1 spectrophotometry
Spectrophotometry is a method to measure how much a chemical / biological substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution.
Most instruments today use filters (photometers), prisms/gratings (spectrometers) to select a narrow range of incident light.
WHAT ARE WE FINDING?
How much light is transmitted
How much light is absorbed
Optical Density/Absorbance
LAWS OF SPECTROPHOTOMETRY
BEER’S LAW
Absorbance is proportional to concentration over a certain concentration range or inversely proportional to the logarithm of the transmitted light.
LAMBERT’S LAW
Absorbance is directly proportional to the length of light path through the sample.
DERIVATION OF BEER-LAMBERT LAW
if materials are exposed to radiation, part of the incident radiation is absorbed, part is scattered and part is transmitted
The fraction of incident light absorbed depends on the thickness of the absorbing medium
The intensity of the light absorbed is related to the concentration of the absorbing substance
The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length
A ∝ cl A = cl
A = absorbance
= molar extinction coefficient /absorptivity coefficient(M-1 cm-1 or mol-1 L cm-1)
c = concentration (M or mol L-1)
l = path length (cm) (width of cuvette)
ABSORPTIVITY COEFFICIENT
ε is the absorption obtained of a 1M solution of a pure compound with light path of 1cm under standard conditions of solvent, temperature and wavelength
Beer- Lambert’s law A(λ) = (λ) cl
This means the absorptivity coefficient is wavelength – specific.
eg. Calculate the molar extinction coefficient of NADH if a 1.37 x 10-4 M solution exhibits an absorbance of 0.85 at a wavelength of 340nm in a 1cm cell.
Answer: 6204 or 6.204 x 10^3
SPECTROPHOTOMETRIC QUANTITIES
There are two quantities that relate the change in the intensity before, I0 , and after, It interaction with matter.
Transmittance
Absorbance
Transmittance, T, is simply defined as the fraction of light that reaches a detector after passing through a sample
T = It/Io
The percent transmittance, %T, is simply
%T = (It/Io) x 100
absorbance Defined as: the negative logarithm of the transmittance
A= log T
A = log (It/Io)
A =log (Io/It)
FACTS OF THE BEER-LAMBERT’S LAW
If A=0, then no photons are absorbed
If A=1.00 then 90% of the photons are absorbed; only 10% reach the detector
If A=2.00, then 99% of the photons are absorbed; only 1% reach the detector
NB: It is the absorbance, however, that displays a simple dependence on the concentration and cell path length (Beer’s law), thus most biochemists choose to report data in terms of absorbance rather than transmittance
LIMITATIONS OF BEER – LAMBERT’S LAW
Deviations in absorptivity coefficients at high concentrations(> 0.01M) due to electrostatic interactions between the molecules in the sample.
Scattering of light due to particles in the sample
Fluorescence or phosphorescence of the sample
Changes in refractive index at high analyte concentration
Non-monochromatic radiation (several wave length)
INSTRUMENTAL FACTORS
This may be due to the fluctuation in power supply or as a result of unstable light source or non-linear response from the detector-amplified system.
The presence of stray light is another problem and can be corrected by painting the interior walls of the monochromator and sample chamber with black non-reflecting paint
CHEMICAL FACTORS
These factors are observed only when concentration changes usually at higher concentrations.
The deviation may be due to the reaction of the chromophore with the solvent.
Examples of reactions are complexation, dissociation, hydration, polymerization and association reactions. The effect is present in both UV and visible determination.
SPECTROPHOTOMETRIC ERRORS
Lamp age
Correction: replace with new lamp.
Dust and fingerprints
Correction: clean with tissue paper and isopropanol.
Scattering of light caused by turbid samples.
Correction: using instruments in which the photodetector is located close to the exit surface of the cell.