Beer Lamberts Law and Spectrophotometry

Principle
I0 Beer Lamberts Law A=bc
b

A b c

absorbance (-) molar absorbtivity with units of L mol-1 cm-1 path length of the sample (cuvette) Concentration of the compound in solution, expressed in mol L-1

Transmittance, T = I / I0, Absorbance, A = log10 I0 / I A = log10 1 / T A = 2 - log10 %T

%T = 100 T

Spectrophotometer

Amount of light that a sample absorbs. Beam of monochromatic light Measuring the intensity of light reaching a detector

Calibration / Effect of Concentration

Note that the Law is not obeyed at high concentrations

Effect of path length

Deviation of Beer Lambert law

Deviations in absorptivity coefficients at high concentrations (> 0.01M) due to electrostatic interactions. Scattering of light due to particulates in the sample Fluoresecence or phosphorescence of the sample Changes in refractive index at high analyte concentration Non-monochromatic radiation

Calibration curve from Lab #2

Spectrophotometer Calibration
2.5 Absorbance (nm) 2 1.5 1 0.5 0 0 20 40 60 80 100 Concentration (mg/L) y = 0.0194x + 0.4542 R2 = 0.7488

Standard

1 2 3 4 5

Concentration of Absorbance (nm) Fluroescien in Standard(mg/L) 80 1.75 40 1.6 20 1.21 8 0.53 2 0.095

Linear fit

Calibration curve from Lab #2

Spectrophotometer Calibration
2 Absorbance (nm) 1.5 1 0.5 0 0 20 40 60 80 100 Concentration (mg/L) y = 0.4843Ln(x) - 0.3036 R2 = 0.9718