Table of Contents

  • Notices and Trademarks
  • Open this section 1. Introduction
    • Open this section 1.1. Installing Chenomx NMR Suite
      • Windows (64-bit)
      • Windows (32-bit)
      • Mac OS X
      • Linux (64-bit)
      • Linux (32-bit)
    • 1.2. Running Chenomx NMR Suite
    • 1.3. Software Updates
    • 1.4. Tutorial and Sample Files
  • Open this section 2. Handling Samples and Spectra
    • Open this section 2.1. Sample Preparation
      • Preparing Filter Tubes for Sample Filtration
      • Filtering the Sample through Microcentrifuge Filter Tube
      • Internal Standard Solution
    • Open this section 2.2. Spectral Data Acquisition
      • Pulse Sequences
      • Required NMR Parameters
    • Open this section 2.3. Spectral Data Processing
      • Data Processed with Other Software
  • Open this section 3. Common Tasks
    • 3.1. Processing a Spectrum
    • 3.2. Profiling a Spectrum
    • 3.3. Identifying Compounds
    • 3.4. Profiling Overlapped Regions in a Spectrum
    • 3.5. Determining Compound Concentrations
  • Open this section 4. The Basics
    • 4.1. Opening Files
    • 4.2. Importing Spectra
    • 4.3. Saving Files
    • 4.4. Closing Files
    • 4.5. Exiting Modules
    • 4.6. Undo and Redo
    • 4.7. Change Columns
    • Open this section 4.8. Sidebar
      • Legend
      • Files
      • Reference Cards
      • Spectrum Details
      • Processing History
      • Compound Sets
      • Simulation
    • 4.9. Spectrum Overlays
    • Open this section 4.10. Spectrum Graph Tools
      • Show Entire Spectrum
      • Set Zoom
      • Undo/Redo Zoom
      • Auto Zoom
      • Increase/Decrease Vertical Zoom
      • Increase/Decrease Horizontal Zoom
      • Spectrum Graph
      • Spectrum Thumbnail
      • Select Region
    • 4.11. Export Spectrum Image
    • 4.12. Copy Spectrum Image to Clipboard
    • 4.13. Display Options
    • 4.14. Preferences
  • Open this section 5. Processor
    • 5.1. Overview
    • Open this section 5.2. Processing Tools
      • Line Broadening
      • Phase Correction
      • Baseline Correction
      • Shim Correction
      • Region Deletion
      • Reverse Spectrum
      • Batch Process
    • Open this section 5.3. Calibration Tools
      • Calibrate CSI
      • Define Custom CSI
      • Calibrate pH
      • Calibration Tool
    • Open this section 5.4. Information Tools
      • Spectrum Details (Sidebar)
      • Processing History (Sidebar)
    • Open this section 5.5. Importing and Exporting Data
      • Batch Import
      • Send to Profiler
      • Export as JCAMP-DX
  • Open this section 6. Profiler
    • 6.1. Overview
    • Open this section 6.2. Profiling Tools
      • Compound Table
      • Custom Colors
      • Pinned Compounds
      • Starred Compounds
      • Remember Selection
      • Quick Searches
      • Concentrations and Compound Fits
      • Fit Automatically
      • Concentration Fitting and Optimization Tool
      • Enforce Transform Windows
      • Scale Concentrations
      • Batch Edit
      • pH Tool
      • Compound Snapper
      • Batch Fit
    • 6.3. Cluster Navigator
    • Open this section 6.4. Compound Set Tools
      • Compound Sets (Sidebar)
    • Open this section 6.5. Importing and Exporting Data
      • Batch Export
      • Export Compound Table
      • Export Profiled Compounds
      • Import Profile
      • Send to Processor
    • 6.6. Spectral Binning
    • Open this section 6.7. Information Tools
      • Legend (Sidebar)
      • Reference Card Panel (Sidebar)
      • Spectrum Details (Sidebar)
      • Concentration Units
  • Open this section 7. Spin Simulator
    • 7.1. Overview
    • Open this section 7.2. Simulating Tools
      • New Simulation
      • Spin Systems
      • Spin Definitions
      • Spin Navigator
      • J-Modifiers
    • Open this section 7.3. Information Tools
      • Spin Definition (Sidebar)
      • Simulation Details
  • Open this section 8. Compound Builder
    • 8.1. Overview
    • Open this section 8.2. Building Tools
      • New Compound
      • Import Simulation
      • Adding Peaks
      • Selecting Peaks and Clusters
      • Delete Selected Peaks
      • Adjusting Peaks
      • Grouping Peaks as a Cluster
      • Optimize Selected Peak Shapes
      • Generate Cluster for Region
      • Transform Windows
      • pH Sensitivities
      • Manage Cluster IDs
      • Cluster Navigator
    • Open this section 8.3. Information Tools
      • Reference Card Panel (Sidebar)
      • Compound Details
      • Information Panel
    • 8.4. Compound Fit Style
  • Open this section 9. Library Manager
    • 9.1. Overview
    • Open this section 9.2. Library Tools
      • Compound Table
      • Quick Searches
      • Add Compounds
      • Export Compounds
      • Send to Compound Builder
      • Remove Compounds
      • Update Compounds
    • Open this section 9.3. Compound Set Tools
      • Compound Sets (Sidebar)
      • Automatic Compound Sets
      • Compound Sets
      • Smart Compound Sets
      • Rename Compound Sets
      • Remove Compound Sets
    • Open this section 9.4. Reference Card Editor
      • Required Details
      • Optional Details
    • Open this section 9.5. Information Tools
      • Reference Card Panel (Sidebar)
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Chapter 2. Handling Samples and Spectra

Accurate analysis of your NMR spectra with Chenomx NMR Suite requires preparing good samples and acquiring high-quality spectra.

2.1 Sample Preparation

Preparing your samples properly helps you get the best possible results from your analyses with Chenomx NMR Suite.

Preparing Filter Tubes for Sample Filtration

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.

Filtering the Sample through Microcentrifuge Filter Tube

Most type of samples like biofluids, tissue culture extracts and cell lysates (except urine) must be purified of macromolecular components by using filter tubes. Pipette the sample into the sample reservoir (upper part) of the prewashed microcentrifuge filter tube.

  • 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.

  • 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.

  • 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.

Internal Standard Solution

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:

  1. 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.

  2. For optional pH measurement in Processor, add one or more of 100 mM imidazole, 100 mM creatinine and 20 mM DFTMP.

  3. 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

  • To adjust the pH of the sample solutions, you can use a buffered internal standard solution.

  • You can purchase the internal standard solution directly from Chenomx.

  • Instead of DSS, you may use 3-trimethylsilylpropionate (TSP or TMSP) or formate as a CSI. However, we do not recommend TSP or formate, as their chemical shifts are sensitive to pH.

  • 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.

  • 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

  • Spectra that are poorly collected or collected from samples that are poorly prepared may be difficult or even impossible to convert, process, and analyze accurately.

  • 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.


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Last modified: 30 Jun 2021