Tips & Tricks for Photometric Measurements

Photometry is a common, easily performed method for the determination of concentrations of substances in solution, and it is also capable of assessing sample purity. However, in cases where values do not make obvious sense, or if they vary considerably, troubleshooting may be complex. It is therefore helpful to eliminate possible sources of error from the outset and pay special attention to the following considerations when setting up and carrying out photometric measurements.

Set-up & Environment

Solvent
The absorbance of a sample is influenced by its pH and the ionic strength of its solvent [1]. Buffers with a low salt content and a neutral or slightly alkaline pH, which prevents variations in pH, are recommended. At the same time, the buffer’s inherent absorbance at the wavelength being measured should be low in order to avoid possible limitations of the measurement range.

Dilution

In the case of highly concentrated sample solutions, dilution may be necessary in order to stay within the linear range of measurement. Dilutions in the range of 1:10 are ideal. Dilutions higher than 1:50 are not recommended due to the increasing risk of inaccuracies introduced via this pipetting step. In principle, a calibrated system of pipette and pipette tips must be used for accurate and precise pipetting.

Mixing

Following the preparation of dilutions, but also after long periods of immobility, local concentration differences may arise within the sample. It is therefore important to thoroughly mix the sample solution before and after each dilution step, as well as immediately prior to measurement. Best results are achieved through vortexing.

Temperature

The environment and the photometer, as well as cuvettes and samples, should be kept at identical temperatures in order to prevent a drift of the measured values.

Equipment & Measurement

Cuvettes
When selecting cuvettes, their transparency at the required wavelengths, as well as their compatibility with the respective photometer, are among the most important deciding factors. This includes, for example, the coordination of the heights of the light paths and ensuring that the cuvettes are filled with sufficient sample volumes. In order to obtain accurate results, the blank, as well as samples, should be measured in the same cuvette, with the cuvette always positioned in the same orientation in the cuvette shaft.

Measurement
Prior to measurement, care must be taken to avoid the formation of air bubbles inside the cuvette that would interrupt the light path (this applies to both the blank and the samples). The photometer settings must be appropriate for the method, the sample and the cuvettes selected. Following the measurement, all results should be checked for plausibility.

References:
[1] Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 1997; 22:474–481.