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 2
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
1-D FID
1-D spectra, each with an incremental variable change
FTs can be performed on the vertical data sets
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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.
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 3
Preparation Evolution Acquisition
t1 t2
Mixing
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 4
90x90x
t1
t2
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• Consider a simple spectrum with one resonance (CHCl3):
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 5
B0z
x
y
M
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• We pulse the sample with our standard 90o
x tilting magnetization into y
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 6
B0z
x
y
90x
M
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• After a short time t the vector begins to evolve around the z-axis
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 7
B0z
x
y
90x
M
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• This vector now has an x-component and y-component
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 8
B0z
x
y
90x
M
Mcoswt1
Msinwt1
t1
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• If a 90o
x pulse is applied again only the y component is rotated to -z
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 9
B0z
x
y
90x
M
Mcoswt1
Msinwt1
t1
90x
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• If we now detect in the xy plane, only the x-component remains
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 10
B0z
x
y
90x
Mx
Msinwt1
t1
90x
Signal:
t2
7.0
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• Repeat the experiment, but let time evolve by a longer increment
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 11
B0z
x
y
90x
M
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• This vector now has a greater x-component than before
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 12
B0z
x
y
90x
M
Mcoswt1
Msinwt1
t1
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• If a 90o
x pulse is applied again only the y component is rotated to -z
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 13
B0z
x
y
M
Mcoswt1
Msinwt1
90x
t1
90x
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• Now we will detect a larger x-component than before
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 14
B0z
x
y
90x
Mx
Msinwt1
t1
90x
Signal:
t2
7.0
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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!
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 15
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 16
FT
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• Now let’s make this more complex; but what occurs now is a simplification!
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 17
B0z
x
y
M
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• Let’s pulse a sample with two nuclei that are spin coupled.
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 18
B0z
x
y
90x
M
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• By T2 relaxation vector begins to diverges as +J/2 and –J/2
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 19
B0z
x
y
M
B0z
x
y
M
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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!!!
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 20
z
x
y
M
z
x
y
M
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 21
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.• Also keep in mind that J values have a sign and the vectors they generate
have a – and + component, so in reality:
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 22
f1
f2
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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.
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 23
O
O
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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!
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 24
O
O
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 25
O
O
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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.
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 26
O
O
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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.
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 27
O
O
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 28
PROTON-PROTON CORRELATION THROUGH J-COUPLING
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
2-D NMR Spectroscopy 6-1
CHEM 430 – NMR Spectroscopy 29
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
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 30
PROTON-HETERONUCLEUS CORRELATION
HETCOR.• Note how we can now determine that two protons are on the same carbon:
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 31
PROTON-HETERONUCLEUS CORRELATION
HETCOR.• The principle disadvantage of HETCOR is total acquisition time. Typically 5-
8 times the length of the corresponding 13C experiment.
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 32
PROTON-HETERONUCLEUS CORRELATION
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!
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 33
PROTON-HETERONUCLEUS CORRELATION
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!
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 34
PROTON-HETERONUCLEUS CORRELATION
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.
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 35
PROTON-HETERONUCLEUS CORRELATION
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!
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 36
PROTON-HETERONUCLEUS CORRELATION
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!
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 37
PROBLEMS - 12-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 38
PROBLEMS - 12-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 39
PROBLEMS - 12-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 40
PROBLEMS - 12-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 41
PROBLEMS - 12-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 42
PROBLEMS - 22-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 43
PROBLEMS - 22-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 44
PROBLEMS - 22-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 45
PROBLEMS - 22-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 46
PROBLEMS – 2 - HSQC2-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 47
PROBLEMS - 32-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 48
PROBLEMS - 32-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 49
PROBLEMS - 32-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 50
PROBLEMS - 32-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 51
PROBLEMS - 32-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 52
PROBLEMS - 42-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 53
PROBLEMS - 42-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 54
PROBLEMS - 42-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 55
PROBLEMS - 42-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 56
PROBLEMS - 42-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 57
PROBLEMS - 52-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 58
PROBLEMS - 52-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 59
PROBLEMS - 52-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 60
PROBLEMS - 52-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 61
PROBLEMS - 52-D NMR Spectroscopy 6-P
CHEM 430 – NMR Spectroscopy 62
PROTON-HETERONUCLEUS CORRELATION
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.
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 63
PROTON-HETERONUCLEUS CORRELATION
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.
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 64
NH2
OH
OH2N
PROTON-HETERONUCLEUS CORRELATION
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.
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 65
PROTON-HETERONUCLEUS CORRELATION
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).
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 66
PROTON-HETERONUCLEUS CORRELATION
NOESY.
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 67
COSY
NOESY
N
N
H H H
PROTON-HETERONUCLEUS CORRELATION
NOESY.
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 68
Overlapped COSY and NOESY
NOESY in faded color
N
N
H H H
PROTON-HETERONUCLEUS CORRELATION
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.
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 69
PROTON-HETERONUCLEUS CORRELATION
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
2-D NMR Spectroscopy 6-2
CHEM 430 – NMR Spectroscopy 70
EXAMPLES
VGSE: Valine-Glycine-Serine-Glutamic acid.
2-D NMR Spectroscopy 6-E
CHEM 430 – NMR Spectroscopy 71
EXAMPLES
VGSE: Valine-Glycine-Serine-Glutamic acid.
2-D NMR Spectroscopy 6-E
CHEM 430 – NMR Spectroscopy 72
EXAMPLES
VGSE: Valine-Glycine-Serine-Glutamic acid.
2-D NMR Spectroscopy 6-E
CHEM 430 – NMR Spectroscopy 73
EXAMPLES
VGSE: Valine-Glycine-Serine-Glutamic acid.
2-D NMR Spectroscopy 6-E
CHEM 430 – NMR Spectroscopy 74
EXAMPLES
VGSE: Valine-Glycine-Serine-Glutamic acid.
2-D NMR Spectroscopy 6-E
CHEM 430 – NMR Spectroscopy 75
EXAMPLES
VGSE: Valine-Glycine-Serine-Glutamic acid.
2-D NMR Spectroscopy 6-E
CHEM 430 – NMR Spectroscopy 76
EXAMPLES
VGSE: Valine-Glycine-Serine-Glutamic acid.
2-D NMR Spectroscopy 6-E
CHEM 430 – NMR Spectroscopy 77