Reading an IR spectrum: an infrared absorption table

An infrared (IR) spectrum shows which wavelengths of infrared light a sample absorbs. Each type of chemical bond vibrates at its own typical frequency, so the dips in the spectrum point to the groups present in a molecule. This guide explains what FTIR is, how to read the axes, gives an IR spectrum table of the most useful absorption bands, and covers the amide bands that every peptide shows.

For how FTIR compares with NMR, Raman and the other methods used on peptides, see NMR, FTIR and amino acid analysis. Terms are defined in the glossary.

What is FTIR?

FTIR stands for Fourier-transform infrared spectroscopy, the standard way IR spectra are recorded today. The instrument records the infrared light that has met the sample as an interference signal, called the interferogram. A mathematical step, the Fourier transform, turns that signal into the familiar spectrum of absorption against wavenumber.

Most modern instruments measure by attenuated total reflectance (ATR). A small amount of solid or liquid is pressed on to a crystal, often diamond, and the infrared beam reflects inside the crystal. At each reflection a little of the light reaches just beyond the crystal surface, about 1–2 µm into the sample for diamond at 1,000 cm⁻¹, so the sample needs firm contact with the crystal but almost no preparation (Spragg, Spectroscopy 2011).

How to read an IR spectrum

An IR spectrum is a graph with two axes:

• The horizontal axis is wavenumber, in cm⁻¹ (waves per centimetre). It usually runs from 4,000 cm⁻¹ on the left to 400 or 500 cm⁻¹ on the right. A higher wavenumber means a shorter wavelength and more energy. For how wavenumber relates to wavelength, see light and the electromagnetic spectrum.
• The vertical axis is usually transmittance, in percent. 100% means all the light passed through. An absorption shows up as a dip pointing downwards. Some instruments plot absorbance instead, and then the bands point upwards.

Each band is described by three things:

• Position: the wavenumber of the dip. This identifies the type of bond.
• Intensity: strong, medium or weak. Bonds that are very polar, such as C=O, absorb strongly.
• Shape: sharp or broad. Hydrogen bonding spreads an O–H or N–H band into a broad, smooth dip.

The four regions

Source: OpenStax, Organic Chemistry 12.7.

The fingerprint region

Below 1,500 cm⁻¹ the spectrum is crowded with bands from many bending and single-bond vibrations. They are hard to assign one by one, but the pattern as a whole is close to unique for each compound. That is why this region is used to compare a sample with a reference spectrum, like matching a fingerprint, rather than to identify individual groups.

IR spectrum table: characteristic absorptions

The values below are typical positions. Real bands shift by a few to tens of wavenumbers depending on the molecule's surroundings, its state (solid, liquid or solution) and hydrogen bonding.

Source: OpenStax, Organic Chemistry, Table 12.1.

A few patterns are worth remembering:

• A broad band above 3,000 cm⁻¹ usually means O–H or N–H. Very broad and running down towards 2,500 cm⁻¹ suggests a carboxylic acid.
• A strong, sharp band near 1,700 cm⁻¹ almost always means a C=O group. Its exact position helps tell an ester from a ketone or an amide.
• Nothing between 2,500 and 2,000 cm⁻¹ is normal. Few molecules have triple bonds, so a band there stands out.

For the groups themselves, see functional groups in peptides.

The amide bands of peptides

Every peptide bond is an amide (–CO–NH–), and the backbone of a peptide repeats it many times. That gives peptides and proteins a set of strong, characteristic bands, called the amide bands.

Sources: amide A, B, I and II from the IMB Jena Image Library; amide III from Ji et al., ACS Omega 2020. Published ranges differ by a few tens of wavenumbers between sources.

Amide I and secondary structure

The amide I band is the most useful for peptides, because its exact position depends on how the backbone is folded. Hydrogen bonds between C=O and N–H groups in a helix or a sheet shift the C=O vibration by a small, consistent amount.

Source: IMB Jena Image Library.

In practice the bands overlap, so the amide I band is split mathematically into its parts to estimate how much of each structure is present. Short peptides in solution often have little fixed structure, so their amide I band is broad. For helices and sheets, see peptide and protein structure.

Two things that get in the way

• Water. Liquid water absorbs strongly near 1,640 cm⁻¹, on top of the amide I band. Samples are measured dry, in heavy water (D₂O), or with careful subtraction of the water signal.
• Trifluoroacetate. Peptides supplied as TFA salts carry a counter-ion that absorbs near 1,670 cm⁻¹, which can distort amide I. See trifluoroacetic acid and counter-ions.

What IR can and can't tell you

IR is quick and needs very little sample. It shows which functional groups are present and gives a fingerprint for comparing a sample with a reference. It doesn't give a sequence, a molecular weight or a purity percentage. For those, labs use mass spectrometry and HPLC. Raman spectroscopy measures the same vibrations by a different route and is covered in NMR, FTIR and amino acid analysis.

Related guides

For where infrared sits among other kinds of light, see light and the electromagnetic spectrum. For the bonds that cause these vibrations, see chemical bonds and polarity. For how to read a drawn structure, see reading a structure diagram. An independent lab tests every lot we sell for purity by HPLC and identity by mass spectrometry; we don't run IR on our lots. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.