Steps in Analysing samples by UV/Vis Spectroscopy
A solvent must be used for preparation of sample solutions.It must be non-flammable and non-toxic and transparent at all wavelengths. Distilled water. Is ideal but it is not suitable organic or non-polar compounds. Therefore other solvents must be used and their cut off wavelengths must be known as to prevent interactions between solute and solvent which leads to absorption band broadening. Conc, pH and temperature can also affect position and intensity of absorption bands.
The solvent must be of the highest possible grade, greater than 99% purity. If the solvent is organic solutes must be dried thoroughly under a vacuum.
Use analytical balances for accurate mass determinations in preparing the standards and the unknown.To make up volumes a volumetric flask must be used.
If the sample has particulate matter this must be filtered to ensure clear homogeneous solutions. The presence of particulate matter causes scattering of incident light
The instrument must be blanked or zeroed and the blank must be prepared in the same way as the standards and the unknown but must not contain the analyte.
Absorbance values should be between 0.1 and 0.9. At higher absorbances the concentrations of the analyte is too high and the analyte particles are too close together and causes scattering of the incident radiation. Therefore all solutions should be sufficiently dilute so that the absorbances would fall within this range.
If the Lamda max is not known can be determined using a full scan
Prepare calibration curve and read of unknown by interpolation.