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. Data
are frequently reported in percent transmission (I/I0 * 100) or in absorbannce [A = log (I/I0)]. The
latter is particularly convenient. [common coefficients of near-ultraviolet absorption bands of
some amino acids and nucleotides]
Sometimes the extinction coefficient is given in other units; for example,
A = E1% * b * c
where the concentration C is in gram per 100 ml of solution. This useful when the molecular
weight of the solute is unknown or uncertain.
Q: Cytosine has a molar extinction coefficient of 6*103 at 270 nm at pH 7. Calculate the
absorbance and percent transmission of 1*10-4 and 1*10-3 M cytosine solution in a 1-mm
cell. [Solution....]
Q: A protein with extinction coefficient E1% = 16 yields an absorbance of 0.73 when
measured in a 0.5-cm cell. Calculate the weight concentration. [Solution....]

Instrumentation
Experimental measurements are usually made in terms of transmittance (T), which is defined
as:
T = I / Io

where I is the light intensity after it passes through the sample and Io is the initial light
intensity. The relation between A and T is:
A = -log T = - log (I / Io).
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:

Io is the intensity entering the sample at z=0, Iz 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 / Iz = -

* N * dz

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

ln(I) - ln(Io) = -

*N*b

or - ln(I / Io) =

* 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 / Io) =

* (6.023x1020 / 2.303) * c * b

- log(I / Io) = A =
where

*b*c

* (6.023x1020 / 2.303) =

* 2.61x1020

Typical cross-sections and molar absorptivities are:

(cm2)
absorption - atoms
molecules
infrared
Raman scattering

-12

10
10-16
10-19
10-29

(M-1 cm-1)
3x108
3x104
3x10
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