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Short answer: Absorbance (A) is the base-10 logarithm of how much light a blank lets through compared with the sample, A = log10(I0/I) = −log10(T), and it has no unit. “OD” (optical density) is often used as a synonym, and for a true absorber the two numbers are the same. They are not interchangeable when the sample scatters light, as bacterial cultures read at 600 nm do.
Definition and formula
A spectrophotometer compares the light intensity passing through a blank (I0) with the intensity passing through the sample (I). Transmittance is T = I/I0, and absorbance is A = log10(I0/I) (Vernier, “Decoding Your Absorbance Readings”). Because it is a logarithm of a ratio, absorbance is unitless; “AU” is common on instrument screens, but BMG Labtech notes that the use of units should be avoided.
The scale is logarithmic, which is why small changes in absorbance are large changes in transmitted light:
| Transmittance T | Absorbance A | Basis |
|---|---|---|
| 100% (1.0) | 0 | A = −log10(T) |
| 10% (0.1) | 1 | A = −log10(T); BMG Labtech: A 1.0 corresponds to 10% transmission |
| 1% (0.01) | 2 | A = −log10(T) |
| 0.1% (0.001) | 3 | A = −log10(T) |
For a full introduction to the instrument, blanks and calibration, see the guide to UV-Vis spectrophotometer basics.
Is optical density equal to absorbance?
Often the number is identical, but the meaning is not. BMG Labtech reports that scientists have used “optical density” interchangeably with absorbance for years, that the term is also used for light scattering, and that IUPAC discourages “optical density” when absorbance is what is meant.
The practical decision rule:
- The sample absorbs light at the chosen wavelength (a dye, a nucleic acid at 260 nm, a coloured assay product): the reading is absorbance, and Beer-Lambert applies.
- The sample mainly scatters light (most bacteria at 600 nm): the reading, usually written OD600, reflects scattering proportional to the number of organisms, not absorption. BMG Labtech adds that pigmented organisms such as cyanobacteria can absorb in this region and skew results. The value depends on the instrument and path length, so state the wavelength, instrument and vessel when you report it. A turbidity-based reference such as the 0.5 McFarland standard addresses the same kind of scattering measurement.
How to measure absorptivity
The Beer-Lambert relationship links absorbance to the concentration of the absorbing species (c), the path length (l) and the absorption coefficient (ε), which BMG Labtech describes as how strongly a substance absorbs light at a specific wavelength. If absorbance, path length and ε are known, concentration can be calculated. Measuring ε runs the same relation the other way:
- Prepare standards of known concentration of the pure absorbing substance, plus a blank of the solvent alone.
- Fix the wavelength and the path length (a cuvette is typically 1 cm; microplate path lengths range from a few hundred micrometres to several millimetres, per BMG Labtech).
- Read each standard against the blank and plot A against c.
- The slope of the line is ε × l, so ε = slope / l.
Illustrative arithmetic, not measured data: if a line through the standards has a slope of 0.50 per mM and l = 1 cm, then ε = 0.50 mM-1 cm-1. Keep the standards inside the range where the plot stays straight (next section), and see calibration curves and linear range for the fitting and back-calculation. An OD600 reading cannot be used to derive ε, because the signal is scattering, not absorption.
What does it mean if absorbance increases?
A higher absorbance means less of the incident light reaches the detector, which for an absorbing sample at a fixed wavelength and path length means more absorbing species, as the Beer-Lambert relation predicts. For a scattering sample it means more scattering, such as more cells or more turbidity. The relationship holds best in a limited window, summarised below.
| Reading | What the sources say |
|---|---|
| 0.1 to 1.0 | Range recommended for the highest accuracy and precision; corresponds to 90% to 10% transmission (BMG Labtech). Vernier gives 0.1 to 1.0 as the useful range for its devices. |
| Above 1 | “Any absorbance reading above 1 can be inaccurate” (Vernier). |
| Around 3 to 4 | Greater error; above 3.0 is not recommended for quantitative work. Dilute the sample and correct for the dilution factor (BMG Labtech). |
The linear relationship can also fail at high concentration, so a very high reading is a prompt to dilute, not to extrapolate.
What does a negative absorbance reading mean?
Because A = log10(I0/I), a negative value means the sample transmitted more light than the blank did (I greater than I0). This follows directly from the formula; the sources above do not discuss negative readings, so the causes below are editorial interpretation rather than sourced findings.
- A small negative number near zero is consistent with ordinary noise or drift around the blank.
- A larger or persistent negative value suggests the blank is not a fair reference for the sample: a blank that absorbs or scatters more than the sample, a mismatched or dirty cuvette or plate well, or a reading taken long after the blank.
Re-blank with the same vessel type and solvent and read again before interpreting the value. Instrument checks are covered in spectrophotometer calibration.
Where it matters for labs
- Reporting: write “absorbance at 260 nm” or “OD600” rather than a bare “OD”, and omit units.
- Plate readers: automatic path-length correction normalises a reading to 1 cm, but BMG Labtech notes it is problematic for OD600 and recommends volume-based correction. See what a plate reader is.
- Range planning: design dilutions so expected readings land between 0.1 and 1.0.
Common misunderstandings
- “OD600 tells me concentration by Beer-Lambert.” It mainly reflects scattering, so it is an empirical proxy for cell number, not an absorption measurement.
- “A reading of 3.5 is more precise because it is larger.” The opposite: error grows at high absorbance.
- “AU is a unit.” Absorbance is unitless.
FAQ
How to measure absorptivity?
Read absorbance for standards of known concentration at a fixed wavelength and path length, plot A against c, and divide the slope by the path length to get ε (Beer-Lambert relation, as described by BMG Labtech).
Is optical density equal to absorbance?
For an absorbing sample the number is the same, but “optical density” is also used for scattering measurements such as OD600, and IUPAC discourages the term when absorbance is meant (as reported by BMG Labtech).
What does it mean if absorbance increases?
Less light is reaching the detector: more absorber for an absorbing sample, more scattering for a turbid one. Accuracy is best between 0.1 and 1.0.
What does a negative absorbance reading mean?
The sample passed more light than the blank. Small values near zero are consistent with noise; larger ones point to a blank or vessel mismatch (editorial interpretation from the formula).
Sources
- BMG Labtech, “Optical density and absorbance measurements”, https://www.bmglabtech.com/en/blog/optical-density-for-absorbance-assays/ (read 2026-11-07).
- Vernier, “Decoding Your Absorbance Readings”, https://www.vernier.com/blog/decoding-your-absorbance-readings/ (read 2026-11-07).
