Practical Session II: Setting frequencies
and calibrating non-1H pulses (15N, 13C)
MSMPH 2055
March 23, 2021
2D 1H-15N HSQC experiment
(with water flipback and WATERGATE solvent suppression and 15N decoupling)
The 2-D 1H-15N HSQC is normally run in 95% H2O/5% D2O; this requires additional waterselective ‘flipback’ and WATERGATE pulses to simultaneously prevent saturation of the
water signal and to suppress it. This requires additional lower power 1H pulses (in addition
to the higher power pulses that lead to uniform excitation of 1Hs).
The 2-D 1H-15N HSQC is also normally run such that 15N-decoupling pulses are applied
while the 1H FID is collected. This eliminates the effect of 1H-15N J-coupling and yields
singlets in the 1H dimension (rather than 1H doublets which would otherwise arise due to
the J-coupling of the detected amide 1H to their directly bonded 15N). This requires
additional lower power 15N pulses (in addition to the higher power pulses that lead to
uniform excitation of 15Ns).
1H Pulse Calibration
The high power 90° and 180° 1H pulses in the HSQC will be applied at
close to the maximal power possible (pl1 = +1.0 dB), which with a non-salty
sample will yield a 90° pulse of about 7 ms.
The HSQC, however, also includes water-selective 90° pulses for water
flipback and WATERGATE water suppression that are applied at lower
power. Typically, a 90° pulse time of 1000 ms is used for WATERGATE.
This pulse can be calibrated using the same method used for high power 1H
90° pulses (demonstrated previously, in which the 360° null was found).
To get an idea of what power level to use, you can calculate how many more
dB you will have to attenuate the 1H transmitter (relative to the +1 dB that
yielded a 7 ms 90° pulse) using the relationship given previously.
1H
Pulse Calibration
Previously, it was indicated that a factor of four change in power changes the pulse time by a
factor of two. Relative pulse powers are specified on a logarithemic ‘decibel’ scale:
DdB = -10log(P1/P2); for P1/P2 = 4, DdB = 6
On Bruker spectrometers, the amplifier power levels are specified in decibels of attenuation,
so if you knew pl1 = +1 dB gave a 7 ms 90° pulse, then you would predict that +7 dB would
give a 14 ms 90°.
Pulses times are directly proportional to voltage (in contrast to the power, which is
proportional to the power squared); hence
DdB = -20log(V1/V2); for V1/V2 =2, DdB = 6
For the specific example of the 1H-15N HSQC, if you knew that the 1H 90° was 7 ms at pl1 =
+1 dB, then you could estimate that a 1000 ms 1H 90 for the WATERGATE pulse would be:
DdB = -20log(V1/V2)= -20log(1000/7) = +43.1 dB higher (than +1 dB)
Hence estimated power level would be +1 dB + 43.1 dB = 44.1 dB
Normally, one would check both high power 1H 90 ° pulse and lower power 1H 90° pulses
(for both WATERGATE and water flipback) by finding the 360° null as shown in the previous
demonstration.
15N Pulse Calibration
The following pulse sequence is used for calibrating 15N 90° pulses. This is
termed an “inverse” method since the 15N 90° pulse is determined indirectly
by 1H detection.
1H
15N
(I)
(S)
In practice, fully attenuate 15N transmitter (pl3 = +120 dB) so that no pulse is
obtained, regardless the value of the pulse. Verify that antiphase doublet is
obtained and phase. Next, reduce 15N transmitter (say pl3 = +1 dB) so that
pulse is now obtained for the duration specified. Adjust power level/pulse
time until the antiphase doublet is eliminated (this corresponds to the 15N 90°
pulse).
15N Pulse Calibration
1. Place 15N urea (in DMSO-d6) in magnet. Lock, tune 15N, tune 1H, shim.
2. Open calibration expt (/opt/topspin, hincklab, calibn)
3. Examine pulse program (“edcpul” command or click on PulseProg tab)
;decp90f3
;avance-version (12/01/11)
;calibration of 90 degree pulse on f3 channel
;
;$CLASS=HighRes
;$DIM=1D
;$TYPE=
;$SUBTYPE=
;$COMMENT=
#include <Avance.incl>
ph1=0 2 2 0 1 3 3 1
ph2=0 2 2 0 1 3 3 1
ph31=0 2 2 0 1 3 3 1
;pl1 : f1 channel - power level for pulse (default)
;pl3 : f3 channel - power level for pulse (default)
;p1 : f1 channel - 90 degree high power pulse
;p21: f3 channel - 90 degree high power pulse
;d1 : relaxation delay; 1-5 * T1
;d2 : 1/(2J(XH))
;cnst2: = J(XH)
;ns: 1 * n, total number of scans: NS * TD0
"d2=1s/(cnst2*2)"
1 ze
Zero memory
30 ms delay
2 30m
Recycle time, typically 1s
d1
Proton pulse on Ch. F1
p1 ph1
1/2JN-H
d2
Nitrogen pulse on Ch. F3
p21:f3 ph2
Collect FID, and loop to line 2
go=2 ph31
30m mc #0 to 2 F0(zd)
exit
;$Id: decp90f3,v 1.10.8.1 2012/01/31 17:56:22 ber Exp $
Check RF Setup using ‘EDASP’
Check spectrometer RF setup using “edasp” (1H should be on f1; 15N on f3)
Setting Frequencies on Bruker Spectrometers
Frequencies applied by spectrometer are specified by SFO1, SFO2, and SFO3 are given in units of MHz.
How do you know how to set these to correspond to a given ppm value (e.g. what SFO3 value should be used to place the
15N carrier on resonance for the test sample, 15N urea)?
This is done using indirect referencing ratios … the indirect referencing ratios are known and basically specify the ratio of
the 0 ppm frequency for 1H to the 0 ppm frequency for other nuclei (e.g. 15N and 13C). The indirect referencing ratios are
independent of field, and hence can be universally applied. The IUPAC-IUB IDRR are provided below:
13C:
15N:
0.251449530
0.101329118
The 0 ppm 1H frequency on the Pitt Structural Biology NMR spectrometers can be measured directly by placing a sample
with DSS in the magnet and by recording a 1D 1H spectrum. On so doing, it will be found that these are very close to
600.230000, 600.130000, 700.110000, 800.300000, and 900.100000 for the 600-1, 600-2, 700, 800, and 900 MHz
spectrometers, respectively.
Thus, to set the 15N carrier to 78.0ppm on the 700 (78.4 ppm is the position of the 15N resonance of 15N urea):
15N 0.0 ppm = 700.110000 MHz x 0.101329118 = 70.9415288 MHz
15N 78.0 ppm = 70.9415288 MHz + (78.4 x 70.9415288 Hz/ppm)/10^6 Hz/MHz = 70.9470906 MHz
The IDRR are “embedded” in the Bruker software, so that rather than needing to calculate the needed frequency for a
given ppm value, you can simply type the command “o3p 78.4” …. this changes SFO3 to a value that would coincide with
78.4 ppm.
Note, the temperature will effect the 0 ppm reference, and hence the above is a bit simplified relative to reality. A more
detailed description can be found at:
http://www.hincklab.structbio.pitt.edu/internal/new-internal-lab-pages/nmr-methods/chemical-shift-referencing-andtemperature-calibration/ (note, you’ll need a password for this, i1ikenmr)
15N
1.
2.
3.
4.
Pulse Calibration
Use “ased” to view parameters
Set pl1 = 1 dB, p1 = 7 us. Set p21=40 us, pl3 = +120 dB.
“ZG”, “EF”, and phase.
Optimize 15N pulse using “POPT”; Set pl3= +1 dB; Vary p21 from 1 to 81 us in
increments in 10 us.
15N
5.
Pulse Calibration
Once approximate 90° pulse is found, re-run “POPT” to find more exact 90°
(37 to 42 us in increments of 0.5 us)
15N
Pulse Calibration
The 90° and 180° pulses in the HSQC will all be applied at the thus
determined values (pl = +1 dB, 90° pulse = 40.0 us).
Note, however, that the HSQC also includes composite pulse decoupling
(CPD) during the acquisition time. The CPD, as discussed, is usually
applied at lower power, however, to minimize sample heating (as discussed).
Typically, a 90° pulse time of 180 us is used for the 15N CPD. This
approximate power level to attain this 90° pulse time can be calculated
using the relation between amplifier attenuation levels and pulse times (as
previously discussed, dB = 20log(V/V’). Specifically, we can calculate how
many more dB we will have to attenuate the 15N transmitter (relative to the -2
dB that yielded a 40 us 90° pulse) using the following:
dB = 20log10 (180 40) =13.1 dB
Hence, for 180 us 90° pulse for CPD, you can estimate that pl3 = +1 dB +
13.1 dB = +14.1 dB. Can check this using the previous procedure.