Spectrophotometric Determination of Iron in a Vitamin Supplement Class Notes

Beer-Lambert Law

The Beer-Lambert Law states that for monochromatic light (one wavelength) absorbance is proportional to length and concentration of the species.

This means that if concentration doubles, absorbance will also double. If length triples then absorbance triples. If concentration is cut by half then absorbance will be cut by half.

We are using the Beer-Lambert Law in a quantitative way, to determine the amount of iron in our vitamin supplement.

Spectrophotometry and the Spec 20

The five main components of the Spec 20 are:

1) The light source

2) The monochromator

3) The Sample Cell

4) The Detector

5) The meter (readout)

A spectrophotometer is a device used to measure light intensities.

Absorbance

Absorbance is the measure of the amount of light being absorbed (not passing through) by a substance at a particular wavelength. Absorbance is specific to the absorbing species. Each species has its own absorbance (maximum absorbance).

Maximum Absorbance

You will plot a graph of the absorbance of light by a sample for various wavelengths. This graph is called the absorbance spectrum of the sample. The wavelength that has the most light absorbed for the sample is called the maximum absorbance wavelength. It is used to set the Spec 20 to test samples for the construction the calibration curve graph.

0-phenanthroline

Pure iron does not absorb light. In order to use the Spec 20 to determine a maximum absorbance, we must first treat iron with 0-phenanthroline to form an orange-red complex that does absorb light. O-phenanthroline has two pairs of unshared electrons that can be used to form coordinate covalent bonds and we have created a coordination complex. (brightly colored, central metal atom, coordinate covalent bonds, can be synthesized)

Hydroxylamine

O-phenanthroline will not react with Fe³⁺. So we must first treat Fe³⁺ with hydroxylamine to reduce Fe³⁺ to Fe ²⁺. O-phenanthroline will react with Fe²⁺. The hydroxylamine will keep iron in the +2 state.

Calibration Curve

Once you have chosen the correct wavelength, from the maximum absorbance, you must construct a calibration curve. This consists of a plot of absorbances versus concentrations for a series of four standard solutions whose concentrations are know accurately. Each standard solution is prepared identically in the same fashion, the only difference between them being their concentrations.

The Beer-Lambert Law implies that when concentration is equal to zero, absorbance must also be zero. This means that the calibration line must pass through zero. Since absorbance is proportional to concentration, the calibration line must be a straight line that passes through the point zero if your data obeys the Beer-Lambert Law.

Once the calibration curve is set up, you can measure the absorbance of any unknown and determine from the calibration curve the concentration of that unknown.

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