2H:
Lock and more…
Rainer Kümmerle
User Meeting Benelux Brussels
29. November 2016
December 5, 2016
Outline
•
Lock
•
Lock regulation
•
Lock optimization
•
Magnetically perturbed environments
•
CryoProbes & Deuterium
•
2H spectra
2
Outline
•
Lock
•
Lock regulation
•
Lock optimization
•
Magnetically perturbed environments
•
CryoProbes & Deuterium
•
2H spectra
3
The lock job...
•
•
Lock regulation – what does the lock need to do?
•
Field stabilization (always)
•
Field Perturbation suppression (only if present!!)
•
(Field homogenisation – maximizing lock level)
Measure & regulate the sample temperature (NMR thermometerTM) !
4
NMR ThermometerTM for bio-NMR
deuterated micelles
2H-spectrum
of chain-perdeuterated DHPC-d22
(dihexanoyl-phosphatidyl choline) in 90% H2O / 10% D2O
D2O
*
*
*
*
*
*
*
*
*
*
DHPC-d22
288 K
298 K
310 K
Sample courtesy of Katrine Bugge, Birthe B. Kragelund and Kaare Teilum, University of Copenhagen,
Department of Biology
5
NMR ThermometerTM for bio-NMR
deuterated micelles
Sample heating due to CC-spinlock
•
monitored temperatures:
target = green
sample internal = yellow
HCCH-TOCSY experiment
50ms spinlock duration
recycle delay = 1 sec
sample: protein in DHPC, acqueous solution
Bruker digital lock
•
Lock
exciting @ 6.67 kHz
•
Lockphase
150 ms
Mx
x/y
My
7
regulation
H0
Lock parameters (1)
lockphase
•
•
Manual:
•
Always adjust on locked sample (shimmed & non-saturated!)
•
Optimize lock level or check lineshape of sharp line (dip at signal
base) in gs mode with a gradient sequence
Autophase:
•
LCB & ELCB with L-TX/L-RX as above
•
ELCB with L-TRX: as above or FFT adjustment (can be done
unlocked)
8
WATERGATE: gradient effects
•
WATERGATE:
•
Lockphase OK
•
Lockphase wrong (30o)
•
Depending on offset
(positive / negative) artifact
can be on right or left side
of signal
8.9
8.8
9
8.7
8.6
8.5
8.4
8.3
8.2
8.1
ppm
Gradient-COSY
•
COSYGPQF:
•
Lockphase wrong by
ca. 40o
ppm
1
2
3
4
5
6
7
8
9
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
10
ppm
Lock parameters (2)
•
Lock power:
peak power of excitation
pulse
•
Lock gain:
receiver („display“) gain
150 ms
11
Lock power adjustment
•
•
Optimal lock power depends on
•
2H
channel efficiency of probe
•
T1 of solvent
•
(Solvent concentration)
Seek for lock power close to saturation
•
No overshoot after gradient pulse (correct shim & phase)
•
Lockpower +1dB & lockgain -1dB -> same level
12
Lockpower – saturation ?
Lockpower +1dB
Lockgain -1dB
Lockpower +1dB
Lockgain -1dB
Same level
No saturation
Lower level
Saturation !
13
Lock Parameters (3)
•
•
Feed-back loop regulation parameter (Proportional–Integral–Derivative
controller – PID controller) triplet:
•
Loop gain
•
Loop time
•
Loop filter
Requires correct triplet settings for correct feed-back loop regulation
14
PID triplets / «lock.x» macros
lock.3
1
2
3
4
5
6
7
8
9
10
11
12
119.3
115.4
110.2
107.2
104.1
99.7
96.0
92.6
89.6
86.0
83.9
82.2
Loop
gain
-17.9
-14.3
-9.4
-6.6
-3.7
0.3
3.9
7.1
9.9
13.2
15.2
16.8
Loop
time
0.681
0.589
0.464
0.384
0.306
0.220
0.158
0.111
0.083
0.059
0.047
0.041
Loop
filter
20
30
50
70
100
160
250
400
600
1000
1500
2000
Lock
gain
15
Feed-back loop regulation (PID)
•
Lock regulation – use predefined PID triplets
•
lock.1
weakest
... lock.12
... strongest
[lock.3 is a good start]
•
Automatic optimization: loopadjust
autophase, then selection of appropriate triplet
16
Feed-back loop regulation (PID)
•
How to choose the appropriate lock regulation PID’s?
•
Magnetically “quiet” environment, e.g. no perturbation:
as gentle as possible (lock.3)
•
Field perturbations present / difference spectra:
as strong as possible (lock.6 … lock.12)
17
Spectrometer in „quiet environment“
2D TOCSY without gradients
•
Strong lock regulation:
•
can increase t1-noise
•
(autoshim may also
increase t1-noise)
increased t1-noise
18
Outline
•
Lock
•
Lock regulation
•
Lock optimization
•
Magnetically perturbed environments
•
CryoProbes & Deuterium
•
2H spectra
19
Magnetic field perturbations:
Spectrometer stability
•
AV III 600 MHz USplus
•
Magnet – Tramway
14m distance
•
Field perturbation:
> 0.6 kHz peak-peak
(measured in the lab)
Unlocked
1 point / sec
20
Magnetic field perturbations:
Spectrometer stability
•
“Ernst-Test”
•
AV III 600 MHz USplus
•
Field perturbation due to
tramway
•
Refocused HMQC
without gradients
•
Ns = 2
21
Magnetic field perturbations:
Pulsed field gradients / lock hold…
•
Strong T1 ridges appear for “weak” lock regulation
•
Lock loss may occur if…
•
•
strong magnetic perturbations during “lock hold”
•
pp with long lock-hold periods are used
•
“aggressive” lock PID’s are used
•
weak / imperfect lock signal (low D-content, ringdown, lock
phase wrong, …)
Extreme cases (e.g. being near a tramway): some pp’s require
optimisation: lock-hold only during gradient pulse
22
23
Outline
•
Lock
•
Lock regulation
•
Lock optimization
•
Magnetically perturbed environments
•
CryoProbes & Deuterium
•
2H spectra
24
CryoProbes & Lock
•
Tune & Match available
•
Cooled RF & preamp on a low gyromagnetic nucleus:
•
•
Increased efficiency (lower lock power required)
•
Increased Signal-to-Noise
•
Increased ringdown
Required amount of D2O to lock on TCI CryoProbe (700 MHz):
•
1 ml – 2 ml (0.2% – 0.3% in a 5mm sample)
25
CryoProbes: ringdown
•
Ringdown on 2H
•
Automatic lock fails (for samples with low 2H content)
•
Lock DC level changes with Lockpower / Lockgain
26
CryoProbes: ringdown assessment
sample up / lock power -8dB
90
100
110
120
130
140
Lockgain values
Note: DC may vary up or down, depending on phase
27
CryoProbes: ringdown
•
•
•
How to reduce / eliminate ringdown (AV III & AV III HD)
•
ELCB with L-TX/L-RX: pulse bank setting / 2H overcoupling
•
ELCB with L-TRX: pulse bank setting / 2H overcoupling / compensated
pulse (40us) setting
Pulse bank setting 1, 2, 3 reduces excitation pulse width
•
important SiNo loss on 2H (use only if required, not default!
•
compensate (in part) with increased lockpower
Compensated Pulse (40us) maintains default excitation pulse width and
therefore SiNo (preferred!)
28
Pulse bank setting
150 ms
•
Default (0)
Compensated Pulse 40us
•
1
•
2
•
3 (high-Q)
29
CryoProbes: ringdown
•
How to overcouple the lock channel on a CryoProbe:
•
De-match in the direction of „lower resonance frequency“ (wobble
curve broadens), recover resonance frequency with tuning
•
Pulse length not significantly increased
•
Signal-to-Noise not significantly reduced
30
CryoProbes: 2H overcoupling
31
CryoProbes: 2H overcoupling
32
CryoProbes: 2H overcoupling
P 90°66 ms
SiNo: 350:1
P 90°66 ms
SiNo: 340:1
33
P 90°67 ms
SiNo: 327:1
Outline
•
Lock
•
Lock regulation
•
Lock optimization
•
Magnetically perturbed environments
•
CryoProbes & Deuterium
•
2H spectra
34
2H
•
•
spectroscopy
2H
spectrum or 2H tuning & matching (CryoProbe)
•
new even expno: edc «600»
•
rpar gradshim1d2h all
•
getprosol
•
xaua
Tune & match or acquire 2H spectrum with «zg2h», locnuc off!
•
to finish «2H mode» and switch back to «1H mode» with lock active
•
odd expno with gradshim1d2h parameters: xaua
35
Summary Lock
•
Correct lockpower
•
Correct lockphase
•
Correct PID parameters depending on
•
Probe, solvent & environment
•
“good” environment: as little lock regulation as possible
•
“bad” environment: as strong lock regulation as possible
36
www.bruker.com
© Copyright Bruker Corporation. All rights reserved.