Question

How can measurements be affected if a blank solution is not used in a Beers Law experiment?

Answer 1

Ans. The color of a solution may be due to-

I. The color of reagents

II. The color of product

In spectrophotometry experiments based on Beer’s Law, it is the color of the product which is directly proportional to the amount of the specified molecule (reactant) present in the sample.

The color of the reagents provides the background color.

So, to cancel out the effect of color of the reagent and account only the color of the product, the reading of the sample tubes is taken against the reagent blank. By doing so, the resultant OD of sample tubes is solely due to the absorbance of the product or color of the product.

Therefore, if a suitable reagent blank is not used, say an air blank is used, the resultant OD is due to the absorbance of both the reagent and the product. Obtaining an inclusive OD (OD of reagent + Product) gives abnormally high value (concentration) of the specified molecule present in the sample.

#2. In case the reagents are colorless, use of a reagent blank or distilled water as blank is preferred. Note that the color of the product is recorded relative to the background color of the solution. If an air blank (a empty cuvette in reference cell) is placed in reference cell and a solution-filled cuvette in sample cell, the OD of the sample cuvette would be taken against the air. Since air and solution (mostly water) have different refractive index – that of air is much lower than water, the resultant OD of sample tubes is much higher than when the same is recorded with a reagent blank in reference cell.

In this case, the resultant concentration of the specified molecule would be abnormally high because the OD would be higher when taken against air in comparison to when taken against water/regent blank.

Note: For reactions where the OD of sample is inversely proportional to the concentration of specified molecule, the recorded OD would also be higher but the calculated concentration of specified molecule would be lower than normal.