Beer-Lambert Law

Introduction

The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as:
A = a() * b * c
where A is the measured absorbance, a() is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as:
A = * b * c
where is the wavelength-dependent molar absorptivity coefficient with units of M^-1 cm^-1.

Instrumentation

Experimental measurements are usually made in terms of transmittance (T), which is defined as:
T = I / I_o
where I is the light intensity after it passes through the sample and I_o is the initial light intensity. The relation between A and T is:
A = -log T = - log (I / I_o).

Absorption of light by a sample

Modern absorption instruments can usually display the data as either transmittance, %-transmittance, or absorbance. An unknown concentration of an analyte can be determined by measuring the amount of light that a sample absorbs and applying Beer's law. If the absorptivity coefficient is not known, the unknown concentration can be determined using a working curve of absorbance versus concentration derived from standards.

Derivation of the Beer-Lambert law

The Beer-Lambert law can be derived from an approximation for the absorption coefficient for a molecule by approximating the molecule by an opaque disk whose cross-sectional area, , represents the effective area seen by a photon of frequency w. If the frequency of the light is far from resonance, the area is approximately 0, and if w is close to resonance the area is a maximum. Taking an infinitesimal slab, dz, of sample:

I_o is the intensity entering the sample at z=0, I_z is the intensity entering the infinitesimal slab at z, dI is the intensity absorbed in the slab, and I is the intensity of light leaving the sample. Then, the total opaque area on the slab due to the absorbers is * N * A * dz. Then, the fraction of photons absorbed will be * N * A * dz / A so,

dI / I_z = - * N * dz

Integrating this equation from z = 0 to z = b gives:

ln(I) - ln(I_o) = - * N * b

or - ln(I / I_o) = * N * b.

Since N (molecules/cm³) * (1 mole / 6.023x10²³ molecules) * 1000 cm³ / liter = c (moles/liter)

and 2.303 * log(x) = ln(x)

then - log(I / I_o) = * (6.023x10²⁰ / 2.303) * c * b

or - log(I / I_o) = A = * b * c

where = * (6.023x10²⁰ / 2.303) = * 2.61x10²⁰

Typical cross-sections and molar absorptivities are:

                          (cm2)         (M-1 cm-1)     
absorption - atoms       10-12           3x108 
             molecules   10-16           3x104 
             infrared    10-19           3x10 
Raman scattering         10-29           3x10-9

Limitations of the Beer-Lambert law

The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of nonlinearity include:

• deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity
• scattering of light due to particulates in the sample
• fluoresecence or phosphorescence of the sample
• changes in refractive index at high analyte concentration
• shifts in chemical equilibria as a function of concentration
• non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band
• stray light

Related Topics

• Introduction to spectroscopy