Ideally the absorbance spectrum of a solution containing a single analyte should be a single absorption band at the wavelength of maximum absorbance. However, in real samples the spectrum gets influenced by presence of other interfering species. Such interferences can be easily eliminated by adopting different approaches covered in the article.
Interferences in UV- Visible spectroscopy result from several factors which can be both physical or chemical in nature. Chemical interferences result from presence of any single or group of compounds that absorb in close vicinity of the primary absorbing molecule. On the other hand physical interferences generally arise from suspended solid impurities in sample which can lead to scattering.
Scattering of light is caused by the presence of suspended impurities in the absorbing solution.It results in a background absorbance which reduces the absorbance of the analyte of interest. Filtering or centrifugation of samples prior to making absorbance measurements appears to be the obvious solution but this is not a practical approach when only μl size samples are available. The loss of absorbance due to scattering can be reduced by reducing the gap between the sample and the detector.
Chemical interferences can result from presence of a single or a group of absorbing entities in the light absorbing solution.
In case an interfering compound is present with known absorbance characteristics then its interference with the main analyte can be eliminated by selecting a wavelength where the interfering compound shows some absorbance as it does at analytical wavelength.On subtracting the absorbance at this wavelength from the absorbance at analytical wavelength the residual absorbance is the correct absorbance of the analyte. This approach is practical if only a single interferent is present and if its maximum absorbance wavelength is far removed from the absorbing wavelength of the analyte.
Multicomponent analysis is applicable when more than one interferent is present and there is considerable spectral overlap with the spectra of main analyte. The absorbance of pure interferent is subtracted from the measured absorbance to arrive at the true absorbance of the analyte of interest,
Three Point Correction
In this method two wavelengths are selected close to the analytical wavelength but on either side of it. The interference of background can be accurately estimated using linear interpolation. The method is applicable particularly for non-linear background absorbances resulting from complex sample matrices.
By far the most convenient approach to background and noise correction is derivative spectroscopy. The inflexion point of the first derivative corresponds to the wavelength of maximum absorbance and the second derivative appears as the tip of the negative shaped peak. This helps differentiate between very closely spaced or overlapping absorbance peaks. The first derivative further eliminates baseline shifts, if any, and this helps improve the accuracy of quantitative analysis.In addition to baseline shift, derivative spectroscopy also helps overcome the effects of scattering from other unidentified interfering compounds.