Friday, 22 September 2017

ELNs and the importance of life analytical data

Setting the scene

Over the last 25 years, during my bachelor's degree, PhD, Post Doc, and now as director of R&D at Mestrelab, I have had the opportunity to interact with many organic chemists. Most of them, although with their own singularities, share relatively similar procedures and workflows, with their strengths and weaknesses. I have witnessed many advances in the way they conduct their research, but I also must say that there are some areas of it that remain firmly rooted in the past.

An example of the latter which I’m still seeing in many labs is the issue of data loss: In the particular case of academia, research teams are typically made up of (pre)doctoral or postdoctoral students whose residence time is usually between 3 and 8 years, roughly speaking.

During that period, they produce an enormous amount of spectroscopic data (NMR, GC/LC/MS, UV/IR, etc.) to characterize their molecules. Whilst some groups have some sophisticated IT infrastructures equipped with either in-house or third party DBs (including Mnova DB for analytical data), I think it is not unreasonable to say that most of them save their spectroscopy data on their personal computers (e. g. laptops) or in shared folders of their research group (e. g. Dropbox). Data leakage is the result as students leave.

If you're a principal investigator, I'm sure you've found yourself in the following situation: one of your students synthesized a compound some time ago. However, for some reason, you are now considering the possibility that the proposed structure may not be the right one. Obviously, to review this structure, you need to have access to the original spectroscopic data, but unfortunately, the student is no longer part of your research group and you have no way of locating the NMR spectra.

In the same plot line, some students only keep the spectroscopic data of the products that they have successfully synthesized but discard the data of those reactions that did not work in the way they had planned.

These are just two examples of what I consider to be a more general problem associated with the difficulty of efficiently managing analytical information in an organic chemistry laboratory.

Nowadays, many labs are moving from paper-based to electronic laboratory notebooks (ELNs) that offer significant benefits for long-term storage. However, most of them lack the capability to understand and handle spectroscopy data in an integrated manner. Some of them are just repository of PDFs of analytical data generated by some specialized software. This is, in my opinion, a very limited, unproductive and inefficient solution to the extent that data generated in this form has been dubbed as “dead data” where all the valuable spectroscopy information has been removed, reducing it to a series of unstructured set of images and text strings. As it is stored today, analytical data is virtually unusable and tasks like the ones listed below are simply impossible to perform:

  1. NMR data could have been processed incorrectly making a comprehensive analysis of the data unfeasible.
  2. Only some parts of the spectrum could have been reported or the resolution is too low to characterize a compound unambiguously. For instance, accurate determination of coupling constants, inspection of possible impurities or side products in a reaction would not be possible.
  3. Spectroscopic data search: Do I have any spectrum that contains a triplet at 3.5 ppm? This is a question that could not be answered with dead data.
  4. Do I have any spectrum similar to this one?

Some ELNs, in addition to PDF or plain images, also store raw data but do not offer a solution with real spectroscopy intelligence capabilities within a searchable and homogeneous environment.

Mbook 2.0: A spectroscopy-aware ELN

Our ELN, MBook 2.0 is our answer to those issues. It has been designed to take advantage of all the power of Mnova which is tightly integrated with Mbook and is responsible for processing the analytical data acquired by the chemist. The scientist only needs to send the data in a zip file and Mnova will automatically recognize the file format (NMR data such as those from Bruker, JEOL, Varian / Agilent, Magritek, Thermo picoSpin, Nanalysis as well as many LC/GC/MS and UVIR files) and process in a fully unattended way. As a result, a new Mnova document is generated on the fly and saved into the ELN.

This file can be accessed and viewed directly from within Mbook with a new spectral viewer which provides basic navigation tools such as zoom-in and out.  

At this present time Mbook 2.0 does not include spectral search capabilities, but we expect to offer this feature shortly once the integration of Mbook with Mnova DB is completed

Saturday, 5 December 2015

Stanning: A new NMR apodization function

Apodization refers to the mathematical processing technique by which the FID is multiplied pointwise by some appropriate function in order to improve the instrumental line shape. The term apodize actually derives from its Greek meaning “removing the feet”. The feet being referred to are actually the side-lobes found in the FT spectrum resulting from zero-filling a truncated FID (this phenomenon is also known as leakage).  
Probably the most widely used apodization function in NMR, especially in 13C spectroscopy, is the Exponential function although other functions such as Hanning are also very popular.
In this short post, I want to introduce a new apodization function, the so-called Stanning function which gives superior results compared to Exponential and Hanning apodization functions.
The name Stanning is a play on words which combines Hanning (which forms the basis of this function) with Stan, the inventor of this apodization function to whom all credit should be given.
The performance of this apodization function is illustrated with a 19F NMR spectrum whose FID is shown in Figure 1.

Figure 1

This FID consisted of ca 59K acquired data points which are then extended by zero filling to a final size of 128K. As the FID has not fully decayed to zero during acquisition, resulting FT spectrum will show the expected truncation artefacts, as shown in Figure 2.

Figure 2
Multiplication of the FID by an exponential function, in this case with a line broadening value of 1.0 Hz results in the following spectrum where the wiggles have been significantly reduced but not in a totally satisfactory way (see Figure 3).

Figure 3

Application of the new Stanning function yields the result depicted in Figure 4. As it can be seen, the truncation artifacts have been further reduced whilst the resolution of the spectrum is slightly better compared to the exponential function.

Figure 4

The mathematical formulation of Stanning as well as some additional illustrative examples will be covered in a future blog post. 

Saturday, 2 May 2015

NMR for iPad and Android: Beta testing

We at Mestrelab are delighted to announce our first iPad / Android app ever, Mnova Tablet. You won’t find it in the google or iPad stores though as it is still in the final Beta testing stage, but from these lines I’d like to welcome anyone willing to test it out. 
Just send me an email at and I’ll be more than happy to give you the details on how to Beta test it for the platform of your choice

There is also an article in Magnetic Resonance in Chemistry which describes the main features of the app and how it was developed from a more technical point of view.


The beauty of this app is that it provides a very simple and enjoyable mobile experience for NMR data processing and viewing, not to mention the fact that it’s free, at least for the basic functionality. This is how it works:

The free version reads all NMR data (including molecules) supported by Mnova (meaning that virtually all NMR data files will be supported) and transform the raw NMR data automatically, if need be. It also allows basic graphical manipulations, including zoom-in, panning, and spectral intensities expansions.
On the other hand, in order to edit or change any processing operations (apodization, phase, baseline, etc) or apply any analysis (peak picking, integration, multiplet analysis), it will be necessary to pay a small fee via in-app purchases in the Google or Apple stores. More details about this as soon as the official release becomes available.

Key features

  • Automatic processing of 1D and 2D NMR data sets in multiplet formats (Bruker, Varian/Agilent, Jeol, Magritek, Oxford Instruments, Nanalysis, Thermo picoSpin, amongst others)
  • Support of 1D arrayed experiments
  • Processing of 2D-NUS spectra
  • Dropbox support
  • Ability to import spectra directly from the email client and share the spectra or images to social media

Screen shots

Friday, 10 April 2015

Mbook: A new Electronic Laboratory Notebook that speaks NMR

When we founded Mestrelab back in 2005, our only commercial product was 100% about NMR data processing / analysis. Over these years, our NMR products have matured with an increasing number of features and robustness. At the same time, we have released other products such as LC/GC/MS and analytical DB software.
This week, we have released a new brand product, Mbook: This is an electronic Lab Notebook which we have been developing in collaboration with the Universities of Santiago de Compostela and Vigo, both in Spain.  
There are many ELNs out there already so why have we ventured into developing a new one? The short answer is that we believed that most of the existing solutions lacked a real integration between chemistry (i.e. reactions) and analytical data (e.g. NMR): One of the unique features of Mbook is that it is tightly integrated with Mnova so that any analytical data supported by the latter (1D & 2D NMR, LC/GC/MS) will be automatically handled by Mbook. Technically speaking, Mbook comes with a special version of Mnova which runs in the background. This means that when you upload, for example, and NMR experiment (i.e. raw FID), Mbook will process it automatically for you (via Mnova) so that you will see the processed spectrum automatically in your reaction. Of course, the raw data will always be available should you want to process it differently, either with your Mnova client or with any other NMR processing software.

Another feature worth mentioning is that Mbook has been designed solely and exclusively for synthetic organic chemists. If you do any other type of chemistry, Mbook will not be for you. If you are an organic chemist and you are looking for a new ELN, please give Mbook a try, we will be very happy to hear your feedback!
Oh! And it will soon be available as a native Android and iOS application, and, on that, we think it might be the first of its kind!

Sunday, 14 December 2014

Quadruplet, triplet … so simple?

In the picture below I’m showing the ‘synthetic’ NMR spectrum of Ethanol. It has been synthesized using Mnova Spin Simulation capabilities and the experimental values (chemical shifts and couplings) taken from the NMR spectrum of ethanol recorded at 600 MHz in water, so the OH signal will not show up.

Nothing new under the sun. This is a very simple spectrum where the two observed multiplets seem to follow very nicely the well-known first order multiplet rules that most chemists use on daily basis. In this case, a very simple A3X2 spin system.
But does this mean that this spectrum is actually composed by only 7 peaks? The answer is, of course not, there are many more peaks! But because of the very limited resolution, most of them are not observed and merge in such a way that only 7 peaks are ultimately observed.
In other words, the number of NMR transitions is usually much larger than the number of peaks we actually observe in the spectrum. Just to give an example: A molecule containing 30 coupled protons will result in a spectrum having 16106127360 (=1.61E+10) transitions. As its corresponding NMR spectrum will show only about 100-200 peaks, that makes it well over eighty million quantum transitions per resolved peak!

For example, let’s magnify the quadruplet and use Mnova unique capabilities to display the individual transitions by simply hovering with the mouse cursor over the atoms in the molecule (CH2 in this case). We can see that there are some ‘hidden peaks’, these are the NMR transitions calculated by diagonalizing the NMR Hamiltonian.

These transitions are so close that they cannot be resolved under the usual NMR resolution conditions. In fact, to separate all these signals, it would be necessary to have a spectral resolution of < 0.01 Hz

Whilst this is far from being feasible experimentally nowadays, it is easy to do numerically. In the figure below I’m displaying the same synthetic spectrum of Ethanol but this time synthesized using a line width of just 0.01 Hz and 1 MB of digital data points. Now the individual transitions can be seen as resolved peaks so in this example a transition will be virtually equivalent to an NMR peak.   

Simply put, an NMR spectrum is just a superposition of all spectral transitions (which can be in the order of millions), transitions compose peaks, peaks group into multiplets, and multiplets compose the spectrum.

The ability of Mnova to show the individual NMR transitions in a synthetic spectrum can be a good teaching tool

For a more theoretical and rigorous discussion on NMR transitions, see A.D. Bain, D.A. Fletcher and P. Hazendonk. "What is a transition?" Concepts in Magnetic Resonance 10 85- 98 (1998) (link)

Saturday, 20 September 2014

From NMR multiplets reports to synthetic spectra

I admit that I was never a fan of the traditional way in which NMR spectra are usually reported in organic chemistry journals, something like:

1H NMR (300 MHz, CDCl3) 7.91 (d, J=8.2 Hz, 2H), 7.31 (d, J=8.2 Hz, 2H), 3.65 (t, J=6.3 Hz, 2H), 3.13 (t, J=6.9 Hz, 2H), 2.95 (p, J=6.9 Hz, 1H), 2.20 (p, J=6.6 Hz, 2H), 1.26 (d, J=6.9 Hz, 6H)

It is not only that there is not a standard format that is strictly followed by all journals. It is also that it does not convey all the NMR information contained in the actual spectrum (reducing a spectrum into a multiplet report results in an irreversible loss of important information) and facilitates the job to those willing to cheat ( see this and this).

Today, in the 21st century, I don’t see any reason why the experimental raw data (i.e. FID+metadata) should not be an integral part of any article where NMR spectra have been used to characterize a chemical structure. In any event, there are millions of articles with NMR spectra in the form of those old fashioned multiplet reports and we thought that it would be a good idea to implement some tools to facilitate the analysis of those reduced spectra.

That is why we developed the Mnova script “Multiplet Report to Spectrum”, a tool which is available in Mnova from the scripts menu:

It is very easy to use: Once this command is issued, you only need to copy to the clipboard your multiplet report from the article (PDF, Word document, etc) and paste it into the Multiplet report edit box at the top of the dialog:

As soon as it is pasted, this application will parse the multiplets and the different fields (chemical shifts, number of protons, multiplicity, solvent, nucleus, etc) will be automatically populated. If for any reason some of those values are not correctly parse, you can manually amend them.
Once you are happy with those values, you can press OK so that Mnova will synthesize a spectrum with those values.

We believe that this is a very useful tool, in particular for organic chemists. It can be used to easily compare an experimental spectrum with a multiplet report from a journal, for example.

Thursday, 31 July 2014

PCA and NMR: Practical aspects

As of version 9.0, it is possible to perform PCA of NMR data sets directly from within the Mnova User Interface without having to resort to third party applications. The basic PCA functionality has been previously covered in this blog (see Chemometrics under Mnova 9 – PCA) and in this entry we are going to discuss in more detail some more practical aspects, particularly on the different binning, filtering and scaling options. 

What follows has been kindly written by Silvia Mari (project leader of the PCA module) and Isaac Iglesias, who programmed this module in Mnova.


Matrix generation from an array of NMR spectra is the core step in chemometric analysis. This procedure involves several options that the user should chose. In this entry we want to focus on the practical aspects concerning matrix preparation from NMR data. Broadly speaking, we can consider three main issues:
  1. Choice of binning method: Sum vs Peak
  2. Filtering or not filtering?
  3. Choice of Scaling strategy

Choice of binning method: Sum vs Peak

When dealing with high resolution NMR spectra it is in general impracticable to work with the entire data points of the spectra which are usually in the order of 32Kb and bigger. The most common strategy used to reduce the number of variables consists in dividing each spectrum in a defined number of regions, the so called bins.  Several binning strategies are available today, from regular binning, where bins have fixed width, to more sophisticated strategies such as gaussian or dynamic adaptive binning [1]. But even for these cases, when dealing with particularly crowded spectra, it usually happens that shifts in peaks close to bin boundaries can cause dramatic quantitative changes in adjacent bins. A good help in solving this problem could come from peak deconvolution strategies.  Generally speaking, a deconvolved peak is a mathematical entity characterized by a chemical shift (frequency), intensity and half-height line width. The integral of a peak can be automatically derived assuming a peak shape (i.e. Lorentzian) and the intensity and line width. For this reason, binning a spectrum of deconvolved peaks reads out virtually completely the problem of bin boundaries as illustrated in figure 1.

 Figure 1 – Binning real peaks versus binning deconvolved peaks

When dealing with an array of NMR spectra, whilst regular binning of a number b of bins over  stacked spectra containing  s spectra will generate a matrix bxs (see figure 2), it is not possible to generate a similar matrix using directly deconvolved peaks (peak list) since the number and position of peaks varies from spectrum to spectrum

Figure 2 – Matrix generation from regular binning or peak list.

To encompass this problem there are two main strategies: (1) provide algorithms for peak alignment over the spectra series, as well as strategies for dealing with missing peaks in order to end up with the same number of peaks and the same peak positions for all the spectra; (2) perform binning over the peak table.

In the PCA module available in Mnova, we adopt the second solution. User can decide whether to use regular binning (Sum) or binning over deconvolved peaks (Peak) from the binning options. An example of better classification is qualitatively represented in figure 3, where score plots are represented for binning using Sum method (panel A) and binning using Peak method (panel B).

Figure 3 – Score plots obtained using same bin width of 0.03ppm; in both cases data were normalized by the sum and pareto scaled. In panel A bins were obtained directly as integration of real spectra; in panel B bins were obtained by binning of the corresponding peak list obtained after global spectral deconvolution.

Filtering or not filtering?

When reducing bin width to approximate spectral resolution, and hence increasing the number of variables, it is generally required to introduce filtering strategies in order to filter out those variables that do not show significantly changes. There are established filtering strategies that are commonly applied to genomics type of data and that could also be successfully used for NMR-based type of data[1].  In the PCA module we have implemented five filtering options, namely: 
  1. Standard Deviation
  2. Median Absolute Deviation
  3. Interquartile Range
  4. Mean Value
  5. Median Value 

In the first three cases a fixed fraction (default 10%) of the bins is discarded (e.g. if the matrix is composed by 100 bins it means that 10 bins are discarded) and the selection is based on the Filter method chosen. In the case of Mean Value or Median Value, user is asked to input a value for the Mean or the Median. By doing so, only bins that display a lower value of the inputted one are discarded. In the following figure, the difference in clustering capability when the filtering is applied or not is illustrated. Finally, it worth noting that very often, NMR data can contain regions which should discarded and included into the so called blind regions; these regions will not be taken into account in the principal component calculation.

Figure 4 - Score plots obtained using same bin width of 0.01ppm; in both cases data were normalized by the sum and pareto scaled. In panel A no filter was applied; in panel B filtering strategy based on Mean Value was applied. A cut-off value of 100 was used.

Choice of Scaling strategy

Scaling is an operation that is performed on the variables (columns) of the matrix. Scaling strategy depends from one hand from the biological information we wish to extract, but on the other hand also on the data analysis method chosen (in our case PCA). As a first approach the so-called Centering is generally applied to every analysis. With Centering all bin values fluctuate around zero instead of around the mean of each bin; therefore Centering is a method that adjusts for differences in the offset between high and low abundant compounds. There are several methods available in literature for scaling [3], and generally centering is applied in combination with these methods. Scaling strategies could be divided in two subclasses:  methods that use data dispersion (such as standard deviation) as scaling factor; and methods that use size measure (such as the mean). For the first group Mnova includes Auto, Pareto and  Vast scaling strategies. For the second group Range and Level scaling are available. Generally speaking, when dealing with PCA analysis, the first group is normally preferred. Figure 5 shows score plot differences between PCA that used Pareto scaling (A panel) in comparison with PCA that used Level scaling

Figure 5 - Score plots obtained using same bin width of 0.05 ppm and normalization by the sum. In panel A Pareto scaling was applied; in panel B Level scaling was applied.


We have focused on some very practical aspects when dealing with PCA analysis. But it is always necessary to think about how good was our experimental design. Quoting Stanley Deming [4] in his overview of Chemometrics of 1986: ”Chemometrics is primarily concerned with the acquisition of data and the extraction of useful information from that data” and again:” In a given situation, it is far better to err on the side of too many pieces of experimental data. If too few data are available, one might not be able to make any conclusion, and the whole set of experiments will have been wasted”.


We are grateful to Dr. Giovanna Musco and Dr. Jose Garcia-Manteiga for providing dataset for testing purposes.


[1] Amber J Hackstadt, Filtering for increased power for microarray data analysis. BMC Bioinformatics 2009, 10:11

[2] Paul E. Anderson, Metabolomics, Volume 7, Issue 2, pp 179-190 (2010)

[3] Robert A van den Berg, Centering, scaling, and transformations: improving the biological information content of metabolomics data. BMC Genomics 2006, 7:142

[4] Stanley N. Deming, Chemometrics:an Overview. CLIN. CHEM. 32/9, 1702-1706 (1986)

Friday, 28 February 2014


Last September, in SMASH 2013, I had the privilege of getting a personal demo from Tim E. Burrow of a cool iPad application he was developing that showed in a very nice way the basics of NMR data processing. This app simulates an FID and shows the effects on the corresponding FT spectrum when different apodization functions, zero filling or other time-domain operations are applied to the FID.

Since that day, I nearly forgot about Learn NMR FID until it was very recently brought to my attention after reading this article in Glenn Facey’s blog.

When I installed this app, I was happy to notice one very nice feature that I had discussed with Tim: The FID can be simulated with two spins (1/2) and add a coupling in such a way that it is possible to see, in an interactive way, the difference between weak (i.e. AX spin system) and strong (i.e. AB spin system) coupling.

I believe this is a great educational tool which I will certainly use in any lecture on NMR processing.  Tim did a great job and I look forward to seeing more cool things from him!

Thursday, 9 January 2014

Chemometrics under Mnova 9 - PCA

(NoteThis entry has been written by Dr. Silvia Mari, from R4R who has helped us to design and implement this module)

Background: spectroscopy and chemometrics

“For many years, there was the prevailing view that if one needed fancy data analyses, then the experiment was not planned correctly, but now it is recognized that most systems are multivariate in nature and univariate approaches are unlikely to result in optimum solutions.”
      Hopke, P. K. (2003). The evolution of chemometrics. Analytica Chimica Acta, 500(1-2), 365–377]
Either we apply analytical chemistry for quality and control or we attempt to a more “system biology” approach for our R&D we do need advanced methods to design experiments, calibrate instruments, and analyze the resulting data. And the “emergence of chemometrics thinking came from the realization that traditional univariate statistics is not sufficient to describe and model chemical experiments”
       Geladi, P. (2003). Chemometrics in spectroscopy . Part 1 . Classical chemometrics, 58, 767–782

With this in mind Mnova 9 now offers to its users a module called PCA which could be found under the main menu “Advanced”. It is the result of our first efforts to include chemometric tools into Mnova and it is meant to give spectroscopist the possibility to interactively work on both stacked spectra and its corresponding statistical plots.

Starting from mid ‘70s where the first paper with chemometrics in the title appeared in 1975 [1], chemometrics has grown up and is now considered a functioning research area in the chemical science. It has expanded widely from its beginnings into a variety of other areas including multivariate calibration, pattern recognition, and mixture resolution and today there are several applications of interest for the NMR spectroscopists [2-5].

PCA module

Principal Component Analysis (PCA) is a procedure which uses orthogonal transformation to convert a set of observations from correlated variables into a set of values of linearly uncorrelated variables (named principal components) [6].

PCA module under Advanced menu is working in two subsequent steps: (1) matrix generation and (2) principal component analysis. The overall workflow can be represented with the following illustration, where general steps available in Mnova are highlighted in blue whilst specific functionalities of this new PCA module are highlighted in yellow.

With the aim to help the spectroscopist to refine and optimize the data matrix to be used for advanced analysis, PCA in Mnova makes it very easy the detection and removal of spectrum outliers, reveal problems in spectral alignment as well as in its phase or baseline. Once the user has properly corrected those regions of interest, the PCA module allows to re-run the analysis, either replacing the previous analysis or creating a new one for comparison.

Interaction with the stacked spectra.

The main effort applied during the design and development could be summarized in one word: SYNCHRONIZATION. PCA plots, PCA tables and stacked plot are always synchronized. By doing so selections of a point in the score plot imply a selection in the stacked plot. 

In the same way, a selection of a point in the loading plots (hence a selection of a variable of the matrix) generate a shadow into the stacked plot according to the bin position and size.

Colors and graphics

When dealing with large dataset, color coding plays a very important role and eventually essential. Even if PCA does not use class definition in its algorithm since it is an unsupervised method, the kind of patterns expected is generally known.
The driving concept here is that colors are assigned on the basis of class belonging. Again, as in the previous section, colors are always synchronized from PCA tables to PCA plots and to stacked spectra as well

Moreover, in the loading plot, the user is allowed to select more than one bin (see flag option in the loading plot table, or multiple selection of table entry using shift or ctrl  key). Visualization of a bin region is obtained with a colored box that is displayed superimposed over the stacked plot. The User can associate different colors to different bins regions

Data filtering and scaling

The results of the analysis depend on the types of filtering and scaling of the matrix that user selects, which therefore must be specified. It can be demonstrated how both factors greatly affect the outcome of the data analysis and thus the rank of the most important variables. PCA module includes several possibilities in terms of data cleaning and scaling.

There is not a general rule in the selection of the type of scaling. For that purposes we recommend the manuscript from van den Berg et. al. [7] which describes extensively how these transformations could improve the information content of the data matrix. Finally, bear in mind that visual inspection and assessment is ultimately one of the most important steps in chemometrics.


We have introduced in Mnova 9 a chemometric module called PCA (Principal Component Analysis). PCA have been shown to be very effective in compressing large volume of noisy correlated data into a subspace of much lower dimension than the original data set. Data pretreatment method is crucial to the outcome of the data analysis. The resulting low dimensional representation of the data set has been shown to be of great utility for analysis or monitoring the system under study, as well as in selecting variables for control or markers of the expected pattern.
The possibility to interactively play with PCA plots and spectra at the same time, and the user friendly interface provided by Mnova will be of great advantages also for spectroscopists that are not familiar with multivariate analysis but would like to learn more and test it.
As has always been for Mnova community, the future of this new first step in chemometrics will be driven by user requirements. For that reason we look forward to get feedback, criticisms, suggestions, comments and lots of requests for future development. So, play with it and have fun at looking at your own datasets from a different perspective!


[1] B.R. Kowalski, Chemometrics: views and propositions, J. Chem. Inf. Comp. Sci. 15 (1975) 201–203
[2] Chemometrics in bioreactor monitoring. Lourenço, N. D., Lopes, J. a, Almeida, C. F., Sarraguça, M. C., & Pinheiro, H. M. (2012). Bioreactor monitoring with spectroscopy and chemometrics: a review. Analytical and bioanalytical chemistry, 404(4), 1211–37. doi:10.1007/s00216-012-6073-9
[3] Metabonomics and chemometrics in food science and nutrition. Kuang, H., Li, Z., Peng, C., Liu, L., Xu, L., Zhu, Y., Wang, L., et al. (2012). Metabonomics approaches and the potential application in food safety evaluation. Critical reviews in food science and nutrition, 52(9), 761–74. doi:10.1080/10408398.2010.508345
[4] Pharmaco-metabonomic phenotyping and chemometrics. Robertson, D. G., Reily, M. D., & Baker, J. D. (2007). Metabonomics in Pharmaceutical Discovery and Development, 526–539.
[5] Metabonomics and chemometrics in drug safety and toxicology. Griffin, J. (2004). The potential of metabonomics in drug safety and toxicology. Drug Discovery Today Technologies, 1(3), 285–293. doi:10.1016/j.ddtec.2004.10.011
[6] Principal component analysis, Svante Wold, Kim Esbensen, Paul Geladi. Volume 2, Issues 1–3, August 1987, Pages 37–52
[7] Van den Berg, R. A., Hoefsloot, H. C. J., Westerhuis, J. A., Smilde, A. K., & van der Werf, M. J. (2006). Centering, scaling, and transformations: improving the biological information content of metabolomics data. BMC genomics, 7(1), 142. doi:10.1186/1471-2164-7-142

Tuesday, 7 January 2014

Chemical Shift, Absolutely!

(Note: This entry has been written by Dr. Mike Bernstein - Thank you, Mike!)

It’s a “given” that for NMR the chemical shift must be reported relative to standard. The most widely used is the 1H signal of tetramethylsilane (TMS) in chloroform, which has an assigned value of exactly zero. This is convention, and we all adhere to it. Correctly referencing 1H NMR spectra is seldom a difficulty, whether we use co-dissolved TMS (or a water-soluble equivalent), or the residual proton signal from the deuterated solvent. Things can get more complex, but this works for the vast majority of us. The chemical shift, d, is defined thus:

Venturing to the “dark side” of NMR – nuclei other than 1H – seldom stretches beyond 13C for most, and a residual solvent signal is very often present that can be used as a secondary chemical shift reference. But beautiful possibilities tempt many of us. Whether you are interested in biomolecular NMR and live and breathe 15N and possibly 31P, or 2H, or an orgametallic chemist with an interest in far more exotic nuclei, each heteronucleus has its charms and challenges. One thing unites NMR of all nuclei: adherence to a convention for chemical shifts. This can be easier said than done, given that some reference materials are difficult to handle, expensive, etc. The chemical shift reference compound for 19F NMR is the banned substance, Freon-11.

Absolute chemical shifts

We get help from a group working under the IUPAC [1] guise for their work in helping us calculate the chemical shift scale for all NMR-active nuclei [2].  That is, they provide us with a standard way to get the chemical shift precisely correct for any and all heteronuclear NMR spectra. That’s amazing - and hugely useful!
So how does it work? Well, it’s quite simple, really. At the heart of the calculation is the absolute frequency of the 1H signal of TMS for your NMR spectrometer hardware (console, probe, etc.) and sample (solvent, temperature, etc.). You need to be able to determine the exact frequency of this reference signal to seven decimal figures, at least. The following equation applies (sometimes expressed as a percentage) and uses a ratio to describe a constant, (Greek capital Xi):

Making it easy with Mnova

We make heavy use of absolute referencing (AR) in Mnova, with the following available:

  • Correctly reference an X-nucleus spectrum when the referenced 1H spectrum is available
  • Apply AR to heteronuclear axes in 2D experiments
  • Allow users to customise the X  values
  • Indirect 1H spectrum referencing using nTMS  for a specific hardware and solvent (locked)

Referencing heteronuclear spectra

Ensure that you have a document having (a) a correctly referenced 1H NMR spectrum, and (b) one or more –nucleus spectra.
Select Analysis è Reference è Absolute reference… and choose the X-axis spectrum/spectra to reference.  

The table of X values

Note that by tapping on the “X values…” button you will be presented with the table of X-nuclei. In the case of 15N, for example, you can choose which reference standard you want to use. By clicking on the blue “+” button you can enter your own, customised value. 

Referencing 2D spectra

When there are 2D spectra in the document then the Absolute reference… selection will reflect this, and allow you to choose which spectrum is used for referencing purposes, and the traces to which this should be applied. Note that you can adjust the referencing of 1H and X-nuclei.

Referencing a 1H spectrum

You can use saved nTMS values to reference another 1H spectrum from the same NMR spectrometer. Start with a correctly-referenced 1H NMR spectrum, and select Analysis è Reference è Edit saved references…  From this dialogue you can add the value for the particular hardware and measurement conditions – solvent, temperature, etc. 

Now, when you select Analysis è Reference è Apply saved reference then the saved value will be used if the criteria are met. 


Absolute referencing is a powerful way to ensure that data are correctly referenced. This is equally important in open-access environments as it is under automation, where it helps processes such as Verify be more robust. 


[1] (a) Harris RK, Becker ED, Cabral de Menzes SM, Goodfellow R, Granger P. Pure Appl. Chem. 2001; 73: 1795
(b) Harris RK, Becker ED. J. Magn. Reson. 2003; 156: 323.