Analytical Method Development Approach When Compound Don’t Have UV Chromophore

High performance liquid chromatography (HPLC) is one of the most important instruments 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:

1.  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.

2.   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.

3.   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.

4.    Addition of reagent to form the complex with formation of colour which can be detected in visible range.

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