Scroll the Support Page to find the Chenomx User Guide, Standard Operating Procedures, Frequently Asked Questions, and the Contact Form for any Technical Issues.
Chenomx NMR Suite Condensed User Guide
The Condensed User Guide is a 3-page document compiling Sample Preparation, NMR Acquisition, as well as a step-by-step Processing, Profiling, and Reporting Procedure. Reviewing and following these steps prior to Acquiring your NMR Data will significantly improve the accuracy of your results.
The User Guide is also included as part of the software download. Also included in that download is a tutorial to help with training of the use of the software.
Copyrighted and developed by Chenomx.
C - 001: Preparing an AMICON ULTRA 0.5ML- 3KDa Filter Tube for Filtration
C - 002: Filtering a Sample through a 3 KDa Filter Tube v1.1
C - 005: Preparing Plasma, Serum or Cell Growth Media Samples for NMR Analysis
C - 004: Preparing Urine Samples for NMR Analysis
L - 001: Transferring NMR Samples into NMR Tubes v1.1
N- 001: NMR Spectra Acquisition using METNOESY Pulse v1.1
S - 001: Preparing Internal Standard Solution
S - 002: Preparing Quality Control (QC1) solution
S - 003: Preparing Quality Control Test Sample
Chenomx NMR Suite is available for:
Chenomx NMR Suite is only available for 64bit OSes since Java no longer supports 32bit architectures.
and processed data in these formats:
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.
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.
The latest version of Chenomx NMR Suite can handle saved data from all previous versions of the software.
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.
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.
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.
We understand that changes to Staff and Students, as well as Hardware Failures can occur. In these instances, we are pleased to issue replacement licenses for your Chenomx Software at no charge. When submitting a request for a Replacement License, we ask that you uninstall your previous license from your old computer.
Please use the following link to request your replacement license: Replacement License Form
Varian users can download the pulse sequence C code here:
metnoesy
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.
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.
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.
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.
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.
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 the user guide.
noesypr1d
the acquisition parameters should be:
temperature= 25C
solvent: 90% H2O/10% D2O
d1= 1s
d8= 100ms (NOE mixing time)
aq= 4s
Always, especially if no phosphate buffers are used.
If the pulse sequence and acquisition parameters differ from how Chenomx acquired its database. We use noesypr1d with aq=4, d8 (noe mixing time)= 100ms, and d1=1. That has consequences on quantification because of different T1 time recovery. It also has consequences of relative heights between different clusters.
The signature of the compounds that you can see in Profiler from the Chenomx library correspond to those recorded at pH 7.0 For maximum compatibility with the library, the pH of the samples should be manually adjusted close to that value or else a saline phosphate buffer (mix of NaH2PO4 (monobasic) and Na2HPO4 (dibasic)) can be added.
It’s a method that is driven by the most dominant peaks and so does not work very well with bio fluids, where usually the special peak density is fairly high.
We strongly recommend importing raw FID files from within your FID folder.
Chenomx software automatically positions your DSS to 0ppm. If your CSI is not positioned at 0 ppm it is likely you are using a different CSI (TSP for instance). The position of TSP is pH-dependent.
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.
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:
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.
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.
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:
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).
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.
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.
Chenomx NMR Suite has a region deletion feature that allows you to delete any region of the spectrum.
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.
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.
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.
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).
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.
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.
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.
‘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.
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.
It is important to remember to set transform windows for all new custom compounds. This can be done by right-clicking on the cluster in the list, choose edit, and then set a transform limit.
TSP/DSS are used as internal standard to calibrate concentrations.
There is no use technically to profile DSS or TSP, since its concentration must be known and entered directly in Processor, first when importing fids and/or later on using “Calibrate CSI”. For instance if you enter 0.42 mM as concentration for DSS, you will profile 0.42 mM in Profiler.
Chenomx software automatically positions your DSS to 0ppm. If your CSI is not positioned at 0 ppm it is likely you are using a different CSI (TSP for instance). The position of TSP is pH-dependent.
The signature of the compounds that you can see in Profiler from the Chenomx library correspond to those recorded at pH 7.0 For maximum compatibility with the library, the pH of the samples should be manually adjusted close to that value or else a saline phosphate buffer (mix of NaH2PO4 (monobasic) and Na2HPO4 (dibasic)) can be added.
It’s basic chemistry. The resonance frequency of each multiplet is influenced by pH. Each compound has its own pH-dependence range if they have acidic and basic chemical moieties. The direction of the resonance frequency, and how the distance over which they travel with pH is unique to each multiplet. Chenomx has built pH-curves calibration (resonance frequencies as a function of pH) for most of the compounds in the library (and each multiplet has its own pH-curve).
On top of that, the pH will influence the protonation state, which in turn can influence the network of J-couplings, and hence the appearance of multiplets. For instance, at a given pH, the resonance of a given proton may be a single peak, while at another pH, it can be a doublet, etc. This is particularly true for N-H groups and their neighbour protons.
The exchange rate of some protons with the solvent is also pH-dependent. A good example of that is urea. Urea will be completely absent at some pH values even if you have 1M of it.
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:
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.
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.
It is important to remember to set transform windows for all new custom compounds. This can be done by right-clicking on the cluster in the list, choose edit, and then set a transform limit.
Ensure your YouTube settings are set to High Resolution, and click the CC icon for subtitles.
‘Tune up’ your NMR spectra to optimize the Chenomx Profiling results. Includes importing NMR data files, phase, shim and baseline correction, CSI and pH calibration.
Identify and measure the concentration of metabolites within the mixture. Many computer assisted functions, batch management of groups of spectra for studies, binning, export of data for further analysis.
Modify or add new compounds to the Chenomx library.
Support for creating library subsets and adding custom entries to the libraries.
Batching allows you to apply uniform changes to groups of spectra for consistent processing and profiling results.
Pack Files allow you to create lists of compound signatures for easy transfer between different Chenomx users or projects.
Spectrum Overlay allows you to easily compare numerous CNX files at once.
If you already have a valid license and are looking to update Chenomx NMR Suite, click the button below.
