NMR Sample



Sample quantity
•NMR has intrinsically poor sensitivity
•Concentration
–Simple spectra (proton 1D) ~ 50 μM
–Multidimensional spectra ~0,5 mM
•Sample tubes
–5 mm, 550 μl
–3 mm, 150 μl
–Shigemi 5 mm, 250 μl
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Solubility and stability
•Aggregation, precipitation
•NA better soluble than proteins
•NMR measurements long
–Run a control spectrum before and after a long experiment (1D, 1H-15N HSQC for labeled proteins)

Molecular weight
•Increasing molecular weight
–Complexity of the spectrum – number of lines, overlap
–Increasing linewidths
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•Remedies
–Isotope labeling
–Multidimensional spectra
–TROSY, MQ

NMR tube
•Choice
–Volume
–Quality (geometry)
•Cleaning
–High quality tubes are expensive, not for single use!
•Drying
–No high temperatures above 50⁰C
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Concentration, solvent exchange
•Lyophylization
–May harm samples (esp. proteins)
•Ultrafiltration
•Gel filtration
•Ultracentrifugation
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Isotope effect, H2O vs D2O
•No D2O is 100%
•Viscosity
•Melting/freezing point of D2O is 3.8⁰C
•pH
–Glass electrode shows 0.4 units difference (7.0 in H2O, 6.6 in D2O), pH* value

Sample parameters
•pH
•Ionic strength
–NMR probes (esp. cryoprobes) do not like high salt concentrations (above 100 mM)
•Buffer type
–Preferably buffer with no NMR signals
•Concentration
–Oligomerization, precipitation, high viscosity
•Temperature
–Sharper lines at higher temperatures, but not for exchangeable protons
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Chemical shift reference
•Internal
–Add reference compound to the sample
•External
–Capillary within the sample tube
•Direct
–Reference compound measured together with the sample
•Indirect
–Reference compound measured separately or the chemical shift is calculated
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Common reference compounds
Wishart et al. (1995) , J. Biomol. NMR 6, 135-140.
•1H
–TMS 0.00 ppm, not soluble in water
–TSP around 0, varies slightly with pH
–DSS 0.00 ppm
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•13C
–TMS 1.7 ppm, not soluble in water
–TSP around 0, varies slightly with pH
–DSS 0.00
•15N
–NH3 0.00 ppm, bio
–CH3NO2 (neat) 381.7, chemistry
•31P
–H3PO4
TMS
tetramethylsilane
DSS
Sodium trimethylsilylpropanesulfonate
(dimethyl silapentane sodium sulfonate)
TSP
Trimethylsilylpropanoic acid

Contamination
•Paramagnetic contamination
–Broadens the lines, destroys resolution
–Cu ,Mn, Cr, Fe, Co
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•Microbial contamination
–Bacteria can eat-up your sample
–Work clean, add sodium azide (toxic!), EDTA
–Bacteria do not strive in 100% D2O
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