11/30/2010 1 Two Dimensional (2D) NMR Spectroscopy Correlation NMR 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 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 ntensity Chemical shift, ppm In 1D spectrum of a protein
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11/30/2010
1
Two Dimensional (2D) NMRSpectroscopyp py
Correlation NMR
The two important parameters obtained from NMR spectraare;
a. Chemical shiftb. Spin-spin coupling constant
Large molecules with numerous atoms nuclear magnetic moment does not permit the determination of thesemoment does not permit the determination of thesefundamental parameters easily.
Some 1D spectra are far too complex for interpretation because signals overlap heavily e.g. cholesterols, protein spectra
nten
sity
Chemical shift, ppm
In
1D spectrum of a protein
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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.
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 interpretationeasier 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.
Common Pulse sequences 1-D:
1D-HNMR
2 2t /T0 2M M sin(2 t )e
1D-13C-NMRDecoupled
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).
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F1
F2
General Presentation of Correlation Spectra
Two frequency axes. F1 and F2 are Fourier transformed frequency axis from a time domain signal.
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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 it’s 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.
HX
HX
H
H
Cl
H
X
COSY Spectrum
X
F1 X
COSY spectrum of a molecule containing just one typeof protons HX.
F2
HAHX
HA
Cl
R2
H
R1
RH
R3
A X
COSY Spectrum
X A
A
F1 HX
X
COSY spectrum of a hypothetical molecule containing just two protons, HA and HX, which are not coupled, is shown.
F2
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HAHX
HA
R3
H
R2
H
R1
R
A X
COSY SpectrumJAX
X A
A
F1 HX
X
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
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
X A A
HX
F1
F2
Diagonal multipletscentered around same F1 and F2.
Cross-peak multipletscenters around different F1 and F2 co-ordinates.
X
In a homonuclear COSY spectrum, the presence of a cross-peakmultiplet 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 nothave given rise to off-diagonal peaks.
COSY spectrum shows which pairs in a molecule areCOSY 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
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Preparation Evolution, t1 Detection, t2
(/2)x (/2)x
Mixing time
Prototype pulse sequence – 2D NMR Prototype pulse sequence – 2D NMR
1 2t /T0 1M M sin(2 t )e
1 2 22t /T0
t /T21M sin(2[M sin(2 t )e t e] )
Data acquired at the end of the an acquisition (after acquisition pulse)is labeled with the time variable labeled t2.0 1( ) 2
The generation of a 2D experiment:
In addition to preparation and detection (done in the 1D experiment) the 2D experiment has an indirect evolutionand a mixing sequence, time t1.
a. Do something with the nuclei (preparation) b. let them precess freely (evolution) t1c do something else (mixing) tc. do something else (mixing) t1e. 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,acquisition time that appears during the acquisition as usual, and the other time domain originates from the variable delay part, t1.
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t1=t
t1=2t
O
frequency data (FID) in one axis (f2, from t2),…
t1=0
1
t2
t1
…
A(t1)
Time domain data in t1. It is periodic; a pseudo FID created for each of the frequencies in f2.
t1
…
t1
f2 (t2)
Decay not shown.
Note all ‘tops’ form one FID, …..
t1
O
t1stacked plot
Appearance:
On the 2D-NMR spectra an additional chemical shift (homonuclear or heteronuclear) is recorded on the third axis.
http://www-keeler.ch.cam.ac.uk/lectures/understanding/chapter_7.pdf
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f
f2
f1
Two dimensional FT yields the 2D spectrum with two f If th t i h l ( i l f
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).
frequency axes. If the spectrum is homonuclear (signals of the same isotope - say 1H, are detected) the spectrum would have a characteristic symmetric topology.
COSY:
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 theacquisition, each with a different value of delay time.
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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 ti th li b t t th t thSometimes the coupling between protons that 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.
time - time
time - frequency
0 200 400 600 800 1000t2 pts
t2
t1
t
Pulegone
frequency - frequency400 500
f2 pts
500 600 700 800 900f2 pts
f2
f2
t1
f1
http://tonga.usp.edu/gmoyna/NMR_EN/NMR_lectures.html
Contour plot
f2
f1
http://tonga.usp.edu/gmoyna/NMR_EN/NMR_lectures.html
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1 2 3 4
d s d-d d
Stick diagram;
1-H NMRSpectrum
4 types of H
1 2 3 4
3
4
axi
s
Each circle represents the center of the multiplet.
1
2
1
2 axis
H4 H3H1
H2 no coupling
C3H8O; U = 0
CH3 – CH2 – CH2 – OH
peak label4 3 2 1
2H 1H 2H 3H
Pick multiplet(s) that can be assigned to a group atoms.
Science Tools
C3H8O: COSY
CH3 – CH2 – CH2 – OH CH3
CH2
CH2 OH
4 3 2 1
Pick a good starting point
H1 H2 H4
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C5H8O2 U = 5 – 8/2 +1 = 2
CH2 - O
d c b a2 2 2 2
CH2 - O|-O-C=O; ester
C5H8O2
CH2 - O
abcd
ba
c
d
Expansion show ba coupling
ba
cba
cbad
CH - O
U = 2, -CO2 group accounts for 1,Therefore other is a ring.
COSY spectrum
1D HNMR; four CH2a CH2 c CH2
b CH2
CH2 - O
13C NMR
No equiv. CCH2OO-C=O
No double/triple bonds
Cyclic structure of 4Cs
d CH2
O
C=O
C8H16O : U = 8 – 16/2 + 1 = 1
4H2H 2H
2H3H
3H
CH2 – CO – CH2
C=OKetone
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C8H16OMe-1
2 34
5
3
5
Me8CH27/5
2 off-peakson same line -overlap
Me1 – 2 - 3 – 4 - 5
7 – Me8
U = 1; 2 Me groups, 3 CH2 groups and 4 aliphatic H’s. CO group accounts for U=1.
COSY spectrum
1D HNMR; five CH2
Two spin systems
CH2 – CO – CH2
; 2
13C NMR
No equiv. CC=O, ketone; accounts for U=1
Me1 – CH22 – CH23 – CH24 CH25
C=O
CH27 Me-8
C11H20O4 U=2
2H 2H
3H 3H
O – CH2 – CH3
CH2 – CH3
X 2
O-C=O
O-C
H-NMR
4 multiplets; area 3:3:2:2
Total H atoms = 20
Symmetrical structure
Chemical shifts: two methylene groups –OCH2CH3 d CH2CH3 Th t t k 10 H tand CH2CH3. That takes 10 H atoms.
13C NMR
6 types of C; also O–C=O and –O–C
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O – CH2 – CH3CH2 – CH3
O = C – O – CH2 – CH3
O = C – O – CH2 – CH3
CH2 – CH3CH3 – CH2 C
C11H20O4methyl
Two spin systems
Ipsenol spectra explanation.
OH
4 CH2
2 aliph., 2 olef.
2 CH
2 spin systems
IpsenolC10H18O
1 4 1 1 1 2 1 1 6
IpsenolC10H18O
1 4 1 1 1 2 1 1 6
OH
DEPT90 CHDEPT135 CH, CH3
CH2
13C NMR
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DQFCOSY
DQFCOSY cleans some clutter on COSY by removingsome high intensity (methyl) peaks.
Point of entry –distinctive peak
OH
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Point of entry –distinctive peak
OH
Point of entry –distinctive peak
OH
Point of entry –distinctive peak
OH
Ipsenol spectra explanation.
DiastereoscopicGeminal, 1 vicinal
Lowest,
OH deshielded
= deshielded
OH DiastereoscopicGeminal, 2 vicinal
Diastereoscopicmethyls, 1 vicinalHighly coupled, overlapped with OH.
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http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch13/ch13-2dnmr-1.html
O
O
http://www.chemistry.ccsu.edu/glagovich/teaching/316/nmr/cosy.html
singlet
COSY 1H-1H COSY (COSY)
13C-1H COSY C Detected (HETCOR)
H Detected COSY (HMQC)
The information on how the H and H are coupled is gleaned from the contour peaks.
H Detected COSY (HMQC)
H- Detected Long Range(HMBC)
The information on how the H are C are correlated is gleaned from the contour peaks.
HETCOR- Heteronuclear Chemical Shift Correlation
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 couplings are removed here Variations of the HETCORH-H couplings are removed here. Variations of the HETCORcan show only CH, or CH and CH3 positive and CH2 negative.
The experiment encodes the proton chemical shift informationinto 13-C signals that are observed. It generates cross peaks for all protons and 13C nuclei that are connected by a 13C-1Hcoupling over one bond.
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HETCOR
1JHC = 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.
HMQC
1JHC = 145 Hz.
Correlates 13C directly attached to H, and the experiment is H detected. Long range couplings eliminated.
ethyl 2-butenoate
-OCH2-
O
-CH3
(HETCOR spectra recorded by D. Fox, Dept of Chemistry, University of Calgary on a Bruker Advance DRX-400 spectrometer
O
O
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H
CH3 H
O
O
CH3
Ethyl Crotonate
H O
O
CH3 H
CH3
Ethyl Crotonate
H
CH3 H
O
O
CH3
Ethyl Crotonate
http://www.tecmag.com/pdf/HETCOR.pdf
Diagonal leads to noinformation.
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HMBC
1/2J can be optimized f diff t
Correlates 13C with 2-bond and 3-bond couplings to H, and the experiment is H detected. Interpretation more difficultBecause of both 2,3-bond (sometimes 4-) correlations are present.
for different coupling constants
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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
NOESY also detects chemical and conformational exchange (EXSY). It is a homo-nuclear 2D plot, with diagonal as thenormal 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 experimentare also present in the NOESY spectrum (effectiveness; r-6)
The peaks additional to COSY peaks are the NOE enhancedpeaks.
NOESY spectrum of codeine
http://www.acornnmr.com/codeine/noesy.htm
Expansion of the up-field region;
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8 - 7, 127 - 18, 18'3 - 5, 105 - 11, 16, 18'9 - 10, 17, 17'10 - 1611 - 18, 16,11 18, 16, 14, 18'18 - 13, 18'16 - 14, 1713 - 14, 17, 17'13' - 17, 17'17 - 17'
' indicates the more up-field of geminal CH2 protons
N-Phenylacetamide
~2Å
A B~2Å
HNMR: Simulated Spectrum N-Phenylacetamide
N
H
O
N-phenylacetamide
A
B
NOSEY: The NOE enhanced peaks (only) for A and B
B
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