SOIL AND PLANT ANALYSIS (1+1)

Experimental Procedure

The following simulation illustrates the procedures for making spectrophotometric measurements.

First, the intensity of light (I0) passing through a blank is measured. The intensity is the number of photons per second. The blank is a solution that is identical to the sample solution except that the blank does not contain the solute that absorbs light. This measurement is necessary, because the cell itself scatters some of the light.

Second, the intensity of light (I) passing through the sample solution is measured. (In practice, instruments measure the power rather than the intensity of the light. The power is the energy per second, which is the product of the intensity (photons per second) and the energy per photon.)

Third, the experimental data is used to calculate two quantities: the transmittance (T) and the absorbance (A).


T =	I I0
A = - log10 T
The transmittance is simply the fraction of light in the original beam that passes through the sample and reaches the detector.
The remainder of the light, 1 - T, is the fraction of the light absorbed by the sample. (Do not confuse the transmittance with the temperature, which often is given the symbol T.)

In most applications, one wishes to relate the amount of light absorbed to the concentration of the absorbing molecule.
It turns out that the absorbance rather than the transmittance is most useful for this purpose.
If no light is absorbed, the absorbance is zero (100% transmittance).
Each unit in absorbance corresponds with an order of magnitude in the fraction of light transmitted.
For A = 1, 10% of the light is transmitted (T = 0.10) and 90% is absorbed by the sample.
For A = 2, 1% of the light is transmitted and 99% is absorbed. For A = 3, 0.1% of the light is transmitted and 99.9% is absorbed.

Using the simulation below, perform the following steps:

• Measure the intensity of light passing through the blank.
• Measure the intensity of light passing through the sample.
• Calculate the transmittance.
• Calculate the absorbance.

Limitation of Beer’Lamberts law

• Deviations in absorptivity coefficients – at high concentrations (>0.01 M) due to electrostatci interactions between molecules in close proximity
• Scattering of light- due to particulates in the sample
• Fluoresence or Phosphorescence of the sample
• Changes in refractive index at high analysis concentration
• Shifts in chemical equilibria as a function of concentration
• Stray light