UV – VIS spectroscopy offers a fast and accurate estimation of analytes in diverse range of applications. The sensitivity of the technique is further enhanced by reaction with reagents that impart colour to otherwise colourless species. Quantitative analysis using UV – VIS spectroscopy has large-scale applications for determination of inorganic analytes including both metals and non-metals, organic compounds, metal – ligand interactions and stability and kinetic studies on light absorbing species.
Beer – Lambert’s law is universally applied in quantitative determinations. In earlier days instruments based on visual comparisons of colour intensities were used but with the advent of high precision spectrophotometers such filter based instruments became obsolete.
UV – Visible spectroscopy offers all the advantages mentioned above but in order to maintain a high degree of accuracy and precision the following precautions need to be taken during analysis.
Generation of absorbing entities
It would be indeed rare that you will come across analytes that can be determined directly due to their strong absorption in the UV- visible region. Mostly you will have to react them with reagents that result in selective colour formation. The conditions employed while carrying out such reactions should be reproducible. You have also to ensure that the reaction with the reagent is fully quantitative.
Each measurement should be made for highest sensitivity at the wavelength of maximum absorbance λ max. Generally the absorbing compounds produce a broad shaped absorbance peak around λ max which is an advantage as any slight variation in absorbance wavelength due to instrumental factors does not affect the sensitivity of determinations. However, λ max should not be close to the absorption maximum signal of the blank solvent or absorption background in the reference cell.
Contributing factors to absorbance variations
Special precautions need to be taken to ensure that sample and reference beams pass through identically controlled sample and reference solutions in respective cells. Special care needs to be taken with regards to matched pair of cell windows, handling of cells, pH and absence of interfering species.
Validation through calibration
Validation of the chosen procedure should be established before proceeding with the analysis. Using different standard concentrations linear plot should be established and estimations should be conducted over the linear portion of the calibration plot.
UV- VIS Spectroscopy is an established technique for quantitative estimations but you can place reliability on results only if the suggested precautions have been exercised during the measurements.