Want good quality results? Follow these steps.!--h2>
John P. Hammond
Check that the spectrophotometer is in a proper state of calibration and is well maintained at all times. Modern spectrophotometers are long-suffering devices which will continue to give approximate readings long after the uncaring user deserves them.
The effective slit width should be correct for the expected natural bandwidth. If accurate absorbance measurements must be made on benzenoid compounds or other compounds with sharp absorption bands (with natural half bandwidths of less than 15 nm), the spectral bandwidth of the spectrophotometer used should not be greater than 1/8th the natural half bandwidth of the compound's absorption.
Ensure that critical absorbance measurements are performed where the expected values lies between 0.8 and 1.5 A. Adjust the cuvette path length rather than the concentration; forget the manufacturer’s ‘hype’ because measurements at the extreme of any range will be subject to increased error and UV/Vis is no different in this respect. Dilution of a highly absorbing species should also be avoided if possible, as solvent interactions may change the absorption characteristics.
Check that stray light is not responsible for negative deviations from the Beer-Lambert law at high absorbances, particularly if the solvent absorbs significantly. Spectrophotometers may have measurement ranges defined to relatively high absorbance values, but what and how the analyte is being measured may result in a significant negative deviation from linearity at higher absorbance values.
Make sure that regular verification of wavelength and absorbance accuracy are carried out and that the stray light is within specification. Wavelength defines the x-axis (abscissa) and absorbance the y-axis (ordinate) scale of any UV/Vis calibration graph, so the accuracy of these scales is vital for any quantitative work. Stray light will increase as instrument optics become dirty or degrade, and source lamp energy decreases, so it therefore provides a good overall ‘health check’ on an instrument.
The environment of the instrument should be clean and free from external interference and particular attention should be paid to electrical interference, thermal variations, and sunlight. Once an instrument is placed in a location, the environment surrounding the equipment will immediately begin to interact with the sensitive components to the degradation of the performance of the system. In an optical spectrophotometer, it’s not surprising that the mirrors constitute these environmentally reactive components. Reducing and minimizing the fluctuation in these external interferences will slow this process.
Observe the manufacturer’s recommendations because specification ranges are quoted to guarantee maximum permissible operating conditions. For the above reasons, do not exceed these, and where possible operate at the median of these ranges.