Determine Number of Protons Attached to Each

Determine Number of Protons Attached to Each Carbon:
Running the DEPT Experiment
Distortionless Enhancement by Polarization Transfer (DEPT) is an experiment that
utilizes a polarization transfer from one nucleus to another, usually proton to carbon or
other X nucleus, to increase the signal strength of the X nucleus. The polarization transfer
is from a nucleus with a relatively larger gyromagnetic ratio,g, to one with a smaller g.
Furthermore, by changing the length of the last proton pulse from 45 to 90 to 135
degrees, the multiplicity of the carbon or X nucleus can be determined (i.e. depending on
the pulse length, the signal for a methine, methylene, or methyl will either be a positive,
negative, or a null response. See table below). Inspection of the spectra will show the
multiplicity of each carbon. Alternatively, addition and subtraction of the various DEPT
spectra will give the multiplicity of each carbon (this can be performed automatically).
Relative Intensities from DEPT
Spectrum Pulse Angle (º)
C
#
(quaternary)
1
45
0.00
2,3
90
0.00
4
135
0.00
CH
(methine)
0.707
1.00
0.707
CH2
(methylene)
1.00
0.00
-1.00
CH3
(methyl)
1.06
0.00
1.06
Preliminary Information
1. DEPT is slightly more sensitive than a regular 13C NMR experiment.
Furthermore, the repetition rate is governed by the T1’s of the protons and NOT
the carbons. Since proton T1’s are usually shorter than carbon T1’s, you can have
a shorter recycle delay (d1) than the carbon experiment. This translates to an
improved Signal-to-Noise (S/N) per unit time for DEPT over a regular 13C NMR
experiment.
2. pw90 and pp: The default values for pw90 (90º pulse width on proton) and pp
(the polarization transfer pulse on carbon) are usually good for most samples.
However, if your sample is ionic, contains paramagnetic materials, or has a
solvent very different from the standard, you will need to calibrate these
parameters for your sample. A usual indication of pp in need of calibration is the
appearance of methylene and methyl peaks in the DEPT 90 spectrum. Please
contact the NMR staff if you need to calibrate these parameters.
3. d1: As stated earlier, the recycle delay is set based on the T1’s of the PROTONS.
This value is usually 1 to 2 times your longest proton T1.
4. nt: By default, nt is set to half the value used in your regular 13C spectrum due to
its higher sensitivity.
Explanation of Types of Commands Found in this Handout:
1. The VNMR software and the UNIX operating system are both case sensitive.
This means that the computer distinguishes whether the letters are entered in
upper case (i.e. CAPITALS) or lower case. The user must be careful to type the
correct case for each letter in a command.
EXAMPLE: jexp1 is not the same as JEXP1
2. Some commands are line commands and are typed in by the user followed by a
hitting the RETURN key.
EXAMPLE: su
Hitting the RETURN key is assumed for all bold text commands.
3. Some commands are executed by clicking a mouse button with its pointer on a
‘button’ found on the screen. The execution of these commands is indicated by a
two-letter designation (LC {left click}, RC {right click}, or CC {center click})
followed by a word or words in bold that would appear in the ‘button’.
EXAMPLE: LC Main Menu
This means to click the left mouse button with its pointer on the ‘button’
that says “Main Menu”.
4. Some commands are executed by the mouse itself. These commands are
indicated by the two-letter designation (LC, RC, or CC) and a description of what
the user should do in parentheses.
EXAMPLE: LC (at 6 ppm)
This means to click the left mouse button with the mouse cursor at 6
ppm.
5. Parameters are entered by typing the parameter name followed by an equal sign,
the value, and a return.
EXAMPLE: nt=16 <rtn>
*
*
*
*
*
*
*
*
COLLECT A 13C SPECTRUM
NOTE: If you have a previously acquired carbon spectrum, load it now and skip ahead to
SETUP THE DEPT EXPERIMENT. Otherwise,
Acquire a 13C spectrum and reference your solvent. Solvent referencing is important
because the solvent peak is nulled in DEPT.
SETUP THE DEPT EXPERIMENT
NOTE: This is an arrayed experiment that will run 4 separate DEPT experiments: a
DEPT 45, 2 DEPT 90’s with slightly different pulse widths, and a DEPT 135.
Because of the chemical shift differences between methyls and methines, it is sometimes
sufficient to run just a DEPT 135, which gives methylenes up and methyls and methines
down. To just run the DEPT 135, go to the next section.
DEPT
nt=#
time
go
macro that sets up the DEPT experiment.
Must be typed as written (i.e. in CAPITAL
LETTERS).
(where # is the number of scans. Use 64 or a larger number if necessary.)
returns the time of the experiment. Make
sure you have enough allocated time to run
the experiment.
a total of 4 FID’s, each having nt scans, will
be acquired.
After acquisition is complete, save your file [i.e. type svf('filename')]
SETUP A DEPT 135 ONLY
NOTE: This is an alternative to running all four DEPT experiments. As stated in the
previous note, a DEPT 135 is sometimes sufficient to fully assign carbon multiplicities.
However, it is recommended to run ALL four DEPT experiments for unknown
compounds.
DEPT
macro that sets up the DEPT experiment.
Must be typed as written (i.e. in CAPITAL
LETTERS).
mult=1.5
sets the DEPT carbon pulse to be 135º
nt=# (where # is the number of scans. Use 64 or a larger number if necessary.)
go
only a DEPT 135 will be acquired.
After acquisition is complete, save your file [i.e. type svf('filename')]
Processing and printing are the same as for a normal 13C spectrum except that automatic
phasing may not work. You may have to do manual phasing.
PROCESS DEPT DATA
wft
performs a weighted Fourier transform of all
4 FID’s.
displays the first spectrum (this is the DEPT
45 spectrum; all peaks are positive).
ds(1)
Phase this spectrum as usual. You may need to phase manually.
dssa
to view all 4 spectra stacked vertically.
You may want/need to scale the spectra to fit. To do this, type ds(#), where # is the
number of the spectrum you wish to scale. Scale the selected spectrum as usual using the
middle mouse button. Return to the stacked plot with dssa.
ds(1)
displays first spectrum. DO NOT
RESCALE.
enters threshold routine.
LC th
Place the yellow threshold line below all the peak tops by left clicking and dragging the
yellow line to the desired position.
fp
dll
this stores the peak frequencies in memory.
displays the line list.
PRINT DEPT DATA WITHOUT ANALYSIS
text(‘add text\\add more text to new line\\etc.’)
pl(‘all’) pscale plext ppf page
plots all spectra.
PRINT DEPT DATA WITH ANALYSIS
DEPTP
this is a macro that automatically processes
and prints the DEPT data.
PRINTING INDIVIDUAL DEPT SPECTRA
Sometimes I have found it beneficial to print the individual DEPT spectra. This is
particularly helpful when the automatic DEPT analysis gives ambiguous results. The
automatic analysis adds and subtracts the different spectra, which can lead to subtraction
artifacts. With the help of the DEPT table above, it is rather straightforward to determine
the carbon multiplicity without the automatic analysis. For example, from the second and
third spectra (to view type ds(2) or ds(3)), which are DEPT 90 spectra, I will get only the
methine carbons and from the fourth spectrum, I get the methylene carbons because they
are the negative peaks.
ds(#) (where # is the number of the spectrum you wish to print).
Manipulate and plot as you would a standard Carbon spectrum.
When completed, be sure to exit VNMR and log-out properly.
Varian DEPT Pulse Sequence. The numerical values are time increments.
An example of a DEPT 135 Spectrum of Menthol. Menthol has 4 CH, 3 CH2, and 3 CH3.