[Matt89]

Hello everyone,

I have a question regarding the use of the mid-IR region to do quantitative analysis.
I was so far able to roughly estimate (quantitative analysis) the protein content of some matrix using a FTIR.

Recently i started to have odd estimates (circa 10% less in amount than before).
Does anyone know what could be the issue. Supplier says heat or humidity is not a factor of variation. What could explain such a variation ?

Thanks

[mjc123]

Can you give some more information about your method and samples? Are your latest samples just lower in concentration than previous ones? Or if they are old samples/standards, have they gone off? Is it the same protein? The same matrix?
Have you checked your wavelength/frequency calibration? Or has the peak maximum shifted in the latest samples? If you are measuring at a single specific frequency, such a shift may cause a change in the apparent calibration. Do you take a full spectrum?
Apart from anything else, how rough is your "rough estimate"? Good enough to be sure a 10% difference is real? Personally I would not call a rough estimate "quantitative analysis".

[Matt89]

Hello,

I'm not a native English speaker so the use of the word "rough" was probably not adapted. I'm more or less within a 2% range with my FTIR method compared to the results from external certified laboratory for protein analysis (most generally Kjeldahl or Dumas method).

I work on a sample type that is a complex heterogeneous matrix, but that does not vary that much in terms of  molecules concentration (lipids, proteins, hydrocarbons, etc... are fairly distributed - we could say it's like having 100 cow milk samples).

It is possible that my latest samples are lower in concentration than previous ones, but i got about 10% lower estimation with my standards as well. They are starting to get old but i keep them in a dried dark place.
I checked the wavelength, and there is no major shift. I got a 3 cm-1 difference for my right and left edges which is, I believe, not a full explanation of the 10% difference.

But what's odd is that I did those analysis again, after the last weekend, and this time I found the same results (+- 0,3%) with my 2 analyzed standards, and a value for my unknown sample 9,8% above the one I considered to be wrong.

I also contacted our supplier which say sunlight and/or humidity is in no case causing trouble to analysis since the instrument has been designed to work properly even in subtropical climate.
I proceeded to do weighing instrument verification, and it seems that the whole deviation was coming from the balance.
I have a technician coming in the next days/week to do some check-up and repairs.
I guess that in the meantime I will have to weigh my samples with an another precision balance. 

[Furanone]

Two points to possibly consider as these affected my FTIR spectra reproducibility.

1) If your FTIR instrument has a compartment where dessicant is kept to absorb internal moisture, then check to see if stones have turned 'purple' colour and if so recharge in oven tp turn back to dry 'blue' colour.

2) If using an ATR attachment on FTIR, the wearing of the ATR crystal from constantly compressing my powder samples caused slight changes in spectra (typically more noise) over time. Have a look at the crystal and see if it is whole or starting to crack and if so replace.

[Corribus]

Hell, ATR spectra can change intensity just depending on how your sample is making contact with the crystal surface.

[Furanone]

Hell, ATR spectra can change intensity just depending on how your sample is making contact with the crystal surface.

Generally speaking, the ATR-FTIR spectra I get from running different powder samples have very good reproducibility (exception, of course, is 600-400 cm-1 range) that would be the envy of most other analytical methods I use. If the powder is coarse grain (many different sugars and salts), I always ground to finer grain as this can definitely cause reproducibility issues.