Using Beer-Lambert Law to Calculate Concentration from Absorbance
UV-Vis spectroscopy is one of the most routine quantification methods in a lab โ measure absorbance, calculate concentration โ but a few details determine whether that number is actually accurate.
The Beer-Lambert law
Where A is absorbance (unitless), ฮต is the molar extinction coefficient (Mโปยนcmโปยน, specific to your compound at a given wavelength), c is concentration (M), and l is the path length (cm, almost always 1 cm for a standard cuvette).
Where to find the extinction coefficient
ฮต is specific to the compound, the solvent, and the wavelength โ it isn't a universal constant. For common biomolecules (DNA, RNA, many proteins at 280 nm) there are standard reference values, but for a specific compound, you may need to determine it experimentally using a calibration curve of known concentrations, or find it in the literature for your exact conditions.
The linear range matters
โ ๏ธ Beer-Lambert only holds within a limited absorbance range โ typically absorbance readings between about 0.1 and 1.0 are considered reliable. Above roughly 1.0-1.5, many spectrophotometers deviate from linearity due to stray light and detector limitations, giving you a concentration reading that's systematically too low. If your sample reads above this range, dilute it and remeasure rather than trusting the raw reading.
Common sources of error
- Not blanking correctly โ the blank should match your sample's solvent/buffer exactly, or background absorbance skews every reading
- Air bubbles in the cuvette โ scatter light and produce falsely elevated absorbance
- Wrong cuvette path length assumed โ most are 1 cm, but some specialty cuvettes aren't; check before calculating
- Using an extinction coefficient from a different wavelength or solvent than your actual measurement conditions
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