The chemical requirements for post-column derivatization are generic.
- Stability of Reagent: The minimum reagent stability sufficient for routine work is one day. This means that the yield and signal-to-noise ratio for a given sample must remain constant for at least 8 hours.
- Completeness of Reaction: The analytical separation is complete when the reagent is mixed with the column effluent. Therefore, in order to minimize band spreading, it is important to keep the volume small between the mixing tee and the detector. If the reaction is slow (in excess of one minute), an elevated temperature can be used to decrease the reaction time.
- Reproducibility: Unless the system consistently produces the same signal for the same sample, quantitation is impossible. Because the reaction is occurring ³on the fly² as the combined column and reagent stream flows toward the detector, the reproducibility is linked to the flow-rate precision of the pumps and to the temperature. Accordingly, even an incomplete reaction will be as repeatable as the retention time for any given species. The completeness of the reaction, then, is not strictly necessary for reproducibility, but it is important for maximum sensitivity.
- Minimal Detector Response of Reagents: The color or background fluorescence of the reagent (or its by-products) represents a continuous noise source. Because the reagent is present in excess relative to the analyte, the analyte’s signal could be obliterated by the reagent’s strong background signal. Pickering’s Chromatographic Grade® eluants and reagents are guaranteed to produce the absolute minimum possible detector background signal in post-column applications.
- Solubility: All species must remain in solution, including the combined components of the eluants and the reagent(s), as well as the newly formed derivative(s). Precipitates can block capillary tubes, burst reactors and foul detector flowcells.
Ninhydrin chemistry provides a good example of multiple solubility considerations. Ninhydrin reagents contain a lithium acetate buffer, ninhydrin, hydrindantin, and a water-miscible organic solvent. The organic solvent is necessary to maintain both the hydrindantin and the new purple chromophore (derivative) in solution. Also the presence of lithium ion in the formula precludes the use of eluants containing phosphate, because lithium phosphate is insoluble and would precipitate at the point of mixing.