UV spectrophotometer is an instrument which makes use of light in the wavelength in the range of visible, near visible, infrared and near infrared range for identification and quantification analysis of pharmaceuticals.
Light consist of many wavelengths in it and when it passes through a solution of a drug, drug molecule tend to absorb part of light and show block the particular wavelength of light in the particular absobstion maxima, the absorption of light is directly proportional to concentration of drug present, a particular drug in a mixture or of many drug absorbs light at very peculiar wavelength a characteristic of that drug molecule. Therefore a particular specific wavelength is mentioned for estimation of a drug by spectrophotometer.
How a drug absorb light in a particular wavelength:
Each drug molecule has its own characteristic molecular formula and its state of electronic configuration, and consists one or more pie electrons and non-bonding electrons, a drug molecule absorb light in a particular wavelength and it is use the energy for excitation of electrons, electrons are excited and take part in sifting from one orbit to other and molecular spinning , and vibrations , more is the molecule able to absorb light energy and transform it to physical change in the state of molecule , more is the absorption of a particular wavelength or a spectrum of wavelength.
Absorbance depends on the concentration of solute in a solution and it is directly proportional to the length of the path through with light passes through, and solution in which a solute is dissolved also affect, hence it should be considered in calculation as a constant factor, or a black reading is determined to eliminate the reading due to solvent being used, at constant temperature and pressure., this is the simple explanation of Beer–Lambert law.
To estimate concentration of a drug a sample is prepared as per directed in monograph in pharmacopoeia, and its absorbance is compared with absorbance of drug with known concentration or purity. Alternatively a graph can be plotted absorption of on Y-Axis against the series of dilution of standard concentration and absorption of a unknown can be extrapolated and concentration its concentration can be found.
It is required that spectroscopes give consistent and reproducible results hence all chances errors must be taken care of , one of the method to ensure this periodic calibration program and sop for calibration of UV/Visible spectrophotometer are designed and adapted.
How UV/Visible spectrophotometer is calibrated.