c) d) e) f) g) h) 2010-

7 HPLC Pump Water 5% MS 95% Solvent (Non-deuterated) UV Detector SPE NMR Column Solvent (Deuterated) : Non-deuterated solvent : Deuterated solvent Fig. 9 Schematic representation of the LC-UV-SPE-NMR/MS LC-NMR NMR-silent 16 NMR NMR MS LC-CD-NMR LC-NMR CD Circular Dichroism LCLC LC-CD-NMR Fig. 10 Pump UV detector NMR Solvent Chiral column Workstation Conventional LC-NMR CD detector Chiral LC-CD-NMR system Fig. 10 Schematic view of chiral LC-CD-NMR system 2010-

8 CDNMR Fig. 11(A) UV 8 LC-CD-NMR UVCD NMR CD Fig. 11(B) (A)UV Peak 1 Peak 1(+) Fig Main Component min (B)CD (C)NMR 8.5 2(+) + 3(+) 4 5 Peak 8 Peak 7 Main component Peak 6 Peak 5 Peak 4 Peak 3 Peak 2 Peak 1 4(+) 6 5( ) 6( ) Main Component (+) + 7(+) 7 8 8( ) ppm Chiral LC-CD-NMR analysis of pyridylalanine derivative + Enantiomer Opposite CD sign Same NMR spectrum H NMR Fig. 11(C) CD 1 H NMR 6 NMR UVNMR CD LC-CD-NMR LC LC 3 Table 2 C LC-NMR Table 2 Comparison of Rs Values between Chiral Columns Retention time(min) Peak width(min) Chiral column A Chiral column B Chiral column C Column Solvent Flow rate Column temperature etc. Enantiomer Retention time(min) Peak width(min) Rs Best separation with Chiral column C 2010-

9 LC-NMR LC- 2D NMR LC- NMR/MS LC-CD-NMR LC-NMR 18) LC-NMR 19) NMR NMR 20) LC-NMR 21) LC-NMR LC-NMR 1) D. L. Norwood, J. O. Mullis and T. N. Feinberg, Sep. Sci. Technol., 8, 189 (2007). 2) N. Watanabe and E. Niki, Proc. Jpn Acad., 54, 194 (1978). 3) K. Albert Ed., On-line LC-NMR and Related Techniques, John Wiley & Sons Ltd. (2002). 4) H Pasch, L. C. Heinz, T. Macko and W. Hiller, Pure Appl. Chem., 80, 1747 (2008). 5),,,,, 11, 897 (1997). 6) G. J. Sharman and I. C. Jones, Magn. Reson. Chem. 41, 448 (2003). 7) J. A. de Koning, A. C. Hogenboom, T. Lacker, S. Strohschein, K. Albert and U. A. T. Brinkman, J. Chromatogr. A, 813, 55 (1998). 8) T. Murakami, N. Fukutsu, J. Kondo, T. Kawasaki and F. Kusu, J. Chromatogr. A, 1181, 67 (2008). 9) A. J. Alexander, F. Xu and C. Bernard, Magn. Reson. Chem., 44, 1, (2006). 10) T. D. W. Claridge, High-Resolution NMR Techniques in Organic Chemistry, Elsevier Science Ltd. (1999), p ) U. G. Sidelmann, U. Braumann, M. Hofmann, M. Spraul, J. C. Lindon, J. K. Nicholson and S. H. Hansen, Anal. Chem., 69, 607 (1997). 12) C. A. Scott, D. A. Cragg, F. Row, D. J. White and P. C. J. White, J. Magn. Reson. 60, 397 (1984). 13) S.H. Smallcombe, S.L. Patt and P.A. Keifer, J. Magn. Reson. A, 117, 295 (1995). 14) K.I. Burton, J.R. Everett, M.J. Newman, F.S. Pullen, D.S. Richards and A.G. Swanson, J. Mass Spectrom., 32, 64 (1997). 15) V. Exarchou, M. Godejohann, T. A. van Beek, I. P. Gerothanassis and J. Vervoort, Anal. Chem., 75, 6288 (2003). 16) J.H. Beynon, R.A.Saunders and A.E. Williams, The Mass Spectra of Organic Molecules, Elsevier (1968). 17) T. Tokunaga, M. Okamoto, K. Tanaka, C. Tode and M. Sugiura, Anal. Chem., 82, 4293 (2010). 18) H.C. Dorn, Anal. Chem. 56, 747A (1984). 19) Y. Kashima and Y. Okabayashi, Chem. Pharm. Bull., 58, 423 (2010). 20) Y. Lin, S. Schiavo, J. Orjala, P. Vouros and R. Kautz, Anal. Chem., 80, 8045 (2008). 21) M.E. Elyashberg, A.J. Williams and G.E. Martin, Prog. Nucl. Magn. Reson. Spectrosc., 53, 1 (2008)

10 PROFILE Takashi TOKUNAGA Masahiko OKAMOTO 2010-