Ultraviolet detection derivatization reagents

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

Higher specificity and selectivity can be obtained by reacting -lactams with suitable reagents to form derivatives with improved ultraviolet chromophores. Thus, precolumn derivatization of penicillins with triazole-mercuric chloride and ultraviolet detection at 325 nm (71, 90, 112, 114, 115, 121, 122), 340 nm (123), or 345 nm (116) has become the method of choice for more selective detection and matrix interferences reduction. An alternative precolumn reaction using iodoacetamide as derivatizing reagent has been described in ceftiofur analysis (124), while imidazole-mercuric chloride has also been suggested for on-line postcolumn derivatization of penicillin G (69). 

Specialized Stationary Phases for Liquid Chromatography Chiral Stationary Phases for Liquid Chromatography Detectors for Liquid Chromatography Ultraviolet Detection of Chromophoric Groups Derivatizing Reagents for HPLC... 

Korte, W.D., 9-(Chloromethyl) anthracene a useful derivatizing reagent for enhanced ultraviolet and fluorescence detection of carboxylic acids with liquid chromatography, J. Chromatogr., 243, 153,1982. 

The detection of the individual C vitamers is complicated by their distinctly different properties. Although AA and DHAA are both ultraviolet (UV) absorbers, the absorbance maximum of DHAA is between 210 and 230 nm (15,18,42,43). For practical detection purposes, this makes DHAA particularly susceptible to interferences from a number of naturally occurring food constituents and limits the choice of reagents and solvents. In contrast, AA exhibits a pH-dependent absorbance maximum of 245-265 nm, which makes UV absorbance an ideal choice for detection. On the strength of its reducing capacity, AA can be detected electrochemically, but DHAA is electrochemically inactive. Neither AA nor DHAA fluoresce naturally. However, DHAA readily forms a fluorescent quinoxaline derivative upon reaction with o-phenylenediamine. As a result, chemical derivatization is often used to achieve the sensitivity needed to detect the naturally occurring vitamin C in food. 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

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