Light and the electromagnetic spectrum: wavelength, UV and absorbance

The electromagnetic spectrum is the full range of electromagnetic radiation, ordered by wavelength from radio waves to gamma rays. Visible light is the narrow band from about 380 to 700 nm. Many lab measurements are really measurements of light. An HPLC detector shines ultraviolet light through the liquid leaving the column. A spectrophotometer measures how much light a solution absorbs. Infrared, Raman and fluorescence spectroscopy each use a different part of the spectrum. This guide explains what wavelength means, how the electromagnetic spectrum is laid out, why peptides are measured in the ultraviolet, and what an absorbance number tells you.

Terms are defined in the glossary.

What is wavelength?

Light travels as a wave of electric and magnetic fields. The wavelength is the distance from one peak of the wave to the next. It is written with the Greek letter λ (lambda).

Three properties are linked:

• Wavelength (λ): the length of one wave, usually given in nanometres (nm) for light. One nanometre is a millionth of a millimetre.
• Frequency (f, sometimes written ν): how many waves pass a point each second, in hertz (Hz).
• Energy: how much energy each packet of light, or photon, carries.

Because all light travels at the same speed, a shorter wavelength means a higher frequency and more energy per photon. Ultraviolet light carries more energy than visible light, and visible light more than infrared. That is why ultraviolet light can break chemical bonds and infrared light mostly warms things.

The wave equation: c = fλ and E = hf

Two short equations tie the three properties together:

• c = fλ. The speed of light (c) equals frequency times wavelength. Since c is fixed at 299,792,458 m/s (BIPM, SI Brochure), knowing one of f or λ gives the other.
• E = hf. The energy of one photon (E) equals the Planck constant (h) times the frequency. The Planck constant is exactly 6.626 070 15 × 10⁻³⁴ J s (NIST, CODATA).

A worked example: ultraviolet light at 220 nm, a wavelength used to detect peptides in HPLC.

1. Convert the wavelength to metres: 220 nm = 220 × 10⁻⁹ m.
2. Frequency: f = c ÷ λ = 299,792,458 ÷ (220 × 10⁻⁹) ≈ 1.36 × 10¹⁵ Hz.
3. Energy of one photon: E = hf = 6.626 × 10⁻³⁴ × 1.36 × 10¹⁵ ≈ 9.03 × 10⁻¹⁹ J.
4. For a mole of photons, multiply by the Avogadro constant (6.022 × 10²³): about 544 kJ per mole.

Halve the wavelength and the frequency and energy double. Infrared light at 6,000 nm (a wavenumber of about 1,667 cm⁻¹) has under a twenty-fifth of the energy per photon of the 220 nm light.

In infrared spectroscopy, light is often described by wavenumber instead: the number of waves per centimetre, in cm⁻¹. A higher wavenumber means a shorter wavelength. See reading an IR spectrum.

The electromagnetic spectrum

All electromagnetic radiation is the same kind of wave. Only the wavelength differs.

The electromagnetic spectrum in order

From the longest wavelength (lowest frequency and energy) to the shortest (highest frequency and energy):

1. Radio waves
2. Microwaves
3. Infrared
4. Visible light (red, orange, yellow, green, blue, violet)
5. Ultraviolet
6. X-rays
7. Gamma rays

The table gives rough ranges. The frequencies are worked out from the wavelengths with c = fλ.

The boundaries are conventions, and sources draw them slightly differently. The human eye typically detects about 380 to 700 nm (NASA). Raman spectroscopy uses a visible or near-infrared laser but reads the same bond vibrations as infrared.

The ultraviolet bands

Ultraviolet light is often split into three bands (WHO):

• UV-A: 315–400 nm
• UV-B: 280–315 nm
• UV-C: 100–280 nm

Below about 200 nm, oxygen in the air absorbs strongly. This region is called the vacuum or far ultraviolet, and instruments working there are flushed with nitrogen.

Why peptides are measured with ultraviolet light

Most peptides are colourless: they don't absorb visible light. They do absorb ultraviolet light, at two main places.

Because every peptide has peptide bonds, detection at 214–220 nm sees almost every peptide-related species in an HPLC run. That is why it is the usual wavelength for purity testing. See how HPLC works. At 280 nm, a peptide without tryptophan or tyrosine is almost invisible. See measuring peptide concentration.

The trade-off of working at short wavelengths is that many other things absorb there too, including some solvents, buffers and dissolved oxygen. That is why HPLC solvents are chosen for their UV cut-off, the wavelength below which they absorb too much to use. See HPLC and LC-MS solvents.

What is a spectrophotometer?

A spectrophotometer measures how much light a sample absorbs at a chosen wavelength. What makes it a spectrophotometer is the monochromator, the part that picks out one wavelength from the light source (LibreTexts, Instrumentation). It has four main parts:

1. A light source.
2. A monochromator, which selects the wavelength. Scanning through wavelengths records a whole spectrum.
3. A sample holder for the cuvette, in a light-tight compartment.
4. A detector, which measures the light that gets through.

A single-beam instrument measures the blank and the sample one after the other. A double-beam instrument switches the light between them continuously, which corrects for drift in the lamp and detector (LibreTexts). An instrument covering both the ultraviolet and visible ranges is called a UV-Vis spectrophotometer, and the method UV-Vis spectroscopy. HPLC UV detectors work on the same principle, with a flow cell in place of the cuvette.

What is absorbance?

A spectrophotometer shines light of one wavelength through a sample and measures how much comes out the other side. Transmittance is the fraction that gets through. Absorbance turns that into a more convenient number:

A = log₁₀(I₀ ÷ I)

where I₀ is the light going in and I is the light coming out.

Absorbance has no units. Because it is a logarithm, each step of 1 cuts the light reaching the detector tenfold. At high absorbance so little light gets through that stray light and detector noise dominate, so readings are most reliable in the lower part of the range. A concentrated sample is diluted to bring it down.

Absorbance rises in proportion to concentration and to the distance the light travels through the sample. That relationship is the Beer–Lambert law, covered in measuring peptide concentration and the Beer–Lambert calculator.

Cuvettes and wavelength

The container matters too. Ordinary glass and most plastics absorb ultraviolet light, so a sample measured at 280 nm or below needs a quartz cuvette or a plastic one sold as UV-transparent. Each reading is taken against a blank: the same cuvette filled with solvent only, so the solvent's own absorbance is subtracted.

Other molecules measured in the ultraviolet

The same approach works for any compound with a UV-absorbing group. The adenine ring of NAD+ absorbs at 260 nm, and its reduced form NADH has a second band at 340 nm, which is how the two are told apart. See NAD+ structure and stability.

Light and stored samples

The same energy that makes ultraviolet light useful for measuring also lets it damage molecules. Tryptophan, tyrosine and phenylalanine absorb ultraviolet light and can break down as a result. Amber glass blocks most ultraviolet and blue light, and a closed box blocks it all. See how to store freeze-dried (lyophilised) peptides.

Fluorescence: light in, different light out

Some molecules give out light after absorbing it, at a longer wavelength. This is fluorescence. Tryptophan absorbs near 280 nm and fluoresces around 350 nm. Measuring the emitted light is far more sensitive than measuring absorbance. See NMR, FTIR and other methods.

Related guides

For reading the trace a UV detector produces, see reading a chromatogram. For the extinction coefficient at 205 and 280 nm, use the extinction coefficient calculator. An independent lab tests every lot we sell for purity by HPLC and identity by mass spectrometry. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.