Alex at 18:36, 11 November 2009

2009-11-11T18:36:12Z

Alex: moved NESG:ChemicalShiftReferencing to Chemical shift referencing

2009-11-01T23:32:07Z

moved NESG:ChemicalShiftReferencing to Chemical shift referencing

← Older revision	Revision as of 23:32, 1 November 2009
(No difference)

Admin: Created page with 'This page describes UIPAC-Recommended referencing procedures. See http://www.bmrb.wisc.edu/ref_info/cshift.html and references therein. Also check the information at the CABM web…'

2009-10-08T15:05:43Z

Created page with 'This page describes UIPAC-Recommended referencing procedures. See http://www.bmrb.wisc.edu/ref_info/cshift.html and references therein. Also check the information at the CABM web…'

New page

This page describes UIPAC-Recommended referencing procedures. See http://www.bmrb.wisc.edu/ref_info/cshift.html and references therein. Also check the information at the CABM website http://www-nmr.cabm.rutgers.edu/labdocuments/nmrprotocols/referencing.html.

= '''Chemical Shift Referencing''' =

Proper chemical shift referencing is of key importance for matching resonances in various spectra. DSS serves as the direct 1H reference standard in liquid state NMR and other spins are referenced indirectly through gyromagnetic ratios. Common referencing methods are internal (~20 uM of DSS is added to the protein sample) and substitution (a separate sample with DSS is used). For substitution method a standard sucrose sample with trace DSS amount can be used.

== '''Measuring zero frequency''' ==

To calibrate the spectra you need to know the frequency of the methyl peak of DSS in a 1D spectrum - this is the proton frequency at 0 ppm <tt>w0</tt>. Zero frequency <tt>w0</tt> remains constant for a given spectrometer until its lock frequency (<tt>lockfreq</tt> on Varian spectrometers) is reset due to <tt>B0</tt> drift. If you are not sure whether <tt>w0</tt> has been determined for the current spectrometer setup measure in a 1D 1H spectrum of DSS.

# '''Buffalo.VNMR (Varian)'''
** Record a standard 1H 1D spectrum of DSS sample and locate the DSS methyl peak.
** Expand the region around it and use <tt>nl</tt> and <tt>movetof</tt> to shift 1H carrier to the peak maximum.
** Invoke <tt>spcfrq</tt> to report <tt>w0</tt> with a 7-digit precision
# '''Topspin (Bruker)'''
** Record a standard 1H 1D spectrum of DSS sample and locate the DSS methyl peak.
** Expand the region around it and use 'calibrate' to set this point to 0 ppm.
** In the 'ProcPar' tab SF parameter will contain the <tt>w0</tt> frequency.

To calculate zero frequencies for 13C and 15N spins use indirect chemical shift referencing ratios from BMRB http://www.bmrb.wisc.edu/ref_info/cshift.html

== '''Temperature effects''' ==

The chemical shift of DSS is largely temperature-independent - after all that in in part why it was chosen as the referencing standard.

However, the exact <tt>B0</tt> field will be different for the same sample and spectrometer, but at different temperatures. It happens because spectrometers are locked by adjusting <tt>z0</tt>, to make the deuterium line of the solvent match a certain predefined frequency. The chemical shift of water, in particular, has a very strong temperature dependence, which leads to <tt>B0</tt> discrepancy.

Therefore, it is crucial that the zero frequency <tt>w0</tt> is measured for a DSS sample at the same temperature as the target sample in the substitution method.

For temperature-scan experiments it may be convenient to have the spectra referenced to the water line and not to the DSS peak. '''Provide a table or formula for 1H chemical shift of water vs. temperature.'''

== '''Calibrating Spectra in Various Software''' ==

=== '''Topspin (Bruker)''' ===

Copy zero frequencies for 1H, 13C and 15N into the SF fields of the 'ProcPar' tab.

=== '''PROSA and XEASY (manual calibration)''' ===

==== '''Varian Data''' ====

Get <tt>sfrq</tt>, <tt>dfrq</tt> and <tt>dfrq2</tt> parameters with 7 decimal digits either by invoking <tt>spcfrq</tt> or parsing the <tt>~/acqfil/procpar</tt> file.

Spectral widths in ppm are calculated as <tt>sw/w0</tt>. PROSA scripts use the parameter <tt>delta = 1/sw</tt>.

Maximum ppm shift of the direct dimension is calculated as

<tt>max. ppm = ( sfrq(MHz)*1000000 + sw/2 - w0(MHz)*1000000 ) / w0(MHz)</tt>

Indirect dimensions are calibrated similarly.

==== '''Bruker Data''' ====

Get <tt>SFO1</tt>, <tt>SFO2</tt> and <tt>SFO3</tt> parameters with 7 decimal digits from Topspin or by parsing the <tt>acqus</tt> file.
Spectral width in the direct dimension in Hz can be read from the <tt>SW_h</tt> parameter.

Indirect spectral widths are determined by the incremented delays. Typically <tt>IN0</tt> is used for the first indirect dimension and <tt>IN10</tt> is used for the second. Usually 2 or 4 incremented delays (<tt>D0</tt> and <tt>d10</tt>) used to sample indirect dimensions determined by parameters <tt>ND0</tt> and <tt>ND10</tt>, respectively. See the pulse sequence file for the particular setup.

Thus the spectral width of the first indirect dimension in ppm is calculated as <tt>sw(ppm) = 1/(ND0*IN0)</tt>.
In PROSA <tt>delta = ND0*IN0</tt>.

Maximum ppm shift of the direct dimension is calculated as

<tt>max. ppm = ( w0(MHz)*1000000 + SW_h/2 - w0(MHz)*1000000 ) / w0(MHz)</tt>

Maximum ppm shift of the first indirect dimension is calculated as

<tt>max. ppm = ( w0(MHz)*1000000 + 1/(2*ND0*IN0) - w0(MHz)*1000000 ) / w0(MHz)</tt>

%COMMENT%

-- Main.GaohuaLiu - 24 Jan 2007

Chemical shift referencing - Revision history

Alex at 18:36, 11 November 2009

Alex: moved NESG:ChemicalShiftReferencing to Chemical shift referencing

Admin: Created page with 'This page describes UIPAC-Recommended referencing procedures. See http://www.bmrb.wisc.edu/ref_info/cshift.html and references therein. Also check the information at the CABM web…'