Spectroscopy and spectrometry have been around for a long time; in the ‘modern’ era, spectroscopic instruments have been with us in one form or another for over 70 years. This is particularly true for that old ‘workhorse’ technique, UV-visible. So, by now it might be expected that there was a standardization and calibration environment to make the assurance of spectral data quality a matter of routine. However, nothing stands still in the application of analytical science to assuring quality. Very often it is the simple steps that are so easily forgotten, but fundamental to obtaining good quality data.
- Ensure that all weighing, volumetric, and temperature errors have been minimized as far as possible and have been accounted for in the solution concentration uncertainty calculations. Given the availability and range of modern analytical balances, it’s worth remembering that the errors associated with a gravimetric procedure may be significantly less than with its volumetric counterpart.
- The analyte must be completely dissolved. Heating or sonication (not in the cell) is often a prudent step as particles smaller than those visible to the human eye will still scatter light.
- The solution should not be turbid, so filter if necessary. Clearly (no pun intended!), a turbid solution will scatter light and produce erroneous readings.
- Make sure that the cuvettes are clean and orientated consistently in the light beam as well as taking care that there are no bubbles on the cuvette windows. The errors and uncertainties associated with a given procedure pale into insignificance if one considers the effect of a thumbprint on the optical faces of a cuvette.
- Biological matrices such as proteins are particularly prone to adsorption onto the cuvette walls so ensure this effect is not occurring.
- Ensure that the reference solution is subjected to exactly the same procedures as the sample solution. In order to achieve a clear and interference-free solution sample, pretreatment may be required. But even a simple procedure such as filtration may inadvertently remove the analyte under test by adsorption.