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 four main components of the Spec 20 are:

1) The light source

2) The monochromator

3) The Sample Cell

4) The Detector

A spectrophotometer is a device used to measure light intensities.


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.


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 from coordinate covalent bonds and we have created a coordinate covalent complex. (brightly colored, central metal atom, coordinate covalent bonds, can be synthesized)


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 you 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 curve the concentration of that unknown.

Calculating Concentration of the Iron Solutions

You are making four standard solutions of increasing concentrations of Fe. You use standard solution amounts of 10.00 mL, 20.00 mL, 30.00 mL, and 40.00 mL. The concentration of the standard solution is 0.02 mg/L. The final volume for each solution you make is 100 mL. Calculate the concentrations of Fe.

In the above equation, M1 is the inital molarity (concentration), V 1 is the original volume, M2 is the concentration you are making, and V2 is the final volume.

Repeat the above calculations replacing 10.00 mL with 20.00 mL, 30.00 mL, and 40.00 mL.

Using a Pipet

Our standard solutions must be prepared identically, so we must use an instrument that will deliver exactly the same amount of liquid each time it is used. That instrument is called the pipet. Our pipet will transfer exactly 10.00 mL of liquid each time. After delivering the liquid from the pipet, wait 20 seconds then touch the tip of the pipet to the inside of the volumetric flask. You will get exactly 10.00 mL of liquid. When you are using the pipet, remember not all liquid comes out. A small amount of liquid is to remain in the tip of the pipet. Do not blow the rest of the liquid from the tip of the pipet.


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last updated: March 9, 2000