High-performance liquid chromatography (HPLC) is one of the most important instruments used in the pharmaceutical industry. Most of the pharmacopeial monograph methods are based on HPLC with a UV-Visible detector and It is a useful analytical technique for qualitative and quantitative purposes. In some compound, there is a lack of or absence of chromophore which result in low UV absorption or no UV absorption. Chromophores are a light-absorbing group that allows the 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 that can be used to enhance UV detection:
- Non-chromophoric compounds can be analyzed by using a suitable derivatizing agent which will be UV active with the help of a UV-Visible detector. Before starting the derivatization method development, the sample must be chirally pure. The procedure of pre-column derivatization is challenging hence it’s a problematic and inconvenient method to reproduce.
- Regular derivatization of chiral compounds is not recommended as it will cause racemization. Even the addition of any reagent in the mobile phase may cause a reduction of resolution in the enantiomer. Hence need to use a chiral derivatization reagent for chiral compounds. Use of 5% beta-cyclodextrin in the mobile phase to improve the selectivity of enantiomers.
- Post-column derivatization technique is used by adding the correlated compound through a T-junction allowing for adjustment of the background absorbance to match the concentration of the analyte. The sensitivity of the method will be depending on the final absorbance of the related mobile phase by optimizing the flow rate.
- The addition of reagent forms the complex with the formation of color which can be detected in the visible range.
- Use of chelating agent during sample preparation to form metallocomplexs to improve UV detection.
- The addition of right light absorbing material into the mobile phase is checked by the measure of decline in the light absorbed by eluent as the sample elutes from the HPLC column.
- Use of the following alternate type of detector separately or in a combination of UV-Visible detector with HPLC for the quantitative estimation of assay and impurities.
- Refractive Index Detector (RID): RI detector is also called a universal detector. It’s non-destructive and concentration dependant bulks property. It’s based on the variation of the refractive index of the eluent from the column with reference to the absolute mobile phase. RI detector is less sensitive and generally used for detection of non-ionic compounds and needs control temperature and is inappropriate for gradient elution.
- Electrochemical Detectors (ECD): It can be used to measure the current related to the oxidation or reduction of solutes. ECD is sensitive to changes in the flow rate or constitution of the eluant and it requires a working electrode, reference electrode, and auxiliary electrode.
- Fluorescence Detection (FLD): It needs the compound to have characteristic or usual fluorescence properties. It usually detected the fluorescence compound with bonded rings or a very conjugated planer system. It is extremely sensitive and very selective.
- Charged Aerosol Detector (CAD): CAD is a universal detector that 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 dependent on the chemical properties of the sample. It is comparatively sensitive and gradient conditions can be used.
- Evaporative Light Scattering Detector (ELSD): ELSD includes nebulization and then vaporization in a drift tube which is measured by the resultant light scattering. The detector response is proportional to the presence of the entire quantity of the sample. ELSD can be used with gradients and is not sensitive to temperature or change in flow rate. A volatile mobile phase is required for ELSD and shows uneven response, even though the 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 physiochemical properties of the analyte even a UV detector can be coupled with a suitable detector to confirm that there will be no peak ignored.
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