Accurate analysis of your NMR spectra with Chenomx NMR Suite requires preparing good samples and acquiring high-quality spectra.
The “Amicon Ultra-0.5 Centrifugal FilterTube” contains trace amounts of Glycerol. If this material interferes with analysis, rinse the device with Milli-Q water before use. Centrifuge the filter tube for 5 minutes at 13,000 rpm or 13,793 rcf. Remove the water from the filtrate receiver (bottom half of the microcentrifuge filter tube) and discard. Repeat steps 2-4 four times to ensure removal of the glycerol preservative.
Caution: Do not allow the membrane in filter devices to dry out once wet. If you are not using the device immediately after rinsing, leave fluid on the membrane until the device is used.
Most types of samples like biofluids, tissue culture extracts and cell lysates (except urine) have molecules larger than 500kDa. In those cases we recommend filtering macromolecular components by using filter tubes before acquiring spectra from the samples. Pipette the sample into the sample reservoir (upper part) of the prewashed microcentrifuge filter tube.
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The reservoir holds a maximum of 500 μL. Because of this limitation, most samples will require two filters for efficient collection of filtered samples. Centrifuge the sample for 30-60 minutes at 13,000 rpm or 13,793 rcf.
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Some samples may need more time in the centrifuge. Centrifuge until you get the maximum amount of sample possible accumulated in the filtrate receiver. Record the time in your laboratory notebook.
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The final filtrate should ideally be clear of any coloration. In most cases, coloration is the result of a filter breakdown, in which case the filtrate should be re-filtered through a new filter.
Transfer the filtered sample into a labeled Eppendorf tube using a pipette.
Your samples must contain an internal standard for accurate quantification using Chenomx NMR Suite. The internal standard is used as an internal reference to calibrate the concentration and chemical shifts of the analytes.
The internal standard solution recommended for use with Chenomx NMR Suite is prepared by the following procedure:
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To the desired volume of 99.9% D2O, add 5 mM (DSS-d6) 3-(Trimethylsilyl)-1-propanesulfonic acid-d6 sodium salt solution as a chemical shape indicator (CSI) and 0.1% w/v sodium azide (NaN3) to inhibit bacterial growth. D2O is used to help with the lock.
Remember that DSS is hygroscopic in the solid form. For accurate concentrations from a Chenomx NMR Suite analysis, you must determine the final DSS concentration accurately.
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For optional pH measurement in Processor, add one or more of 100 mM imidazole, 100 mM creatinine and 20 mM DFTMP.
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Adjust the resulting solution to about pH 6.5.
Add the internal standard as a 1 in 10 (10% v/v) spike to each of your samples. For example, for a 700 μL NMR analysis preparation, add 70 μL of Chenomx ISTD to 630 μL of acquired sample or an ISTD volume equivalent to 10% of the total sample volume to each of filtered samples. Vortex the sample for 30 seconds for uniform mixing. Transfer the sample containing Chenomx ISTD into a NMR tube. It is recommended to add 185 μL and 560 μL into 3mm and 5 mm NMR tubes, respectively.
Tips and Tricks
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To adjust the pH of the sample solutions, you can use a buffered internal standard solution.
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You can purchase the internal standard solution directly from Chenomx.
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DSS is recommended for your CSI, but 3-trimethylsilylpropionate (TSP or TMSP) or formate are also acceptable. Please note: TSP or formate, as their chemical shifts are sensitive to pH.
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For accurate sample analysis with Chenomx NMR Suite, the pH of your samples should be between 4 and 9, and ideally close to 7 for manual analysis.
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For using “Spectral Binning” feature, it is recommended to adjust the pH of all of the samples at a specific number to have an accurate analysis
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If you choose not to add any pH indicators to your samples, you still need to supply a measured pH value for proper analysis with Chenomx NMR Suite (see “Calibrate pH”). More accurate pH values for your samples allow you to make better use of pH-sensitive signatures to profile spectra of the samples.