I acquire 1D 1H NMR spectra of metabolite extract using 100% D2O in 700MHz Bruker Avance set up . Now I am facing an interesting problem.-TSP peak height/ intensity seems to be different in every spectrum. Can you please guide me whether it is accepted or how to solve this? here I am stating how I prepare sample for spectra acquisition-
- I resuspend the dried material in 10mM NaP buffer in D2O.
- I add required volume of TSP to get 30uM concentration from 5mM stock (in D2O) for intracellular sample or 300uM for extracellular sample followed by ultracentrifugation @ 12000 rpm for 5 min.
- I take the clear sup in NMR tube and run it.
I am using same TSP stock every time, and I do tap and give a short spin to have a homogeneous solution.each time I get the spectrum and overlap it with other ones, I see TSP peaks are not exactly overlapping (intensity). And it is necessary to input exact TSP concentration in Chenomx to analyse. Wrong TSP concentration will lead to erroneous quantification.Can you please help me? Another thing is that when I run a standard sample with known concentration, twice with fresh sample (with same composition, method, parameters) there is difference in intensity between two spectra. is it normal or what is the reason behind it?
Your comments will be really helpful for my experiments.
Intensity is always based on your compound. In other words, the peak height is always relative (to your compound's quantity). When the amount of your compound is small, your TSP peak looks high. For example, imagine you have a compound at 30 nM, in your tube A. And another tube (B) has the same compound at 30 pM. Since the concentration of TSP is 30 uM, in a tube A, 9H from TSP : 9H from your compound should be 1,000 :1, while in tube B, it is 1,000,000 : 1. You can change the scale of your vertical axis to fix the height of TSP to a constant, then you can compare the amount of the compounds, each of your samples have.
Thank you Kohei.
but how can I change the scale of vertical axis to fix the height of TSP to a constant ? Do I need to do it every time I acquire spectrum?
Another thing Kohei Torikai , What you said goes for different samples. But when I am running same conc. sample (say 10mM sucrose) two times with fresh solution, then also I can see difference in peak intensity in sample peaks as well as TSP peaks between two spectrum. It should overlap exactly, right ( same conc. sample, same parameters, same magnetic field)? Otherwise, its imposssible to quantify unknown molecules. How can I solve this?
> Do I need to do it every time I acquire spectrum?
Yes, you do.
> how can I change the scale of vertical axis to fix the height of TSP to a constant ?
Please check how to do it by yourself, because it depends on the software you are using.
> I can see difference in peak intensity in sample peaks as well as TSP peaks between two spectrum. It should overlap exactly, right ( same conc. sample, same parameters, same magnetic field)?
Yes it should, only if you fix the constant vertical axis. In NMR, intensity is always a relative value. The only constant value is the ratio. I think there is a standardization function in your software, so I recommend you to ask a software customer center.
Okay, thank you Kohei. I will look into it.
By the way, I use Bruker Topspin software to process spectrum. Do you use the same?
And, can it be done post acquisition or during acquisition?
Hi, This is a very interesting and relevant question for the entire research community working in metabolomics. Please also feel free to register (for free to create a login with your Email ID!) and join the International Metabolomics Society’s Forum: http://www.metabolomics-forum.com/index.php?action=forum;?action=forum#c12 to ask more specific questions any software, database, approach and platform (mass spectrometry, NMR) issues where you can get very specific responses from expert users and researchers! Thanks again, Biswa
- Badges
- Science topic
- Similar topics
- Philosophy
- Ethics