Good NMR Spectroscopy Practiceslab.pharm.uic.edu/nmr/3.Good_NMR_Spectroscopic_Practices_Get_Better_Data.pdfInnovation with Integrity Good NMR Spectroscopy Practices: How to Continually

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


Especially True with experiments such as HSQC/TOCSY were the decoupling or spinlock can heat the sample

Sucrose in D2O HSQC


No Temperature

regulation


“xf2” does the FT in the direct dimension only Temperature dependent changes can be seen over time

Sucrose in D2O HSQC


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?


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



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



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


“tune”

• Iterative process of using the lock to optimize Lock Phase X, Y, XY, YZ, XZ shims


“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.



• 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


pulsecal with a single strong signal

FT

PW = correct


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




and only noise is collected, thus decreasing S:N



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


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



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

1HUU$qU.UqUN 13CUUUU1U1UUUU


Selective excitation in the indirect dimension



Comparison with conventional experiment


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


• 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


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…


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

Documents

Good NMR Spectroscopy Practiceslab.pharm.uic.edu/nmr/3.Good_NMR_Spectroscopic_Practices_Get_Better_Data.pdfInnovation with Integrity Good NMR Spectroscopy Practices: How to Continually