Measuring the blank – and how to get it right Determining blank, or zero, values is an important step in all photometric measurements. It serves the calibration of the photometer, which is thus set to “zero”. The blank value should encompass all those components which are included in the measurement of the sample, with the sole exception of the substance to be analyzed. Blank measurements are carried out in the same instrument, under the same experimental conditions, as the actual sample measurement. This approach will minimize any possible influence that the instrument, as well as the environment, may exert on the determination of the absorbance value of the sample. In addition, auto-absorbance of both the cuvette and the medium, in which the analyte is dissolved, is automatically subtracted from the result. While cuvettes are specially designed to be as transparent as possible for the light of photometric wavelengths, every material is subject to auto-absorbance that could potentially distort the result. The same is true for the solvent used.
The following must be considered for materials as well as for the experimental approach:
Cuvettes should display the lowest possible auto-absorbance for the wavelengths utilized in order to avoid compromising the photometric measurement range. This may be checked and verified via determination of the blank value of the cuvette. To this end, the cuvette is filled with demineralized water and measured against a blank value which is obtained by measuring the empty cuvette shaft. When using glass cuvettes, special care should be taken to detect and avoid possible damage as well as contamination of the surfaces. In order to obtain the most accurate value possible, blank and sample should be measured using the same cuvette. It is also important to ensure that the orientation of the cuvette inside the cuvette shaft remains the same for each subsequent measurement. Markings on the sides of the cuvettes, which identify the transparent windows, allow correct and consistent orientation of the cuvette inside the cuvette shaft (figure 1).