Chlorophyll Concentration Calculator
Converts spectrophotometer absorbance readings at 645 nm and 663 nm into total chlorophyll concentration in a plant extract. Use it in plant physiology and ecology labs to assess photosynthetic pigment content.
About this calculator
Chlorophyll a and b have distinct but overlapping absorption spectra. Mackinney's equations (refined by Arnon) use absorbance at two wavelengths — 663 nm (chlorophyll a peak) and 645 nm (chlorophyll b peak) — to estimate total chlorophyll concentration. The combined formula for total chlorophyll (a + b) in µg/mL is: C_total = 20.2 × A₆₄₅ + 8.02 × A₆₆₃. To express the result per gram of fresh or dry tissue, multiply by the extract volume (mL) and divide by 1000: Total chlorophyll (mg) = (20.2 × A₆₄₅ + 8.02 × A₆₆₃) × volume / 1000. Readings must be taken against a pure solvent blank (typically 80% acetone). Accurate measurement requires absorbance values within the spectrophotometer's linear range (ideally 0.1–0.8 AU).
How to use
A leaf extract in 80% acetone gives A₆₄₅ = 0.45 AU and A₆₆₃ = 0.62 AU, with an extract volume of 25 mL. Step 1 — Enter 0.62 in 'Absorbance at 663 nm'. Step 2 — Enter 0.45 in 'Absorbance at 645 nm'. Step 3 — Enter 25 in 'Extract Volume'. Step 4 — The calculator computes: (20.2 × 0.45 + 8.02 × 0.62) × 25 / 1000 = (9.09 + 4.97) × 25 / 1000 = 14.06 × 25 / 1000 = 0.3515 mg total chlorophyll.
Frequently asked questions
Why are wavelengths 645 nm and 663 nm used for chlorophyll measurement?
These wavelengths correspond to the absorption maxima of chlorophyll b (645 nm) and chlorophyll a (663 nm) in organic solvents such as 80% acetone. Using two wavelengths allows the simultaneous estimation of both pigments in a mixed extract through simultaneous equations, because each pigment has a different molar absorptivity at each wavelength. The Arnon method's coefficients (20.2 and 8.02) were empirically derived to deconvolute the overlapping spectra. Other extraction solvents (ethanol, methanol) shift the peaks slightly and require different coefficients.
How does the choice of extraction solvent affect chlorophyll concentration measurements?
The solvent used to extract chlorophyll changes the positions and intensities of the absorption peaks. The Arnon formula with coefficients 20.2 and 8.02 is specific to 80% acetone. If you use 95% ethanol, the peaks shift (e.g., chlorophyll a absorbs maximally at around 665 nm) and different empirical coefficients must be applied. Mixing solvents or using degraded samples containing pheophytin can also introduce errors. Always match your formula and coefficients to the solvent used, and ensure extractions are performed rapidly and in low light to prevent pigment degradation.
What factors can cause inaccurate chlorophyll readings from a spectrophotometer?
Several sources of error can skew results: absorbance values above ~0.8 AU fall outside Beer-Lambert linearity, so dilute samples before reading. Contamination from carotenoids or other pigments overlapping at the chosen wavelengths will inflate readings. Poorly blanked instruments (blank not measured with the same solvent and cuvette) introduce baseline error. Pigment degradation to pheophytin — triggered by light, heat, or acidic conditions — reduces measured absorbance. Finally, cuvettes must be clean and scratch-free, as surface scattering increases apparent absorbance.