= Derivatization
\b[Derivatization] chemically converts an <analyte> into a related product whose properties are more suitable for a measurement or separation. A useful change might improve <fluorescence> detection or alter chromatographic behavior. It is a chemical transformation of the analyte, rather than merely diluting the sample or increasing the detector gain.
A primary <capillary electrophoresis> experiment by <James W. Jorgenson> and <Krynn DeArman Lukacs> used a fluorescence detector for separated amino acids, peptides and urinary amines; its abstract explicitly states that fluorescent derivatives were necessary for detection.<Free-zone electrophoresis in glass capillaries by J. W. Jorgenson and K. D. Lukacs>[Clinical Chemistry 27, 1551–1553, abstract: fluorescence detector and required derivatives]{ref} The observation demonstrates the dependence of a successful separation-and-detection system on the chosen chemical and optical response, not universal fluorescence of the unmodified analytes.
The order of reaction and separation matters. IUPAC defines <post-column derivatization> as reaction of separated components after they leave the column and before detection, and contrasts this with derivatization before the sample enters the separation medium.<IUPAC Compendium of Chemical Terminology (Gold Book) by International Union of Pure and Applied Chemistry>[Entry P04771, Post-column derivatization; originating chromatography Recommendations 1993, p. 827]{ref}
A simple mass-balance model exposes <derivatization yield bias>. Let $n$ be the initial analyte amount and let a 1:1 reaction convert a fraction $y$ to a detectable product. With background-subtracted signal $S=kyn$, a calibration made at yield $y_c$ gives
$$
\widehat n=\frac{S}{ky_c}=\frac{y}{y_c}n.
$$
For stipulated sample yield $y=0.80$ and calibration yield $y_c=1.00$, an actual $10\,\mathrm{nmol}$ gives an apparent $8\,\mathrm{nmol}$. A perfectly repeatable detector can therefore support a biased original-analyte result. This model assumes one detected product per converted molecule and equal detector response for that product.
Reagent blanks, product identity, competing reactions and the <sample matrix> address different parts of that inference. Measuring the derivative accurately does not by itself prove complete conversion or establish the amount originally present.
Back to article page
