If you can't find the answer to your question on this page,  please contact our support team.

May 3, 2017: We have found that Chenomx NMR Suite is not compatible with the new release Windows Creator. It seems as if Microsoft is delaying the release of Windows Creator for compatibility reasons but some people already have the update. If you have installed that update we ask that you uninstall the Creator version and go back to the previous version of Windows until either Microsoft fixes the compatibility or we find out the issue ourselves and make a new update.

General Technical Questions

On which operating systems can I run Chenomx NMR Suite?

Chenomx NMR Suite is available for:

  • Windows 10 / 8 / 7 / Vista (32 and 64-bit)
  • Mac OS X 10.12 – 10.8.3 (64-bit)
  • Linux x86 (32 and 64-bit)
What version of Java do I need to run Chenomx NMR Suite?
Newer versions of Chenomx NMR Suite (version 7.6 or higher) no longer require that you install Java. If you are using an older version of Chenomx NMR Suite you will need to download and install the latest version of Oracle’s Java Runtime Environment. Note that Chenomx NMR Suite does not support older OpenJDK Java Runtime Environments.
What NMR data formats can Chenomx NMR Suite handle?
Chenomx NMR Suite currently supports raw data in these formats:
  • Agilent (fid)
  • Bruker (fid)
  • JEOL (.jdf)
  • NMRPipe (.fid)

and processed data in these formats:

  • Agilent (phasefile)
  • Bruker (1r)
  • JEOL (.jdf)
  • NMRPipe (.ft2)
  • JCAMP-DX (.jdx, version 5.1 and higher)
  • Mestrelab MNOVA
Which spectrometer frequencies does the Chenomx Compound Library support?

The Chenomx Compound Library supports spectra at 400, 500, 600, 700, and 800 MHz. Chenomx also supports spectra at 750, 850, 900, 950, 1000, 1100, and 1200 MHz with simulated compounds. Custom compounds at other frequencies can be created using Compound Builder. The latest version of the Chenomx Compound Library is included with the software but older versions may be downloaded here.

Can I select multiple files when exporting concentrations?

Yes, in fact many other features such as converting spectra in Chenomx Processor, overlaying spectra and binning spectra in Chenomx Profiler, all have the ability to run on multiple files. In any of these operations, when the file chooser is shown, you can either select a folder, or you can select multiple files by choosing a range of files using the SHIFT key, or specific set of files by holding the CTRL key and clicking.

I have spectra that were profiled using an older version of Chenomx NMR Suite. Are they compatible with the latest version?

The latest version of Chenomx NMR Suite can handle saved data from all previous versions of the software.

How can I license Chenomx NMR Suite for multiple users on a single computer?

Windows: When activating the software with a license file select the “License extends to other users on this computer” checkbox.

OS X 10.5 – 10.6: When activating the software with a license file select the “License extends to other users on this computer”.

OS X 10.7 – 10.8: Before running the software use the Finder to navigate to the “/Library/Application Support” folder. Create a new folder named “Chenomx” inside “Application Support”. The finder will ask you for your password since this directory can only be written to by administrators. Once the folder is created, acitvate the software with a license file and select the “License extends to other users on this computer” checkbox.

Linux: You must run Chenomx NMR Suite as root to license it for all users. Some file managers in Linux will allow you to right click on the application launchers in the installed ChenomxNMRSuite folder and choose “Open as administrator”. If this is not available open a terminal window and navigate to the installed ChenomxNMRSuite folder. Then run the software with the command:

sudo ./Application/ChenomxNmrSuite.sh

Once the software is running under root, activate the software and select the “License extends to other users on this computer” checkbox. After activating the software you no longer need to run it as root.

Can Chenomx NMR Suite be run from a networked location?
While not recommended, it is possible to install and run Chenomx NMR Suite from a networked location. Users running the software this way will not have any file associations and/or shortcuts on their local system.

Chenomx NMR Suite will still check for activation on each local system, when running Chenomx NMR Suite from a networked location. A separate activation key will need to be generated and separate license file will need to be installed on each computer. Individual preferences will also be stored separately on each computer.

While this type of installation is possible, it is recommended and much easier to install Chenomx NMR Suite on each computer separately.

I have multiple users in my lab, can I run Chenomx NMR Suite with a floating license?

Chenomx NMR Suite is licensed on a per seat (computer) basis. Each computer system, will generate it’s own activation code and will need it’s own unique license file. Multiple users in your lab are free to use Chenomx NMR Suite on the same computer system, but if you need to run Chenomx NMR Suite on different systems, then separate licenses will be required.

Processing Spectra

Why does my spectra seem to be shifted by approximately 0.01ppm after processing and calibration? OR When I use TSP as a CSI, why is the TSP peak not at 0 ppm?

The Chenomx Compound Library is based on the characteristics of DSS, since its chemical shift does not change with pH; the chemical shift of TSP does change with pH. When you select TSP as a CSI, your spectra are rereferenced as if they had been acquired using DSS to allow accurate comparison with the compound library.

This results in your spectra being shifted by about 0.01ppm compared to setting the TSP signal to 0 ppm. This may appear odd if you are used to referencing to TSP but it does allow you to accurately calibrate your spectra with TSP and still use our library compounds.

How can I reference spectra that contain no CSI?

We recommend that you add a supported CSI to all samples intended for use with Chenomx NMR Suite (currently, we support DSS, TSP and formate). You should consider adding a CSI to any new samples that you intend to analyze using Chenomx NMR Suite. That being said, you can still extract some information from existing spectra of samples with no CSI.

When you open a spectrum of a sample that contained no CSI, you can now choose “none” as a CSI option. The software will try to automatically determine the best location for reference.

Although, we suggest that you still calibrate the spectrum manually to no CSI by referencing the ppm location to a known peak in the spectrum. In Processor, load your spectrum and calibrate your CSI. While calibrating, you can drag the x-axis at any ppm location that you wish to reference to.

When you have manually referenced spectra using this technique, please remember:

  1. Absolute compound concentrations measured in Profiler will be inaccurate, but relative concentrations will still be reasonably accurate. For example, if you measure acetate at 1 mM and alanine at 2 mM, those numbers will not necessarily reflect the ‘real’ concentrations of those compounds in the sample. However, it would be fair to say that alanine is present at twice the concentration of acetate.
  2. The transform windows may not be optimal for all compounds; you may not be able to move a cluster to exactly where you would like it to be. To fine tune your manual referencing, you can click Jump to Processor, tweak the CSI position in the CSI Editor, and then click Jump to Profiler to continue profiling.
How can I consistently choose reasonable CSI parameters for spectra with no CSI?

In the absence of a supported CSI, you will need to set reasonable values for the position (chemical shift) and width of the lines when calibrating your spectrum with a CSI in Processor.

  1. Referencing the chemical shift to another compound requires knowledge of the expected chemical shift of the alternate compound. Zoom in to this known peak. Drag the x-axis by clicking on the ppm location this known peak is known to resonate at. A red line will appear at the ppm location. Drag the axis until the red line lines up with the known peak.
  2. To set the width, start by setting the CSI width to about 1.2 Hz. Most of the compounds in the Chenomx library were originally fit using spectra with about this linewidth, so this is a good starting point for many high-quality spectra. If you realize while analyzing the spectrum in Profiler that most compound signatures are considerably narrower or wider than the corresponding features in the spectrum, switch to Processor (use the Jump to Processor feature), adjust the CSI width accordingly, and switch back to Profiler (the Jump to Profiler feature lets you do this with the click of a button).

Remember, this technique will not allow accurate absolute quantification, but you will still be able to identify compound. Relationships among concentrations within the same sample will work (you should be able to tell that creatinine was twice the concentration of alanine in sample X), comparisons across multiple samples will work, so long as they were acquired at the same time with no changes to the NMR equipment.

Why do I need to set all of these values for the CSI? I just want to set the chemical shift!

In addition to setting the chemical shift, the CSI in Chenomx NMR Suite allows calculating concentrations of compounds in your compound library. The ‘extra’ information that you need to enter about the CSI is what allows these calculations to occur.

Calculating the absolute concentration of any compound based on its signal intensities in an NMR spectrum requires the following information:

  1. The number of protons contributing to the signal from the compound
  2. The number of protons contributing to the signal from the reference compound
  3. The intensity of a signal from a reference compound
  4. The concentration of the reference compound
  5. The intensity of a signal from the compound

When you are using Chenomx NMR Suite, item #1 is stored in the compound signatures distributed in the Chenomx Compound Library, or any signatures that you might create yourself using Spin Simulator and Compound Builder.

#2 is built into the definitions for the supported CSI compounds (DSS, TSP and formate).

You define #3 and #4 when you process a spectrum in Processor, via the CSI editor. You set #3 when you fit the red CSI line to the spectrum using the CSI Editor, and #4 when you enter a CSI concentration in mM.

Finally, you manipulate #5 as you fit the compound in Profiler.

In short, the concentrations that you determine in Profiler depend on the number of protons (as you might expect), but they also necessarily depend on the concentration of the reference compound (as may be less obvious). It is not possible to calculate absolute concentrations using NMR without involving the concentration of a reference compound (in Chenomx NMR Suite, the CSI).

How can I open JCAMP files exported from MestReNova (MNova) in Chenomx NMR Suite? I keep getting error messages.

The problem described appears in Chenomx NMR Suite v6.1 or earlier. All subsequent releases should handle MestReNova JCAMP files natively.

If you are using an affected version of Chenomx NMR Suite, you can adjust JCAMP files to allow them to be opened. Simply open the file in a text editor (e.g., Notepad, Wordpad, vim, Emacs, etc.), and replace all instances of ‘NMRSPECTRUM’ with ‘NMR SPECTRUM’ and ‘NMRFID’ with ‘NMR FID’ (i.e., insert a space after ‘NMR’). You can use the relevant search and replace functions in your text editor as needed.

If you have a large number of affected JCAMP files, you can use tools like grep (Linux and Mac) or grepwin (Windows) to change the text strings in all of the files in a single operation. Please be sure to backup your data before attempting any large scale changes of this nature.

Can I import plain text files into Chenomx NMR Suite as spectra? Can I use a third-party product to produce Chenomx spectrum files (.cnx) from plain text files?

Chenomx NMR Suite’s .cnx files are proprietary, so there are no third-party products that directly create or read these files. However, the JCAMP-DX file format is stored as plain text. Chenomx NMR Suite does support importing and exporting JCAMP spectrum files.

Can I use the Processor module to delete regions of an NMR spectrum other than the water region?

Chenomx NMR Suite has a region deletion feature that allows you to delete any region of the spectrum.

When I change the line broadening for a spectrum in Processor, then take the spectrum back to Profiler, the library signatures do not seem to reflect the new line broadening. What is happening?

The CSI lineshape is the key to matching library signatures to patterns in your experimental spectrum. The more closely your CSI settings in Processor match a spectrum, the better the library compounds will match while profiling the spectrum.

Specifically, when you make changes to your spectrum in Processor (like adding or changing line broadening), you need to update your CSI settings to reflect the changes. If you are using DSS or TSP as a CSI, you can usually just switch to the CSI Editor in Processor and click ‘Find Automatically’. If the automatic method does not work, simply adjust the red CSI peak to better fit the spectrum, exactly as you would adjust peaks and clusters in other modules.

Profiling Spectra

What is the maximum compound concentration that I can measure in Profiler?

Profiler can measure concentrations up to 5000 mM, or 5000000 μM. If you are using mg/dL as your concentration units, the maximum concentration will vary depending on the molecular weight of the compound, but is equivalent in every case to 5000 mM.

I can change the CSI line width when processing spectra in Processor. Can I similarly change the line width of the signature line in Profiler, setting different line widths for different compounds to get better fits?

In short, no, you cannot change the line width of the signature line in Profiler.

The line width of the signature lines in Profiler is directly calculated from the line width of the CSI that you set in Processor, and is applied equally to every compound that you fit in Profiler. Thus, it is not possible to set different line widths for different compounds.

You may get better results with a different CSI (we recommend DSS), or if you are working with samples that have some protein content, you may want to try filtering your samples using a 3kDa molecular weight cutoff filter to remove the proteins. Also, some variation in line widths may occur due to varying shimming technique during data acquisition, especially if more than one person is involved in acquiring the spectra.

Can I see NMR peak assignments in ppm using Profiler? What about the integrated area under each peak?

Profiler works exclusively with clusters (groups of peaks), not individual peaks. It is possible to see cluster centers in ppm using Profiler. Simply select a cluster and hover the mouse cursor over it; the display in the top right corner of the spectrum includes the center of the selected cluster.

You can measure area under a cluster using the Select Region tool; double-click on the spectrum, then click and drag to select a region. The areas appear in the top right corner of the spectrum. ‘Spectrum Line Area’ indicates the area under the black line (acquired spectrum), while ‘Sum Line Area’ indicates the area under the red line (your current analysis of the spectrum).

When performing spectral binning in Profiler, there are two normalization methods available, total area and standardized area. Which one should I use?

You should select a normalization method based on the nature of the dataset that you are binning. Use total area to reduce the influence of dilution effects among the samples in your dataset; this is the most common scenario. If you can assume that dilution effects are insignificant, use standardized area.

What minimum threshold of peak heights should I consider when profiling a spectrum?

You must decide for yourself what minimum threshold you will consider acceptable for profiling. There is no minimum level of absolute peak heights that applies to every sample. As a rule of thumb, if the peaks are small enough that you question whether the compound is present at all, you will not obtain reliable concentration measurements for that compound.

I expect compound X to be present in most samples for my current study, but Profiler shows a maximum concentration of zero. What’s going on?

The simplest possibility to consider is that the compound in question is just not present. Having excluded that, for example by supplementary analysis confirming that the compound is indeed present, there are some other considerations.

If you are running reconstituted samples in pure D2O, Profiler may erroneously assign some compounds a maximum concentration of zero. In pure D2O, exchangeable protons are completely replaced by deuterium, effectively removing the signal for those protons from the NMR spectrum by reducing its intensity to near zero. When calculating maximum concentrations, Profiler only considers values that do not allow any cluster of a compound to exceed the measured intensity of the spectrum. If an expected cluster is effectively zero intensity, Profiler will predict a maximum concentration of zero for that compound.

Two other factors can also influence Profiler’s calculation of maximum concentration. Incorrect CSI settings can reference the spectrum to the wrong chemical shift or result in Profiler incorrectly interpreting intensity ratios with which it calculates all compound concentrations. Also, incorrectly setting the spectrum pH can result in clusters or transform windows starting in the wrong positions, meaning that Profiler will not use the correct spectrum locations in calculating maximum concentrations.

What is the “Maximum Concentration” column for in Profiler?

‘Maximum Concentration’ is a column in the compound table in Profiler. It is meant
to be used as a guideline to help the user select the compounds that likely have the highest
concentrations in the mixture.

For a given compound, it is the maximum concentration that compound can be set at such that
none of its peak clusters, within their respective transform window will exceed the experimental
spectrum signals. This does not consider potential overlapping compounds so will always be an
amount larger than the final value calculated after fitting all the signals.

Sorting the compound table by this column can provide a helpful order for fitting. Once all the compounds have been fit the maximum concentration for that compound is no longer a useful parameter.

Are there any issues with Glucose quantification?

There are some issues while measuring Glucose concentrations in a mixture.

Glucose exists in solution as 2 anomers (alpha, beta) that are capable of interconverting in solution. In fact all cyclic structures of monosaccharides exhibit anomeric versions. The equilibrium between the 2 forms and therefore their relative proportion is dictated by pH since the interconversion is catalyzed by acid or base.

The signature of all the sugars in the Chenomx library reflect the proportion that exists at neutral
pH and is therefore a weighted average.

We do not have separate entries for the pure forms.

Sample Preparation

What internal standard should I use for my samples?

We recommend (3-trimethylsilyl)​propanesulfonic acid (DSS) as a CSI, at a concentration of about 0.5 mM in the analyzed sample. You may also want to include [difluoro​(trimethylsilyl)​methyl]​phosphonate (DFTMP) as a pH indicator, at a concentration of about 2 mM in the analyzed sample.

How should I acquire my NMR spectra?

You will get the best results analyzing spectra acquired with NMR parameters similar to those used to build the metabolite library. The Chenomx metabolite library was acquired between pH 4 and 9 at a temperature of 298 K (25 °C), with an acquisition time of 4 s and recycling delay of 1 s. The recycle delay includes a 990 ms saturation pulse on water. Our 1D pulse sequence is NOE-based, with a short mixing time of 100 ms. The mixing time also includes a water saturation pulse. The length of the proton 90° pulse (pw) was calibrated to maximize the intensity of the DSS peak.

I received a pulse sequence from Chenomx, and I am not familiar with the name of the pulse sequence you sent. Why did you name it ‘metnoesy’?

The pulse sequence is called ‘metnoesy’ because it is derived from an older pulse sequence of the same name that was originally written for 2D work. In our variant, we do the equivalent of the first increment in the 2D experiment, yielding a 1D spectrum.

In the metnoesy pulse sequence that I received from Chenomx, I noticed that you do not use gradients to improve water suppression. Why is that?

Adding gradients would give somewhat better water suppression, but can introduce distortions if the gradients are not refocused properly. Using gradients also adds more parameters to the setup of the instrument, and we wanted to keep the pulse sequence as simple as possible.

Can I use chemical shape indicators (CSI) other than TSP or DSS? I normally add compound X as an internal chemical shift reference, since TSP interacts with the proteins in the sample.

Chenomx NMR Suite has three predefined compounds for use as a CSI: DSS, TSP and formate.

In addition, Chenomx NMR Suite also allows you to define your own custom CSI. To define your own custom CSI, you must create a calibration spectrum with known quantities of your custom CSI and a known quantity of one of the predefined CSIs.

This calibration to a predefined CSI is necessary because, the CSI serves not only as a shift reference, but also a line shape reference; the shape of the CSI peak is used to correct shimming issues in spectra and to calculate expected line widths for all compounds. The CSI peak is also used as an internal standard for quantification. All of this information must be correctly defined for our advanced library features.

It is possible that DSS or TSP can bind to certain species, typically proteins, causing broadening of the DSS/TSP resonance and complicating the calculation of expected line widths. Methods of compensating for this binding effect are available. For serum samples, ultrafiltration using 3 kDa molecular weight cutoff filters can remove proteins and large lipids, reducing or eliminating their binding effects on DSS and TSP.

When I acquire spectra of blood samples, NOESY spectra have humps in the alkyl region that make it difficult to analyze the spectrum. Is it better to use J-resolved or CPMG spectra to reduce or remove the hump?

The large humps that you see in the NOESY spectra are due to macromolecules in the samples (mostly proteins and lipids). Alternative pulse sequences such as CPMG or J-resolved spectroscopy can help to remove these humps, but the effects of the proteins on the signals of small molecules will still be present. Most notably, you will need to adjust the linewidth of DSS or TSP to obtain reasonable fitting results, as both DSS and TSP bind to protein.

Note that our signature libraries have been calibrated to match the NOESY spectra and are not calibrated to the results from CPMG pulse sequences. While the results will be relative to other results in a particular study the actual values of the concentrations are not always accurate.

For more details on preparing blood serum and plasma samples for analysis using Chenomx NMR Suite, please refer to our application note.

 

Some compound signatures that I see while profiling blood samples have a shape different from the original NMR spectrum. For example, a doublet in the original spectrum appears as a wide singlet in the compound signature.

The most common cause of very broad peaks in a plasma sample is high protein content. Even when you use special pulse sequences like CPMG to reduce the appearance of protein signals, some compounds will still bind to the protein, resulting in broadening of specific signals from the affected compounds. We strongly recommend microfiltration of plasma samples using a 3 kDa molecular weight cutoff filter to remove proteins before acquiring spectra. See here for more details.

Compound Libraries

How can I add compounds to my library?

To add compounds to the library, you need to acquire spectra of the compounds under conditions similar to those you expect to encounter in your experiments. Once you have these spectra:

  1. Process the spectra in Processor and save them as .cnx files
  2. Overlay a .cnx file in Spin Simulator and fit it (see the Spin Simulator tutorial in the User Guide for details). Save the simulation as a .xss file.
  3. Import the .xss file in Compound Builder, and overlay the .cnx file. Refine the fit to match the acquired spectrum (see the Compound Builder tutorial in the User Guide for details). Save the signature as a .xcpd file.
  4. Import the .xcpd file using Library Manager. Make sure that the new compound appears in at least one Compound Set.
  5. Use the new signature in Profiler. Make sure that at least one compound set containing the new compound is selected to see the compound in Profiler.
I have a library in Bruker’s sbase format which has been built using Bruker software. Is there an easy way to import spectra from this library to a Chenomx NMR Suite compatible library?

There is no method available to import Bruker sbase data into a Chenomx library. The Chenomx library format is not simply a collection of raw spectra, but rather a collection of mathematical models (called compound signatures). The models are based on raw spectra, but contain additional information that allows calibrating the models to better match experimental line widths, solution pH, ionic strength, and so on. The tutorial sections of the user guide will help you apply this process to your own compounds, but creating new compound signatures cannot readily be automated, as it requires informed human input.

If you have access to the original spectra, you can create your own compound signatures using the Spin Simulator and Compound Builder modules, as described in the user guide tutorials. You can create a ‘quick and dirty’ set of signatures by generating clusters in Compound Builder, but signatures created this way will be significantly less flexible than properly simulated and calibrated signatures in fitting arbitrary spectra. We recommend using them only in limited testing scenarios, and not for production analysis.

Another option is to let us prepare proper compound signatures via contract services. You can send pure samples of the compounds, or spectra that you have acquired, and have us prepare signature files for you to add to your library.

Why do the Chenomx Reference Compounds not contain DMSO?

DMSO is a popular solvent for use in extraction of metabolites from certain types of samples.   It
has no real biological value as part of the results for metabolomics studies and, therefore, Chenomx does
not have a signature for DMSO in the Chenomx Reference Compounds.