Innovation with Integrity
Good NMR Spectroscopy Practices: How to Continually Get Good Data from Your NMR Clemens Anklin Bruker Pre-ENC Workshops and Symposium Orlando, FL – April 2018
Nothing Magical or Secret : Just Possibly Lost or Forgotten Over Time
Overview • Sample preparation
Solvents Tubes Weighing/Dissolution/Purification
• Instrument preparation Temperature calibration and regulation Locking Tuning Shimming Pulse calibration
• Data acquisition
Acquisition Time
Receiver Gain
Pulsing Too Fast
Sample Preparation
Sample Preparation
With few exceptions, sample prep is a manual process, thus you have a lot of influence over the quality of NMR sample
Factors that affect quality include:
Solvents
Tubes
Dissolution / Mixing / Purification
NMR Solvents Are Usually Deuterated
Why Deuterate?
Provide a signal for the field/frequency lock
To avoid a very large solvent signal in the spectrum
What Makes a Good NMR Solvent?
Sample Solublity & Stablility
One or few 1H resonances
Strong Lock Signal
Chloroform – CDCl3 Advantages Good Solvent Low Price Single 1H/2H resonance (+ H2O near 1.6 ppm) Easily Removed Disadvantages Light sensitive
• 2 CDCl3 + O2 2 COCl2 + 2 DCl
Can contain HCl • Can be removed by filtering through activated basic alumina
Toxic, carcinogenic Weak lock signal Evaporates over time
hν
Physical characteristics: • δ (1H) = 7.24 ppm, • δ (13C) = 77 ppm • BP = 60.9 C, • MP = -64 C, • density = 1.5 g/cm2
Advantages Good Solvent Low Price Single 1H/2H resonance ( + H2O near 3.33 ppm)
Disadvantages Strong lock signal Hard to remove More viscous
• Slightly higher linewidths possible
High freezing point • Might be solid if room is too cold (20*C), additional water raises freezing point
Hygroscopic • In some cases D2O has been added to hide water content
Dimethylsulfoxide – DMSO-d6
Physical characteristics: • δ (1H) = 2.54 ppm, • δ (13C) = 39.5 ppm • BP = 190 C, • MP = 20.2 C, • density = 1.19 g/cm2
Advantages Good for the water soluble materials either pure or
H2O/D2O mix if exchangeable protons are to be observed Very Low Price Single resonance
Disadvantages Hygroscopic Not easy to remove Temperature dependent chemical shift Spectrum will shift if locked on D2O and temperature is not
stable!
Deuteriumoxide (Heavy Water) – D2O
Physical characteristics: • δ (1H) = 4.7 ppm, • δ (13C) = • BP = 101.4 C, • MP = 3.8 C, • density = 1.1 g/cm2
Advantages Medium price Easy to remove
Disadvantages Temperature dependent -OD chemical shift 2 signals Can exchange OH and NH Expands on freezing, almost always cracks the
tube
Methanol – CD3OD
Physical characteristics: • δ (1H) = 3.3/4.8 ppm, • δ (13C) = 49.15 • BP = 65 C, • MP = -99 C, • density = .89 g/cm2
Advantages Good solvent for moderately polar substances Medium price Single resonance Easy to remove
Disadvantages Can react with solutes Very flammable
Acetone – C3D6O
Physical characteristics: • δ (1H) = 2.05 ppm, • δ (13C) = 29.9/206.7 • BP = 55 C, • MP = -93.8 C, • density = .872 g/cm2
Advantages HPLC solvent Single Resonance
Disadvantages Expensive
Acetonitrile – CD3CN
Physical characteristics: • δ (1H) = 2.05 ppm, • δ (13C) = 29.9/206.7 • BP = 55 C, • MP = --93.8 C, • density = .872 g/cm2
Advantages Specific solubilities Toluene-d8 as a Benzene (C6D6) alternative CD2Cl2 for acid sensitive materials TFE, TFA for polymers or co-solvents
Disadvantages Usually expensive
Other NMR Solvents
Solvent 1H Shift 13C Shift BP MP Density C6D6 7.28 ppm 128 ppm 79.1 6.8 0.95
Toluene-d8
2.09/6.98/7.0/7.09 ppm
20.4/125.49/128.33/ 129.24/137.86 ppm
109 -85 0.94
CD2Cl2 5.32 ppm 54 ppm 39.5 -97 1.362
Pyridine-d5
7.22/7.58/8.74 ppm
123.87/135.91/ 150.35 ppm
114 -41 1.02
C2D2Cl4 5.91 ppm 74.2 ppm 146 -43 1.7
TFE 3.88/5.02 ppm 61.6/126.3 ppm 77 -44 1.42
TFA 11.5 ppm 116.6/164.2 ppm 71 -15 1.5
Choose The Best Type of NMR Tube Maximize the # of Spins inside the
NMR Coil
Choose The Best Type of NMR Tube
10 mm Tube • Only for 10 mm probe • Largest sample volume
4 mL • Great for solubility
limited situations • Very viscous samples
5 mm Tube • Most common tube • Medium Sample volume
• 550 - 600 μL
Choose The Best Type of NMR Tube
Shigemi Tube • Best for sample limited
situations • Smaller Sample volume
• 300 μL • Retain the full filling
factor in a 5 mm probe, but center all the sample/spins inside the coil
3 mm Tube • Best in 3 mm probe but can
be used in 5 mm probe • Small sample volume
200 μL • Good for sample limited
situations • Only a benefit in a 5mm
probe if you use a shorter sample (150 μL) • Small loss in sensitivity due
to “filling factor” • But due to smaller
diameter, can get away with meniscus closer to the coil
More About Shigemi Tubes
• 2 part NMR tube • Glass is susceptibility matched to
solvent D2O (Clear) DMSO (Green) CDCl3 (Clear) CD3OD (Blue)
• Sample capacity is between the bottom length and plunger
• Make sure to get the bottom length compatible with your instrument Bruker – 5mm Jeol – 12mm Agilent – 16mm
General NMR Tube Characteristics
OD
ID
Diameter Concentricity ID tolerance Camber
General NMR Tube Characteristics Dimensions and tolerances for different types of NMR tubes:
Tubes Crosssection (mm2) or Volume/mm height (ul) Variation of Volume
Vendor 1 Price Inner Diameter ID tolerance Avg Min Max
Lower limit Upper limit
Cheap 5mm ~ $ 6.00 4.21 0.13 13.92048 13.07405 14.79345 93.92 % 106.27 % Medium 5mm ~ $ 9.50 4.206 0.0065 13.89404 13.85113 13.93701 99.69 % 100.31 % Ultra thin wall ~ $ 45.00 4.496 0.0065 15.87605 15.83018 15.92199 99.71 % 100.29 % Worst 3mm ~ $ 9.65 2.413 0.13 4.573035 4.093565 5.079051 89.52 % 111.07 % Best 3mm ~ $ 18.00 2.413 0.0065 4.573035 4.548431 4.597705 99.46 % 100.54 %
Vendor 2 Worst 5mm ~ $ 4.50 4.16 0.01 13.59179 13.52652 13.65721 99.52 % 100.48 % Best 5mm ~ $ 16.00 4.2 0.006 13.85442 13.81487 13.89404 99.71 % 100.29 %
Vendor 3 Best 5mm ~ $ 23.00 4.2 0.006 13.85442 13.81487 13.89404 99.71 % 100.29 % Worst 5mm ~ $ 10.00 4.2 0.03 13.85442 13.65721 14.05305 98.58 % 101.43 %
Best 3mm 2.41 0.006 4.561671 4.538986 4.584413 99.50 % 100.50 % Worst 3mm 2.41 0.03 4.561671 4.448809 4.675947 97.53 % 102.51 %
Vendor 4 5 mm ~ $ 2.50 4.19 0.05 13.78853 13.46141 14.11957 97.63 % 102.40 % 3 mm ~ $ 2.50 2.36 0.03 4.374354 4.263848 4.486273 97.47 % 102.56 %
NMR Tubes do’s and don’ts Do’s Wipe the outside of the tube after touching Set the cap on straight Discard chipped or cracked tubes Dry tubes in a vacuum oven at low temperature
Don’ts • Dry tubes in an oven at high temperatures lying on a rack. • Rinse tubes with aggressive media • Reuse tubes you dropped on the floor
Weighing Your Sample
To Weigh or Not To Weigh? If you can weigh or pipet your sample – do so
• Knowing the amount of material will make it easier to predict experiment times
If possible weigh into the container you will use to dissolve the sample – using multiple containers can lead to transfer losses
Weighing directly into the NMR tube requires a very slim spatula and certainty that all material will be soluble in the right amount of solvent • No further purification is possible
Dissolving Your Sample
Dissolve your sample in 600-650 µl of solvent (5mm tube) • An optimally filled tube has a solvent height of 40-45 mm
corresponding to 550-625µl of solution • The small amount of extra solution helps with transfer losses • Stay with minimum volume if sample limited.
Shake well Shake well Shake well
• Good mixing is an argument against dissolving the sample in the NMR tube
Do not leave hygroscopic solvents exposed to air. Cap the vial and work expeditiously
Purification of Your Sample
Simple, easy way of sample filtration: • Pure a small piece of medical cotton or Kimwipe
into a Pasteur pipette • Use a second pipette to pack it down • Transfer solution into pipette and push into NMR
tube with bulb
• You can rinse the cotton with a small amount of NMR solvent first
• You can also add activated charcoal to top of cotton if desired
After the Transfer into the Tube
Label your sample: a) With a fine tipped marker on the tube and
cover with parafilm b) With these handy labels from Sigma Aldrich c) With small hang tags Don’t: a) Place tape… on top of tape… on top of tape… b) Leave large pieces of tape hanging
After the Transfer into the Tube
Make sure your sample: is well mixed is free of air bubbles contains no solids is all the way to the bottom of the tube
Do: a) Wipe the outside of the tube
before/after inserting into spinner to avoid dirt build-up in spinner and probe
a) Set the depth correctly
Center Line
Sample volume is centered about the
line, not just pushed all the way down
During transfer to the magnet
Do: a) Hold the tube and not the spinner b) Avoid touching here c) And here d) If you did, wipe it
e) Dirty spinners will lead to this:
Picture courtesy of @spectracular
Instrument Preparation
Before You Insert The Sample What do I want to learn about my sample? Which experiments do I need to run? If I have a choice of instruments/probes which one is best for me?
Field Probe 1H (0.1% EB)
13C (ASTM)
400 MHz Smart Probe 500:1 200:1
400 MHz Prodigy 1050:1 475:1
400 MHz BBI 600:1
400 MHz 3 mm BBI 190:1
400 MHz 10mm BBO 500:1
700 MHz TXI 1700:1 400:1
700 MHz TCI Cryoprobe 7800:1 1400:1
Why All These Steps?
1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse
Temperature Regulation – Why?
What is the current temperature of the probe? Is it compatible with your solvent?
No matter what the temperature, stability is key!
Sucrose in D2O COSY
With stable temperature
Started collecting data before the temperature
stabilized
Temperature Regulation – Why?
Especially True with experiments such as HSQC/TOCSY were the decoupling or spinlock can heat the sample
Sucrose in D2O HSQC
With stable temperature
No Temperature
regulation
Temperature Regulation – Why?
“xf2” does the FT in the direct dimension only Temperature dependent changes can be seen over time
Sucrose in D2O HSQC
With stable temperature
No Temperature
regulation
VT Unit Heater
Sensor Thermocouple Pt-100
Variable Temperature System
• The sample is heated from the bottom
• Without adequate airflow you will have a temperature gradient in the NMR tube
The EDTE Window
• Is it on?
• What is the airflow?
Temperature Stability • How long has the temperature been at the set point and stable?
Remember – this is the “sensor temperature” NOT your sample!
VT Unit Heater
Sensor Thermocouple Pt-100
Temperature Calibration
• The VT Sensor can be calibrated for accurate sample temperature values
• The –OH of MeOH is temperature sensitive
• The AU program calctemp measures the splitting and determines actual sample temp
Neat Methanol T [K] = 409.0 - 36.54 Δδ - 21.85 (Δδ)2
Δδ
J. Magn. Reson. 1982, 46, 319-321
Temperature Calibration - calctemp
• High Temp = Ethylene Glycol 300-380 K
• Room/Low Temp = Methanol 180 – 330K
Cryoprobes especially 99.8% Methanol-d4
Temperature Calibration / Correction A simple 2 point correction can be created and enabled
1. Set and measure the temperature at 2 targets
2. Suggest 30 – 40 degrees apart.
3. This is the correction for this range
4. Add another correction for other ranges
Temperature Calibration / Correction Enable the correction appropriate for your temperature range
Temperature Stability Calibration isn’t the only important part of Temperature Stability
1) Set point ~5 deg above incoming air temperature
2) Proper “selftune” values
Set Point Actual Temperature Heater Power
Temperature Calibration / Correction Enable the Appropriate Self Tune parameters for your situation
Temperature range
Incoming Air
• Room Temp
• LN2 accessory
• Flow Rate
What is the next step?
1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse
Locking – Choose the correct solvent
• Choose your solvent
• If your solvent isn’t there, create a new one: edsolv
Creating a New Solvent - Why?
Lock and Shim parameters are solvent dependent • Chemical shift dependent
• DMSO w/ 6 2H’s has more signal that CDCl3 with 1 2H
• Mixed solvents
• With more than one 2H signal • With large 1H signal and less 2H signal
Adding A New Solvent
How To Add A New Solvent • Click “Edit • From the pull-down menu
select “Add new Solvent • Enter information
• One can also edit existing solvents with the “Edit Solvent” option
Adding A New Solvent - Locking
• If your new solvent will be used as the lock solvent, then the lock information must be entered
• Click on “Lock” tab to access the lock parameter table
• Right-click on new solvent
Choose “Edit lock parameters”
Adding A New Solvent - Locking
• Lock power value ranges -60 to 0 dB • The easiest starting place for lock & loop
values is the default values for the compound in your new solvent that will be used to lock
• Lock Phase = -1 indicates the current value stored in the BSMS will be used
• Signals: Calibration of the spectrum via the lock
requires chemical shift information An example: MeOD – two 2H signals -CD3 = 3.3 ppm ‒ Used for lock because more
intense -OD = 4.8 ppm
Auto Lock Parameters Through BSMS
Rules Of Thumb For Loop Parameters
“loopadj” will automatically find best filter/gain/time values as well as optimize lock phase
Adding A New Solvent - Referencing
• Calibration of the spectrum via the lock takes place automatically according to the parameters in the lock tab under “Shift” Further experiment specific calibration
can be done with the “sref” command • Solvent Regions are areas where there are
no solvents, gaps between regions are were solvent should be found • Used for peak picking/integration to
avoid picking solvent peaks An example: MeOD Program will search around zero from
-0.5 to +0.5 ppm for a peak. If found, it will set that to 0 ppm
Regions without solvents are defined
Adding A New Solvent - Shimming
• If you want to use TopShim with your new solvent additional parameters need to be defined
• Topshim allows for automated gradient shimming on either 1H or 2H depending on what signal is strongest When samples are >50% protonated solvent, use 1H shimming
• Topshim allows for hard or selective pulses to be used If more than 1 signal present, use selective pulse for shimming
Mixed solvents and/or solvents with more than one signal • To start the setup, type topshim solvcal and follow the
instructions
Problems With Incorrect Lock Parameters
Strychnine in CDCl3
Strychnine in CDCl3 Locked on DMSO
Problems With Incorrect Lock Parameters
2mM Sucrose in 90:10 H20:D2O noesygppr1d
Correct Lock Phase
2mM Sucrose in 90:10 H20:D2O noesygppr1d
Lock Phase off by ~20º
How To Correct Lock Phase
1. Auto phase during locking: With an ELCB / L-TRX board,
an improved “Spectrum” algorithm is available Make sure it is on Make sure the Calibration
has been done
What is the next step?
1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse
Probe Tuning – Why?
The radiofrequency coil in the probe both delivers the rf pulse to the sample, and picks-up the signal from the affected NMR nuclei.
Tuning the probe Adjusting the “tune” and “match” capacitors in the probe circuit to match the inductive resistance of the circuit at a given frequency. Our samples are part of the circuit Samples with different conductance and dielectric constants will change
the inductance and it is not longer matched and there is reflected power When there is reflected power, the circuit is not as effective
A properly tuned probe provides: 1. The shortest/most effective pulse 2. The maximum sensitivity from your probe
Samples Affect Probe Tuning
3% CHCl3 in Acetone-d6 25 mM Cyclosporine in C6D6 4% MeOH in MeOD Urea & Methanol in DMSO 2mM Sucrose in H2O:D2O 500 mM NaCl in D2O
Started with 0.1% EB in CDCl3, tuned the probe. Changed samples and looked at WOBB
Probe Tuning Affects Efficiency: 90º Pulse
Properly Tuned Probe
90º = 9.75μs
Mistuned Probe 90º = 9.87μs
• 1H pulse from pulsecal
• Results from BBFO probe
• Inverse probe would have had a bigger difference
Probe Tuning Affects Efficiency: Signal:Noise 2mM Sucrose – 2 hour 13C spectrum
Probe tuned (1H &13C) from CDCl3 sample
Properly Tuned Probe
What is the next step?
1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse
Using TopShim via Graphical Interface
• Type “topshim gui”
Or
• From the Topshim Tab Pull Down
Using TopShim via Graphical Interface
Common Problem in TopShim
“too many points lost during fit”
• In the presence of a thermal gradient, low viscosity solvents start to develop convection currents
• This is problematic when you are trying to map and correct spatial homogeneity
• topshim convcomp can help compensate
Common Problem in TopShim
“not enough valid points”
• TopShim compares the signal of 2 different gradient echo experiments
• If the shims are way off – there won’t be signal for it to adequately compare Need better starting shims!
Useful TopShim Parameter
“convcomp”
• Uses a gradient echo sequence that compensates for convection currents Very useful for non-viscous
solvents that are prone to convection currents
Only draw back to always using it is it might take slightly longer
Useful TopShim Parameter
“tune”
• Iterative process of using the lock to optimize Lock Phase X, Y, XY, YZ, XZ shims
Useful TopShim Parameter
“ordmax”
• What # of Z shims to modify
• ordmax=8 Z1-Z8 • ordmax=3 Z1-Z3
• Useful & sometimes necessary for
probes with longer coils
Smart Probe
Newer CryoProbes
Useful TopShim Parameter • ordmax= Sets the maximum total order of shim functions (default = 5)
ordmax=3 limits shimming to Z-Z3 topshim ordmax=8 (SmartProbe) topshim 3d ordmax=8,7 ([on-axis],[off-axis])
• 1H or 2H Explicitly sets shimming nucleus
• lockoff Enables shimming with system unlocked • o1p= Explicitly sets excitation frequency in PPM topshim 1H lockoff o1p=2.49 (DMSO-h6)
• selwid= Enables selective excitation of a bandwidth expressed in
ppm; useful when shimming on a solvent with multiple strong signals topshim o1p=1.93 selwid=0.5 (CD3CN+D2O) • convcomp Used to activate convection compensation; useful when using low
viscosity solvents susceptible to convection
Useful TopShim Parameter • durmax= maximum duration per 1D field map acquisition (expressed in seconds)
default = 7 (try 15, 30 or even 120)
• rga force receiver gain optimization before shimming topshim rga (Can be used if a low s/n situation exists)
• tune also shim on the lock before and/or after gradient shimming (tuneb shims X,Y,Z,XZ,YZ before running gradient shimming)
topshim tuneaz (shims Z after running gradient shimming)
• shigemi Used to eliminate unreliable data at axial Shigemi tube walls when 1D shimming
• zrange sets the range in cm in the Z direction used for shimming topshim zrange=-0.8,0.8 (short sample)
• plot Saves data after completion in <TopSpin_home>/data/topshimData … read about more in the Topshim manual! Type help topshim from the Topspin command line.
What is the next step?
1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse
• The prosol table allows a 90º pulse to be calibrated once, and stored for use in subsequent experiments
• Most of the time, these values are correct from sample to sample If your probe is tuned! If the sample isn’t salty
• Inverse geometry probes are more susceptible to changes and need calibration more often sample to sample
Prosol Table
Why The 90º Pulse is Important
DEPT135 (CH/CH3 positive, CH2 negative) DEPT90 – 1H pulse off by 1μs DEPT90 – Correct 1H pulse (CH only)
Strychnine in CDCl3
Why The 90º Pulse is Important
Properly Calibrated Pulses 13C pulse off by 1μs
13C Adiabatic pulses help, but not the solution
as there are still 90º pulses
Strychnine in CDCl3
HSQCEDETPG
HSQCEDETPG
HSQCEDETPGSP
Calibrating The 90º Pulse Manually
• Acquire multiple spectra with increasing p1 values
• Find the 360º null
• Divide by 4 for the 90º
Calibrating The 90º Pulse with PULSECAL
pulsecal is and AU program that uses a nutation experiment to automatically determine the 90º pulse
Calibrating The 90º Pulse with PULSECAL
pulsecal with a single strong signal
FT
PW = correct
Calibrating The 90º Pulse with PULSECAL
pulsecal without a single strong signal
FT
PW = uncertain / too short
Overview
• Sample preparation Solvents Tubes Weighing/Dissolution/Purification
• Instrument preparation Temperature calibration and regulation Locking Tuning Shimming Pulse calibration
• Data acquisition
Acquisition Time
Receiver Gain
Pulsing Too Fast
Things to look out for during acquisition
During data acquisition Maintaining stability and resolution
Obtaining good data quality over long periods of time will require stability of the system and the environment.
Avoid changing the environment during long experiments. Such changes could involve room temperature changes, introduction of vibration or also loud noises.
Features such as autoshim can assist in keeping the resolution and lineshape at an optimum. For reliable operation during gradient experiments a relaxation delay of 1 second is recommended.
During data acquisition Maintaining stability and resolution
Do’s and Dont’s
Do keep the environment stable mainly with respect to temperature.
Avoid traffic with metal objects around the magnet
Avoid activities that could introduce vibrations
Abstain from blasting loud music
Stay away from activities that could lead to a reboot of your workstation
Parameters that Affect Data Quality
• FID form 13C spectrum. AQ = 3.25 seconds T2= 0.33 seconds
Signal Noise
Truncation
Resolution
Data Acquisition- Acquisition Time (AQ)
Setting the Acquisition Time
Parameters that Affect AQ
The FID is a set of descrete data points
Time Between Points (DW) = 1/SW
Increase SW DW decreases
AQ Decreases
Decrease SW DW Increases
AQ Increases
Increase TD
AQ Increases
Acquisition Time Too Short – Truncation AQ = 5 seconds AQ = 0.75 seconds
Sinc wiggles Loss of resolution
Strychnine in CDCl3 – 1H
Acquisition Time Too Short – Truncation AQ = 3.5 seconds AQ = 0.5 seconds
Cyclosporine in C6D6 – 13C
Acquisition Time Too Short – Truncation AQ = 3.5 seconds AQ = 0.5 seconds
Cyclosporine in C6D6 – 13C
Line-broadening can help with the Sinc Wiggles, but will only decrease resolution further
Acquisition Time Too Long – No Benefit
There is a point of diminishing return where there is no additional gain
AQ = 5 seconds D1 = 10 seconds
AQ = 14.5 seconds D1 = 0.5 seconds
Acquisition Time Too Long – Lower S:N
There is a point of diminishing return where there is no additional gain
AQ = 5 seconds D1 = 10 seconds
AQ = 14.5 seconds D1 = 0.5 seconds
and only noise is collected, thus decreasing S:N
Acquisition Time Too Long – Lower S:N
There is a point of diminishing return where there is no additional gain
AQ = 5 seconds D1 = 10 seconds S:N = 3400:1
AQ = 14.5 seconds D1 = 0.5 seconds S:N = 2000:1
and only noise is collected, thus decreasing S:N
Acquisition Time Too Long – Lower S:N
Appropriate Line-broadening will fix by minimizing the later part of the FID (noise)
AQ = 5 seconds D1 = 10 seconds S:N = 8800:1
AQ = 14.5 seconds D1 = 0.5 seconds S:N = 8700:1
What if I acquired too much ?
• TDeff to reduce acquistion time after the fact.
TD
TDeff
Acquisition Time Too Long
AQ is not a substitute for D1, especially when decoupling 1H and starting with
AQ = 3 seconds D1 = 2 seconds
AQ = 4.9 seconds D1 = 0.1 seconds
1H in pulse sequence : DEPT
Data Acquisition – Receiver Gain (RG)
RG Too High – Clipped Fid
1D Spectrum of Strychnine RG set too high
Auto processing not as robust Large distortions in baseline
RG and Signal Intensity
101
RG Value Same Ibuprofen sample at different RG values
71.8
45.2
22.5
5.0
S/N versus RG
• Low receiver gain = ADC noise dominant Signal changes, noise is constant • High receiver gain = probe (system noise) dominant Signal and noise change
• Transition is instrument dependent
0
20
40
60
80
100
120
0 20 40 60 80 100 120
RG and Signal Intensity & Integrals
Integrals defined and calibrated RG=101
Using integral ranges and calibration from previous RG=45.2
Data Acquisition – Recycle Delay (D1)
D1 Too Short - Saturation Missing quaternary carbons
Increase D1 5x – More complete relaxation, they are observable
zgpg 13C observe with 90º pulse and 1H decoupling during D1 and AQ
Ernst Angle – Smaller Flip Angle
cos 𝜃𝜃 = 𝑒𝑒−𝐷𝐷𝐷+𝐴𝐴𝐴𝐴𝑇𝑇1
Here: D1+AQ / T1 = 0.33
Ernst Angle = 45º More signal from
quaternary carbons, Less signal over all
90º
45º
Ernst Angle – Smaller Flip Angle
cos 𝜃𝜃 = 𝑒𝑒−𝐷𝐷𝐷+𝐴𝐴𝐴𝐴𝑇𝑇1
90º - Long D1
45º - Short D1/More Scans
Here: (D1+AQ) / T1 = 0.33
Ernst Angle = 45º More S:N when NS is
increased compared to experiment time with longer D1
D1 Too Short – Artifacts
D1 = 1 D1 = 0.1
Not your grandparents NMR experiments
Stay up to date with modern experiments
Do not run your grandparents experiments for ever.
Just like almost anything else in life NMR experiments also evolve. What used to be great a few years ago might not be state of the art now.
Use these innovations to get better more informative data.
Overview
Better experiments
Improved pulse sequences
Phase sensitive versus magnitude mode acquisition
Improving resolution in the indirect dimension
NUS, semi-selective experiments, folding
Improving resolution in the direct dimension
Longer acquisition times
Pure shift
Homonuclear correlation COSY and TOCSY
A simple basic example Homonuclear correlation spectroscopy COSY To this day many of these
experiments are run with old sequences and using limited data sizes.
COSY 45 was state of the art in the 1980’s
Resolution is ok
Artefacts are plentyful
COSY
Why run a magnitude COSY if phase sensitive can give you much more information.
COSY-45 COSY DQF
COSY
Looking at details the phase sensitive COSY allows the coupling constants to be extracted.
COSY-45 COSY DQF
COSY
Using larger data sizes and linear prediction improves resolution.
Original data no zero filling 2048 x 2048 points, linear predicted
TOCSY
The TOCSY (Total Correlation SpectroscopY) provides correlations along entire spin systems. TOCSY COSY
TOCSY
Run a high resolution TOCSY with Z-filter to get more detailed multiplet information
TOCSY – MLEV TOCSY – DIPSI with z-filter
Heteronuclear correlation HMQC, HSQC and HMBC
HMQC versus HSQC
Compared to a simple absolute value HMQC the multiplicity edited HSQC is now the experiment of choice
HMQC versus HSQC
HMQC vs HSQC: no special processing
HSQC processing techniques
Introducing zero filling and linear prediction
Original 1k x 200 Linear predicted and zero filled 4k x 4k
HSQC acquisition techniques
Reduction of sweep width and folding.
HMBC
A modern semi phase-sensitive HMBC provides superior in the carbon dimension compared to the original sequence
old new
HMBC
A modern semi phase-sensitive HMBC provides superior in the carbon dimension compared to the original sequence
old new
HMBC acquisition techniques
Selective excitation in the indirect dimension
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HMBC acquisition techniques
Selective excitation in the indirect dimension
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HMBC acquisition techniques
Comparison with conventional experiment
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What Can I do to Collect Data Faster?
Getting The Second Dimension
FT
Non-Uniform Sampling (NUS)
FT
How Can I Benefit From NUS?
• Acquire an nD spectrum in less time or • Acquire a spectrum with much higher resolution in the indirect dimension(s) or • Some combination of the above … and more!
Shorter experiment
time
Higher resolution spectra
How Can I Benefit from NUS?
“Regular” HSQC NS = 2 TD = 256 Expt = 20 minutes
Less Time “NUS” HSQC NS = 2 TD = 256 NUS @ 10 % Expt = 2 minutes
Higher Resolution
“NUS” HSQC NS = 2 TD = 2048 NUS @ 10 % Expt = 20 minutes
Acquiring NUS Data – In TS 3.0 and Newer
change FnTYPE from “traditional(planes)” to “non-uniform_sampling”
NUS acquisition in TopSpin
Additional Acquisition parameters
An HSQC with 256 final points will be processed after acquiring only 64 increments (25%)
How sparsely do you want to sample? • Effective TD = 256 (128 complex points)
• You can set either NusAMOUNT[%] or NusPOINTS (Complex Pairs)
Faster Acquisition
NUS acquisition in TopSpin
Additional Acquisition parameters
An HSQC with 8192 final points will be processed after acquiring only 256 increments (3.125%)
How sparsely do you want to sample? • Effective TD = 256 (128 complex points)
• You can set either NusAMOUNT[%] or NusPOINTS (Complex Pairs)
Higher Resolution
How Sparsely Can I Sample?
• NusAMOUNT/NusPOINTS: rules of thumb
- For time savings: ~25 – 50 % per dimension 25 – 50 % for 2D 10 – 25 % for 3D 5 – 10 % for 4D
- For resolution enhancement keep total number of transients constant -> will result in equal or better S/N
• But it’s really more complicated…
How Sparsely Can I Sample?
• NusAMOUNT/NusPOINTS: rules of thumb
• Another relevant question is: How many FID’s do I need to acquire? • It largely depends on complexity of sample/spectrum:
- How many expected frequencies (peaks)
More peaks Acquire more fids to appropriately define the spectrum
- What kind of dynamic range of expected peaks
Large Dynamic range = more artifacts in processing Acquire more fids to minimize artifacts from calcualtions
NUS processing in TopSpin
But first a couple words on Licenses…
• No special NUS licenses are needed for data acquisition
• Prior to TopSpin3.5pl3, a special NUS license was required for processing in Topspin
• In TS3.5pl3 and newer, basic 2D processing is free … but please make sure you have at least TS3.5pl6!
Non Uniform Sampling - Licenses
• Topspin & NUS processing • What is included, what needs a license
Starting with Topspin 3.5 pl 3
Dimensions Methods Options
2D IST (CS) -
2D, 3D, 4D IRLS (CS), MDD Virtual Echo
NUS processing in TopSpin
• Usually no need to change the NUS processing parameters.
• Just process the way you would any other 2D dataset • xfb
or
NUS Processing with No License
But I keep getting this error message…
No license avaible…
Processing CONTINUES with parameters…
Dimensions Methods Options
2D IST (CS) - 2D, 3D,
4D IRLS (CS),
MDD Virtual Echo
NUS processing in TopSpin
1. Make sure Mdd_mod = cs
2. Set the “hidden parameters” from Topspin command line:
• Mdd_CsALG = ist • Mdd_CsVE = false
Getting rid of the NUS license message…
NUS processing in TopSpin
OK, now I can process my spectrum, but I can’ t phase!
NUS processing in TopSpin OK, now I can process my spectrum, but I can’ t phase!
• Imaginary data isn’t kept after the NUS reconstruction. But we can re-create it with a Hilbert transform
• … or type xht2 at the TopSpin command line
• Phasing now works normally!
NUS processing in TopSpin Recommendation: re-process spectrum after phasing
NUS reconstruction works better when 1D spectra are properly phased
xfb; Hilbert transform; phase phase correct first, then xfb
50mM cyclosporine in benzene-d6 25% of TD=256
Comparing Processing Algorithms
Cyclosporine – hsqcedetgpsisp2.3 - 2K x 1K @25% NUS
No Virtual Echo
Virtual Echo
IST IRLS
Comparing Processing Algorithms
Cyclosporine – hsqcedetgpsisp2.3 - 2K x 1K @ 3.125% NUS
No Virtual Echo
Virtual Echo
IST IRLS
Another NUS Processing Option
Cholesterol Acetate – hsqcedetgpsisp2.3 - 2K x 1K @ 3.125%
• IRLS Algorithm • mdd_csalg
• Virtual Echo • mdd_csve
• # Iterations • mdd_csniter # of iterations performed in
reconstruction Smaller value faster but
more artifacts Default value = 0 process
until convergence An option even without license,
but reduced data quality with IST algorithm
Virtual Echo? Algorithm # Iterations Time
No IST / pl5 0 60:00
No IST / pl6 0 00:50
Yes IST 0 00:50
No IRLS 0 00:40
Yes IRLS 0 00:40
Yes IRLS 2 00:12
Faster Is Not Always Better
Cholesterol Acetate – hsqcedetgpsisp2.3 - 2K x 1K IRLS & VirtualEcho
NUS = 12.5% mdd_csniter 2
NUS = 3.15% mdd_csniter 2 12 seconds
NUS = 6.25% mdd_csniter 2
NUS = 3.15% mdd_csniter 8 22 seconds
Data Acquisition Parameters
What parameters can I optimize for better data?
Acquisition Time TD / SW
RG
Recycle Time (D1)
Non Uniform Sampling
??? PULPROG ???
Innovation with Integrity
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