Why and How To Validate UV – Visible spectrophotometer
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's
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 Practicesand it is equally important to maintain documentary records of all suchactivities. 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
Wavelength accuracy establishes the closeness of the recorded wavelength
to the true value. It can be established by using either absorption or emission
standards
Absorption 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.
Emission standards
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
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.
Photometric accuracy
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
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.
Spectrophotometer
ReplyDeleteCan you suggest a good Spectrophotometer. Thanks.
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