Simultaneous determination of photometric accuracy during circular dichroism measurements

Abstract

Photometric accuracy is determined by the difference between the measured absorbance and the established standard value. Most quantitation applications using UV-Vis would involve the measurement of the standards and samples of comparable concentrations in rapid succession on the same instrument. Photometric accuracy is critical for measurement; it compares the extinction coefficients between different instruments, for any photometric inaccuracy will lead to errors in quantitation. In Circular Dichroism (CD) measurements it is important and often a requirement to also monitor the absorbance of the sample. However, it is common to make separate measurements to determine the absorbance which is less accurate and time consuming. This study shows that the simultaneous measurement of ordinary UV/vis absorbance spectra from the photomultiplier high voltage output during a circular dichroism measurement is accurate and reliable. The photometric accuracy and linearity of various spectrometers is determined using standard solutions of potassium dichromate. The mathematical treatment of the signal representing the high voltage applied to the detector is presented here. An ordinary UV-vis spectrophotometer was used to measure the direct UV absorbance for comparison and to assign a reference value (Z-Value) to each CD spectrometer. The results proved to be accurate and reliable.