2d nmr spectroscopy

8/22/2019 2D NMR Spectroscopy

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Two Dimensional (2D) NMRS ectrosco

Correlation NMR

The two important parameters obtained from NMR spectraare;

a. Chemical shiftb. Spin-spin coupling constant

Large molecules with numerous atoms nuclear magnetic

fundamental parameters easily.

Some 1D spectra are far too complex for interpretationbecause signals overlap heavily e.g. cholesterols, proteinspectra

ntensity

Chemical shift, ppm

I

1D spectrum of a protein


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Nonequivalent proton groups can have nearly the samechemical shift and/or complex splitting patterns making1H NMR spectra complicated even for relatively simplemolecules.

The introduction of additional spectral dimensions simplifies thespectra and provides more information.

Two-dimensional (2D) NMR techniques can be used to solvesuch sophisticated structural problems.

2-D spectra simplify the complexity arising from overlappingof peaks.

David E. Alonso* and Steven E. Warren, NMR Analysis of Unknowns: An Introduction to 2D NMR

Spectroscopy, J ournal of Chemical E ducation 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 asa function of two frequencies, usually represented as F1and F2.

There are two ways to present 2D spectra; stack plotsand contour plots.

2-D spectra are presented usually as a contour plot,where, the intensity of the peaks are represented bycontour lines (recall topographical maps).


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F1

F2

General Presentation of Correlation Spectra

Two frequency axes. F1 and F2 are Fourier transformedfrequency 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 protonspectrum 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 theF2 co-ordinates of the peaks correspond to those found inthe normal 1-D spectrum. (Often 1-D spectrum is presentedon the horizontal F2 axis).

F1 co-ordinates of the peaks also correspond to those of thenormal 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

F1X

COSY spectrum of a molecule containing just one type

of protons HX.

F2

HAHX

HA

Cl

R2

H

R1

R

H

R3

A X

COSY Spectrum

X AA

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 SpectrumJ AX

X AA

F1 HX

X

COSY spectrum of a hypothetical molecule containing justtwo 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 ofindividual peaks often forming of a square or rectangular outline.Multiplets form a square or rectangle with two vertices on thediagonal.

HA

X AA

HX

F1

F2

Diagonal multipletscentered around sameF1 and F2.

Cross-peak multiplets

centers around differentF1 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 Xat 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 s ectrum shows which ai rs in a molecule are

coupled (thro bond coupling, hence connectivity).

Recognition of the preceding fact is the essence for theanalysis COSY spectra.

From a single COSY spectrum it is possible to trace out thewhole 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 /TM M sin(2 t e

1 2 22t /T0

t /T21M sin(2[M sin(2 t )e t e] )

Data acquired at theend of the an acquisition(after acquisition pulse)is labeled with the timevariable labeled t .

The generation of a 2D experiment:

In addition to preparation and detection (done in the 1Dexperiment) 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) t1. 1

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 multiplepulse 1D sequence with a systematic variation of the evolutionand mixing times, t1, and then plotting Fourier transformed FID.

This generates two time domains, one of which, t2, is theac uisition time thata ears durin the ac uisition as usualand the other time domain originates from the variable delaypart, t1.


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t1=t

t1=2t

O

frequency data (FID) in one axis (f2, fromt2),

t1=0t2

t1

A(t1)

Time domain data in t1. It isperiodic; a pseudo FID created foreach of the frequencies in f2.

t1

t1

f2 (t2)

Decay not shown.

Note all tops form one FID, ..

t1

O

t1

stacked 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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f2

1

Two dimensional FT yields the 2D spectrum with two

In a real molecule where J coupling exist, during the mixingmagnetization can be transferred from one nucleus to asecond one. Mixing sequences utilize two mechanisms formagnetization transfer, namely scalar coupling or dipolarinteraction (NOE).

requency axes. e spec rum s omonuc ear s gnas othe same isotope - say 1H, are detected) the spectrumwould have a characteristic symmetric topology.

COSY:

1D- double resonance experiment that is often used to findrelationships between protons, the protons are irradiated oneby one.

COSY generates all information from a series of doubleresonance experiments in one output (2D spectrum).

The pulse sequence for a COSY experiment contains avariable delay time as well as an acquisition time. Theexperiment is repeated with different and incremented delaytimes, and the data collected during the acquisition are storedin the computer. The value of the delay time is increased by

regular, small intervals for each experiment, so that the datathat 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 byscalar coupling.

In the COSY spectrum of a molecule where all possibleoff-diagonal peaks are generated; the result is a completedescription of the coupling partners in a molecule.

ome mes e coup ng e ween pro ons a are more anThree chemical bonds apart can be seen.

Signals on the diagonal divides the spectrum in two equalhalves. Signals symmetrical to the diagonal called crosssignals (peaks).

The diagonal results from contributions of the magnetizationthat has not been changed by the mixing sequence.

The cross signals originate from nuclei that exchangedmagnetization during the mixing time. They indicate aninteraction of these two nuclei. The cross signals containthe information of 2D NMR spectra.

time - time

time - frequency

0 200 400 600 800 1000t 2 pt s

t2

t1

Pulegone

frequency - frequency400 500

f 2 pt s

500 600 700 800 900f 2 pt s

f2

f2

1

f1

http://tonga.usp.edu/gmoyna/NMR_E N/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

axis

Each circle represents thecenter of the multiplet.

1

2

1

2 axis

H4 H3H1

H2 no coupling

C3H8O; U =0

CH3 CH2 CH2 OH

peak label

4 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 OHCH3

CH2CH2 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 a

2 2 2 2

CH2 - O|

-O-C=O; ester

C5H8O2

CH2 - O

abcd

ba

c

d

Expansion showba coupling

ba

cba

cbad

-

U =2, -CO2 group accounts for 1,Therefore other is a ring.

COSY spectrum

1D HNMR; four CH2a CH2 c CH2

b CH2

2 -

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

2H

3H

3H

CH2 CO CH2

C=O

Ketone


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C8H16OMe-1

2 345

3

5

Me8CH27/5

2 off-peakson same line -overlap

Me1 2 - 3 4 - 5

7 Me8

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

COSY spectrum

1D HNMR; five CH2

Two spin systems

CH2 CO CH2

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 OCH2CH3an . a a es a oms.

13C NMR

6 types of C; also OC=O and OC


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O CH2 CH3CH2 CH3

O = C O CH2 CH3

O = C O CH2 CH3

CH2 CH3CH3 CH2 C

C11H20O4 methyl

Two spin systems

Ipsenol spectra explanation.

OH

4 CH22 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)

The information on how the H and H arecoupled is gleaned from the contour peaks.

H- Detected Long Range(HMBC)

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

HETCOR- Heteronuclear Chemical Shift Correlation

HETCOR gives the correlations between protons and othernuclei such as 13-C or 15N. Two versions exist absolute valueHETCOR and phase sensitive HETCOR. A related experimentis the HMQC experiment

- .can 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

1J HC =145 Hz.

Correlates 13C directly attached to H , large 1J CH couplings(polarization transfer) and the frequency domains are fromdifferent nuclei . F2 is 13C and F1 is 1H. Therefore nodiagonal symmetry.

HMQC

1J HC =145 Hz.

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

ethyl 2-butenoate

-OCH2- -CH3

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

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

Correlates 13C with 2-bond and 3-bond couplings to H, andthe experiment is H detected. Interpretation more difficultBecause of both 2,3-bond (sometimes 4-) correlations arepresent.

or erencoupling constants


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

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. Informationgleaned from the "cross-peaks", which appear at thecoordinates 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, 16,14, 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

~2

A B~2

HNMR: Simulated Spectrum N-Phenylacetamide

N

H

O

N-phenylacetamide

A

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

2d nmr spectroscopy

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