Fourier transform infrared spectroscopy (FTIR) is an extremely sensitive technique for measuring the absorption and intensity of electromagnetic radiation in the infrared region of the spectrum of either a solid, liquid or gas sample.
You can use FTIR to:
- quantify unknown compounds
- identify unknown compounds
- study the detailed structured coordination of compounds
How Does Fourier Transform Infrared Spectroscopy Work?
Briefly, FTIR measures how well a sample can absorb infrared (IR) radiation with high spectral resolution. This allows you to compare “molecular fingerprints” of known or model compounds to those of unknown compounds because the spectral absorptions of IR cause molecular vibrations to occur in characteristic regions of the spectra.
The molecular vibrations correspond to specific bending and stretching modes between chemical bonds that are unique to each molecule. The stretching frequencies of molecules are generally reported in terms of wavenumbers with units of cm-1. For example, free CO has a stretching frequency of 2143 cm-1, but will decrease in stretching frequency as it binds to compounds.
There are specific molecular vibrations that occur in characteristic regions of the spectra as mentioned above, however, no two compounds have the same exact IR frequency since the specific molecular vibration depends on the bonded atoms. Moreover, the intensities of IR signals do not quantitatively correspond to the number of bonds.
However, some rules do apply when using FTIR spectroscopy. For example, if there is a net change in the molecule’s dipole moment the sample is able to absorb the energy and said to be ‘IR active,’ but if the compound doesn’t, it is ‘IR inactive.’ See below for some examples of IR active and IR inactive compounds.
IR Active vs. IR Inactive Compounds
Symmetrical stretches that can produce an IR spectrum:
- Carbon monoxide
- Iodine chloride
Symmetrical stretches that cannot produce an IR spectrum:
- CO2 is inactive* (if there is no change in the dipole moment of the molecule)
Where Does FTIR Take Place?
The basic components of an IR spectrometer are similar to an UV/vis spectrophotometer:
- Source of electromagnetic energy
- Sample chamber
Tips for Preparing Liquid Samples for Fourier Transform Infrared Spectroscopy
Liquid samples are prepared in a liquid cell apparatus, which is essentially a sandwich of two windows (CaF2), lined with a Teflon spacer held together by metal plates and screws in order to load into the sample chamber.
It is important to prepare excess sample for loading to avoid air bubbles. I’m superstitious and tend to always load my samples on one side of the window, in addition to using very concentrated protein (500 µM).
Do not pre-chill samples before purging with a gas like carbon monoxide to prevent any solubility issues.
Equilibrate the sample inside the chamber for at least 10- 15 minutes to purge water vapors and carbon dioxide prior to collecting the data. However, the amount of time necessary for equilibration will depend on the stability of your sample.
Immediately after you collect data from your sample, you should collect a background measurement using only buffer or even just the air inside the chamber. This step helps to correct for any instrumental properties in addition to other light-absorbing/reflecting properties of the buffer.
The resolution of the IR spectrum depends on several conditions, such as: the number of scans collected, purity of the sample, concentration of the sample, and a maximum optical path difference (OPD, which is generally set for the instrument). I tend to collect 1000 scans for each sample, which can increase the resolution or signal to noise ratio.
Loading the sample and preventing bubbles in the middle of the window is probably one of the most difficult parts about this technique and if this goes wrong, you risk ruining the whole experiment. Depending on the cell apparatus, there are several ways of trying to get around this, but the most important one is to have very clean windows to begin with!
Biological Applications for Fourier Transform Infrared Spectroscopy
FTIR is useful for identifying unknown compounds, whether solid, liquid or gas, and is an attractive technique because of its exquisitely sensitive nature. Popular applications for FTIR include, but are not limited to:
- Environmental: for monitoring air quality, water quality, soil contamination
- Forensics: to identify materials at crime scenes, drugs, counterfeit goods
- Pharmaceuticals: formulation and development of drug compounds
- Biological: study biological molecules structure and function whether protein, lipid, organic or inorganic molecules.
Additional benefits of FTIR include: you don’t need to label your samples prior to analysis and qualitative and/or quantitative analysis are possible. FTIR may be an important technique to use in industry where qualitative analysis and quality control (QA/QC) are very important.
There is an endless list of FTIR applications with some less well known than others. I’d love to hear what you investigate with FTIR! Share your experiences by writing in the comments section.