Hi all,
I am having a issue with my FTIR spectra as some of the peaks seem to be inverted (I repeated this experiment in a different day and the peaks seemed to be normal then).
The main problem seems to be the high background "absorption" that the spectrum with the inverted peaks. What you actually measure with ATR is the reflectance, which is supposed to be unity in the absence of absorption. If you convert the reflectance by -log10(R/R0) to reflectance absorbance, for your R0 which is your background spectrum, total reflection need to be assured, which means that your ATR crystal must be clean (and the proper angle of incidence must be chosen, but I assume that your instrument does not allow to vary it). Since you obtained a good spectrum the other day, I assume that you did not make a common mistake which is to use e.g. a ZnSe ATR crystal with an angle of incidence of 45° for a charcoal sample. In case of the latter the index of refraction is too high, so that 45° is too low to reach the total reflection regime. What kind of sample do you have and what is the ATR crystal and angle of incidence of your ATR accessory?
Dear Thomas, thank you so much for your reply! I am new to FTIR, the instrument is not used often, so I am still trying to understand the theory behind it. The instrument is Nicolet 380, with a smart orbit accessory, so, the crystal it's diamond and, from what I could found, the incidence angle is 45degrees.
My sample is composed of skin cells that are glued to tape, so I was trying to measure this directly: the tape with the cells on it.
Also, I have been told the background can be, for example, my tape, so that I could discount it from my sample, so that is what I have done. But when I read online it is said backgroung should be done with nothing on the crystal, so I am bit confused!
And final question, I tried to make some preliminary measurements in water, having water as background, but I got a spectrum that was mainly noise. Is this correct to do? Or are there any references on how to work in liquid with the ATR?
Thank you so much for your help!
Dear Ana, I guess with your samples you should safe, but in ATR I would always use the empty crystal as reference. Then I would measure the unloaded tape (is it sticky? in any way, always clean the crystal) or destilled water and afterwards the tape with cells or the water with analyte. Absorbance is in a first approximation additiv, so I would then subtract from the latter spectra the former and you should obtain good spectra. Concerning the ATR spectroscopy of liquids, this is something I have never done myself, so I hope somebody else can jump in and provide you some references. Maybe you find some under https://www.researchgate.net/profile/Boris-Mizaikoff .
Infrared spectrum is a composite of absorptance and refractive index spectra. Absorptance looks like the usual IR spectrum if measured correctly. However, the refractive index spectrum is like a derivative spectrum. Usual IR spectrum is dominated by the absorptance spectrum despite having the refractive index spectrum contribution. Unfortunately, when the heterogeneous sample, such as hydrophobic powder was made into a KBr pellet or having a poor contact between ATR crystal and sample, the refractive index spectrum contribution increases significantly due to the poor contact at the interface. The absorption peak deformation due to the poor contact at the interface of heterogeneous sample is called the Christiansen effect, that is caused by the existence of air interface. Since your sample has refractive index of a usual material, 1.2-1.4, air is 1.0, and diamond crystal is 2.4, there is a significant reflectance at the interface. Since refractive index spectrum is below the baseline on the left half of the peak and above the baseline on the right half of the peak, you will see a negative absorption (due to the left half of the derivative peak) as a result. To minimize this, you can use an IR transparent coupling liquid on the surface of the ATR crystal. There are two typically used IR transparent liquid, nujol mull and fluorolube used. The former is completely transparent in the 4000-3100 cm-1 region and any other frequency where CH2 groups do not absorb. On the other hand, fluorolube is transparent in the range 4000-1400 cm-1. This coupling liquid will allow you to eliminate the presence of air and thus minimize the refractive index mismatch without being disturbed by the absorption peaks of the coupling liquid in the above frequency range. You should obtain much less spectrum distortion.
Could it have something to do with the blank spectrum? If you record the blank spectrum it is then subtracted from your sample spectra. If you had some residues or contamination still on your crystal it will show up as a negative peak in your sample spectrum.
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