Raw Data and Noise in Spectrophotometry

Spectrophotometers are ubiquitous in chemical and biological science; however, their precision limits are under-appreciated. Rules-of-thumb and IUPAC referenced guidance restricting the range of absorbance to minimize uncertainty are based on historically important instruments which are no longer as widely used. Instrumentation advances over the last half-century have changed the nature of spectrophotometric “raw” data while enabling opportunities to better evaluate their performance. Current IUPAC refenced guidance indicates that absorbance be limited to between 0.1 and 1.0 a.u. and that optimal performance (minimum relative standard deviation (RSD)) will be obtained at 0.43 a.u. or 0.86 a.u. depending on the type of limiting noise. We characterised noise in UV-Vis spectrophotometers across the spectrum and found wavelength-dependent variation in optimal performance. Optimal RSD approached neither extreme with minima varying depending on wavelength. We could find no evidence justifying guidance restricting absorbance to between 0.1 and 1.0 a.u. Measured RSD and light intensity are more important than absorbance values for assuring good quality measurements. Recovering light intensity estimates is a difficult inverse problem when I and I0 are not available, and the modern commercial instruments tested did not provide these. Based on this work, we recommend IUPAC modernise the references in its Gold Book with up-to-date articles and press instrument makers to provide access to instrument raw data.