The main purpose of derivatization in HPLC is to improve detection specifically when determining traces of solutes in complex matrices, for example :
(i) Pharmaceutical substances lacking an UV-chromophore in the 254 nm region but possessing a reactive functional group,
(ii) Biological fluids e.g., blood, serum, urine ; cerebrospinal fluid (CSF); and
(iii) Environmental samples.
Derivatization may be accomplished by two means, namely :
(a) Pre-column off-line derivatization.
(b) Post-column on-line derivatization.
These two methods shall be discussed briefly at this juncture :
This technique has the following merits :
(a) Requires no modification to the instrument i.e., a plus point when compared to the post-column methods, and
(b) Imposes fewer limitations with regard to reaction-time and conditions.
The demerits include :
(a) Formation of a stable and well-defined product is an absolute necessity,
(b) Presence of excess reagent or by products may invariably interfere with separation, and
(c) Very often derivatization may altogether change the chromatographic properties of the sample which facilitated separation.
The following experimental parameters should be maintained, namely :
(a) Derivatization performed in a special-reactor strategically positioned between the column and the detector,
(b) Reaction must be completed rapidly at moderate temperatures,
(c) Derivatization reaction need not even go to completion provided it can be made reproducible,
(d) No detector-response should exist due to any excess reagent present, and
(e) Reaction must be carried out in a medium other than the mobile-phase.
The main merit of post-column-on-line derivatization is that ideally the separation and detec-tion processes can be optimized individually.
There are potentially viable reagents available that may be employed for the derivatization of compounds either for enhancing UV/visible radiation (called chromatags) or for reaction of non-fluorescent reagent molecules (called fluorotags) with solutes to yield fluorescent derivatives.
Examples : (i) Derivatization for UV-Detectors :
Ninhydrin (a chromatag is commonly employed to yield corresponding derivatives of amino acids that show absorption specifically at about 570 nm as shown in the following reaction :
(ii) Derivatization for Fluorescence Detectors :
Dansyl Chloride (a fluorotag) is invariably used to obtain fluorescent derivatives of proteins, amines and phenolic compounds, the excitation and emission wavelengths being 335 to 365 nm and 520 nm respectively.