Pulse width calibration - Revision history

Xianzhon: /* 13C 90° Pulse Width Calibration */

2012-09-03T03:38:18Z

13C 90° Pulse Width Calibration

← Older revision		Revision as of 03:38, 3 September 2012
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	If you protein completely lacks aromatic residues, you may use aliphatic signals instead. It is then recommended to shift the carrier from 35 ppm to 25, since it is mostly the methyl signals you will be looking at.		If you protein completely lacks aromatic residues, you may use aliphatic signals instead. It is then recommended to shift the carrier from 35 ppm to 25, since it is mostly the methyl signals you will be looking at.

	You can use two macros <tt>cof</tt> and <tt>nof</tt> to set <tt>dof</tt> and <tt>dof2</tt> precisely to a given ppm position. The syntax is like <tt>cof(H1 center in ppm, desired C13 center in ppm)</tt>. For example, <tt>cof(4.73,35)</tt> if one wants to set the center of ~~C15~~ as 35ppm. The same holds true for <tt>nof</tt>. <tt>cof</tt> and <tt>nof</tt> are stored in the <tt>vnmrsys/maclib</tt>.		You can use two macros <tt>cof</tt> and <tt>nof</tt> to set <tt>dof</tt> and <tt>dof2</tt> precisely to a given ppm position. The syntax is like <tt>cof(H1 center in ppm, desired C13 center in ppm)</tt>. For example, <tt>cof(4.73,35)</tt> if one wants to set the center of C13 as 35ppm. The same holds true for <tt>nof</tt>. <tt>cof</tt> and <tt>nof</tt> are stored in the <tt>vnmrsys/maclib</tt>.

	You can also keep <tt>calC=1.0</tt>. In this case the correct pwC will correspond to maximum intensity. This may be the better method for diluted samples, while the zero crossing method is preferred for concentrated ones.		You can also keep <tt>calC=1.0</tt>. In this case the correct pwC will correspond to maximum intensity. This may be the better method for diluted samples, while the zero crossing method is preferred for concentrated ones.

Alex: /* 13C 90° Pulse Width Calibration */

2010-03-11T19:04:55Z

13C 90° Pulse Width Calibration

← Older revision		Revision as of 19:04, 11 March 2010
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	=== 13C 90° Pulse Width Calibration ===		=== 13C 90° Pulse Width Calibration ===

	#Load a [13C, 1H]-HSQC dataset (<tt>~~gChsqc~~</tt> or <tt>~~gCfhsqc~~</tt>~~). Select the proper type: aliphatic, aromatic, etc.~~		#Load the <tt>gChsqc</tt> [13C, 1H]-HSQC dataset
	#Set <tt>nt=2 ss=4 ni=1 phase=1 calC=1.0</tt>		#Select aromatic carbon type by entring <tt>arom='y' aliph='n' alphaC='n' allC='n' lambda=0.0015</tt>
	#Acquire the first increment, process it with FT and phase to pure absorption~~. Expand~~ the region ~~of interest.~~		#Set F1 detection mode to ZZ-filter by typing <tt>'ZZ='y' SE='n'</tt>
	#~~Array~~ <tt>pwC</tt> ~~from~~ around the expected 90° pulse width		#Set <tt>nt=2 ss=2 ni=1 phase=1 calC=1.0 N15refoc='n'</tt>
	#Acquire arrayed spectra and process them with <tt>wft dssh dssl</tt>~~. The intensity maximum~~ will correspond to he true 90° pulse width.		#Acquire the first increment, process it with FT and phase to pure absorption and expand the aromatic region
			#Set <tt>calC=2.0</tt> and array <tt>pwC</tt> around the expected 90° pulse width
			#Acquire arrayed spectra and process them with <tt>wft dssh dssl</tt> - the zero crossing will correspond to the true 90° 13C pulse width
	#Update the probefile with the new <tt>pwC</tt> value, if necessary		#Update the probefile with the new <tt>pwC</tt> value, if necessary

	~~You can also set <tt>calC=2~~.~~0</tt>~~ - ~~in this case~~ the ~~correct pwC will correspond to~~ zero ~~intensity~~.		Focusing on aromatic signals only is the most accurate way to calibrate pwC, since adverse effects due to off-resonance irradiation and non-uniform spin multiplicities are avoided. Along the same lines, ZZ-filter is preferred over gradient-sensitivity enhancement to avoid spurious magnetization transfer, which may obscure the zero crossing point.

	If ~~looking at the methyl region~~ you may ~~want~~ to ~~change <tt>dof</tt> to point~~ to 25 ~~ppm instead of default 35 ppm~~.		If you protein completely lacks aromatic residues, you may use aliphatic signals instead. It is then recommended to shift the carrier from 35 ppm to 25, since it is mostly the methyl signals you will be looking at.

	You can use two macros <tt>cof</tt> and <tt>nof</tt> to set <tt>dof</tt> and <tt>dof2</tt> precisely to a given ppm position. The syntax is like <tt>cof(H1 center in ppm, desired C13 center in ppm)</tt>. For example, <tt>cof(4.73,35)</tt> if one wants to set the center of C15 as 35ppm. The same holds true for <tt>nof</tt>. <tt>cof</tt> and <tt>nof</tt> are stored in the <tt>vnmrsys/maclib</tt>.		You can use two macros <tt>cof</tt> and <tt>nof</tt> to set <tt>dof</tt> and <tt>dof2</tt> precisely to a given ppm position. The syntax is like <tt>cof(H1 center in ppm, desired C13 center in ppm)</tt>. For example, <tt>cof(4.73,35)</tt> if one wants to set the center of C15 as 35ppm. The same holds true for <tt>nof</tt>. <tt>cof</tt> and <tt>nof</tt> are stored in the <tt>vnmrsys/maclib</tt>.

			You can also keep <tt>calC=1.0</tt>. In this case the correct pwC will correspond to maximum intensity. This may be the better method for diluted samples, while the zero crossing method is preferred for concentrated ones.

	15N and 13C 90° pulse widths are much less sensitive to salt concentration than 1H 90° pulse width.		15N and 13C 90° pulse widths are much less sensitive to salt concentration than 1H 90° pulse width.
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	Longer than usual pulse widths may indicate a poorly tuned and matched channel.		Longer than usual pulse widths may indicate a poorly tuned and matched channel.

	If the calibrated pulse widths are consistently calibrated long for different samples, it indicates a deteriorating probe.		If the calibrated pulse widths are consistently calibrated long for different samples, it indicates a deteriorating probe.

	== Automated Calibration of 90° Pulse Widths (BioPack) ==		== Automated Calibration of 90° Pulse Widths (BioPack) ==

Alex: /* Automated Calibration of 90° Pulse Widths (BioPack) */

2010-01-08T11:13:11Z

Automated Calibration of 90° Pulse Widths (BioPack)

← Older revision		Revision as of 11:13, 8 January 2010
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	Upon completion of each calibration step related parameters (e.g. decoupling pulse widths, adiabatic pulses) are re-calculated and probefile is automatically updated with new values.		Upon completion of each calibration step related parameters (e.g. decoupling pulse widths, adiabatic pulses) are re-calculated and probefile is automatically updated with new values.


	Automated calibration should be verified by analyzing its output. The output is controlled with the parameter <tt>BPplot</tt>:		Automated calibration should be verified by analyzing its output. The output is controlled with the parameter <tt>BPplot</tt>:
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	* <tt>BPplot='off'</tt> - no output. (not recommended)		* <tt>BPplot='off'</tt> - no output. (not recommended)

			[[Image:plotting_mode.png\|400px\|right]]
	You can also use the radio buttons in the '''Setup''' -> '''Calibrations''' tab, under '''Plotting Mode'''.		You can also use the radio buttons in the '''Setup''' -> '''Calibrations''' tab, under '''Plotting Mode'''.
	~~<img src="%ATTACHURLPATH%/plotting_mode.png" alt="plotting_mode.png" width='343' height='28' />~~


	In the plotted arrayed spectra check the following:		In the plotted arrayed spectra check the following:
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	=== 1H 90° pulse width only ===		=== 1H 90° pulse width only ===

			[[Image:1h90only_2.png\|400px\|right]]
	Use this method if you sample is unlabeled (at natural abundance) or if its concentration is very low.		Use this method if you sample is unlabeled (at natural abundance) or if its concentration is very low.

	In VNMRJ window go to the '''Setup''' -> '''Calibrations''' tab and select '''1H pw90 only''' from the pull-down menu.		In VNMRJ window go to the '''Setup''' -> '''Calibrations''' tab and select '''1H pw90 only''' from the pull-down menu.

	~~<img src="%ATTACHURLPATH%/1h90only_2.png" alt="1h90only_2.png" width='408' height='248' />~~

	=== 1H and 15N 90° pulse widths ===		=== 1H and 15N 90° pulse widths ===

			[[Image:13cand1honly.png\|400px\|right]]
	Use this method for 15N- or 15N/13C-labeled samples. '''Do not''' use for natural abundance samples.		Use this method for 15N- or 15N/13C-labeled samples. '''Do not''' use for natural abundance samples.

	In VNMRJ window go to the '''Setup''' -> '''Calibrations''' tab and click on the '''1H and 15N PW90 Only''' button. This method uses the <tt>gNfhsqc</tt> pulse sequence and looks for the signal maximum.		In VNMRJ window go to the '''Setup''' -> '''Calibrations''' tab and click on the '''1H and 15N PW90 Only''' button. This method uses the <tt>gNfhsqc</tt> pulse sequence and looks for the signal maximum.

	~~<img src="%ATTACHURLPATH%/13cand1honly.png" alt="13cand1honly.png" width='405' height='248' />~~

	=== 1H and 13C 90° pulse widths ===		=== 1H and 13C 90° pulse widths ===

			[[Image:usegcfhsqc.png\|400px\|right]]
	Use this method for 13C- or 15N/13C-labeled samples. '''Do not''' use for 5% 13C-labeled or natural abundance samples.		Use this method for 13C- or 15N/13C-labeled samples. '''Do not''' use for 5% 13C-labeled or natural abundance samples.

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	Depending on the <tt>BPquick</tt> parameter (see the corresponding checkbox under '''Setup''' -> '''Globals&Probefile''' tab) it will use either <tt>gCfhsqc</tt> or <tt>hcch_tocsy</tt> pulse sequence and looks for the signal maximum.		Depending on the <tt>BPquick</tt> parameter (see the corresponding checkbox under '''Setup''' -> '''Globals&Probefile''' tab) it will use either <tt>gCfhsqc</tt> or <tt>hcch_tocsy</tt> pulse sequence and looks for the signal maximum.

	~~<img src="%ATTACHURLPATH%/usegcfhsqc.png" alt="usegcfhsqc.png" width='614' height='130' />~~

	=== 1H, 13C and 15N 90° pulse widths ===		=== 1H, 13C and 15N 90° pulse widths ===

			[[Image:rfandgradients.png\|400px\|right]]
	Use this method for 15N/13C-labeled samples. '''Do not''' use for 5% 13C-labeled or natural abundance samples.		Use this method for 15N/13C-labeled samples. '''Do not''' use for 5% 13C-labeled or natural abundance samples.

	In VNMRJ window go to the '''Setup''' -> '''Calibrations''' tab and select '''RF and Gradients (Fastest)''' from the pull-down menu. This method will use the <tt>ghn_co</tt> pulse sequence for calibration. It will also calibrate coherence selection gradient amplitudes.		In VNMRJ window go to the '''Setup''' -> '''Calibrations''' tab and select '''RF and Gradients (Fastest)''' from the pull-down menu. This method will use the <tt>ghn_co</tt> pulse sequence for calibration. It will also calibrate coherence selection gradient amplitudes.

	~~<img src="%ATTACHURLPATH%/rfandgradients.png" alt="rfandgradients.png" width='405' height='199' />~~

	Coherence gradients do not usually change with time and do not depend on the sample. <tt>ghn_co</tt> is also less sensitive than <tt>gCfhsqc</tt> and <tt>gNfhsqc</tt>, and this method takes quite some time to run. It is more convenient to run 1H/13C, and 1H/15N calibrations in succession.		Coherence gradients do not usually change with time and do not depend on the sample. <tt>ghn_co</tt> is also less sensitive than <tt>gCfhsqc</tt> and <tt>gNfhsqc</tt>, and this method takes quite some time to run. It is more convenient to run 1H/13C, and 1H/15N calibrations in succession.

	However, this is probably the only method to calibrate 13C 90° pulse width for DCN-labeled samples.		However, this is probably the only method to calibrate 13C 90° pulse width for DCN-labeled samples.

Alex: /* Automated Calibration of 90° Pulse Widths */

2010-01-07T20:51:52Z

Automated Calibration of 90° Pulse Widths

← Older revision		Revision as of 20:51, 7 January 2010
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	If the calibrated pulse widths are consistently calibrated long for different samples, it indicates a deteriorating probe.		If the calibrated pulse widths are consistently calibrated long for different samples, it indicates a deteriorating probe.

	== Automated Calibration of 90° Pulse Widths ==		== Automated Calibration of 90° Pulse Widths (BioPack) ==

	For every sample you should calibrate 90° pulse widths before acquisition.		For every sample you should calibrate 90° pulse widths before acquisition.

Alex at 20:51, 7 January 2010

2010-01-07T20:51:09Z

← Older revision		Revision as of 20:51, 7 January 2010
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	== ~~'''~~Calibration of 1H Carrier Frequency (tof)~~'''~~ ==
			== Calibration of 1H Carrier Frequency (tof) ==

	To achieve the best water suppression <tt>tof</tt> has to be centered on the H2O resonance. To optimize <tt>tof</tt>:		To achieve the best water suppression <tt>tof</tt> has to be centered on the H2O resonance. To optimize <tt>tof</tt>:
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	It may be tempting to run a small tip angle 1D and simply take the water peak maximum as the <tt>tof</tt> value. This method usually yields a different value and worse water suppression than the procedure above.		It may be tempting to run a small tip angle 1D and simply take the water peak maximum as the <tt>tof</tt> value. This method usually yields a different value and worse water suppression than the procedure above.

	== ~~Manual~~ Pulse ~~Width Calibration~~ ==		== Automated Calibration of 90° Pulse Widths ==

	The 1H 90° pulse width is very sensitive to the salt content of the sample. Below are two methods for manual calibration.		The 1H 90° pulse width is very sensitive to the salt content of the sample. Below are two methods for manual calibration.
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	If the calibrated pulse widths are consistently calibrated long for different samples, it indicates a deteriorating probe.		If the calibrated pulse widths are consistently calibrated long for different samples, it indicates a deteriorating probe.

	== ~~'''~~Automated Calibration of 90° ~~pulse widths'''~~ ==		== Automated Calibration of 90° Pulse Widths ==

	For every sample you should calibrate 90° pulse widths before acquisition.		For every sample you should calibrate 90° pulse widths before acquisition.
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	* optimal pw90 is correctly determined from the maximum.		* optimal pw90 is correctly determined from the maximum.

	=== ~~'''~~1H 90° pulse width only~~'''~~ ===		=== 1H 90° pulse width only ===

	Use this method if you sample is unlabeled (at natural abundance) or if its concentration is very low.		Use this method if you sample is unlabeled (at natural abundance) or if its concentration is very low.
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	<img src="%ATTACHURLPATH%/1h90only_2.png" alt="1h90only_2.png" width='408' height='248' />		<img src="%ATTACHURLPATH%/1h90only_2.png" alt="1h90only_2.png" width='408' height='248' />

	=== ~~'''~~1H and 15N 90° pulse widths~~'''~~ ===		=== 1H and 15N 90° pulse widths ===

	Use this method for 15N- or 15N/13C-labeled samples. '''Do not''' use for natural abundance samples.		Use this method for 15N- or 15N/13C-labeled samples. '''Do not''' use for natural abundance samples.
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	<img src="%ATTACHURLPATH%/13cand1honly.png" alt="13cand1honly.png" width='405' height='248' />		<img src="%ATTACHURLPATH%/13cand1honly.png" alt="13cand1honly.png" width='405' height='248' />

	=== ~~'''~~1H and 13C 90° pulse widths~~'''~~ ===		=== 1H and 13C 90° pulse widths ===

	Use this method for 13C- or 15N/13C-labeled samples. '''Do not''' use for 5% 13C-labeled or natural abundance samples.		Use this method for 13C- or 15N/13C-labeled samples. '''Do not''' use for 5% 13C-labeled or natural abundance samples.
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	<img src="%ATTACHURLPATH%/usegcfhsqc.png" alt="usegcfhsqc.png" width='614' height='130' />		<img src="%ATTACHURLPATH%/usegcfhsqc.png" alt="usegcfhsqc.png" width='614' height='130' />

	=== ~~'''~~1H, 13C and 15N 90° pulse widths~~'''~~ ===		=== 1H, 13C and 15N 90° pulse widths ===

	Use this method for 15N/13C-labeled samples. '''Do not''' use for 5% 13C-labeled or natural abundance samples.		Use this method for 15N/13C-labeled samples. '''Do not''' use for 5% 13C-labeled or natural abundance samples.

Alex at 20:48, 7 January 2010

2010-01-07T20:48:34Z

@@ Line 17: / Line 17: @@
 It may be tempting to run a small tip angle 1D and simply take the water peak maximum as the <tt>tof</tt> value. This method usually yields a different value and worse water suppression than the procedure above.
-== 1H 90° Pulse Width Calibration  ==
+== Manual Pulse Width Calibration  ==
 The 1H 90° pulse width is very sensitive to the salt content of the sample. Below are two methods for manual calibration.
@@ Line 49: / Line 49: @@
 You can also set <tt>calH=2.0</tt> - in this case the correct pw90 will correspond to zero intensity.
-== 15N and 13C 90° Pulse Width Calibration  ==
+=== 15N and 13C 90° Pulse Width Calibration  ===
 #Load a [15N, 1H]-HSQC dataset (<tt>gNhsqc</tt> or <tt>gNfhsqc</tt>)
@@ Line 60: / Line 60: @@
 You can also set <tt>calN=2.0</tt> - in this case the correct pwN will correspond to zero intensity.
-== 13C 90° Pulse Width Calibration  ==
+=== 13C 90° Pulse Width Calibration  ===
 #Load a [13C, 1H]-HSQC dataset (<tt>gChsqc</tt> or <tt>gCfhsqc</tt>). Select the proper type: aliphatic, aromatic, etc.
@@ Line 81: / Line 81: @@
 If the calibrated pulse widths are consistently calibrated long for different samples, it indicates a deteriorating probe.
-<br> <br>
+== '''Automated Calibration of 90° pulse widths''' ==
--- Main.AlexEletski - 25 Feb 2008
+For every sample you should calibrate 90° pulse widths before acquisition.

Alex at 18:30, 11 November 2009

2009-11-11T18:30:19Z

← Older revision		Revision as of 18:30, 11 November 2009
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	#Update the probefile with the new <tt>tof</tt> value, if necessary		#Update the probefile with the new <tt>tof</tt> value, if necessary

	~~<img src="%ATTACHURLPATH%/tof1~~.jpg~~" alt="tof1.jpg" width='556' height='231' />~~		[[Image:Tof1.jpg]]


	It is important to use a single increment (<tt>nt=1</tt>), since phase cycling will likely produce additional water suppression and obscure the optimum <tt>satfreq</tt>. The solvent subtraction filter should obviously be disabled (<tt>ssfilter='n'</tt>). Absolute-value mode (<tt>av</tt>) is better here since you don't have to phase the spectrum.		<br> It is important to use a single increment (<tt>nt=1</tt>), since phase cycling will likely produce additional water suppression and obscure the optimum <tt>satfreq</tt>. The solvent subtraction filter should obviously be disabled (<tt>ssfilter='n'</tt>). Absolute-value mode (<tt>av</tt>) is better here since you don't have to phase the spectrum.

	The water signal profile can be indicative of shimming quality: good shimming will result in a symmetric profile, while bad shimming will lead to an asymmetric profile.		The water signal profile can be indicative of shimming quality: good shimming will result in a symmetric profile, while bad shimming will lead to an asymmetric profile.

	It may be tempting to run a small tip angle 1D and simply take the water peak maximum as the <tt>tof</tt> value. This method usually yields a different value and worse water suppression than the procedure above.		It may be tempting to run a small tip angle 1D and simply take the water peak maximum as the <tt>tof</tt> value. This method usually yields a different value and worse water suppression than the procedure above.

	== 1H 90° Pulse Width Calibration ==		== 1H 90° Pulse Width Calibration ==

Alex at 18:29, 11 November 2009

2009-11-11T18:29:12Z

Show changes

Alex: moved NESG:ManualPulseWidthCalibration to Pulse width calibration

2009-11-01T23:24:42Z

moved NESG:ManualPulseWidthCalibration to Pulse width calibration

← Older revision	Revision as of 23:24, 1 November 2009
(No difference)

Admin: Created page with '== '''Manual Calibration of Pulse Widths and 1H Carrier Offset''' == === '''Calibration of 1H Carrier Frequency (`tof`)''' === To achieve the best water suppression `…'`

2009-10-08T14:57:34Z Created page with '== '''Manual Calibration of Pulse Widths and 1H Carrier Offset''' == === '''Calibration of 1H Carrier Frequency (<tt>tof</tt>)''' === To achieve the best water suppression <tt>…' New page == '''Manual Calibration of Pulse Widths and 1H Carrier Offset''' == === '''Calibration of 1H Carrier Frequency (<tt>tof</tt>)''' === To achieve the best water suppression <tt>tof</tt> has to be centered on the H2O resonance. To optimize <tt>tof</tt>: # Load a <tt>presat</tt> or <tt>water</tt> dataset and set the following parameters:<br/> <tt>nt=1 ss=2 satmode='y' ssfilter='n' av</tt> # Acquire a 1D spectrum with <tt>ga</tt> and expand the region around the H2O signal # Array the parameter <tt>satfrq</tt> around the current value of <tt>tof</tt>; a range of +/- 10 Hz with a step of 1-2 Hz will suffice # Acquire the arrayed spectra. Process and display with <tt>wft dssh dssl</tt>. The intensity of the water signal should fall, reach a minimum and then rise again. The value of <tt>satfreq</tt> at the minimum is the correct <tt>tof</tt> # Update the probefile with the new <tt>tof</tt> value, if necessary <img src="%ATTACHURLPATH%/tof1.jpg" alt="tof1.jpg" width='556' height='231' /> <br/> It is important to use a single increment (<tt>nt=1</tt>), since phase cycling will likely produce additional water suppression and obscure the optimum <tt>satfreq</tt>. The solvent subtraction filter should obviously be disabled (<tt>ssfilter='n'</tt>). Absolute-value mode (<tt>av</tt>) is better here since you don't have to phase the spectrum. The water signal profile can be indicative of shimming quality: good shimming will result in a symmetric profile, while bad shimming will lead to an asymmetric profile. It may be tempting to run a small tip angle 1D and simply take the water peak maximum as the <tt>tof</tt> value. This method usually yields a different value and worse water suppression than the procedure above. === '''1H 90° Pulse Width Calibration''' === The 1H 90° pulse width is very sensitive to the salt content of the sample. Below are two methods for manual calibration. ==== '''1H 90° Pulse Width from 1D''' ==== To accurately calibrate 1H pw90 with a 1H 1D experiment you have to find the correct 1H pw360 and then take pw90 as pw360/4. Follow these steps: # Load a <tt>presat</tt> or <tt>water</tt> dataset and set the following parameters:<br/> <tt>nt=1 ss=2 satmode='y' ssfilter='n' ph</tt> # Acquire a 1D spectrum with <tt>ga</tt> and phase it to pure absorption. Expand the region of interest (either amide or aliphatic), excluding the water line # Array the parameter <tt>pw</tt> around the expected value of 4*pw90 # Acquire the arrayed spectra. Process and display with <tt>wft dssh dssl</tt>. The intensity should be negative for <tt>pw</tt> less than pw360, go through zero at pw360 and become positive at <tt>pw</tt> greater than pw360. # Record a spectrum with <tt>pw</tt> set to pw180. If pw360 is determined correctly the result should be near zero. # Update the probefile with the new <tt>pw90</tt> value, if necessary Arraying <tt>pw</tt> directly with the aim of observing maximum signal at pw90 will not yield the true 90° pulse width. Instead it will determine the so-called "Ernst angle", which depends on the recycle delay <tt>d1</tt> and the T1 relaxation time of the sample. However, when working with an unknown sample it is a good idea to use the Ernst angle pulse as an estimate pw90 and then use it to properly set the array range in the pw360 method. ==== '''1H 90° Pulse Width from [15N, 1H]- or [13C, 1H]-HSQC''' ==== You can also calibrate 1H pw90 with an HSQC dataset (<tt>gNhsqc</tt>, <tt>gNfhsqc</tt>, <tt>gChsqc</tt> or <tt>gCfhsqc</tt>) # Load an HSQC dataset. If using <tt>gChsqc</tt> select the proper type: aliphatic, aromatic, etc. # Set <tt>nt=2 ss=4 ni=1 phase=1 calH=1.0</tt> # Acquire the first increment, process it with FT and phase to pure absorption. Expand the region of interest. # Array <tt>pw</tt> from 0 in steps of 1 us # Acquire arrayed spectra and process them with <tt>wft dssh dssl</tt>. The intensity maximum will correspond to pw90. # Update the probefile with the new <tt>pw</tt> value, if necessary You can also set <tt>calH=2.0</tt> - in this case the correct pw90 will correspond to zero intensity. === '''15N and 13C 90° Pulse Width Calibration''' === # Load a [15N, 1H]-HSQC dataset (<tt>gNhsqc</tt> or <tt>gNfhsqc</tt>) # Set <tt>nt=2 ss=4 ni=1 phase=1 calN=1.0</tt> # Acquire the first increment, process it with FT and phase to pure absorption. Expand the region of interest. # Array <tt>pwN</tt> from around the expected 90° pulse width # Acquire arrayed spectra and process them with <tt>wft dssh dssl</tt>. The intensity maximum will correspond to the true 90° pulse width. # Update the probefile with the new <tt>pwN</tt> value, if necessary You can also set <tt>calN=2.0</tt> - in this case the correct pwN will correspond to zero intensity. === '''13C 90° Pulse Width Calibration''' === # Load a [13C, 1H]-HSQC dataset (<tt>gChsqc</tt> or <tt>gCfhsqc</tt>). Select the proper type: aliphatic, aromatic, etc. # Set <tt>nt=2 ss=4 ni=1 phase=1 calC=1.0</tt> # Acquire the first increment, process it with FT and phase to pure absorption. Expand the region of interest. # Array <tt>pwC</tt> from around the expected 90° pulse width # Acquire arrayed spectra and process them with <tt>wft dssh dssl</tt>. The intensity maximum will correspond to he true 90° pulse width. # Update the probefile with the new <tt>pwC</tt> value, if necessary You can also set <tt>calC=2.0</tt> - in this case the correct pwC will correspond to zero intensity. If looking at the methyl region you may want to change <tt>dof</tt> to point to 25 ppm instead of default 35 ppm. You can use two macros <tt>cof</tt> and <tt>nof</tt> to set <tt>dof</tt> and <tt>dof2</tt> precisely to a given ppm position. The syntax is like <tt>cof(H1 center in ppm, desired C13 center in ppm)</tt>. For example, <tt>cof(4.73,35)</tt> if one wants to set the center of C15 as 35ppm. The same holds true for <tt>nof</tt>. <tt>cof</tt> and <tt>nof</tt> are stored in the <tt>vnmrsys/maclib</tt>. 15N and 13C 90° pulse widths are much less sensitive to salt concentration than 1H 90° pulse width. Longer than usual pulse widths may indicate a poorly tuned and matched channel. If the calibrated pulse widths are consistently calibrated long for different samples, it indicates a deteriorating probe. %COMMENT% -- Main.AlexEletski - 25 Feb 2008