11/30/2010

The two important parameters obtained from NMR spectra are; 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

Two Dimensional (2D) NMR p py Spectroscopy

1D spectrum of a protein

In ntensity

Chemical shift, ppm

11/30/2010

Nonequivalent proton groups can have nearly the same chemical shift and/or complex splitting patterns making 1H 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.

Simplification of NMR spectra makes their interpretation easier and sometimes the only way possible. The interaction of nuclear spins (1H with 1H, 1H with 13C, etc.) are plotted in two dimensions Examples: COSY information concerning coupled (homonuclear) systems. HETCOR, HMBC connectivity between protons and carbons. NOSEY and ROSEY configuration of a molecule. INADEQUATE constitution of a molecule without 1H-NMR.

David E. Alonso* and Steven E. Warren, NMR Analysis of Unknowns: An Introduction to 2D NMR Spectroscopy, Journal of Chemical Education 82,1385 (2005)

Common Pulse sequences 1-D: M M 0 sin(2t 2 )e 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).

1D-HNMR

1D-13C-NMR Decoupled

11/30/2010

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

11/30/2010

H-H Correlation Spectroscopy (COSY) In a COSY experiment, the chemical shift range of the proton spectrum is plotted on both axes. The Diagonal of a H-H COSY is its 1-D H-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. (Often 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 H-H COSY.

COSY Spectrum X

HX
X

COSY Spectrum A X

HX

HA

Cl H H
F1

H Cl R

HX

HA

R1 R2 R3
F1
X X

HX

F2 COSY spectrum of a molecule containing just one type of protons HX.

F2 COSY spectrum of a hypothetical molecule containing just two protons, HA and HX, which are not coupled, is shown.

11/30/2010

COSY Spectrum A X

HX

HA JAX

H H R R1 R2
F1

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.

HA

R3

HA

HX
F1
X

HX

F2 COSY spectrum of a hypothetical molecule containing just two types of protons, HA and HX, which are coupled is shown. COSY spectrum has some symmetry about the diagonal, F1=F2, which shown above. F2 Diagonal multiplets centered around same F1 and F2.

Cross-peak multiplets centers around different F1 and F2 co-ordinates.

In a homonuclear COSY 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. If there had been no coupling, their magnetizations would not have given rise to off-diagonal peaks. COSY 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 COSY spectra. From a single COSY spectrum it is possible to trace out the whole coupling network in the molecule.

H-H COSY

11/30/2010

Prototype pulse sequence 2D NMR

Prototype pulse sequence 2D NMR

M [M 0 sin(2t1 )e t1 /T2 ] sin(2t 2 )e t 2 /T2

Preparation

Evolution, t1

Detection, t2

Mixing time

M M 0 sin(2 ( t1 ) )e t1 /T2

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 t1. a. Do something with the nuclei (preparation) b. let them precess freely (evolution) t1 c do something else (mixing) t1 c. e. and detect the result (detection, of course). After preparation the spins can precess freely for a given time t1. 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, t1, and then plotting Fourier transformed FID.

This generates two time domains, one of which, t2, is the acquisition time that appears during the acquisition as usual, and the other time domain originates from the variable delay part, t1.

11/30/2010

O
t1=2t

frequency data (FID) in one axis (f2, from t2),

t1 A(t1)

t1=t t1

Time domain data in t1. It is periodic; a pseudo FID created for each of the frequencies in f2. Decay not shown. Note all tops form one FID, ..

t1=0

t2

f2 (t2)

t1

Appearance:
O

On the 2D-NMR spectra an additional chemical shift (homonuclear or heteronuclear) is recorded on the third axis. t1

t1 stacked plot
http://www-keeler.ch.cam.ac.uk/lectures/understanding/chapter_7.pdf

11/30/2010

f1

f2

COSY: 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 (NOE). 1D- double resonance experiment that is often used to find relationships between protons, the protons are irradiated one by one. COSY generates all information from a series of double resonance experiments in one output (2D spectrum). The pulse sequence for a COSY 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.

Two dimensional FT yields the 2D spectrum with two f frequency axes. If the th spectrum t is i homonuclear h l (signals ( i l of f the same isotope - say 1H, are detected) the spectrum would have a characteristic symmetric topology.

11/30/2010

In the COSY experiment, the magnetization is transferred by scalar coupling. In the COSY 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. S Sometimes ti the th coupling li b between t protons t th that t are more than th 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 t1

Contour plot

0 t2

200

400

600

800

1000 pts

t2

time - frequency
500 f2 600 700 800 900 pts

t1

f2 f1 f2 frequency - frequency f2
f2 400 500 pts

f1

http://tonga.usp.edu/gmoyna/NMR_EN/NMR_lectures.html

http://tonga.usp.edu/gmoyna/NMR_EN/NMR_lectures.html

11/30/2010

1 Stick diagram; 1-H NMR Spectrum 4 types of H d s d-d d Each circle represents the center of the multiplet. H4 H3H1 H2 no coupling 1 2 3 4

2 axis

C3H8O; U = 0
CH3 CH2 CH2 OH

C3H8O: COSY
4 CH3 CH2 CH2 OH
Pick a good starting point

2 CH2

CH2 OH

1 CH3

peak label 4 3

2H

1H

2H

3H

H1 H2 H4
Pick multiplet(s) that can be assigned to a group atoms.

Science Tools

3 1 axis

10

11/30/2010

CH2 - O

C5H8O2
CH2

U = 5 8/2 +1 = 2

C5H8O2
a b

b a

-O d
2

c
2

b
2

a
2

c Expansion show ba coupling

COSY spectrum b a c b a c b a d 1D HNMR; four CH2

C8H16O : U = 8 16/2 + 1 = 1
U = 2, -CO2 group accounts for 1, Therefore other is a ring.
2H CH2 CO CH2 4H 3H 2H 2H 3H

b CH2 a CH2 d CH2 O c CH2 C=O

13C NMR
No equiv. C CH2O O-C=O

Ketone C=O

11

11/30/2010

Me-1 4 2 3

C8H16O

COSY spectrum
Me1 2 - 3 4 - 5 7 Me8 Two spin systems

U = 1; 2 Me groups, 3 CH2 groups and 4 aliphatic Hs. CO group accounts for U=1.

1D HNMR; ; five CH2 2 off-peaks on same line overlap

CH2 CO CH2

Me1 CH22 CH23 CH24

CH25 C=O CH27 Me-8

13C NMR
No equiv. C C=O, ketone; accounts for U=1
CH27/5 Me8

C11H20O4 U=2
X2 2H O CH2 CH3 CH2 CH3 2H 3H 3H

H-NMR 4 multiplets; area 3:3:2:2 Total H atoms = 20 Symmetrical structure Chemical shifts: two methylene groups OCH2CH3 and d CH2CH3. CH2CH3 Th That tt takes k 10 H atoms. t

O-C O-C=O

13C NMR 6 types of C; also OC=O and OC

12

11/30/2010

C11H20O4
O CH2 CH3 O = C O CH2 CH3 CH3 CH2 C CH2 CH3 CH2 CH3

methyl

Ipsenol spectra explanation.

O = C O CH2 CH3

HO

4 CH2 2 aliph., 2 olef. Two spin systems 2 CH 2 spin systems

Ipsenol C10H18O

Ipsenol C10H18O

HO
1 4 1 1 1 2 1 1 6

1 1

2 11

DEPT90 CH DEPT135 CH, CH3 CH2

13C NMR

13

11/30/2010

Point of entry distinctive peak

DQFCOSY

DQFCOSY cleans some clutter on COSY by removing some high intensity (methyl) peaks.
HO

14

11/30/2010

Point of entry distinctive peak

Point of entry distinctive peak

HO

HO

Point of entry distinctive peak

Ipsenol spectra explanation.

= deshielded OH deshielded

Diastereoscopic Geminal, 1 vicinal

Lowest,

HO

Diastereoscopic Geminal, 2 vicinal

HO

Diastereoscopic methyls, 1 vicinal Highly coupled, overlapped with OH.

15

11/30/2010

O O

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

HETCOR- Heteronuclear Chemical Shift Correlation COSY

COSY (COSY) The information on how the H and H are coupled is gleaned from the contour peaks. COSY C Detected (HETCOR) H Detected COSY (HMQC) H- Detected Long Range (HMBC)

13C-1H

HETCOR gives the correlations between protons and other nuclei such as 13-C or 15N. Two versions exist absolute value HETCOR and phase sensitive HETCOR. A related experiment is the HMQC experiment H-H H H couplings are removed here here. Variations of the HETCOR can show only CH, or CH and CH3 positive and CH2 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-1H coupling over one bond.

The information on how the H are C are correlated is gleaned from the contour peaks.

16

11/30/2010

HETCOR

HMQC

1J

HC

= 145 Hz.

1J

HC

= 145 Hz.

Correlates 13C directly attached to H , large 1JCH couplings (polarization transfer) and the frequency domains are from different nuclei . F2 is 13C and F1 is 1H. Therefore no diagonal symmetry.

Correlates 13C directly attached to H, and the experiment is H detected. Long range couplings eliminated.

-OCH2ethyl 2-butenoate

-CH3

O O

(HETCOR spectra recorded by D. Fox, Dept of Chemistry, University of Calgary on a Bruker Advance DRX-400 spectrometer

17

11/30/2010

O H H3C H O CH3

Ethyl Crotonate

O H H3C H O CH3

Ethyl Crotonate
O H H3C H O CH3

Ethyl Crotonate

Diagonal leads to no information.

http://www.tecmag.com/pdf/HETCOR.pdf

18

11/30/2010

HMBC

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

19

11/30/2010

NOESY 1H-1H NOESY (Nuclear Overhauser Effect SpectroscopY) signals the signals arising from protons that are close to each other in space regardless of bonding. A NOESY spectrum arises from through space correlations via spin-lattice relaxation. Provides a means to establish 3-D structural relationships of a molecule. NOESY 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 NOE correlation.

The COSY cross peaks that would arise from the experiment are also present in the NOESY spectrum (effectiveness; r-6) The peaks additional to COSY peaks are the NOE enhanced peaks.

NOESY spectrum of codeine

Expansion of the up-field region;

http://www.acornnmr.com/codeine/noesy.htm

20

11/30/2010

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' ' indicates the more up-field of geminal CH2 protons

N-Phenylacetamide

~2

~2

NOSEY: The NOE enhanced peaks (only) for A and B

HNMR: Simulated Spectrum N-Phenylacetamide

H N O

A B

N-phenylacetamide

21