The Thermo Evolution 300 UV-Visible Spectrophotometer, is used for optical absorbance measurements between 190 and 1100 nm, covering the UV and visible ranges and part of the near IR range.
It is a double-beam instrument with separate positions for sample and reference cuvettes. The light source is a xenon lamp and the detector is a silicon photodiode. A monochromator based on a diffraction grating is used to separate the different wavelengths of light. The instrument is very fast, permitting data acquisition at a rate of 50 data points per sec.
To measure absorbance, a beam of light with intensity I0 is aimed at the tested solution placed in a cuvette. The intensities of the entering beam - I0 and the emerging beam I1 are measured, and the absorbance - A - is calculated from the ratio of the two.
A = -log (I1/I0)
Concentration measurements. The concentration of the analyte in the sample is calculated from the measured absorbance, using Beer's law. The instrument is calibrated using standard solutions with known analyte concentrations.
Time Scan. Changes in absorbance are measured as a function of time at a chosen wavelength. This mode is used to monitor reaction rates, where changes in reactant or product concentration involve changes in optical absorbance.
Wavelength Scan. An Absorbance spectrum of a sample can be obtained by monitoring the absorbance as a function of wavelength. 'Absorbance Spectra' of chlorophyll a and chlorophyll b solutions are presented below.