NMR at UNC: Tips, Tricks, and Techniques

Transcription

1 NMR at UNC: Tips, Tricks, and Techniques Laura Adduci UNC-Chapel Hill Graduate student Gagné Lab NMR Assistant Chemistry Department

2 Topics and Examples 1. 2D spectra 2. Selective 1D spectra 3. Homonuclear and heteronuclear decoupling experiments 4. Spectrometers at UNC Compounds in example spectra: HO HO O HO O O OH OH HO HO O OH OH OH HO HO O OH OH OH HO OH HO O OMe OH HO OH OH

3 Standard 1D spectrum Each resonance has: 1. Chemical shift 2. Integration 3. Multiplicity/coupling constant (e.g. 3 J H,H = 6 Hz) 4. Phase Negative phasing:

4 2D COSY and TOCSY COSY: Correlates coupled protons (usually 2 J H,H and 3 J H,H ) TOCSY: Correlates protons in the same spin system Default: 5 minutes Default: 75 minutes COSY TOCSY 14, ,15 14, , COSY 7 TOCSY

5 2D NOESY NOESY: Correlates protons that are near each other in space , 15 14, 15 Default: 45 minutes , 6 1, 3

6 2D HSQC and phase-sensitive HSQC HSQC: Correlates protons and the carbons to which they are directly bound ( 1 J C,H ) Phase-edited: CH 3 and CH peaks appear as one phase, CH 2 peaks appear as different phase Default: 15 minutes Default: 15 minutes HSQC C H O 8 OH HSQC HMQC , , , ,15 13 HSQC 12,7 12,7 HSQC HMQC

7 2D HMBC HMBC: Correlates protons with carbons that are 2 and 3 bonds away ( 2 J C,H and 3 J C,H ) , 15 Default: 15 minutes 14, , H O 8 OH 1 J C-H coupling: appears as weak doublets, usually visible for sharp/strong proton peaks only 1,3 4,

8 Choosing parameters for 2D experiments Parameters that can be varied: 1. Number of scans (type ns ): affects signal-to-noise ratio Increase if you have low sample concentration 2. Time domain in F1 (type td ): affects resolution in the F1 dimension (vertical) Increase if your peaks are close together TD(F1) NS 3. Experiment-specific parameters TOCSY: TOCSY mixing time (d9) Affects the number of bonds through which magnetization is transferred Increase to see correlation between distant protons in the same spin system Typical values: 10 ms 400 ms NOESY: NOESY mixing time (d8) Affects NOE buildup between correlated nuclei Typical values: 10 ms 900 ms

9 Alternative to 2Ds: selective 1D experiments 1. Selectively irradiate one peak in a 1D spectrum 2. Apply pulse sequence analogous to a certain 2D experiment 3. Only nuclei that are correlated to the irradiated peak by the applied method are observed in the new 1D spectrum Example: 1. Irradiate here 2. Apply COSY-like pulse sequence 3. Expect to see peak for this proton in addition to the initially irradiated proton

10 Selective 1D COSY 1 1 6,6' HO 5 HO 4 O 1 2 OMe 3 OH OH Me Default: 1 minute

11 Choosing selective 1D s vs 2D Selective 1D s are beneficial if: 1. Limited sample concentration 2. Overlapping peaks 3. You want to see shape of correlated peaks, not just whether or not there is a correlation 4. Many correlations expected, but only a few are of interest

12 Selective 1D NOESY example Mike Geier Default: 3 minutes

13 Selective 1D TOCSY of sucrose G HO HO HO O F HO O O G OH OH OH OH Separation of peaks from two different spin systems G, F F F F G G G G G G G G G G F F F F Default: 1 minute

14 Selective 1D TOCSY with two products Z E Separation of peaks from two different compounds in sample Trandon Bender Z E Z E

15 Selective 1D TOCSY: step by step

16 Selective 1D TOCSY: step by step

17 Selective 1D TOCSY: step by step

18 Selective 1D TOCSY: step by step

19 Selective 1D TOCSY: step by step

20 Selective 1D TOCSY: step by step

21 Selective 1D TOCSY: step by step

22 Selective 1D TOCSY: step by step

23 Selective 1D TOCSY: step by step

24 Selective 1D TOCSY: step by step

25 Selective 1D TOCSY: step by step

26 Selective 1D TOCSY: step by step

27 Selective 1D TOCSY: step by step

28 Selective 1D TOCSY: step by step

29 Selective 1D TOCSY: increasing mixing time TOCSY mixing time Mixing time affects the number of bonds through which magnetization is transferred 1 d9 = 10 ms 2 d9 = 20 ms 3 d9 = 40 ms 4 d9 = 60 ms 5 d9 = 120 ms 6 6

30 Homonuclear decoupling Selectively irradiate at one frequency. Coupling to the peak at that frequency is not observed in other peaks Example: irradiate at H 2. Coupling to H 2 is suppressed from H 1 (triplet becomes singlet) and H Should never conflict with COSY data!

31 Heteronuclear decoupling: 1 H{ 31 P} 31 P decoupling

32 Spectrometers at UNC Signal to noise ratios 400 NB 400 WB 500 (BBO) Varian (CryoQNP) 1 H C Remember: signal-to-noise increases as the square of the number of scans example: to multiply signal-to-noise by 2, must multiply number of scans by 4 Drop off NMR on the Bruker 600: Samplechanger + spreadsheet Tuesdays & Fridays at 12:00 pm Thursdays at 6:00 pm (overnight) Holder # Onyen Data set name Solvent Experiment Title Advisor Grant number Mail Data/Notify to: Directory 11 bezier DB834ALLEXP C6D6 Proton_C13 512_COSY_HMQC_HMBC_ DB834ALLEXP Brookhart bezier@live.unc.edu C:\data\bezier 12 samander SA2_151_pure CDCl3 Proton_C SA2_151_pure PADI PO You samander@ .unc C:\data\samand 13 dabrowje JAD I 067 2F7 14 CDCl3 C JAD I 067 2F7 14 Gagne dabrowje@live.unc.edc:\data\dabrow 14 bvass BFV 27 [Ru(b)(phen)(COCD2Cl2 PROTON BFV 27 [Ru(b)(phen)(C Schauer bvass@live.unc.edu C:\data\bvass 17 rrwatkin RW 1 12 p vinylphenyladmso PROTON UAA after deprotectio Brustad rrwatkin@live.unc.ed C:\data\rrwatk 18 wilger DW_02_053_A CDCl3 PROTON DW_02_053_A Nicewicz wilger@live.unc.edu C:\data\wilger 19 wilger DW_02_053_B CDCl3 PROTON DW_02_053_B Nicewicz wilger@live.unc.edu C:\data\wilger 20 wilger DW_02_053_C CDCl3 PROTON DW_02_053_C Nicewicz wilger@live.unc.edu C:\data\wilger 21 wilger DW_02_053_D CDCl3 PROTON DW_02_053_D Nicewicz wilger@live.unc.edu C:\data\wilger 22 wilger DW_02_053_E CDCl3 PROTON DW_02_053_E Nicewicz wilger@live.unc.edu C:\data\wilger 23 tbender TAB2 76 A DMSO DMSO Proton_C13 512_COSY_HSQC TAB2 76 A DMSO Gagne tbender@live.unc.educ:\data\tbende

33 Summary 2D experiments help with compound characterization 1D selective experiments complement 2D experiments and sometimes provide more information 1D selective TOCSY spectra can be used to separate peaks from two different species in solution Selective decoupling can help elucidate coupling constants Thanks to Ben Giglio Dr. Marc ter Horst Mike Geier Trandon Bender

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35 Extra slides

36 Heteronuclear decoupling: 1 H{ 19 F} Irradiate one nucleus coupling to that nucleus is suppressed. Most common: proton-decoupled carbon spectrum Can use this method for other nuclei as well. Ben Giglio Standard 1 H spectrum -CH 2 - -OH 1 H spectrum with 19 F decoupling

37 J-resolved spectrum HO 1' OH OH 2' ' OH OH OH Horizontal axis is proton spectrum. Vertical axis is peak separation in Hz 3 J H,H = 6.5 Hz Hz 1Hz

38 13 C APT and DEPT spectra 13 C{ 1 H}: all peaks positive A B C D G A E C B F D E F G 13 C APT: carbons with 1 or 3 attached protons are negative, carbons with 0 or 2 protons are positive (and a deuterium is not a proton!) DEPT 135: carbons with 1 or 3 protons are positive, carbons with 2 protons are negative, carbons with 0 protons are attenuated DEPT 90: carbons with 1 proton are positive, all others are attenuated

39 Quantification of 13 C NMR D G O A E C B What factors affect peak integration? 1. Number of attached protons: NOE during decoupling gives NOE enhancement Solution: can use inverse gated decoupling. Decoupler is on during acquisition so that peaks appear decoupled but off during delay so that there is no time for the NOE to build up F 2. Relaxation delay: peaks with T1 s that are longer than the relaxation delay are attenuated Solution: add relaxation agent to shorten all T1 s, or lengthen relaxation delay 128 scans Regular 1H decoupling Inverse gated decoupling Without Cr(acac)3 With Cr(acac)3 d1(s) time 2:47 3:14 4:20 6:32 24:08 46:08 2:43 3:10 4:16 6:28 24:04 46:04 Int A Int B Int C Int D Int E Int F Int G d1(s) time 2:47 3:14 4:20 6:32 24:08 46:08 2:43 3:10 4:16 6:28 24:04 46:04 Int A Int B Int C Int D Int E Int F Int G

40 Overlapping Kinetics Using the Samplechanger Staggered kinetics runs Start one run, then a second (third, fourth ) during the delay between timepoints Allows multiple kinetics runs to be acquired in the same amount of time as one run Minimum time between samples: 5 minutes + acquisition time Works best when samples are similar (same solvent) Sample 1 Sample Sample 2 30 min 30 min 30 min 30 min min Sample Sample 1 Sample 3