NMR tube choices: If you have adequate amount of sample, use a regular NMR tube; If you have limited amount of sample, usage a shigemi NMR tube; If your sample has high salt concentration , use a 3mm NMR tube.
Sample volume should be a minimum of 250 uL for a Shigemi tube, 160uL for a 3mm/5mm tube, or 500 uL for a regular NMR tube. Insufficient volume may cause bad shimming result, and consequently spectra with poor resolution and line shape. In many cases the sample volume should weight more than the concentration for a good NMR measurement.
The NMR sample should be homogeneous, free of air bubbles and insoluble substances. High salt concentration and paramagnetic ions should be avoided if possible
High quality NMR tubes should be used at BioNMR facility, in particular on the 800 MHz spectrometer.
For volatile organic solvent, or long term use sample, sealing the sample NMR tube with parafilm is highly recommended.
For aqueous samples,
D2O concentration should be equal to or higher than 7%, and kept equal to or higher than 7% during a titration.
NaCN 0.02% is recommended for sample buffer to prevent bacterial growth and bio sample degradation.
Degasing the buffer or the sample to reduce the chance of forming air bubbles in the solution.
Labeling a NMR tube
Sample Spinners
Type “lock” and choose the correct deuterated solvent that is with the sample. Contact the NMR facility manager if you can not find your solvent in the solvent table.
For samples requiring no solvent/water suppression, pulse program zg or zg30 can be used to run a simple 1D 1H experiment. The zg30 pulse needs much shorter relaxation delay d1 since the rotation angle is only 30 degrees. For quantitative NMR analysis, d1 needs to be set long enough to ensure full relaxation recovery.
There are multiple methods to suppress water NMR signal. Excitation sculpting (zgesgp) and watergate (zggpwg) are effective in suppressing a large amount of water in a NMR sample, while pre-satuation methods such as zgcppr or zggppr are effective in suppressing trace amount of water in organic solvents.
Type "rpar P31CPD" in a new data set, or use "edc" command to copy from an exisiting 1D 31P experiment. Another option is "rpar P31" depending on decoupling choice.
Use "getprosol" command to load the default phosphorous power parameters.
In the acquisition parameter window check and adjust td, ns, ds, sw, d1.
Estimate receiver gain using command rga, and then start the experiment using commandzg ;
Process the FID by typing “efp”, and phase the spectrum when necessary.
Copy an existing 1D experiment (pulse program: zgwg.df) by typing “edc”.
Type eda, in the acquisition parameter window check and adjust td, ns, ds, sw, O1P and O2P.
Type ased, in the pulse program parameter window, enter calibrated p1 and pL1 values; set pcpd2 220 us and corresponding pL12 12 dB if applying decoupling. Otherwise pL12 should be 120 dB.
Start the experiment by rga and zg;
Process the FID by typing “dfp”, and phase the spectrum when necessary.
Go to an existing HSQC experiment (pulse program: hsqcetfpf3gp), type “edc” to copy its parameters and generate a new experiment number. Or in a new experiment number "rpar HSQCETFPF3GP".
Type “eda”, set parameters td, ns, ds, sw, O1P and O3P. For 13C and 15N labeled sample, ZGOPTNS should be "-DLABEL_CN".For only 15N labeled sample, leave ZGOPTNS blank.
Type “ased” to set power length and power level for both 1H and 15N, including p1, pL1, p21, pL21, pL12, and pcpd2. Or type "getprosol 1H A B" to set power related parameters. A is the pulse length in µs, and B is the power leverl in dB.
Estimate receiver gain using rga command, and Start the experiment using command zg;
Process the FID by typing “xfb”. Phase the spectrum when necessary.
Go to an existing experiment (pulse program: hsqcetgp), type “edc” to copy its parameters and generate a new experiment number. Or in an new experiment number "rpar HSQCETGP".
Type “eda”, set parameters td, ns, sw, O1P and O2P, CNST2. Note that CNST2 can be different for different C-H bond.
Type “ased” to adjust power length and power level for both 1H and 13C, including p1, pL1, p3, pL2, pL12, and pcpd2. Or type "getprosol 1H A B" to set power related parameters. A is the pulse length in µs, and B is the power leverl in dB.
Start the experiment by rga and zg; Process the FID by typing “xfb”. Phase the spectrum when necessary.
Go to an existing SOFAST experiment (pulse program: sofast), type “edc” to copy its parameters and generate a new experiment number. Or "rpar SFHMQCF3GPPH" in an new experiment entry.
Type “eda” to check and adjust parameters such as td, ns, sw, O1P and etc.
Type “ased” to adjust power length and power level for both 1H and 15N, including, pL1, p3, pL2, pL12, and pcpd2. Or type "getprosol 1H A B" to set power related parameters. A is the pulse length in µs, and B is the power leverl in dB.
To calibrate shape pulse sp15 and sp20, type “stdisp” to open shapetool window, and calculate PC9 (3000 us at 120°) and Reburp1000 (2000us at 180°) pulses. sp value = shapetool value + p1 in dB.
Shape Pulse Calculation Steps: choose the shape pule in shapte tool window, then go to Analysis/Integrate Shape, put in correct shape pulse length, angle, and 90 degree pulse length, hit "Enter" key. The calculated shapetool value will be shown as "Change of power level"
Note that for Bruker pulse programs, you may use "getprosol 1H pulselength powerlevel" command, to let TopSpin calculate the power related parameter for you.
Start the experiment by rga and zg;
Process the FID by typing “xfb”. Phase the spectrum when necessary.
Go to an existing 2D-Tocsy experiment (pulse program: dipsi2esfbgpph), type “edc” to copy its parameters and generate a new experiment number. Or in a new experiment, "rpar DIPSI2ESFBGPPH".
Pulse program dipsi2esfbgpph is based on dipsi2esgpph and have f2 and f3 channels included.
Type "edasp" to check /adjust spectrometer routing.
Type “eda”, set parameters td, ns, ds, sw, O1P, O2P and O3P if necessary.
In acquisition window, if sample is unlabeled, leave ZGOPTNS blank. If sample is N15 labeled, set ZGOPTNS "-DLABEL_N". If sample is N15 and C13 labeled, set ZGOPTNS "-DLABEL_CN".
Type “ased”, adjust power length and power level for 1H. If sample is labeled, you will need to check 15N and/or 13C power level and power length according. Or type "getprosol 1H A B" to set power related parameters. A is the pulse length in µs, and B is the power leverl in dB.
Type "pulse" to calculate pL10 based on targeting p6 value. Use p6 35 us.
Set d9 (Tocsy mixing time) 90 ms for a protein sample; set pL32 70dB for water presaturation
In gs mode, adjust O1P and p12 to achieve minimum FID area integral for water suppression.
Start the experiment by rga and zg.
Process the FID by typing “xfb”. Phase the spectrum when necessary.
Go to an existing 2D-Noesy experiment (pulse program: noesyesfbgpph), type “edc” to copy its parameters and generate a new experiment number. Or in a new experiment, "rpar noesyesfbgpph".
*Pulse program noesyesfbgpph is based on noesyesgpph and have f2 and f3 channels included.
Type "edasp" to check and adjust spectrometer routing.
Type “eda”, set parameters td, ns, ds, sw, O1P, O2P and O3P if necessary.
In acquisition window, if sample is unlabeled, leave ZGOPTNS blank. If sample is N15 labeled, set ZGOPTNS "-DLABEL_N". If sample is N15 and C13 labeled, set ZGOPTNS "-DLABEL_CN".
Type “ased”, adjust power length and power level for 1H. If sample is labeled, you will need to check 15N and/or 13C power level and power length according. Or type "getprosol 1H A B" to set power related parameters. A is the pulse length in µs, and B is the power leverl in dB.
Set d8 (NOESY mixing time) around 90 ms for biomolecules, or 300-500 ms for small molecules; set pL32 70dB for water presaturation.
Users may consider using 2D-ROSEY for medium size molecules,
MW 1000-3000 Da, mixing time 200ms.
In gs mode, adjust O1P and p12 to achieve minimum FID area integral for water suppression.
Start the experiment by rga and zg.
Process the FID by typing “xfb”. Phase the spectrum when necessary.
Your may copy a data set from "expts" folder, or use rpar command to import a paramenter set from the bruker library.
Make sure the constants (cnst21, cnst22, cnst23) are set consistently through out all your 3D Triple resonance experiments, and also set O2P the value suggested in the pulseprogram. If one constant is different from one experiment to another, the resulting spectra may show discrepancy in carbon chemical shift of the same signal. Note that that discrepancy may not be correctable.
Reference values: CNST21=173, CNS22=53, CNST23=42
It is highly recommended to run a 2D 1H-13C plane of the 3D experiment first, just to make sure all the paramenters are set correctly. Signals in HNCA, HNCOCA, HNCACB, and CBCACONH should overlap well. Signals in HNCO and HNCACO should overlap well.
To process 3D data, check Fourier Transform REVERSE settings by looking at the processed 2D plane. Sometimes the default "FALSE" setting can be wrong resulting in a reversed spectrum. In general the 2D plane should be extracted and processed first, and then the optimized processing parameters are used to process the 3D data.
The best application of STD-NMR is ligand/drug screening, to identify ligand(s) that interacts with the host from a compound pool (multiple compounds) . In this system, the host is usually larger molecules, such as proteins and nucleic acids.
Samples: (using a DNA sample and one ligand as an example)
NMR Procedure:
NMR result:
The BBFO probe has only two coils (two channels). One is designated to 1H, and and the other is adjustable to various nuclei from 15N to 31P. Therefore, the BBFO is limited to 1D and 2D expeirments.
1H-15N HSQC. Set channel F2 instead of F3 to 15N. Use Bruker dataset HSQCETGP_15N (rpar HSQCETGP_15N) or modify a 13C HSQC experiment.
BBFO Probe Experiment library. Users can use those experiments as templates to set up their own experiments.