NMR Spectroscopy CHEM 430. P ROTON -P ROTON C ORRELATION T HROUGH J-C OUPLING 2D NMR Basics. In actuality, the techniques we have already covered 1 H,

NMR Spectroscopy

CHEM 430Spring

2014

PROTON-PROTON CORRELATION THROUGH J-COUPLING

2D NMR Basics.• In actuality, the techniques we have already covered 1H, 13C, and DEPT are 2-

D (frequency vs. intensity) however, by tradition the intensity component is dropped when discussing dimensionality

• In 2-D techniques, many FIDs (proto-NMR spectra) are taken one after another, with some acquisition variable or pulse sequenced varied by small increments

• Since each FID is a collection of digitized data points in the first dimension (say 10 points to make a spectrum) if 10 spectra are accumulated with an incremental change in variable, an FT can be performed in the other dimension

2-D NMR Spectroscopy 6-1

CHEM 430 – NMR Spectroscopy

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1-D FID

1-D spectra, each with an incremental variable change

FTs can be performed on the vertical data sets


2D NMR Basics.• The first perturbation of the system (pulse) is called the preparation

of the spin system.

• The effects of this pulse are allowed to coalesce; this is known as the evolution time, t1 (NOT T1 – the relaxation time)

• During this time, a mixing event, in which information from one part of the spin system is relayed to other parts, occurs

• Finally, an acquisition period (t2) as with all 1-D experiments.



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Preparation Evolution Acquisition

t1 t2

Mixing


2D COSY.• H-H COrrelation SpectroscopY (COSY):

• The pulse sequence for COSY is as follows:

• A 90o pulse in the x-direction is what we used for 1-D 1H NMR

• Here, after a variable “mixing” period, a second 90o pulse is performed, followed by acquisition of a spectrum



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90x90x

t1

t2


2D COSY.• Consider a simple spectrum with one resonance (CHCl3):



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B0z

x

y

M


2D COSY.• We pulse the sample with our standard 90o

x tilting magnetization into y



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B0z

x

y

90x

M


2D COSY.• After a short time t the vector begins to evolve around the z-axis



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B0z

x

y

90x

M


2D COSY.• This vector now has an x-component and y-component



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B0z

x

y

90x

M

Mcoswt1

Msinwt1

t1


2D COSY.• If a 90o

x pulse is applied again only the y component is rotated to -z



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B0z

x

y

90x

M

Mcoswt1

Msinwt1

t1

90x


2D COSY.• If we now detect in the xy plane, only the x-component remains



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B0z

x

y

90x

Mx

Msinwt1

t1

90x

Signal:

t2

7.0


2D COSY.• Repeat the experiment, but let time evolve by a longer increment



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B0z

x

y

90x

M


2D COSY.• This vector now has a greater x-component than before



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B0z

x

y

90x

M

Mcoswt1

Msinwt1

t1


2D COSY.• If a 90o

x pulse is applied again only the y component is rotated to -z



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B0z

x

y

M

Mcoswt1

Msinwt1

90x

t1

90x


2D COSY.• Now we will detect a larger x-component than before



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B0z

x

y

90x

Mx

Msinwt1

t1

90x

Signal:

t2

7.0


2D COSY.• In the COSY experiment a new ‘spectrum’ is acquired at increments of t1

• If we stack the array of spectra evolving at t1 increments, notice how we now have a new FID of sorts in the orthogonal coordinate!



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2D COSY.• If we carry out a Fourier Transform in this other coordinate, we generate a 2-

D spectrum where the CHCl3 peak shows up at 7.27 on both axes.

• The peak is more a sharp cone in shape



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FT


2D COSY.• Now let’s make this more complex; but what occurs now is a simplification!



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B0z

x

y

M


2D COSY.• Let’s pulse a sample with two nuclei that are spin coupled.



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B0z

x

y

90x

M


2D COSY.• By T2 relaxation vector begins to diverges as +J/2 and –J/2



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B0z

x

y

M

B0z

x

y

M


2D COSY.• The two nuclei are precessing at different w and decaying at different T1

BUT they share a frequency of oscillation of resultant x-component about the z-axis!!!



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z

x

y

M

z

x

y

M


2D COSY.• So in the 1st dimension, the spectra appear “normal”, but in the second

dimension these two nuclei share a oscillation frequency of x-component

• If we stack the spectra we see that they show an artifact of related spin at the frequency of their coupling partner

• Rather than view the 3-D map, it is customary to interperet the 2-D spectrum as viewing from overhead



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2D COSY.• Also keep in mind that J values have a sign and the vectors they generate

have a – and + component, so in reality:



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f1

f2


How to use 2D COSY.• Consider the COSY spectrum

of butyl propanoate

• Remember all resonances share their own variation of x-component in the COSY experiment.

• The peaks along the diagonal in the COSY spectrum show this relationship and can be ignored.



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O

O


How to use 2D COSY.• The ‘normal’ 1-D 1H

spectrum is placed on the F1 and F2 axes as a reference.

• This spectrum is obtained separately!



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O

O


How to use 2D COSY.• To find a pair of coupling

partners simply find the cross peak relationships

• For example the resonance at d 4.05 we can identify by

chemical shift as being adjacent to the sp3 oxygen of the ester.

• We find it is coupled to the resonance at d 1.65



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O

O


How to use 2D COSY.• We now use the d 1.65

resonance to find the next coupling partner

• We see it is coupled to the adjacent resonance at d 1.4

• Likewise the d 1.4 resonance is coupled to the resonance at d 0.95

• This resonance only has the d 1.4 as a coupling partner, so the chain ends.



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O

O


How to use 2D COSY.• Likewise we can deduce the

ethyl chain attached to C=O as a separate coupled family

• The only drawback of the COSY experiment is it cannot ‘see through’ parts of the molecule that have no 1H-1H coupling (2JHH or 3JHH)

• These include 4o carbons, C=O, 3o amines, -O-, -S-, etc.



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O

O


DQFCOSY.• Double quantum filtered - an extra

pulse is added after the second COSY pulse, and phase cycling converts multiple quantum coherences into observable magnetizations.

• The resulting 2D spectrum lacks all singlets along the diagonal

• The experiment has surplanted the COSY-45 in the text



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DQFCOSY.• An important feature of the

experiment is that double quantum filtration allows both diagonal and cross peaks to be tuned into pure absorption at the same time.

• This reduces the size of all diagonal signals and permits cross peaks close to the diagonal to be observed.

• The only disadvantage of DQF– COSY is a reduction in sensitivity



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PROTON-HETERONUCLEUS CORRELATION

HETCOR.• The 13C-1H COSY (HETeronuclear CORrelation) experiment correlates 13C

with directly attached 1H via 1JCH couplings.

• Since the frequency domains F1 and F2 are for different nuclei we do not observe diagonal peaks as in 1H-1H COSY

• During the evolution time the large 1JCH is used for polarization transfer, so only 13C directly bound to 1H are detected



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HETCOR.• Note how we can now determine that two protons are on the same carbon:



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HETCOR.• The principle disadvantage of HETCOR is total acquisition time. Typically 5-

8 times the length of the corresponding 13C experiment.



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HSQC/HMQC.• One way around the sensitivity problem with HETCOR is to observe the 1H

in the 13C-1H system and study 1JHC rather than the non-abundant 13C.

• The 2D HSQC (Heteronuclear Single-Quantum Correlation) experiment permits to obtain a 2D heteronuclear chemical shift correlation map between directly-bonded 1H and X-heteronuclei (commonly, 13C and 15N).

• One interesting artifact of the HSQC experiment is the ability to create a low-resolution 1-D 13C spectrum from the 2-D data!



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HSQC/HMQC.• The data is interpreted like HETCOR; the 13C spectrum on the F1 axis is

acquired separately and actually takes longer than the 2D acquisition!



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HMBC.• The 2D HMBC experiment permits to obtain a 2D heteronuclear chemical

shift correlation map between long-range coupled 1H and heteronuclei (commonly, 13C).

• It is widely used because it is based on 1H-detection, offering high sensitivity when compared with the 13C-detected.

• In addition, long-range proton-carbon coupling constants can be measured from the resulting spectra.



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HMBC.• The power of this technique is it allows us to “see” through a quarternary

carbon center!

• The drawback is that for small molecules with tight ring or fused-ring systems (like many of the unknowns) everything may be correlated to everything else!



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HMBC.• In this example we see ipsenol and

each of the ‘5-C domains’ that make up the molecule.

• For example C-4 is within 3JHC of H-3, H-3’, H-2, OH, H-5 and H-5’

• C-7 can be seen to be within 3 bonds of 5/5’ through the 4o center at C-6!



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PROBLEMS - 12-D NMR Spectroscopy 6-P


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PROBLEMS – 2 - HSQC2-D NMR Spectroscopy 6-P


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TOCSY.• TOtal Correlation SpectroscopY ( TOCSY). By spin locking the protons

during the second COSY pulse, the chemical shifts of all the protons may be brought essentially into equivalence.

• The initial pulse and the period occur as usual, but the second pulse locks the magnetization along the y-axis so that all protons have the spin lock frequency.

• All coupled spins within a spin system then become closely coupled to each other, and magnetization is transferred from one spin to all the other members, even in the absence of J couplings.



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NOESY.• The spectrum on the right shows the TOCSY

experiment for lysine.

• TOCSY has particular advantages for large molecules, including enhanced sensitivity and, if desired, the phasing of both diagonal and cross peaks to the absorption mode.

• The process of identifying resonances within specific amino acid or nucleotide residues is considerably simplified by this procedure. Each residue can be expected to exhibit cross peaks among all its protons and none with protons of other residues.



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NH2

OH

O

H2N


NOESY.• After the second pulse in COSY the NOE mechanism can modulate the cosine

component of the magnetization along the z axis.

• The frequency of modulation is the frequency of the magnetization transfer partner from dipolar relaxation

• After a suitable fixed period (tm, the mixing period), during which this modulation is optimized, the cosine component may be moved to the xy plane by a third pulse and may be detected along the y axis during a acquisition period.



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NOESY.• Because the frequency of magnetization of some nuclei during moves to

another value during t1 and is observed at the new frequency during tm , the 2D representation of this experiment exhibits cross peaks.

• When the cross peaks derive from magnetization transfer through dipolar relaxation, the 2D experiment is called NOESY (NOE SpectroscopY).

• The duration of the fixed time tm depends on T1 and the rate of NOE buildup.

• In the NOESY experiment valuable information can be ascertained about the distance between various protons within a molecule (< 5Å).



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NOESY.



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COSY

NOESY

N

N

H H H


NOESY.



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Overlapped COSY and NOESY

NOESY in faded color

N

N

H H H


NOESY.At least three factors complicate the analysis of NOESY spectra.

1. COSY signals may be present from scalar couplings and may interfere with interpretations intended to be based entirely on interproton distances.

2. In small molecules, the NOE builds up slowly and attains a theoretical maximum of only 50% — Because a single proton may be relaxed by several neighboring protons, the maximum is much less than 50%.

3. In addition to its transfer directly from one proton to an adjacent proton, magnetization may be transferred by spin diffusion. In this mechanism, magnetization is transferred through the NOE from one spin to a nearby 2nd spin and then from the 2nd to a 3rd spin that is close to the 2nd spin, but not necessarily to the first one. These multistep transfers can produce NOESY cross peaks between protons that are not close together.



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ROESY.• The Rotating frame Overhauser Effect SpectroscopY utlizes spin-locking

(like TOCSY) to ameliorate the drawbacks of NOESY for small molecules.

• The pulse sequence is as follows –data interpretation is the same as NOESY



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EXAMPLES

VGSE: Valine-Glycine-Serine-Glutamic acid.

2-D NMR Spectroscopy 6-E


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Documents

NMR Spectroscopy CHEM 430. P ROTON -P ROTON C ORRELATION T HROUGH J-C OUPLING 2D NMR Basics. In actuality, the techniques we have already covered 1 H,