Spectrophotometer Beer Lambert Calculator
Our bio laboratory calculator computes spectrophotometer beer lambert accurately. Enter measurements for results with formulas and error analysis.
Reviewed by Daniel Agrici, Founder & Lead Developer
Formula
A = epsilon * l * c = -log10(It/I0)
Where A is absorbance (unitless), epsilon is molar absorptivity (L/mol/cm), l is path length (cm), c is molar concentration (mol/L), I0 is incident light intensity, and It is transmitted light intensity. Transmittance T = It/I0.
Worked Examples
Example 1: Determining Protein Concentration
Problem:A protein sample in a 1 cm cuvette shows incident intensity of 100 units and transmitted intensity of 25 units. The molar absorptivity at 280 nm is 6,420 L/mol/cm. Find the concentration.
Solution:Transmittance T = 25/100 = 0.25\nAbsorbance A = -log10(0.25) = 0.6021\nBeer-Lambert: A = epsilon x l x c\n0.6021 = 6,420 x 1 x c\nc = 0.6021 / 6,420 = 9.38 x 10^-5 mol/L
Result:Concentration: 9.38 x 10^-5 M (93.8 micromolar) | Absorbance: 0.602
Example 2: Verifying Dye Concentration
Problem:A dye solution at known concentration 2.5 x 10^-5 M in a 1 cm cell transmits 35% of 520 nm light. Calculate the molar absorptivity.
Solution:Transmittance T = 0.35\nAbsorbance A = -log10(0.35) = 0.4559\nA = epsilon x l x c\n0.4559 = epsilon x 1 x 2.5 x 10^-5\nepsilon = 0.4559 / (2.5 x 10^-5) = 18,237 L/mol/cm
Result:Molar absorptivity: 18,237 L/mol/cm | Absorbance: 0.456
Frequently Asked Questions
How do you prepare a calibration curve using Beer-Lambert Law?
Preparing a calibration curve involves measuring the absorbance of a series of standard solutions with known concentrations at a specific wavelength, then plotting absorbance versus concentration. First, prepare at least five standard solutions spanning the expected concentration range of your unknown samples, plus a blank containing only the solvent. Measure the absorbance of each standard at the wavelength of maximum absorption (lambda-max) for your analyte. Plot concentration on the x-axis and absorbance on the y-axis. If Beer-Lambert Law holds, the points will form a straight line passing through the origin with a slope equal to epsilon times l. Use linear regression to determine the best-fit equation. Then measure your unknown sample absorbance and use the calibration equation to calculate its concentration. Always verify that your unknown absorbance falls within the calibrated range and not beyond it.
References
Reviewed by Daniel Agrici, Founder & Lead Developer · Editorial policy