CH 143 LAB REPORT

WEEK 5
Vincentius Jeremy Suhardi

Purpose :
The first experiment is dedicated to observe the effect of NOE due to the dipole-
dipole relaxation mechanism that will cause signal enhancement. The second
experiment is used to observe the effect of irradiation of certain nuclei in enhancing
peaks of the nearby nuclei, thuis showing the interaction between proton nuclei.
Third experiment is dedicated to observe the general interaction between one
proton to other protons in the molecule using the elaborated technique on the
second experiment in 2D manner to show the interaction between proton nuclei by
NOE.

Material :

1. Menthol in CDCl3 in NMR tube

2. Sucrose in D2O

3. NMR sepctrometer with spin is turned on

Procedure :
First Experiment :

1. Mentol sample in CDCl3 is inserted to the NMR probe and after locking and
shimming,(nt=4, gain=20), 13C NMR spectra is acquired. The spectra range is
readjusted (0 and 100 ppm) and then the spectra is reacquired.

2. Pw=16, tpwr=57, dpwr=36, lb=0.5, d1=10, and ss=4, then the decoupler
time is arrrayed to get both the incerse gated decoupling and the NOE setup.
Spectra is reacquired.

3. Integral value of both the arrayed spectra is displayed. Both spectra is

observed and analyzed.

Second Experiment :

1. Proton NMR of sucrose sample in D2O is acquired with parameter as follow :

d1=4, nt=4, gain=2, tpwr=57, pw=90, and at=4. Spectra range is then
adjusted to 2 until 7 ppm, and then spectra is reacquired.
 2. Resulting spectra was moved to a new experiment and peak at 5.26 and 5.28
ppm are irradiated. Tau is set to 0.1 , dm=’ynn’, ss=4, dpwr=12, nt =32 and
spectra is reacquired.

3. Vp was set to 120, dscale(-10) and the spectra is ploted and analyzed.

Third Experiment :

1. 2D NOESY spectra is acquired with sucrose in D2O as the sample.

2. Spectra is plotted and analyzed.

Observation and Results :

First Experiment :

General explanation of the technique used :

The inverse gated decoupling is used to measure the intensities of the peak without
the presence of NOE. This technique is applied by turning on the decoupler only in
the acquisition period. Since the build up of the NOE is slow, then what we will see
in the spectra is decoupled peak but without the presence of any NOE. After the
acquisition period, the decoupler is turned offagain, to reduce the NOE back to zero
again, and then the spectra is re-acquired.

Explanation of the experimental result :

The first spectra (left) has lower intensities of peak compared to the corresponding
peak in the same chemical shift on the second spectra. Both spectra is proton
decoupled, thus the difference of the intensity of the peak is due to the presence of
NOE in the second spectra. As explained in the general technique above, the first
spectra is acquired with inverse gated decoupling method, thus causing the peak
intensity to be in the normal intesnity with proton decoupled pattern. The second
spectra is acquired using continuous decoupling technique. Thus we can see the
efffect of NOE on the second spectra. Integrating the intensity of both spectra, we
can see that the second spectra has 2.7403 times greater intensity compared to the
corresponding peak in the same spectra.

By the equation :

Since the gyromagnetic rato between the proton and the carbon is positive, thus as
expected, the NOE will have positive valuse, and thus enhancing the intensity of the
peak.
Second Experiment :

General explanation of the technique used:

The NOE-difference spectrum is acquired to be able to measure the NOE interaction

between two protons in a molecule. Irradiation is done to a specific nuclesu at
specific frequency by giving 180o pulses to invert the normal population distribution.
This irradiation will cause the targeted proton nuclei to be excited from the αα to βα
and αβ to ββ. However the relaxation from ββ to αα predominates and hence
depleting the ββ state and restoring the αα state. This will cause the transition
corresponding to other nuclei that couple with the irradiated nulcei ( correspond to
the αα to αβ transition and also βα to ββ transition) to be enhanced and NOE is
observed. At the end of the irradiation period, 90o pulse is applied to the sample to
generate the FID. The irradiated nucleus practically does not give any signel,
because it average Mz is near zero in the start of the 90o pulse, and the nuclei that
are close to it in space give signals that are enhanced by a few percent over their
normal peak intesnities. However, the difference between the control spectrum and
the irradiated spectrum is often too difficult to be observed, thus the
reference(control) spectrum is then mathematically subtrated from the NOE
spectrum at every point to yield a difference spectrum. In this difference spectrum,
the irradiated peak will be inverted down, while the peak with NOE will appear as
weak positive peaks.

Peak tables (1H NMR)control spectrum:

ppm Assigned proton

3.35 M

3.42 L

3.54 K

3.58 J

3.62 G

3.69 F

3.72 E

3.76 D

3.92 C

4.08 B
 5.28 A

Explanation of the difference spectrum :

Irradiation of the peak at 5.28 ppm cause the peak to appear as inverted in
the NOE difference spectrum. This peak at 5.28 correspond to the anomeric proton
which is chosen because this proton has chemical shift that is far away from the
other proton in the molecule, so that there is no saturation of any other resonances
of other nucleus in the molecule. Since the irradiation practically reduce or even
annihiliate the peak at the 5.28 ppm, thus thiswill cause the appearance ofnegaive
peak at the difference spectrum. Going upfield along the spectrum we can see
many dispersion pattern ( 4.08 ppm, 4.06 ppm, 3.76 ppm, 3.69 ppm, 3.62 ppm, and
3.58 ppm ) that was caused by the imperfect horizontal allginment of the peaks
between the reference spectra and the NOE spectra. Subtraction of the two peak
then cause a dispersive appearance. As we can see form the difference spectra,
there exist doublets at 3.42 and 3.58 ppm. These positive peaks correspond to
proton K and L, which located near the irradiated proton, thus assuring the
existence of positive NOE of both of these protons. The existence od small positive
peak at 3.92 ppm indicate that this proton also interact slightly with the irradiated
proton, causing the existence of small positive NOE.

Third Experiment :

The NOESY spectra is very much ike the COSY spectra, but there are some peaks
that can be seen in the NOESY spectra that cannot be seen in the COSY spectra.
NOESY spectra is very much like the second experiment but now the NOE is shown
not only for a nucleus only, but each nucleus is irradiated and the resulting NOE-Diff
spectra for each nuclei is arranged in 2D manner to show the relation between each
proton to the rest of the protons. NOESY is ued to asign the relative realtion
between protons in certain conformers, such as 1,3 diazial interaction in
cyclohexane ring and the interaction between proton in protein structure.