A UV – Visible spectrophotometer can be seen in almost any laboratory be it a college lab, research lab or an industrial laboratory. Due to its ruggedness and large-scale presence anyone can be tempted to take it is performance on face value but it is important to keep in mind that a spectrophotometer like any other sophisticated instrument needs periodic calibration against prescribed standards to provide data of unquestionable integrity.
Validation is a fundamental requirement under Good Laboratory Practices and it is equally important to maintain documentary records of all such activities. It is mandatory to validate the performance parameters as suggested in the article. Besides these some additional performance tests will ensure results that would be acceptable in any other laboratory.
Wavelength accuracy establishes the closeness of the recorded wavelength to the true value. It can be established by using either absorption or emission standards
Holmium oxide glass or Quartz filters provides a quick check on wavelength accuracy. The filter provides only sharp absorbing bands in the visible region.In order to establish wavelength accuracy over both the UV and visible region of solution holmium oxide in perchloric acid is recommended in most pharmacopoeia’s. Another reference standard recommended in pharmacopoeia is didymium glass.
The deuterium lamp gives a sharp emission line at 656.1 nm and a weaker line at 486.0 nm. By switching off the visible lamp the position of either or both of the lines can establish the wavelength accuracy. Mercury lamp offers more lines in the UV region in comparison to the deuterium lamp but the deuterium lamp is more convenient to use as an emission standard.
Resolution tells us how well two closely appearing absorbance bands are resolved by the spectrophotometer.For the purpose, a solution of 0.02% w/v of toluene in hexane is used. The ratio of peak at 269 nm to trough at 266 nm should be equal to or greater than 1.5.
Metal or quartz filters have been used for keeping check on photometric or absorbance accuracy. Such filters are convenient to use but lead to complications due to back reflection caused by the reflective coating applied to the quartz plate.
A potassium dichromate solution offers reliable absorbance accuracy standard which is recommended by Pharmacopoeia’s. Potassium dichromate is hygroscopic so it should be dried before weighing. 57.0 to 63.0 mg of the solid is diluted to 1000ml with 0.005M sulphuric acid and absorbance is recorded at 235, 257,313 and 350nm. Since 2005 an additional reference wavelength of 430 nm has been included. The dilution in such case is made 100ml instead of 1000 ml
Stray light results from presence of other wavelengths besides the wavelength isolated by the monochromator.It is an important performance specification as it can lead to noticeable interference particularly at high absorbance values.
As per European pharmacopoeia specification the absorbance of a 12 g/l solution of potassium chloride solution at 198 nm should be 2 or higher.
Additional test parameters
In addition to the stipulated tests additional tests can be performed and these provide useful information on overall performance of the instrument and its component parts. Such tests can include absorbance stability, baseline stability, and noise at zero or at other specified absorbance values. It is equally important that solvents from reliable sources of good quality are used and optically matched pair of absorption cells are used for conducting validation studies.