Reference solution:

A proper reference solution contains color reagent plus sample buffer. The difference between the reference and a sample is that the concentration of the assayable substance in the reference solution is zero. The reference tube transmits as much light as is possible with the assay solution you are using. A sample tube with any concentration of the assayable substance absorbs more light than the reference, transmitting less light to the photometer. In order to obtain the best readability and accuracy, the scale is set to read zero absorbance (100% transmission) with the reference in place. Now you can use the full scale of the spectrophotometer. If you use a water blank as a reference, you might find that the assay solution alone absorbs so much light relative to distilled water that the usable scale is compressed, and the accuracy is very poor.