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High performance liquid chromatography (HPLC) is one of the most important instrument used in pharmaceutical industry. Most of the pharmacopeial monographs methods is based on HPLC with UV-Visible detector and It is useful analytical technique for the qualitative and quantitative purpose. In some of compound there is lack of or absence of chromophore which result into low UV absorption or no UV absorption. Chromophores are light absorbing group that allows detection of analytes.
When there is low or no chromophore in that case it’s very challenging to detect at low concentrations or even at higher content of molecule.
Following are some methods which can be used to enhance the UV detection:
- Non-chromophoric compounds can be analysed by using suitable derivatising agent which will be UV active with the help of UV-Visible detector. Before starting derivatisation method development, sample must be achirally pure. Procedure of pre-column derivatization is challenging hence it’s problematic and inconvenient method to reproduce.
- Regular derivatization of chiral compounds is not recommended as it will cause racemisation. Even addition of any reagent in mobile phase may cause reduction of resolution in enantiomer. Hence need to use a chiral derivatization reagent for chiral compounds. Use of 5% beta-cyclodextrine in mobile phase to improve the selectivity of enantiomers.
- Post column derivatisation technique is used by adding the correlated compound through a T-junction allows to adjust the background absorbance to match the concentration of analyte. The sensitivity of the method will be depending on the final absorbance of the related mobile phase by optimise flow rate.
- Addition of reagent to form the complex with formation of colour which can be detected in visible range.
- Use of chelating agent during sample preparation to form metallocomplexs to improve UV detection.
- Addition of right light absorbing material into mobile phase and it is checked by measure of decline in the light absorbed by eluent as the sample elutes from the HPLC column.
- Use of following alternate type of detector separately or in combination of UV-Visible detector with HPLC for the quantitative estimation of assay and impurities.
- Refractive Index Detector (RID): RI detector also called as a universal detector. It’s non-destructive and concentration dependant bulk property. It’s based on the variation of the refractive index of the eluent from the column with reference to absolute mobile phase. RI detector is less sensitive and generally used for detection of non-ionic compounds and needs control temperature and inappropriate to gradient elution.
- Electrochemical Detectors (ECD): It can be used to measure the current related with the oxidation or reduction of solutes. ECD is sensitive to changes in the flow rate or constitution of the eluant and it requires working electrode, reference electrode and auxiliary electrode.
- Fluorescence Detection (FLD): It needs the compound have characteristic or usual fluorescence properties. It usually detected the fluorescence compound with bonded rings or very conjugated planer system. It is extremely sensitive and very selective.
- Charged Aerosol Detector (CAD): CAD is universal detector which can be generally used for the estimation of non-volatile or semi-volatile samples, including: peptides, proteins, polymers, lipids, carbohydrates, steroids, surfactants and oligosaccharides. The response of CAD is not dependant on chemical properties of the sample. It is comparatively sensitive and gradient conditions can be used.
- Evaporative Light Scattering Detector (ELSD): ELSD includes nebulisation and then vaporised in a drift tube which is measured the resultant light scattering. Detector response is proportional to the present of entire quantity of the sample. ELSD can be used with gradients and is not sensitive to temperature or change in flow rate. Volatile mobile phase is required for ELSD and shows uneven response, even though compound is structurally similar.
The UV-Visible detector is common and inexpensive but, in some cases, different detection methods or detectors need to be used to detect the non-chromophore compound. The selection of an appropriate technique or detector for the non-chromophore compound depends on the physio-chemical properties of the analyte even a UV detector can be coupled with a suitable detector to confirm that there will be no peak is ignored.
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