Good NMR Spectroscopy Practices: How to Continually Get Good Data from Your NMR

Transcription

1 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 Innovation with Integrity

2 Nothing Magical or Secret : Just Possibly Lost or Forgotten Over Time

3 Overview Sample preparation Data acquisition Solvents Acquisition Time Tubes Receiver Gain Weighing/Dissolution/Purification Pulsing Too Fast Instrument preparation Temperature calibration and regulation Locking Tuning Shimming Pulse calibration

4 Sample Preparation

5 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

6 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 1 H resonances Strong Lock Signal

7 Chloroform CDCl 3 Advantages Good Solvent Low Price Single 1 H/ 2 H resonance (+ H 2 O near 1.6 ppm) Easily Removed Disadvantages Light sensitive 2 CDCl 3 + O 2 2 COCl DCl Can contain HCl hν Can be removed by filtering through activated basic alumina Toxic, carcinogenic Weak lock signal Evaporates over time Physical characteristics: δ ( 1 H) = 7.24 ppm, δ ( 13 C) = 77 ppm BP = 60.9 C, MP = -64 C, density = 1.5 g/cm 2

8 Dimethylsulfoxide DMSO-d6 Advantages Good Solvent Low Price Single 1 H/ 2 H 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 Physical characteristics: δ ( 1 H) = 2.54 ppm, δ ( 13 C) = 39.5 ppm BP = 190 C, MP = 20.2 C, density = 1.19 g/cm 2

9 Deuteriumoxide (Heavy Water) D 2 O Advantages Good for the water soluble materials either pure or H 2 O/D 2 O 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! Physical characteristics: δ ( 1 H) = 4.7 ppm, δ ( 13 C) = BP = C, MP = 3.8 C, density = 1.1 g/cm 2

10 Methanol CD 3 OD 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 Physical characteristics: δ ( 1 H) = 3.3/4.8 ppm, δ ( 13 C) = BP = 65 C, MP = -99 C, density =.89 g/cm 2

11 Acetone C 3 D 6 O Advantages Good solvent for moderately polar substances Medium price Single resonance Easy to remove Disadvantages Can react with solutes Very flammable Physical characteristics: δ ( 1 H) = 2.05 ppm, δ ( 13 C) = 29.9/206.7 BP = 55 C, MP = C, density =.872 g/cm 2

12 Acetonitrile CD 3 CN Advantages HPLC solvent Single Resonance Disadvantages Expensive Physical characteristics: δ ( 1 H) = 2.05 ppm, δ ( 13 C) = 29.9/206.7 BP = 55 C, MP = C, density =.872 g/cm 2

13 Other NMR Solvents 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 Solvent 1H Shift 13C Shift BP MP Density C 6 D ppm 128 ppm /6.98/7.0/7.0 9 ppm 20.4/125.49/ / / ppm CD 2 Cl ppm 54 ppm Toluened8 Pyridined5 7.22/7.58/8.74 ppm /135.91/ ppm C 2 D 2 Cl ppm 74.2 ppm TFE 3.88/5.02 ppm 61.6/126.3 ppm TFA 11.5 ppm 116.6/164.2 ppm

14 Choose The Best Type of NMR Tube Maximize the # of Spins inside the NMR Coil

15 Choose The Best Type of NMR Tube 5 mm Tube Most common tube Medium Sample volume μl 10 mm Tube Only for 10 mm probe Largest sample volume 4 ml Great for solubility limited situations Very viscous samples

16 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

17 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

18 General NMR Tube Characteristics Diameter Concentricity ID tolerance Camber ID OD

19 General NMR Tube Characteristics Dimensions and tolerances for different types of NMR tubes: Tubes Crosssection (mm 2 ) or Volume/mm height (ul) Variation of Volume Vendor 1 Price Inner Diameter ID tolerance Avg Min Max Lower limit Upper limit Cheap 5mm ~ $ % % Medium 5mm ~ $ % % Ultra thin wall ~ $ % % Worst 3mm ~ $ % % Best 3mm ~ $ % % Vendor 2 Worst 5mm ~ $ % % Best 5mm ~ $ % % Vendor 3 Best 5mm ~ $ % % Worst 5mm ~ $ % % Best 3mm % % Worst 3mm % % Vendor 4 5 mm ~ $ % % 3 mm ~ $ % %

20 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

21 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

22 Dissolving Your Sample Dissolve your sample in µl of solvent (5mm tube) An optimally filled tube has a solvent height of mm corresponding to µl 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

23 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

24 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

25 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 Center Line 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 Sample volume is centered about the line, not just pushed all the way down

26 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

27 Instrument Preparation

28 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 1 H (0.1% EB) 13 C (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

29 Why All These Steps? 1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse

30 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

31 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

32 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

33 Variable Temperature System The sample is heated from the bottom Without adequate airflow you will have a temperature gradient in the NMR tube Sensor Thermocouple Pt-100 Heater VT Unit

34 The EDTE Window Is it on? What is the airflow?

35 Temperature Stability How long has the temperature been at the set point and stable? Remember this is the sensor temperature NOT your sample!

36 Temperature Calibration The VT Sensor can be calibrated for accurate sample temperature values The OH of MeOH is temperature sensitive Δδ Sensor Thermocouple Pt-100 Neat Methanol T [K] = Δδ (Δδ)2 The AU program calctemp measures the splitting and determines actual sample temp Heater VT Unit J. Magn. Reson. 1982, 46,

37 Temperature Calibration - calctemp High Temp = Ethylene Glycol K Room/Low Temp = Methanol K Cryoprobes especially 99.8% Methanol-d4

38 Temperature Calibration / Correction A simple 2 point correction can be created and enabled 1. Set and measure the temperature at 2 targets 2. Suggest degrees apart. 3. This is the correction for this range 4. Add another correction for other ranges

39 Temperature Calibration / Correction Enable the correction appropriate for your temperature range

40 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

41 Temperature Calibration / Correction Enable the Appropriate Self Tune parameters for your situation Temperature range Incoming Air Room Temp LN 2 accessory Flow Rate

42 What is the next step? 1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse

43 Locking Choose the correct solvent Choose your solvent If your solvent isn t there, create a new one: edsolv

44 Creating a New Solvent - Why? Lock and Shim parameters are solvent dependent Chemical shift dependent DMSO w/ 6 2 H s has more signal that CDCl3 with 1 2 H Mixed solvents With more than one 2 H signal With large 1 H signal and less 2 H signal

45 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

46 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

47 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 2 H signals -CD 3 = 3.3 ppm Used for lock because more intense -OD = 4.8 ppm

48 Auto Lock Parameters Through BSMS

49 Rules Of Thumb For Loop Parameters loopadj will automatically find best filter/gain/time values as well as optimize lock phase

50 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

51 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 2 H depending on what signal is strongest When samples are >50% protonated solvent, use 1 H 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

52 Problems With Incorrect Lock Parameters Strychnine in CDCl3 Strychnine in CDCl3 Locked on DMSO

53 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

54 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

55 What is the next step? 1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse

56 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

57 Samples Affect Probe Tuning Started with 0.1% EB in CDCl3, tuned the probe. Changed samples and looked at WOBB 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

58 Probe Tuning Affects Efficiency: 90º Pulse Mistuned Probe 90º = 9.87μs Properly Tuned Probe 90º = 9.75μs 1 H pulse from pulsecal Results from BBFO probe Inverse probe would have had a bigger difference

59 Probe Tuning Affects Efficiency: Signal:Noise 2mM Sucrose 2 hour 13 C spectrum Properly Tuned Probe Probe tuned ( 1 H & 13 C) from CDCl 3 sample

60 What is the next step? 1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse

61 Using TopShim via Graphical Interface Type topshim gui Or From the Topshim Tab Pull Down

62 Using TopShim via Graphical Interface

63 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

64 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!

65 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

66 Useful TopShim Parameter tune Iterative process of using the lock to optimize Lock Phase X, Y, XY, YZ, XZ shims

67 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

68 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

69 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.

70 What is the next step? 1. Temperature Regulation 2. Lock 3. Tune 4. Shim 5. 90º Pulse

71 Prosol Table 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

72 Why The 90º Pulse is Important DEPT135 (CH/CH 3 positive, CH 2 negative) DEPT90 1 H pulse off by 1μs DEPT90 Correct 1 H pulse (CH only) Strychnine in CDCl3

73 Strychnine in CDCl3 Why The 90º Pulse is Important HSQCEDETPG HSQCEDETPG 13 C Adiabatic pulses help, but not the solution as there are still 90º pulses HSQCEDETPGSP Properly Calibrated Pulses 13 C pulse off by 1μs

74 Calibrating The 90º Pulse Manually Acquire multiple spectra with increasing p1 values Find the 360º null Divide by 4 for the 90º

75 Calibrating The 90º Pulse with PULSECAL pulsecal is and AU program that uses a nutation experiment to automatically determine the 90º pulse

76 Calibrating The 90º Pulse with PULSECAL pulsecal with a single strong signal PW = correct FT

77 Calibrating The 90º Pulse with PULSECAL pulsecal without a single strong signal PW = uncertain / too short FT

78 Overview Sample preparation Data acquisition Solvents Acquisition Time Tubes Receiver Gain Weighing/Dissolution/Purification Pulsing Too Fast Instrument preparation Temperature calibration and regulation Locking Tuning Shimming Pulse calibration

79 Things to look out for during acquisition

80 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.

81 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

82 Parameters that Affect Data Quality

83 Data Acquisition- Acquisition Time (AQ) FID form 13C spectrum. AQ = 3.25 seconds T2= 0.33 seconds Signal Noise Truncation Resolution

84 Setting the Acquisition Time

85 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

86 Strychnine in CDCl3 1 H Acquisition Time Too Short Truncation AQ = 5 seconds AQ = 0.75 seconds Sinc wiggles Loss of resolution

87 Cyclosporine in C6D6 13 C Acquisition Time Too Short Truncation AQ = 3.5 seconds AQ = 0.5 seconds

88 Cyclosporine in C6D6 13 C Acquisition Time Too Short Truncation AQ = 0.5 seconds AQ = 3.5 seconds Line-broadening can help with the Sinc Wiggles, but will only decrease resolution further

89 Acquisition Time Too Long No Benefit There is a point of diminishing return where there is no additional gain AQ = 14.5 seconds D1 = 0.5 seconds AQ = 5 seconds D1 = 10 seconds

90 Acquisition Time Too Long Lower S:N There is a point of diminishing return where there is no additional gain and only noise is collected, thus decreasing S:N AQ = 14.5 seconds D1 = 0.5 seconds AQ = 5 seconds D1 = 10 seconds

91 Acquisition Time Too Long Lower S:N There is a point of diminishing return where there is no additional gain and only noise is collected, thus decreasing S:N AQ = 14.5 seconds D1 = 0.5 seconds S:N = 2000:1 AQ = 5 seconds D1 = 10 seconds S:N = 3400:1

92 Acquisition Time Too Long Lower S:N Appropriate Line-broadening will fix by minimizing the later part of the FID (noise) AQ = 14.5 seconds D1 = 0.5 seconds S:N = 8700:1 AQ = 5 seconds D1 = 10 seconds S:N = 8800:1

93 What if I acquired too much? TDeff to reduce acquistion time after the fact. TD TDeff

94 Acquisition Time Too Long AQ is not a substitute for D1, especially when decoupling 1 H and starting with 1 H in pulse sequence : DEPT AQ = 4.9 seconds D1 = 0.1 seconds AQ = 3 seconds D1 = 2 seconds

95 Data Acquisition Receiver Gain (RG)

96 RG Too High Clipped Fid 1D Spectrum of Strychnine RG set too high Auto processing not as robust Large distortions in baseline

97 RG and Signal Intensity RG Value Same Ibuprofen sample at different RG values

98 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

99 RG and Signal Intensity & Integrals Integrals defined and calibrated RG=101 Using integral ranges and calibration from previous RG=45.2

100 Data Acquisition Recycle Delay (D1)

101 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

102 Ernst Angle Smaller Flip Angle cos θθ = ee DDD+AAAA TT 1 90º Here: D1+AQ / T1 = 0.33 Ernst Angle = 45º 45º More signal from quaternary carbons, Less signal over all

103 Ernst Angle Smaller Flip Angle cos θθ = ee DDD+AAAA TT 1 90º - Long D1 Here: (D1+AQ) / T1 = 0.33 Ernst Angle = 45º 45º - Short D1/More Scans More S:N when NS is increased compared to experiment time with longer D1

104 D1 Too Short Artifacts D1 = 1 D1 = 0.1

105 Not your grandparents NMR experiments

106 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.

107 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

108 Homonuclear correlation COSY and TOCSY

109 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

110 COSY Why run a magnitude COSY if phase sensitive can give you much more information. COSY-45 COSY DQF

111 COSY Looking at details the phase sensitive COSY allows the coupling constants to be extracted. COSY-45 COSY DQF

112 COSY Using larger data sizes and linear prediction improves resolution. Original data no zero filling 2048 x 2048 points, linear predicted

113 TOCSY The TOCSY (Total Correlation SpectroscopY) provides correlations along entire spin systems. TOCSY COSY

114 TOCSY Run a high resolution TOCSY with Z-filter to get more detailed multiplet information TOCSY MLEV TOCSY DIPSI with z-filter

115 Heteronuclear correlation HMQC, HSQC and HMBC

116 HMQC versus HSQC Compared to a simple absolute value HMQC the multiplicity edited HSQC is now the experiment of choice

117 HMQC versus HSQC HMQC vs HSQC: no special processing

118 HSQC processing techniques Introducing zero filling and linear prediction Original 1k x 200 Linear predicted and zero filled 4k x 4k

119 HSQC acquisition techniques Reduction of sweep width and folding.

120 HMBC A modern semi phase-sensitive HMBC provides superior in the carbon dimension compared to the original sequence old new

121 HMBC A modern semi phase-sensitive HMBC provides superior in the carbon dimension compared to the original sequence old new

122 HMBC acquisition techniques Selective excitation in the indirect dimension 1HUU$qU.UqUN 13CUUUU1U1UUUU

123 HMBC acquisition techniques Selective excitation in the indirect dimension 1HUU$qU.UqUN 13CUUUU1U1UUUU

124 HMBC acquisition techniques Comparison with conventional experiment 1HUU$qU.UqUN 13CUUUU1U1UUUU

125 What Can I do to Collect Data Faster?

126 Getting The Second Dimension FT

127 Non-Uniform Sampling (NUS) FT

128 How Can I Benefit From NUS? Shorter experiment time Higher resolution spectra 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!

129 How Can I Benefit from NUS? Higher Resolution NUS HSQC NS = 2 TD = % Expt = 20 minutes Regular HSQC NS = 2 TD = 256 Expt = 20 minutes Less Time NUS HSQC NS = 2 TD = % Expt = 2 minutes

130 Acquiring NUS Data In TS 3.0 and Newer change FnTYPE from traditional(planes) to non-uniform_sampling

131 NUS acquisition in TopSpin Additional Acquisition parameters How sparsely do you want to sample? Effective TD = 256 (128 complex points) You can set either NusAMOUNT[%] or NusPOINTS (Complex Pairs) An HSQC with 256 final points will be processed after acquiring only 64 increments (25%) Faster Acquisition

132 NUS acquisition in TopSpin Additional Acquisition parameters How sparsely do you want to sample? Effective TD = 256 (128 complex points) You can set either NusAMOUNT[%] or NusPOINTS (Complex Pairs) An HSQC with 8192 final points will be processed after acquiring only 256 increments (3.125%) Higher Resolution

133 How Sparsely Can I Sample? NusAMOUNT/NusPOINTS: rules of thumb - For time savings: ~25 50 % per dimension % for 2D % 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

134 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

135 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!

136 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

137 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

138 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

139 NUS processing in TopSpin Getting rid of the NUS license message 1. Make sure Mdd_mod = cs 2. Set the hidden parameters from Topspin command line: Mdd_CsALG = ist Mdd_CsVE = false

140 NUS processing in TopSpin OK, now I can process my spectrum, but I can t phase!

141 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!

142 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

143 Cyclosporine hsqcedetgpsisp2.3-2k x NUS Comparing Processing Algorithms IST IRLS No Virtual Echo Virtual Echo

144 Cyclosporine hsqcedetgpsisp2.3-2k x 3.125% NUS Comparing Processing Algorithms IST IRLS No Virtual Echo Virtual Echo

145 Another NUS Processing Option 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 Cholesterol Acetate hsqcedetgpsisp2.3-2k x 3.125%

146 Cholesterol Acetate hsqcedetgpsisp2.3-2k x 1K IRLS & VirtualEcho Faster Is Not Always Better NUS = 12.5% mdd_csniter 2 NUS = 6.25% mdd_csniter 2 NUS = 3.15% mdd_csniter 2 12 seconds NUS = 3.15% mdd_csniter 8 22 seconds

147 Data Acquisition Parameters What parameters can I optimize for better data? Acquisition Time TD / SW RG Recycle Time (D1) Non Uniform Sampling??? PULPROG???

148 Innovation with Integrity