Derivatization for selective and sensitive detection

The availability of both GC and HPLC detectors in SFC means that derivatization to enhance selectivity and sensitivity should be an important analytical tool. Thus, David and Novotny showed [25] how nitrogen thermionic detection of quinoxalinol derivatives of a-keto acids was sensitive at the pg level with a response linear over 3 4 orders of magnitude. The derivatives were formed by reaction with o-phenylenediamine  

The thermionic detector was used [26] in phosphorus mode for the capillary SFC analysis of thiophosphinic esters derived from steroids by reaction with dimcAhyl-thiophosphinic chloride in the presence of 4-dimethyl-aminopyridine catalyst. 

Figiire 7. SFC separation of quinoxalinol derivatives of a-keto acids of human urine. Tentative peak assignments 1, methyl 2, ethyl 3, n-propyl 4, benzyl quinoxalinols. Conditions I0mx50/ m ID open-tubular column SE-30 stationary phase NjO mobile phase temperature 110-200 °C thermionic detection. (Reprinted with permission from Ref 25). 

A sensitivity of 120fgs was observed for the bis(thiophosphinic) ester of pregnanediol. The SFC analysis of steroids from human urine and plasma as thiophosphinic esters was demonstrated. 

Derivatization has also been applied to generate UV-absorbing compounds from amphetamines [27]. The reagent used was FMOC, which reacts with primary and secondary amines to form derivatives which provided selective and sensitive UV detection during packed column SFC with modified COj as mobile phase. David and Sandra noted [28] that COj is able to react with amines at elevated temperatures and pressures to give carbamic acid derivatives insoluble in supercritical CO2. Formation of the trifluoroacetyl derivatives, however, gave excellent peak shapes in capillary SFC for a variety of Cift-Cii amines with COj mobile phase and FID. 

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