Two Dimensional (2D) NMR Spectroscopy

The two important parameters obtained from NMR spectra are; Two Dimensional (2D) NMR Spectroscopy py Correlation NMR a. Chemical shift b. Spin-spin coupling constant Large molecules with numerous atoms nuclear magnetic moment does not permit the determination of these fundamental parameters easily. Some 1D spectra are far too complex for interpretation because signals overlap heavily e.g. cholesterols, protein spectra 1D spectrum of a protein In ntensity Chemical shift, ppm 1

Nonequivalent proton groups can have nearly the same chemical shift and/or complex splitting patterns making 1 NMR spectra complicated even for relatively simple molecules. The introduction of additional spectral dimensions simplifies the spectra and provides more information. Two-dimensional (2D) NMR techniques can be used to solve such sophisticated structural problems. 2-D spectra simplify the complexity arising from overlapping of peaks. David E. Alonso* and Steven E. Warren, NMR Analysis of Unknowns: An Introduction to 2D NMR Spectroscopy, Journal of Chemical Education 82,1385 (2005) Simplification of NMR spectra makes their interpretation easier and sometimes the only way possible. The interaction of nuclear spins ( 1 with 1, 1 with 13 C, etc.) are plotted in two dimensions Examples: CSY information concerning coupled (homonuclear) systems. ETCR, MBC connectivity between protons and carbons. NSEY and RSEY configuration of a molecule. INADEQUATE constitution of a molecule without 1 -NMR. Common Pulse sequences 1-D: 1D-NMR 1D-13C-NMR Decoupled M M sin(2 t )e 0 2 t 2/T2 1-D spectra are plots of intensity vs a frequency (chemical shift). In 2-D spectra the intensity is plotted as a function of two frequencies, usually represented as F1 and F2. There are two ways to present 2D spectra; stack plots and contour plots. 2-D spectra are presented usually as a contour plot, where, the intensity of the peaks are represented by contour lines (recall topographical maps). 2

General Presentation of Correlation Spectra Two frequency axes. F1 and F2 are Fourier transformed frequency axis from a time domain signal. F2 F1 3

- Correlation Spectroscopy (CSY) In a CSY experiment, the chemical shift range of the proton spectrum is plotted on both axes. The Diagonal of a - CSY is it s 1-D -NMR spectrum! Each peak is specified by the two frequency co-ordinates (F1, F2). 2-D NMR spectra are always arranged so that the F2 co-ordinates of the peaks correspond to those found in the normal 1-D spectrum. (ften 1-D spectrum is presented on the horizontal F2 axis). F1 co-ordinates of the peaks also correspond to those of the normal 1-D spectrum (1-D spectrum plotted on the F1 axis) in - CSY. CSY Spectrum X CSY Spectrum X A X Cl X F1 X X R A Cl R 1 R 2 X R 3 F1 X A A X A X CSY spectrum of a molecule containing just one type of protons X. F2 F2 CSY spectrum of a hypothetical molecule containing just two protons, A and X, which are not coupled, is shown. 4

CSY Spectrum A X R R 3 R 1 R 2 F1 X X F2 A A A X J AX A X In 2-D spectra the idea of a multiplet consists of an array of individual peaks often forming of a square or rectangular outline. Multiplets form a square or rectangle with two vertices on the diagonal. F1 X A A X A X CSY spectrum of a hypothetical molecule containing just two types of protons, A and X, which are coupled is shown. CSY spectrum has some symmetry about the diagonal, F1=F2, which shown above. Diagonal multiplets centered around same F1 and F2. F2 Cross-peak multiplets centers around different F1 and F2 co-ordinates. In a homonuclear CSY spectrum, the presence of a cross-peak multiplet F1 = A, F2 = X indicates that the two protons A and X at chemical shifts A and X are scalar coupled. - CSY If there had been no coupling, their magnetizations would not have given rise to off-diagonal peaks. CSY spectrum shows which pairs in a molecule are coupled (thro bond coupling, hence connectivity). Recognition of the preceding fact is the essence for the analysis CSY spectra. From a single CSY spectrum it is possible to trace out the whole coupling network in the molecule. 5

Prototype pulse sequence 2D NMR Prototype pulse sequence 2D NMR ( /2) x ( /2) x M [M sin(2 t )e ] sin(2 t ) e 0 1 t/t 1 2 t 2/T2 2 Preparation Evolution, t 1 Detection, t 2 Mixing time M M sin(2 t )e Data acquired at the end of the an acquisition (after acquisition pulse) is labeled with the time t/t 1 2 0 ( 1) variable labeled t 2. The generation of a 2D experiment: In addition to preparation and detection (done in the 1D experiment) the 2D experiment has an indirect evolution and a mixing sequence, time t 1. a. Do something with the nuclei (preparation) b. let them precess freely (evolution) t 1 c. do something else (mixing) t 1 e. and detect the result (detection, of course). After preparation the spins can precess freely for a given time t 1. During this time the magnetization is labeled with the chemical shift of the first nucleus. The basic 2D spectrum would involve repeating a multiple pulse 1D sequence with a systematic variation of the evolution and mixing times, t 1, and then plotting Fourier transformed FID. This generates two time domains, one of which, t 2, is the acquisition time that appears during the acquisition as usual, and the other time domain originates from the variable delay part, t 1. 6

frequency data (FID) in one axis (f 2, from t 2 ), t 1 t 1 =2 t t 1= t t 1 A(t 1 ) Time domain data in t 1. It is periodic; a pseudo FID created for each of the frequencies in f 2. Decay not shown. t 1 =0 t 2 f 2 (t 2 ) Note all tops form one FID,.. t 1 Appearance: n the 2D-NMR spectra an additional chemical shift (homonuclear or heteronuclear) is recorded on the third axis. t 1 stacked plot t 1 http://www-keeler.ch.cam.ac.uk/lectures/understanding/chapter_7.pdf 7

f 1 f 2 In a real molecule where J coupling exist, during the mixing magnetization can be transferred from one nucleus to a second one. Mixing sequences utilize two mechanisms for magnetization transfer, namely scalar coupling or dipolar interaction (NE). Two dimensional FT yields the 2D spectrum with two frequency axes. If the spectrum is homonuclear (signals of the same isotope - say 1, are detected) the spectrum would have a characteristic symmetric topology. CSY: 1D- double resonance experiment that is often used to find relationships between protons, the protons are irradiated one by one. CSY generates all information from a series of double resonance experiments in one output (2D spectrum). The pulse sequence for a CSY experiment contains a variable delay time as well as an acquisition time. The experiment is repeated with different and incremented delay times, and the data collected during the acquisition are stored in the computer. The value of the delay time is increased by regular, small intervals for each experiment, so that the data that collected consist of a series of FIDs collected during the acquisition, each with a different value of delay time. 8

0 200 400 600 800 1000 t2 f2 f2 500 600 700 800 900 pts 400 500 pts 11/30/2010 In the CSY experiment, the magnetization is transferred by scalar coupling. In the CSY spectrum of a molecule where all possible off-diagonal peaks are generated; the result is a complete description of the coupling partners in a molecule. Sometimes the coupling between protons that t are more than Three chemical bonds apart can be seen. Signals on the diagonal divides the spectrum in two equal halves. Signals symmetrical to the diagonal called cross signals (peaks). The diagonal results from contributions of the magnetization that has not been changed by the mixing sequence. The cross signals originate from nuclei that exchanged magnetization during the mixing time. They indicate an interaction of these two nuclei. The cross signals contain the information of 2D NMR spectra. Pulegone time - time Contour plot t pts 1 t 2 time - frequency t 1 f 2 f 1 f 2 frequency - frequency f 1 f 2 http://tonga.usp.edu/gmoyna/nmr_en/nmr_lectures.html http://tonga.usp.edu/gmoyna/nmr_en/nmr_lectures.html 9

1 2 3 4 Stick diagram; 1 2 3 4 1- NMR Spectrum 4 types of d s d-d d Each circle represents the center of the multiplet. 3 4 axis 4 3 1 2 no coupling 1 2 1 2 axis C 3 8 ; U = 0 C 3 C 2 C 2 C 3 8 : CSY 4 3 2 1 C C 3 2 C 3 C 2 C 2 C 2 Pick a good starting point peak label 4 3 2 1 2 1 2 3 1 2 4 Pick multiplet(s) that can be assigned to a group atoms. Science Tools 10

C 5 8 2 U = 5 8/2 +1 = 2 C 5 8 2 C 2 - d c b a C 2 - d c b a 2 2 2 2 b a c Expansion show b a coupling --C=; ester C 2 - d CSY spectrum b a c b a c b a d U = 2, -C 2 group accounts for 1, Therefore other is a ring. b C 2 C 8 16 : U = 8 16/2 + 1 = 1 3 2 C 2 C C 2 4 3 2 2 1D NMR; four C 2 C 2 - a C 2 c C 2 No double/triple bonds d C 2 C= Cyclic structure of 4Cs 13C NMR No equiv. C C 2 -C= Ketone C= 11

C 8 16 5 4 2 3 Me-1 CSY spectrum Me1 2-3 4-5 7 Me8 U = 1; 2 Me groups, 3 C 2 groups and 4 aliphatic s. C group accounts for U=1. Two spin systems 1D NMR; five C 2 2 off-peaks on same line - overlap 5 3 C 2 C C 2 13C NMR No equiv. C C=, ketone; accounts for U=1 Me1 C 2 2 C 2 3 C 2 4 C 2 5 C 2 7 C= Me-8 C 2 7/5 Me8 2 C 2 C 3 C 11 20 4 U=2 C 2 C 3 2 3 3 X 2 -NMR 4 multiplets; area 3:3:2:2 Total atoms = 20 Symmetrical structure Chemical shifts: two methylene groups C2C3 and C2C3. That takes 10 atoms. -C= -C 13C NMR 6 types of C; also C= and C 12

= C C 2 C 3 C 11 20 4 C 2 C 3 C 2 C 3 methyl Ipsenol spectra explanation. C 3 C 2 C C 2 C 3 = C C 2 C 3 Two spin systems 4 C 2 2 aliph., 2 olef. 2 C 2 spin systems Ipsenol C 10 18 Ipsenol C 10 18 1 4 1 1 1 2 1 1 6 1 4 1 1 1 2 1 1 6 DEPT90 C DEPT135 C, C 3 C 2 13C NMR 13

DQFCSY Point of entry distinctive peak DQFCSY cleans some clutter on CSY by removing some high intensity (methyl) peaks. 14

Point of entry distinctive peak Point of entry distinctive peak Point of entry distinctive peak Ipsenol spectra explanation. = deshielded deshielded Diastereoscopic Geminal, 1 vicinal Lowest, Diastereoscopic Geminal, 2 vicinal Diastereoscopic methyls, 1 vicinal ighly coupled, overlapped with. 15

singlet http://www.chem.ucalgary.ca/courses/351/carey5th/ch13/ch13-2dnmr-1.html http://www.chemistry.ccsu.edu/glagovich/teaching/316/nmr/cosy.html CSY 1-1 CSY (CSY) The information on how the and are coupled is gleaned from the contour peaks. 13 C- 1 CSY C Detected (ETCR) Detected CSY (MQC) - Detected Long Range (MBC) The information on how the are C are correlated is gleaned from the contour peaks. ETCR- eteronuclear Chemical Shift Correlation ETCR gives the correlations between protons and other nuclei such as 13-C or 15N. Two versions exist absolute value ETCR and phase sensitive ETCR. A related experiment is the MQC experiment - couplings are removed here. Variations of the ETCR can show only C, or C and C 3 positive and C 2 negative. The experiment encodes the proton chemical shift information into 13-C signals that are observed. It generates cross peaks for all protons and 13C nuclei that are connected by a 13C-1 coupling over one bond. 16

ETCR MQC 1 J C = 145 z. 1 J C = 145 z. Correlates 13C directly attached to, large 1 J C couplings (polarization transfer) and the frequency domains are from different nuclei. F2 is 13C and F1 is 1. Therefore no diagonal symmetry. Correlates 13C directly attached to, and the experiment is detected. Long range couplings eliminated. -C2- -C3 ethyl 2-butenoate (ETCR spectra recorded by D. Fox, Dept of Chemistry, University of Calgary on a Bruker Advance DRX-400 spectrometer 17

3 C C 3 Ethyl Crotonate C 3 C 3 Ethyl Crotonate 3 C Ethyl Crotonate C 3 Diagonal leads to no information. http://www.tecmag.com/pdf/etcr.pdf 18

MBC 1/2J can be optimized for different coupling constants Correlates 13C with 2-bond and 3-bond couplings to, and the experiment is detected. Interpretation more difficult Because of both 2,3-bond (sometimes 4-) correlations are present. 19

NESY 1-1 NESY (Nuclear verhauser Effect SpectroscopY) signals the signals arising from protons that are close to each other in space regardless of bonding. A NESY spectrum arises from through space correlations via spin-lattice relaxation. Provides a means to establish 3-D structural relationships of a molecule. The CSY cross peaks that would arise from the experiment are also present in the NESY spectrum (effectiveness; r -6 ) The peaks additional to CSY peaks are the NE enhanced peaks. NESY also detects chemical and conformational exchange (EXSY). It is a homo-nuclear 2D plot, with diagonal as the normal 1-D spectrum and projections on each axis. Information gleaned from the "cross-peaks", which appear at the coordinates of 2 protons which have an NE correlation. NESY spectrum of codeine Expansion of the up-field region; http://www.acornnmr.com/codeine/noesy.htm 20

8-7, 12 7-18, 18' 3-5, 10 5-11, 16, 18' 9-10, 17, 17' 10-16 11-18, 16, 14, 18' 18-13, 18' 16-14, 17 13-14, 17, 17' 13' - 17, 17' 17-17' N-Phenylacetamide ~2Å A B ~2Å ' indicates the more up-field of geminal C2 protons NMR: Simulated Spectrum N-Phenylacetamide NSEY: The NE enhanced peaks (only) for A and B N N-phenylacetamide A B 21