Spectrophotometer Calculators

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A spectrophotometer is an instrument that measures the amount of light absorbed or transmitted by a sample at a specific wavelength. It is one of the most ubiquitous analytical instruments in biology and chemistry laboratories — used for quantifying nucleic acids (A260), proteins (A280, Bradford, BCA), enzyme activity (A340 for NADH), bacterial growth (A600/OD600), and any colored compound. The Beer-Lambert law provides the mathematical foundation relating absorbance to concentration, path length, and molar absorptivity.

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How a Spectrophotometer Works

  1. Light source: Tungsten (visible range) or deuterium (UV range) lamp generates broad-spectrum light
  2. Monochromator: Prism or diffraction grating selects a specific wavelength from the broad spectrum
  3. Sample: Light passes through a cuvette containing the sample (typically 1 cm path length)
  4. Detector: Photodetector (photodiode or photomultiplier) measures transmitted light intensity
  5. Readout: Absorbance A = log₁₀(I₀/I), where I₀ = incident light and I = transmitted light

Beer-Lambert Law

A = ε × c × l

Where A = absorbance (dimensionless); ε = molar absorptivity (L mol⁻¹ cm⁻¹); c = concentration (mol/L or M); l = path length (cm).
Rearranged for concentration: c = A / (ε × l)

Valid when A is between 0.1 and 1.0 (linear range).

Key Wavelengths in Biology

  • 260 nm (A260): Nucleic acid absorbance — purines/pyrimidines absorb strongly. ε(dsDNA) = 50 μg⁻¹mL cm⁻¹; 1 OD₂₆₀ ≈ 50 μg/mL dsDNA
  • 280 nm (A280): Protein absorbance — tyrosine and tryptophan. A260/A280 ratio assesses nucleic acid purity (pure DNA: ratio ≈ 1.8; pure RNA: ≈ 2.0)
  • 340 nm: NADH absorbance (ε = 6,220 M⁻¹cm⁻¹) — enzyme kinetics assays
  • 600 nm (OD600): Bacterial turbidity — cell density measurement
  • 595 nm: Bradford protein assay (Coomassie dye-protein complex)

Glossary

Spectrophotometer
An instrument measuring light absorbance at specific wavelengths. Consists of light source, monochromator, sample cuvette, and photodetector. Used for quantifying nucleic acids (A260), proteins (A280), NADH (A340), and cell density (OD600).
Beer-Lambert Law
A = ε × c × l. Absorbance equals molar absorptivity × concentration × path length. Linear relationship between A and c. Used to calculate unknown concentration: c = A/(ε×l). Valid for A between 0.1 and 1.0.
A260/A280 Ratio
Ratio of absorbance at 260 nm to 280 nm used to assess nucleic acid purity. Pure dsDNA: ~1.8; pure RNA: ~2.0. Values below 1.7 indicate protein contamination. Values above 2.0 may indicate RNA contamination in DNA samples.

Frequently Asked Questions

A light source generates broad-spectrum light; a monochromator (prism or diffraction grating) selects a specific wavelength; light passes through the sample cuvette (typically 1 cm path length); a photodetector measures transmitted light intensity. Absorbance A = log₁₀(I₀/I), where I₀ = incident and I = transmitted intensity. A = 0 means no absorbance (100% transmission); A = 1 means 90% of light absorbed; A = 2 means 99% absorbed.

A = ε × c × l, where A = absorbance, ε = molar absorptivity (L mol⁻¹ cm⁻¹), c = concentration (mol/L), l = path length (cm). A linear relationship between absorbance and concentration — used to calculate unknown concentration from absorbance: c = A/(ε×l). Valid when A is 0.1–1.0. Outside this range, deviations occur due to scattered light effects (high A) or detector noise (very low A).

dsDNA absorbs strongly at 260 nm due to the purine and pyrimidine bases. The relationship: 1 OD₂₆₀ (absorbance of 1.0 in a 1 cm cuvette) corresponds to ~50 μg/mL dsDNA; ~40 μg/mL ssRNA; ~33 μg/mL ssDNA. DNA concentration (μg/mL) = A260 × 50 × dilution factor. The A260/A280 ratio assesses purity: pure dsDNA ≈ 1.8; pure RNA ≈ 2.0. Values below 1.7 indicate protein contamination.

OD600 (optical density at 600 nm) measures the turbidity of a bacterial suspension — light scattered by bacterial cells reduces transmitted intensity. Higher OD600 = more cells = less transmitted light = higher absorbance reading. A linear relationship holds between OD600 ~0.05–0.8; above this, the relationship becomes non-linear due to cell-to-cell light scattering interactions. OD600 is used to track bacterial growth curves, determine log-phase timing, and standardize inocula for experiments.