NMR: PRACTICAL ASPECTS

Transcription

1 NMR: PRACTICAL ASPECTS Pedro M. Aguiar Sample Preparation Well prepared sample can yield high quality spectra Poorly prepared sample typically yields low quality spectra Tubes of appropriate quality Higher fields require higher quality tubes Contact local facility manager for specifics Sample conc. at typical field instruments (MWt. < 000) H NMR: 3 C NMR: >= 0 mm > = 0 mm Filter sample to remove insoluble material No signals Hampers ability to detect signal of soluble components Dry using vacuum and store in ovens no warmer than 80 C

2 Sample Preparation Good 3- mm Assumed detection region for shimming ideal reality 3- Sample Preparation Short < 3 mm Good 3- mm Assumed detection region for shimming ideal reality Poor shimming Weak signal (not in detector) 3-

3 Sample Preparation Short < 3 mm Good 3- mm Long > mm Assumed detection region for shimming ideal reality Poor shimming Weak signal (not in detector) May impact shimming Weak signal (dilute) 3-3 Sample Volume: mm NMR tube 300 µl volume poorly-shimmed Poor lineshape 600 µl volume Well-shimmed Good lineshape

4 Limited Sample volume In cases of Limited sample volume partially filled mm tube yields poor data Alternatives Smaller NMR tubes 3mm tubes (80-00 µl volume) Coaxial Inserts 0-00 µl volumes Susceptibility-matched (Shigemi) tubes µl Relative Chemical Shifts 6-

5 Relative Chemical Shifts Downfield 6- Relative Chemical Shifts Downfield Upfield 6-3

6 Relative Chemical Shifts Downfield Upfield 6- Relative Chemical Shifts Downfield Higher frequency Upfield Lower frequency 6-

7 Relative Chemical Shifts Downfield Higher frequency Upfield Lower frequency 6-6 Relative Chemical Shifts Downfield Higher frequency De-shielded or Less shielded Upfield Lower frequency Shielded or More shielded 6-7

8 Relative Chemical Shifts Downfield Higher frequency De-shielded or Less shielded 6-8 Upfield Lower frequency Shielded or More shielded Apodisation and Processing FT 7

9 Apodisation and Processing e πlbt Exponential Apodisation (line broadening) is most common for D Optimum is to use LB of 0.- times FWHM of peaks 8 Apodisation and Processing FT 9

10 Quantitative NMR? The integrated signal intensity in NMR can be reflective of the number of nuclei of a given type Quantitative NMR? The integrated signal intensity in NMR can be reflective of the number of nuclei of a given type Caveats Signal intensities not enhanced artificially (e.g., noe, INEPT/DEPT, ph ) Often 3 C, 3 P, B and 9 Si on walk-up instruments not quantitative All nuclei must be allowed to reach equilibrium before spectrum acquired Pre-experiment/recycle/relaxation delay must be long enough 3- times T H (-0s), 9 F (-0 s), 3 C (0-600 s), 9 Si (-000 s)

11 Quantitative spectra Nucleus Experiment Quantitative Comments H 9F 3C Single-pulse Single-pulse Single-pulse w/ H decoupling Recycle delay long enough (-0s) Recycle delay long enough (-60s) Background interference noe enhances signals of sites with hydrogens 3P Single-pulse w/ H decoupling Alternative is to use Inverse-gated decoupling Allow long relaxation time before each scan (0-60s) noe enhances signals of sites with hydrogens Alternative is to use Inverse-gated decoupling Allow long relaxation time before each scan (-30s) Baseline Correction Accurate intgration requires good definition of zero intensity (i.e., baseline) Automatic Routines (polynomial or spline) work well for most small distortions In some cases may need other methods After BC Portion of signal below zero contributes a negative value to sum (integral) 3

12 Baselines due to stuff in probe NMR probes are made of stuff Borosilicate glass and quartz ( 0/ B and 9 Si) Fluoropolymers ( 9 F and sometimes 3 C) Metals in the detection coil ( 63/6 Cu, 9 Pt) Result in obtrusive background signals/baselines 9F Broad signals, short FIDs Solution is to remove offending points Then, use backwas linear prediction

13 Linear Prediction Time-domain (FID) fitting routines to replace missing data Existing points used as a basis set Backwas linear prediction generates point at beginning Useful if long delay before acquisition (alternative to very large ph) x time 6 9F Removal of initial points & Backwas Linear Prediction As-collected 7

14 Are integrals Accurate? Integrals in most software packages done numerically Works well when signal-to-noise is high Alternative is lineshape fitting Works will even if signal-to-noise is low Numerical Integration Lineshape Fitting 8 Real Example lineshape Fits: Pt-9 NMR High signal-to-noise signal Integration and lineshape similar result Low signal-to-noise signal Integration yields erratic result Lineshape fit yields consistent result 9 Samples Courtesy: Imelda Silalahi and Duncan Bruce, University of York

15 Bandwidth and Integrals Probe and Pulses utilised have intrinsic bandwidths Peaks very far apart may be affected in different ways Can be issue for 9 F if very large shifts are present E.g., Ar-F (30 ) vs. M-F (-30 ) Coupling to Quadrupolar Nuclei Quadrupolar nuclei can often have Ts comparable to /J [(R) P) Co-H Magnetically equivalent phospines P-3 (N.A. 00%, I-/) Cobalt Co-9 (N..A. 00%, I-7/) Expect octet of triplets Observe a blob?

16 Coupling to Quadrupolar Nuclei Quadrupolar nuclei can often have Ts comparable to /J /J is a measure of time it takes a nucleus to tell another nucleus what state its in. If T comparable to /J, then the spin-state can change before a nucleus has time to tell another nucleus what state it is in Coupling to Quadrupolar Nuclei Quadrupolar nuclei can often have Ts comparable to /J [(R) P) Co-H Magnetically equivalent phospines P-3 (N.A. 00%, I-/) Cobalt Co-9 (N..A. 00%, I-7/) H spectrum with 9 Co decoupling results in triplet (due to 3P J- coupling) 3

17 DEPT-3 vs 3 C D Correlation Spectroscopy (i.e., D NMR) Collect a series of D spectra where some period of additional evolution occurs Evolution of states during (often linked via J-coupling) -

18 Correlation Spectroscopy (i.e., D NMR) Collect a series of D spectra where some period of additional evolution occurs Evolution of states during (often linked via J-coupling) something st evol. something else nd evolution - Correlation Spectroscopy (i.e., D NMR) Collect a series of D spectra where some period of additional evolution occurs Evolution of states during (often linked via J-coupling) something st evol. something else nd evolution something st evol. something else nd evolution -3

19 Correlation Spectroscopy (i.e., D NMR) Collect a series of D spectra where some period of additional evolution occurs Evolution of states during (often linked via J-coupling) something st evol. something else nd evolution something st evol. something else nd evolution something st evolution something else nd evolution - Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity

20 Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity 0 Connectivity limited to or bonds Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity 0 Connectivity limited to or bonds

21 Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity

22 Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity

23 Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity

24 Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity

25 Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity

26 Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity Correlation Spectroscopy (i.e., D NMR) COrrelation SpectroscopY (COSY) One of most basic D experiments Information about connectivity

27 Total Correlation Spectroscopy (TOCSY) Provides all correlations irrespective of magnitude Allows distinction of coupling networks Does not permit identification of neighbouring spins 0 Homonuclear Hartman- Hahn (HOHAHA) mixing Mixing times 0-30 ms: - bonds 0-00 ms: -0 bonds 3 mix TOCSY vs COSY COSY Correlations between individual coupled spins Limited to bonds TOCSY Correlations between all spin in a spin-system Beyond bonds Correlations limited by presence of non-h bearing atoms Follow coupled spins sequentially to understand connectivity Allow identification of groups of spins from specific parts of molecule e.g., sugar rings in polysaccharide Sidechains in peptide/protein 3

28 Spin or chemical Exchange : NOESY/EXSY Nuclear Overhauser effect Dipolar cross-relaxation COSY-type signals may also be present 0 No RF applied during mixing Mixing times ms Negative correlations = noe Positive correlations = Xchng 3 mix Spin or chemical Exchange : NOESY/EXSY Nuclear Overhauser effect Dipolar cross-relaxation COSY-type signals may also be present 0 No RF applied during mixing Mixing times ms Negative correlations = noe Positive correlations = Xchng 3 mix

29 Spin or chemical Exchange : NOESY/EXSY Nuclear Overhauser effect Dipolar cross-relaxation COSY-type signals may also be present 0 No RF applied during mixing Mixing times ms Negative correlations = noe Positive correlations = Xchng 3 mix Distance Information from noe noe inensity is related to the distance between the two nuclei Acquisition of spectra at different mixing times can allow extraction of distances Figure from: P. Brocca, P. Berthault, S. Sonnino; Biophysical Journal (998), 7(), 309 3

30 When NOESY doesn t work Nuclear Overhauser effect near zero for molecules of MWt (depending on solvent) Small molecules Use roe (rotating frame Overhauser effect) Mixing times -00 ms D spectrum similar to NOESY TOCSY-like peaks may be present large molecules mix 3 Heteronuclear Correlation Used for probing connectivity between two different types of nuclei H /J /J HMQC X H /J /J /J /J HSQC X 36

31 Some Common H-X J-coupling ranges 3 C One-bond: 0-70 Hz Two-bond: <30 Three-bond: < 3 P One-bond: Two-bond: <0 Three-bond: <0 N One-bond: 6-00 Hz Two-bond: < Hz Three-bond: < Hz 37 HSQC vs HMQC HSQC Heteronuclear Single-Quantum Correlation H-detected H-X correlation spectrum Typically used to probe one-bond correlations Can be higher resolution Variants with multiplicity (XH, XH, XH 3 etc.) selection HMQC Heteronuclear Multiple-Quantum Correlation H-detected H-X correlation spectrum Typically used to probe one-bond correlations can be higher sensitivity 38

32 HSQC vs. HMQC HSQC capable of intrinsically higher resolution than HMQC H{ 3 C} HSQC H{ 3 C} HMQC Edited HSQC Adds a DEPT-like selection XH and XH 3 positive XH negative 0

33 Weak signals in HSQC Weak signals in HSQC can arise from various sources Minor impurities/byproducts Multiple-bond correlations 60 x Multiple-Bond Correlation Heteronuclear Multiple-bond correlation experiment Multiple-bond correlations have smaller J-couplings HMBC effectively an HMQC with /J set to optimize for smaller J-coupling Often have an additional evolution to minimize signals from correlations between sites with large J-couplings Use together with HSQC/HMQC to determine C-C connectivity

34 HMQC/HSQC plus HMBC Use HMQC/HSQC together with HMBC to determine connectivity HMQC/HSQC plus HMBC Use HMQC/HSQC together with HMBC to determine connectivity Single-bond correlation

35 HMQC/HSQC plus HMBC Use HMQC/HSQC together with HMBC to determine connectivity Single-bond correlation HMQC/HSQC plus HMBC Use HMQC/HSQC together with HMBC to determine connectivity Single-bond correlation

36 HMQC/HSQC plus HMBC Use HMQC/HSQC together with HMBC to determine connectivity Single-bond correlation HMQC/HSQC plus HMBC Use HMQC/HSQC together with HMBC to determine connectivity ? 00 Single-bond correlation

37 Linear Prediction: indirect dimension 7 min min NO Forwa Linear Prediction 7 min min With Forwa Linear Prediction Relevant Literature Books Modern NM Techniques For Chemistry Research, A.E. Derome High-Resolution NMR Techniques in Organic Chemistry, T.D.W. Claridge Nuclear Magnetic Resonance, P.J. Hore (Oxfo Primer) Spin Dynamics, M.H. Levitt Journals/Book Series Concepts in Magnetic Resonance Annual Reports in NMR (Chemistry Library) Encyclopedia of NMR (DH Coffee room)

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