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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.^[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.^[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 $y_c$ givesFor 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.

At chemical equilibrium, ${\mu }_{\mathrm{AB}}={\mu }_{\mathrm{A}}+{\mu }_{\mathrm{B}}$. Using the number-concentration form givesHere $\Delta {\epsilon }_0$ is the reaction energy between the chosen molecular reference states. Rearranging,Define the dimensionless concentration equilibrium constant byThusThe standard concentration $c^{\circ }$ has the same units as $c_i$. It converts concentration ratios to ideal thermodynamic activities; changing it changes the numerical dimensionless $K_c$ for this dissociation, which creates an extra particle. The unnormalized quotient $c_{\mathrm{A}}{c}_{\mathrm{B}}/c_{\mathrm{AB}}$ instead has concentration units.^[ref]

First construct the energy cost for heterolysis from the molecular potential minimum. Dissociation to neutral atoms costs $D_e$, ionizing caesium costs $I$, and attachment of the electron to iodine releases EA. ThereforeFor the ground-state reference convention used above, the initial molecule already has vibrational zero-point energy. Thus its ground-state dissociation energy is $D_0=D_e-\frac{1}{2}{k}_B{\theta }_{\mathrm{vib}}$ andAt $3000\mathrm{K}$, $k_{B}T=0.258520\mathrm{eV}$, givingAlternatively, retaining the molecular minimum as reference gives $\Delta {\epsilon }_{\min }=4.36\mathrm{eV}$ and a Boltzmann factor $4.7372\times 10^{-8}$. That convention must use the full vibrational partition function, including $e^{-{\theta }_{\mathrm{vib}}/(2T)}$, in the next part. The ground-state convention instead uses $q_{\mathrm{vib}}^0$. Either consistent convention gives the same final equilibrium constant; using the ground-state reaction energy with the full zero-point factor would count the correction twice. The distinction between $D_e$ and $D_0$ is a distinction between initial states, not a change in the dissociation products.^[ref]

Volumetric analysis determines an amount of substance from the measured volume of a reagent of known strength that reacts with it in a known proportion. A measured aliquot is placed in a suitable vessel, the standard solution is delivered from a burette, and the volume required to reach the chosen titration endpoint is read. A pipette measures the aliquot; a volumetric flask prepares or dilutes the standard solution.

For a reaction $aA+bB⟶\text{products}$, if the concentration of the titrant $B$ is $c_B$ and the corrected volume delivered is $V_B$, thenWith volume in litres and concentration in moles per litre, the answer is an amount in moles. The corresponding mass is $m_A=M_A{n}_A$, using the appropriate molar mass. For an unknown solution of aliquot volume $V_A$,These are modern symbols for the same proportional reasoning that a nineteenth-century analyst could express with equivalent weights and a specified standard solution.

A suitable reaction is sufficiently rapid, complete and selective, with a recognisable endpoint. In an acid-base titration, an indicator can change colour near neutralization; other reactions use precipitation, redox or a colour intrinsic to a reagent. The observed titration endpoint must represent the required equivalence point closely enough. The reagent's nominal preparation alone is insufficient if its concentration changes in storage; standardisation of a solution, a reagent blank where needed, and correct reading of the meniscus control systematic error. A titration measures the intended analyte only insofar as interfering substances have been excluded or accounted for.