Department of Chemistry, University of Alberta              February 1999
NMR News 99-02
News and tips for users of the Varian NMR systems in the Department.

There are no fixed publishing dates for this newsletter; its appearance solely depends on whether there is a need to present information to the users of the spectrometers or not.

Other content of this NMR News is no longer meaningful and has been removed April 2010.


FAQ 99-02.1 when to phase and when not to phase spectra?

FAQ 99-02.1 when to phase and when not to phase spectra?
In general, NMR spectra are either phase-sensitive, in which case they need to be phased correctly for a good result, or
absolute value
in which case phasing is neither necessary nor possible. An overview of the available techniques in the
system with regard to phase sensitivity:

absolute value - no phasing

phase-sensitive - phasing required

homonuclear 2D: GCOSY, GTOCSY
heteronuclear 2D: GHMQC, GHMBC

C13: 1D, APT
others: F19, P31, B11
homonuclear 2D: TROESY
heteronuclear 2D: GHSQC

Phasing is typically trivial for 1D spectra, but it can be more difficult in 2D techniques, especially in cases where C13 signals
are involved because the signal-to-noise ratio in the first spectrum might be too low for signals to be phased adequately.
HMQC and GHMBC provide absolute value data and cannot be phased by design of the pulse sequence. GHSQC by contrast
is case-sensitive and instructions on phasing are found here.
If only correlations are required absolute value is not a problem
but if coupling constants are to be measured it is better to record a phase-sensitive experiments with its inherent sharper lines.

For TROESY, correct phasing is essential for success. The procedure is described here in some detail.

In EZ-NMR P+P use button WFT 1st incr (it is located in the Fourier Transform section) which will produce a spectrum of the
first increment in the 2D matrix, i.e. it
is the FT of the first of the series of FIDs that were collected (as indicated by ni, for
256). It is not uncommon that the phase of this spectrum is quite off.

penta_TROESY_1st_wrong_phase.gif (4020 bytes)

penta_TROESY_1st_correct_phase.gif (3775 bytes)

However, this can easily be corrected. Just use the Phase button which is in the Phasing section of EZ-NMR P+P. Use
the left mouse button to phase the spectrum until it looks like the one shown on the right above. When done, use the
button to transform the entire 2D spectrum.

penta_TROESY_wrong_phase.gif (7534 bytes)

first increment not phased correctly

penta_TROESY_correct_phase.gif (36782 bytes)

after correct phasing of the first increment


penta_TROESYexp_correct_phase.gif (11255 bytes)

The difference in the quality of the spectrum is dramatic. When correctly phased, even peaks close to the diagonal can be seen clearly as shown with arrows in the expansion on the left.

If phasing is desired directly on a F2 trace taken from the 2D spectrum then pmode='full' has to be entered before processing. By default pmode='partial' which saves disk space and speeds up the FT but does not allow phasing of the 2D spectrum after WFT2D. Even when using the full option, the correct phasing as described here before WFT2D is still an excellent idea.

Phase sensitivity is a prerequisite for correct TROESY data interpretation. All NOE/ROE-type of experiments suffer
to some extent from undesired effects which are created through interactions via bonds rather than through space (see also
NMR News 98-08). Phase sensitive recording makes it possible to distinguish the two contributions. True through-space
correlations are of opposite phase of the diagonal, whereas undesired effects are of the same phase.
This is best seen
in the colors displayed on the monitor, i.e. diagonal peaks are by default phased positive and displayed with yellow/orange/red
colors ("warm" colors) whereas the negative levels are in blue/purple ("cold" colors). Sometimes cross peaks show a mixed
phase, that is they have a lot of purple but also some yellow in them. This is a sign that some distortion of the NOE through
coupling interactions is present. This is not a problem for qualitative or semi-quantitative interpretations, but it precludes such
peaks from integration for quantitative studies like inter-proton distance measurements.

An example (from a different data set than the one shown before) is depicted here. The mixed phase is clearly evident (blue is NOE, yellow is J-distortion).

TROESY_mixed_phase_Xpeak.jpg (53368 bytes)

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