Fluorescent derivatives, limitations

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

Other limitations found in fluorescent derivatives include quenching by oxygen, metals, or other species, chemical or photochemical instability, and biodegradability. 

A -3-Ketosteroids, e.g. progesterone in plasma 150°C, 20 min Conversion of A -3-ketosteroids into fluorescent derivatives (Xn = 440 nm). Relatively selective for progesterone at 150°C, detection limit 2-5 ng. 

Fluorescence detection is sensitive to naturally fluorescent analytes or to fluorescent derivatives. Amperometric detection is sensitive to analytes that can be oxidized or reduced at an electrode (Figure 26-29). Conductivity detection with ion-exchange suppression of the background electrolyte (as in Figure 26-4) can detect small analyte ions at 1-10 ng/mL. Electrospray mass spectrometry (Figure 22-18) provides low detection limits and gives qualitative information about analytes.33... 

Derivatization is never used in the analysis of the different phospholipid classes present, but is very widely used in the analysis of molecular species. The main reason for (mostly precolumn) derivatization in this case is to reduce significantly the detection limits. To this end, both UV-ab-sorbing and fluorescent derivatives are frequently used, as described in more detail in Secs. IV.B and V.3. 

The main limitation of direct FILAs is the limited availability of fluorescent target molecules. The preparation of a fluorescent derivative is time-consuming and requires the presence in the molecule of an appropriate functional group. On the other hand, derivatization may compromise MIP selectivity. 

The first work in this field was probably that of Piletsky et al. [84] that described a competitive FILA for the analysis of triazine using the fluorescent derivative 5-[(4,6-dichlorotriazin-2-yl)amino]fluorescein. The fluorescence of the supernatant after incubation was proportional to the triazine concentration and the assay was selective to triazine over atrazine and simazine. The same fluorescent triazine derivative was applied to competitive assays using atrazine-imprinted films [70]. To this end an oxidative polymerization was performed in the presence of the template, the monomer(s) 3-thiopheneboronic acid (TBA) or mixtures of 3-amino-phenylboronic acid (APBA) and TBA (10 1) in ethanol-water (1 1 v/v) where the template is more soluble. The polymers were grafted onto the surface of polystyrene microplates. The poly-TBA polymers yielded a detection limit of 8 pM atrazine whereas for the poly-TBA-APBA plates it was lowered to 0.7 pM after 5 h of incubation. However, a 10-20% decrease in the polymer affinity was observed after 2 months. 

The selectivity of fluorescence is one of its most important advantages as an analytical method. Selectivity is obtained mainly through structural restrictions. Only certain types of molecules possess the ability to fluoresce and often only under specified conditions. However, this fact is also the main reason for the limited use of fluorescence for analysis. Nevertheless, the formation of fluorescent derivatives of compounds which do not fluoresce permits the method to be extended into most areas of concern to the analytical chemist. 

Chloro-7-nitrobenz-2,l,3-oxadiazole (NBD-C1) reacts with primary and secondary aliphatic amines to produce intensely fluorescent derivatives. Although anilines, phenols and thiols react with NBD-C1, they produce derivatives which do not fluoresce or which are only weakly fluorescent. This limits the types of compounds which can be analyzed with this reagent and thus makes the technique more selective. This selectivity is desirable in trace analysis because of a lower degree of interference from co-extractives. The general reaction scheme for formation of the fluorescent amine derivatives is shown in Fig.4.50. 

These detectors are often used to detect components of a fluorescent derivative prepared to increase the detection sensitivity of compounds with poor UV absorptions. Both variable and filter variable, fixed-wavelength fluorom-eters are available for HPLC, with the same limits of lamp life and sensitivity seen in comparable UV detectors. 

N-Methylcarbamate insecticides have been labelled with dansyl chloride (28). The procedure involves hydrolysis with aqueous base to form a phenol and methylamine. The reagent reacts rapidly with both compounds and the newly formed fluorescent derivatives can then be applied and separated by tic (29, 30). A typical reaction scheme is shown in Figure 7. Detection limits are good ( 1 ng per spot) and dansylation of pesticides can be accomplished with extracts from water and soil samples (31J. ... 

Goto et al. (67) synthesized the sucdnimidyl ester [14] of (—)- -methoxy-a-methyl-l-naphthaleneacetic acid for the normal-phase LC resolution of chiral amines. The reagent permitted the determination of the enantiomers of an amphetamine derivative in blood plasma after administration of racemic drug to rabbits. With detection at 280 nm, the lower limit of sensitivity was 5 ng/mL for each enantiomer (67). Several chiral acids from the "profen" group of nonsteroidal antiinflammatory drugs have been adapted as CDAs. One of these, naproxen, [15], is the S enantiomer and is commercially available as the resolved acid several of these acids have the advantage of providing fluorescent derivatives (68,69). 

By reaction of natamycin (or its decomposition products containing the mycosamine group) with NBD-chloride (4-chloro-7-nitrobenzo-2-oxa-l,3-diazole) a fluorescent derivative was obtained. Upon chromatography on silica gel the spots were quantified by means of a spectrofluorometer. The detection limit on the plate was 8 ng [27]. This procedure has been applied for the determination of residual natamycin in food. 

The separation power and sensitivity of CE is illustrated in Figure 21.23. A mixture of 18 amino acids is separated in 30 min, at quantities ranging from 2 to 7 attomoles. The amino acids were derivatized with fluorescein isothiocyanate (Fl i C) to form fluorescent derivatives, and a fluorometric detector system was employed. The detection limits ranged down to 10""2o mol in 1 nL, corresponding to 10 " M and 6000 molecules ... 

Zika (1977) compared two precolumn reagents — fluorescamine and d2insyl chloride — and found the latter to be more suitable for seawater determinations. The long reaction times and the solvent extraction step involved prior to injection, together with the bulky nature of the derivative (leading to a loss of resolution), and the fact that the reagent and the reaction by-product fluoresce themselves, limits the usefulness of the method. 

For this purpose detectable levels in the range of 1 pg per ml of plasma are needed. In order to achieve this sensitivity it is necessary to make a fluorescent derivative of sulfisoxazole. One such derivative is the highly fluorescent "Dansylsulfonamlde" which is formed by the reaction shown in Figure 9. The fluorescence yield of this compound is sufficient to measure about 1 yg of sulfisoxazole per ml of plasma. An even more sensitive derivative is formed when sulfisoxazole is reacted with 4,5-methylene-dioxy-phthalaldehyde to form a phthalimidine derivative (Figure 9). The sensitivity limit for this derivative is from 1 X 10 to 1 X 10 10 mole/ml (22). 

A fluorescent derivative of clofazimine was formed following reduction with titanous chloride (Dill et al., 1970). The fluorescence was measured at 366m i emission. The limits of... 

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