Malz, F., & Jancke, H. (2005). Validation of quantitative NMR. Journal of Pharmaceutical and Biomedical Analysis, 38(5),

Transcription

1 Malz, F., & Jancke, H. (2005). Validation of quantitative NMR. Journal of Pharmaceutical and Biomedical Analysis, 38(5),

2 Malz, F., & Jancke, H. (2005). Validation of quantitative NMR. Journal of Pharmaceutical and Biomedical Analysis, 38(5),

3 Frequency dependence of 90 pulse Z magnetization B1=2500 Hz Frequency offset (Hz)

4 Ernst optimium angle

5 Malz, F., & Jancke, H. (2005). Validation of quantitative NMR. Journal of Pharmaceutical and Biomedical Analysis, 38(5),

6 Phase and baseline corrections were done manually. This manual mode was used also for the signal integration (choice of integration limits (generally without the 13C satellites) and if needed the BIAS- and SLOPE-functions for the integral calculation due to improper baseline corrections). Integral region 64 times linewidth for 99% area. (0.1ppm for a 1 Hz MHz) Malz, F., & Jancke, H. (2005). Validation of quantitative NMR. Journal of Pharmaceutical and Biomedical Analysis, 38(5),

7 Malz, F., & Jancke, H. (2005). Validation of quantitative NMR. Journal of Pharmaceutical and Biomedical Analysis, 38(5),

8 Malz, F., & Jancke, H. (2005). Validation of quantitative NMR. Journal of Pharmaceutical and Biomedical Analysis, 38(5),

9 Integral region 64 times linewidth for 99% area. (0.1ppm for a 1 Hz MHz) Giraudeau, P. (2014). Quantitative 2D liquid-state NMR. Magnetic Resonance in Chemistry, 52(6),

10 Giraudeau, P. (2014). Quantitative 2D liquid-state NMR. Magnetic Resonance in Chemistry, 52(6),

11 Calibration

12 Lewis, I. A., Schommer, S. C., Hodis, B., Robb, K. A., Tonelli, M., Westler, W. M., Markley, J. L. (2007). Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra. Analytical Chemistry, 79(24),

13 INEPT revinept x,y S evol. Lewis, I. A., Schommer, S. C., Hodis, B., Robb, K. A., Tonelli, M., Westler, W. M., Markley, J. L. (2007). Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra. Analytical Chemistry, 79(24),

14 Lewis, I. A., Schommer, S. C., Hodis, B., Robb, K. A., Tonelli, M., Westler, W. M., Markley, J. L. (2007). Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra. Analytical Chemistry, 79(24),

15 Lewis, I. A., Schommer, S. C., Hodis, B., Robb, K. A., Tonelli, M., Westler, W. M., Markley, J. L. (2007). Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra. Analytical Chemistry, 79(24),

16 Better pulse sequences

17 Antiphase amplitude J=125Hz J=140Hz J=170Hz Time

18 Reverse concatenation A (B=0) C 1/2JCH3 A+C=1/2JCH3 A B C 1/2JCH3 A-B+C=1/2Jarom To collect the HSQC requires taking several spectra with different values of B to cover the different coupling constants. 18

19 INEPT revinept x,y Variation ~2% S evol. Sum of 2.94 ms, 2.94, 2.94, and 5.92 ms (1/2J) Heikkinen, S., Toikka, M. M., Karhunen, P. T., & Kilpeläinen, I. A. (2003). Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: Application to wood lignin. Journal of the American Chemical Society, 125(14),

20 Heikkinen, S., Toikka, M. M., Karhunen, P. T., & Kilpeläinen, I. A. (2003). Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: Application to wood lignin. Journal of the American Chemical Society, 125(14),

21 180 adiabatic { Equivalent to: CH3 CHaliph A 1/2JCH3 CHarom B C

22 CPMG 1H-1H Decoupling Errors of up to 17% with a 7.3% standard deviation. The error increased even more when the 13C carrier was set off-resonance. Koskela, H., Heikkilä, O., Kilpeläinen, I., & Heikkinen, S. (2010). Quantitative two-dimensional HSQC experiment for high magnetic field NMR spectrometers. Journal of Magnetic Resonance, 202(1),

23 HSQC0 Hu, K., Westler, W. M., & Markley, J. L. (2011). Simultaneous quantification and identification of individual chemicals in metabolite mixtures by two-dimensional extrapolated time-zero 1H- 13C HSQC (HSQC 0). Journal of the American Chemical Society, 133(6),

24 Hu, K., Westler, W. M., & Markley, J. L. (2011). Simultaneous quantification and identification of individual chemicals in metabolite mixtures by two-dimensional extrapolated time-zero 1H- 13C HSQC (HSQC 0). Journal of the American Chemical Society, 133(6),

25

26 Sample spiking

27 ITOCSY Lewis, I. A., Karsten, R. H., Norton, M. E., Tonelli, M., Westler, W. M., & Markley, J. L. (2010). NMR method for measuring carbon-13 isotopic enrichment of metabolites in complex solutions. Analytical Chemistry, 82(11),

28 Lewis, I. A., Karsten, R. H., Norton, M. E., Tonelli, M., Westler, W. M., & Markley, J. L. (2010). NMR method for measuring carbon-13 isotopic enrichment of metabolites in complex solutions. Analytical Chemistry, 82(11),

29 Spiking can also be done using 1H NMR, Measure area before and after a known amount of compound is added. Lewis, I. A., Karsten, R. H., Norton, M. E., Tonelli, M., Westler, W. M., & Markley, J. L. (2010). NMR method for measuring carbon-13 isotopic enrichment of metabolites in complex solutions. Analytical Chemistry, 82(11),

30 Simulations

31 Bingol, K., Zhang, F., Bruschweiler-Li, L., & Brüschweiler, R. (2013). Quantitative analysis of metabolic mixtures by twodimensional 13C constant-time TOCSY NMR spectroscopy. Analytical Chemistry, 85(13),

32 Errors up to 25% Bingol, K., Zhang, F., Bruschweiler-Li, L., & Brüschweiler, R. (2013). Quantitative analysis of metabolic mixtures by twodimensional 13C constant-time TOCSY NMR spectroscopy. Analytical Chemistry, 85(13),