Basic One-and Two Dimensional NMR Spectroscopy by malj


									Basic Two Dimensional NMR

             M. Manickam

       M. Manickam@ bham. ac. uk

          Semester- 1; Week-4

         Second year: CHM2C3
        First and Second Semester
                  Basic of 2D NMR
                  1H-1H Correlation spectra
                  1H-13C Correlation spectra
                  13C DEPT Spectra

                  HMBC, HSQC and NOESY
                  Few examples
                  2h Workshop and Pro-Forma


          Given the expected compound-interpret the spectra

  Pro-forma- 1 week to hand in with usual penalties for late submission
Haworth Room No 214 (administration office, between 12 noon and 2 pm)
 Application of Organic Spectroscopy
                        Why is it needed? What is it used for?
                             Structure Determination

Chemists synthesises new and known materials and they need to know the structure
To characterise materials
Is the structure as expected? Or is it different? Some interesting new reactions and
materials have been discovered from unexpected results
To enable the physical and chemical properties to be related to the structure. This
facilitates the synthesis of better materials, for example, drugs, liquid crystals,
pesticides, polymers etc
Safety reasons dictate that the structure of a materials is known so that any hazards can
be related to structure and so that a material can be safely used and disposed of
The progress of reactions can be monitored by spectroscopy, usually NMR and this
technique allows the perfect timing of reactions to provide optimum results and can
prevent unwanted further reaction
Reaction can be carried out in the NMR instrument to enable instant analysis of
structure which allows the structure of intermediates to be determined and reaction
mechanisms to be established
The purity of materials can be determined by NMR, routine checking of structures
                       Why NMR
        A    +     B                        Product

To find out required and side products

To analysis and confirm the natural products structures.

We use a variety of spectroscopic techniques

Mass spectroscopic: Gives a compound’s mass (little

IR spectroscopic: Functional groups information

UV spectroscopic: Chromospheres and conjugated systems

NMR spectroscopic :Gives great detailed structural information
  and the most powerful spectroscopic method used by organic
    NMR: Nuclear Magnetic Resonance

                   Basic principle

   1HNMR spectroscopy provides information about the
    environments of the H atoms in a molecule

 It is based on the same principles as in 13C NMR spectroscopy

 The 1H nucleus has nuclear spin 1/2, so when placed in a strong
  magnetic field, it can exist in higher or lower energy states.
  NMR: Nuclear Magnetic Resonance

 When the nucleus is irradiated, it absorbs radio frequency
  radiation, and nuclei in the lower energy spin states are
  promoted to higher energy spin states

 There are important differences between 1H and 13C NMR

 The 1H atom has 99.98% abundance in naturally occurring H, so
  1H NMR spectra can usually be measured by a single scan, so

  FT methods are only used in exceptional circumstances

 As a result, the peaks are proportional to the number of H
  atoms that the peak represents - this is very valuable when
  analysing spectra.
     Carbon 13 (13C) NMR Spectrum

Basically the same in principle to proton NMR obviously,
  precessional frequencies of carbons are different to those of
  protons but this is no problem
                  low sensitivity
Major problem is that 13C is only 1.1% of carbon additionally the
  magnetic moment of 13C is 4x weaker than for 1H
Overall 13C signals are 6000x weaker than 1H signals
However, using pulse FT-NMR 30,000 pulses can be made
  reasonably quickly to give an excellent spectrum
                   high resolution
Useful advantage is that typically 13C signals are spread over 200 
  units and so there is less chance of coincidence-hence-
  13CNMR is 20x more resolved than 1H NMR

  Chemical shift () values are determined in the same way as for
  proton signals-shielding and deshielding.
                    NMR Spectrum

From each signal you should be able to obtain three pieces information:

 • From the Chemical shift, the environment of the proton-
   containing group;

 • From the integration, the relative number of protons in the
   proton- containing group;

 • From the splitting, the number of protons on an adjacent
   carbon atom.
                       1 Dimensional NMR
• These are the most essential NMR spectra

• Spectra have one frequency axis and one intensity axis (see

•   1H and 13C NMR spectra must contain all the required
    resonances for the expected compound.

•   1H   NMR: - Integration-all protons must be accounted for
             - Chemical shifts must be correct.

           - Protons on adjacent carbon atoms will couple to
    produce multiplets.

•   13C NMR: number of peaks shows number of carbon atoms
    (accounting for overlap and equivalency)

• Oxygen atoms, nitrogen atoms do not appear in NMR spectra
  but their presence is implied in the chemical shift
General regions of Chemical Shifts
13C   Chemical Shifts
1H-1H   COSY (Correlation Spectroscopy)

 2-D NMR spectra have two frequency axes and one
   intensity axis. The most common 2-D spectra
   involve 1H-1H shift correlation; they identify protons
   that are coupled (i.e., that split each other’s signal).
   This is called 1H-1H shift- correlated spectroscopy,
   which is known by the acronym COSY.

  1H-1H correlation spectra
  2-D dimensional plot with 1H spectrum along each axis and
   on the diagonal
  Protons coupling to one another produce off diagonal correlations
 This allows assignment of proton groups that are connected
   in the molecule
  Shows connectivity in the compound
COSY spectrum of Ethyl Vinyl Ether
  Fig:1 Stack plot   Fig: 2 Contour plot


    1H-1H      COSY Spectrum of Ethyl Vinyl
Fig:1. It looks like a mountain range viewed from the air because intensity
   is the third axis.

These “mountain-like” spectra (known as stack plots) are not the spectra
  actually used to identify a compound.

Instead, the compound is identified using a contour plot Fig:2, where
   each mountain in Fig:1 is represented by a large dot (as if its top had
   been cut off). The two mountains shown in Fig:1 correspond to the
   dots labelled B and C in Fig: 2

Fig:2, the usual one-dimensional 1H NMR spectrum is plotted on both the
   x- and y- axes.

To analyze the spectrum, a diagonal line is drawn through the dots that
  bisect the spectrum.
 1H-1H       COSY Spectrum of Ethyl Vinyl
The dots that are not on the diagonal (A, B, C) are called cross
  peaks. Cross peaks indicate pairs of protons that are coupled.

For example, if we start at the cross peak labeled A and draw a
  straight line parallel to the y-axis back to the diagonal, we hit
  the dot on the diagonal at ~ 1.1 ppm produced by the Ha

If we next go back to A and draw a straight line parallel to the x-
    axis back to the diagonal, we hit the dot on the diagonal at ~
    3.8 ppm produced by the Hb protons. This means that the Ha
    and Hb protons are coupled.
1H-1H    COSY Spectrum of Ethyl Vinyl
If we then go to the cross peak labelled B and draw two
    perpendicular line back to the diagonal, we see that the Hc and
    He protons are coupled; the cross peak labelled C shows that
    the Hd and He protons are coupled.

Notice that we used only cross peaks below the diagonal; the
  cross peaks above the diagonal give the same information.

Notice also that there is no cross peak due to the coupling of Hc
  and Hd, consistent with the absence of coupling for two protons
  bonded to an sp2 carbon.
   HETCOR Spectrum or (1H-                        13C   COSY)
2-D NMR spectra that show 13C-1H shift correlation are called HETCOR
   from heteronuclear correlation) spectra. HETCOR spectra indicate
   coupling between protons and the carbon to which they are

Example: 2-methyl-3-pentanone
The 13C NMR spectrum is shown on the x-axis and the 1H NMR
  spectrum is shown on the y-axis. The cross peaks in a HETCOR
spectrum identify which hydrogens are attached to which carbons.

For example, cross peak A indicates that the hydrogens that shows a
  signal at ~ 0.9 ppm in the 1H NMR are bonded to the carbon that
  shows a signal at ~ 6 ppm in the 13CNMR spectrum.
Cross peak C shows that the hydrogens that show a signal at ~ 2.5
  ppm are bonded to the carbon that shows a signal at ~ 34pp
HETCOR spectrum of 2-methyl-3-
            CH   CH2             CH3

          DEPT       13C   NMR SPECTRA

13C   DEPT spectra enable different carbon
      (CH3, CH2, CH, and quaternary)

Types to be identified

DEPT 90:     only CH peaks visible?

DEPT 135: -CH2 peaks negative
          -CH and CH3 peaks positive

PENDANT: -CH2 and quaternary peaks negative
         -CH3 and CH peaks positive
             DEPT      13C    NMR SPECTRA
• DEPT: stands for distortionless enhancement by polarization

• This technique to distinguish among CH3, CH2, and CH group

• It is now much more widely used than proton coupling to
  determine the number of hydrogens attached to a carbon.

• DEPT 13C spectrum does not show a signal for a carbon that is
  not attached to a hydrogen.

• For example: 13C NMR spectrum of 2-butanone shows 4 signals
  because it has 4 nonequivalent carbons, whereas the DEPT 13C
  NMR of 2-butanone shows only three signals because the
  carbonyl carbon is not bonded to a hydrogen, so it will not
  produce a signal.

        4    3     2   1           Normal 13C NMR gives 4 signals
        CH3-CH2-CO-CH3              DEPT 13C NMR gives 3 signals
 DEPT      13C   NMR Spectra of Ipsenol
In CDCl3 at 75.6 MHz:
Subspeectrum A, CH up.                       5           7

Subspectrum B, CH3 and CH up, CH2       HO           3

   down. The conventional 13C NMR                    2
   spectrum is at the bottom.

                                         3X CH-

                                         3X CH, 2X CH3

                                    4X CH2
    Other Types Of 1H-13C COSY

1. HMBC: 1H-13C several bond correlation

2. HSQC: 1H-13C carbon and protons direct   correlation

3. HMQC: correlation between protons and other nuclei
   such as 13C or 15N
  HMBC SPECTRUM (Hetronuclear Multiple-
          Bond CH Correlation)
 This is a 2D experiment used to correlate, or connect, 1H and 13C
  peaks for atoms separated by multiple bonds (usually 2 or 3).
 The coordinates of each peak seen in the contour plot are the 1H
  and 13C chemical shifts. This is extremely useful for making
  assignments and mapping out covalent structure.

 Heteronuclear Multiple Bond Correlation
 13C-1H Correlations over Several Bonds
 Typically over 2 or 3 bonds can be seen.
 Possible because of sensitivity of the powerful magnets of today's
  NMR spectrometers.
 Can be used to establish connectivity across barriers such as O
  atoms or quaternary carbon atoms

    1H-C-13C   (Two-bond)             1H-C-C-13C   (Three- bond)
                           HMBC of Codeine






  H-8 to aromatic carbons C-1            H-9 to aromatic carbons C-1,
  and C-6 ( both are three bond          C-3 and C-4 ( both are three
  coupling                               bond coupling
HSQC (Heteronuclear Single-Quantum

13C-1H   correlation spectra

2 Dimensional plot -1H spectrum on one axis, 13C on the other

Shows Correlations between carbons and protons
directly attached to one another

allows further connectivity within the molecule to be established
   Nuclear Overhauser (NOESY) Spectrometry
           Proximity Through Space

A proton that is close in space to the irradiated proton is affected by
   the NOE whether or not it is coupled to the irradiated proton; if it
   is coupled, it remains at least partially coupled because the
   irradiation is week in comparison with that used for a decoupling

NOESY for very large molecules, ROESY for mid-size molecules
These spectra are used to locate protons that are close together in space

       Can be a 1D or 2D NOESY technique

       nOe is a through space effect

       It has nothing to do with connectivity in the molecule
   Nuclear Overhauser Enhancement


• Elucidation of molecular constitution and conformation

   Is used to solve geometric problems within a molecule
   Relative stereochemistry can be seen
   Regiochemistry can be seen
   Powerful technique for 3D study of proteins and other

• Aiding assignments

• Investigating molecular motions
 Nuclear Overhauser Enhancement
                                            Which methyl signal belongs
                                            to which group?
                   Ha   OCOCH3                                         +17%
Cl     Hb
Cl                                      H3C     H
                                            N C
 Cl                                     H3C                      H3C               H
                                                O                      C       C
 Cl                                                             H3C                COOH
  Cl                                           - 2%
              Cl                                                         - 4%
        semiclathrate               Dimethylformamide                      3-methylcrotonic
                                       two Me groups are non-              acid
                                    equivalent owing to hindered
                                    rotation about the C-N bond.
                                Both Me signals are therefore found
                            At  2.79 and  2.94, together with a singlet
                        at  8.0 for the formyl proton. If one now saturates
                        the Me signal at  2.94, the intensity of the formyl
                        proton signal increases by 18%. When instead the
                        other methyl signal is saturated, a decrease of 2%
                                             is observed.
   Nuclear Overhauser Effect Difference
  (NOESY) Spectrometry, 1H 1H Proximity
             Through Space
             O                         O                   O
                                                            2           readily
  H3C                            R                 H3C 3
                 O                         O                   O1       available
     H               H           H             H     H4    5        H
             R                         CH3                 CH3

                     natural product       2                     3

NOE difference spectrometry determined the substitution pattern of a
natural product, whose structure was either 1 or 2.

NOE difference spectrometry of compound 3 will help us to
settle the final structure
      NOE difference spectrometry for
                Compound 3
                                                          H3C 3
                                                             H4       5       H

Irradiation of the 5-Me group resulted in enhancement of both H-4 and H-6,
whereas irradiation of the 3-Me group enhanced only H-4; the assignments of
these entities to the absorption peaks is now clear.
2-D   13C-13C     Correlations: INADEQUATE

(Incredible Natural Abundance DoublEQUAntum Transfer Experiment)

  2-D INADEQUATE provides direct carbon connectivities
  enabling us to sketch the carbon sheleton unambiguously

  2-D INADEQUATE has very limited applicability because of
  its extremely low sensitivity
                Further Reading

1.   Spectroscopic Methods in Organic Chemistry (fifth
     edition): Dudley H. Williams and Ian Fleming
2.   Spectrometric Identification of Organic
     compounds (six edition): Robert M. Silverstein and
     Francis X. Webster
3.   Basic One- and Two- Dimensional NMR
     Spectroscopy : Horst Friebolin

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