Hi,
I just started to do some peak-fitting for my IR spectra of proteins. I am using the technic described in the article ' Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca2+- ATPase', where the basic steps are :
1) calculate the second derivative of the absorbance spectrum, and take note of the band number and positions
2) do the peak-fitting by setting the bands at the wavenumbers found in 1), and adjust manually or automatically the fitting (I am using Grams AI)
3) once I have a 'clean' coherent fitting, I calculate its second derivative, and compare it to the second derivative of the experimental absorbance spectrum.
What do you think about this technic ? I do not intend to have my work rely only this peak-fitting, as I know some people think that it has flaws, but with a rigourous method such as this one, I should be fine right ?
I attached a figure of the result for one of my spectrum.
Gray and blue spectra : fitting and its second derivative
black and red : original spectrum and its second derivative.
Please let me know what are your thoughts about that !
If you want to learn a little bit more about peak fitting and its connection to the much more advanced method called "dispersion analysis", I suggest: Article Quantitative Evaluation of Infrared Absorbance Spectra - Lor...
The best way to check the result is to use the gained optical constants to simulate an indipendent experiment and to compare modelled and experimental spectrum, but this is certainly not possible with the primitive implementations in spectroscopic software...
Benjamin,
The method you suggest is Ok to start with. However, it must be applied with care, as a subjective decision making ("manual tuning", for example) needs to be made during the analysis. To improve your analysis results and prevent you from flaws, there are the following recommendations (sorry, they are too general):
- If possible, make clear and scientifically backed up criteria for the manual tuning of the spectra.
- Investigate what happens with your spectra, spectra fits, and your scientific conclusions, if you intentionally moderately abuse your spectra analysis procedure. You must be sure that your procedure and scientific conclusions are "robust enough for your purpose", i.e. your scientific conclusions do not change if spectra tuning procedure is performed in a slightly different way.
- if possible, use an alternative techniques to verify your IR conclusions.
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