Hi Folks. I’ve been away from blogging while enjoying the holidays but now I am back. I’m going to start off the New Year talking about a pet peeve of mine: FTIR myths. These are totally unfounded pieces of “wisdom” that I hear all too frequently from FTIR users, and worse yet from people who have never touched an FTIR. After looking at the title of this blog post you may be asking yourself, “Where’s Debunking FTIR Myths I?” It’s already been written, but was not given the proper title because at the time I did not realize this was going to become a series. The first entry in this series was my most recent blog post originally entitled “FTIR vs. GC-MS Smackdown.” It is now called “Debunking FTIR Myths I: “FTIR Can’t Identify Things in Mixtures.” Go read it now if you haven’t already.
This post’s myth has to do with spectral subtraction. Spectral subtraction makes use of specialized software to simplify mixture spectra and make mixture analysis easier. The attached figure illustrates the utility of spectral subtraction. The bottom spectrum in red is of the amino acid glutamine dissolved in water, and the middle spectrum in blue is of pure liquid water. The glutamine peaks in the bottom spectrum are very small and masked in part by the strong, broad absorbances of liquid water. It would be hard to tell from the bottom spectrum whether there was anything dissolved in the water at all. Using a spectral subtraction program I subtracted the spectrum of pure liquid water, known as the reference spectrum, from the sample spectrum. The subtraction result, in green, is at the top of the figure. The water peaks have now either been removed or greatly reduced in size, simplifying the mixture spectrum. The glutamine peaks are now apparent for all to see. It would be difficult or impossible to identify the glutamine in this sample without the use of subtraction.
The criticisms I hear of subtraction are that its “arbitrary”, that you are “adding something” to the spectrum when subtracting or that you can “make the result say anything you want.” This is all baloney. Spectral subtraction is like anything else in life, if it is USED PROPERLY it has great utility and legitimacy of purpose. The “arbitrary” critique probably stems from the fact that subtraction involves user interaction. Most of the time when two spectra are being subtracted from each other their absorbances are different because of differences in pathlength or concentration between the two samples. To compensate for this the absorbances of the reference spectrum are multiplied times a number called the “subtraction factor” or “scale factor” which adjusts the reference spectrum absorbances so they match those of the sample. When the subtraction is performed the reference spectrum absorbances subtract out. There is nothing arbitrary about adjusting the subtraction factor; there are rules and procedures to follow that are clearly described in books and training courses on FTIR (including my own). If done properly, there is nothing arbitrary in the setting of the subtraction factor.
The “adding something” myth is the most ridiculous. Subtraction and addition are completely opposite mathematical operations. You can’t “add” something to a spectrum by subtracting something from it; nothing is added during a spectral subtraction. The “you can make a subtraction say anything you want” criticism is the most damning, and the most wrong. If performed properly a spectral subtraction will allow you to more clearly see that which was already present in the sample. In the attached example by following proper procedures I was able to decide upon the best subtraction factor (0.96 in this case), and the result is that the glutamine peaks that were difficult to see before are now obvious. I didn’t make the result say anything I wanted, the result simply shows me more clearly that which was already present in the spectrum.
In the hands of a properly trained user spectral subtraction is a legitimate tool to simplify mixture spectra and make mixture analysis easier. Books and training courses to help you to perform subtractions properly exist (www.spectros1.com), so there is no excuse for not using subtraction in your work, and there is certainly no excuse for badmouthing a useful spectroscopic technique.
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