Absorbance

A logarithmic measure of how much a sample attenuates transmitted light relative to a reference.

After a nucleic acid sample produces a reading at 260 nm, it is tempting to convert it straight into a concentration. First, however, we need to establish how the number is derived from a light-intensity ratio, and under what conditions it can represent the absorbing species in the sample.

Start with reference and transmitted light

Spectrophotometry first measures radiant power P₀ under reference conditions, then radiant power P after light passes through the sample. Transmittance T is P/P₀; absorbance converts this ratio to a base-ten logarithmic quantity. Weaker transmitted light means a smaller T and higher absorbance.

The logarithmic expression makes the successive attenuation of light through a sample additive, and helps establish comparisons across path lengths and concentrations. But a recorded change in light intensity does not automatically represent absorption alone: scattering, reflection, fluorescence and unwanted light in the optical system can all affect the result.

When absorbance can estimate concentration

When the sample is homogeneous, the chemical state of the target species is stable, the path length is known, and spectral bandwidth and instrument response meet the method requirements, the Beer–Lambert relationship connects absorbance with concentration, path length and molar absorption coefficient. UV absorbance quantification of nucleic acids and proteins is a common application.

Concentration calculations also depend on the chosen absorption coefficient or conversion factor, sample composition and Blank. Equal absorbance does not mean that all samples have the same concentration. Numbers cannot be compared independently of the wavelength, path length, buffer system and substance being measured.

Both high and low readings have limits

At high absorbance, very little of the intended transmitted light reaches the detector. Stray light and the detector's low-signal limitations then become more significant, often causing an apparent underestimate of absorbance or a restricted linear range. Low absorbance is more susceptible to baseline drift, Blank differences and random noise.

Scattering from turbid samples, particles, bubbles or an unstable liquid column also reduces transmitted light, but does not directly establish the target concentration. For an unusual spectrum, first check the full spectrum, Blank, sample condition and path length before deciding whether to dilute, repeat the measurement or use a more suitable method.

The next step in understanding absorbance is to connect it with transmittance, the Beer–Lambert law, path length and stray light within the same measurement process, rather than considering only a single-wavelength result.