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Some compounds isolated from biological media were tested as fungicides in biotests where Phytium ultimum and Cladosporium cucumerinum were the fungal strains (108). Moreover, nemato-cidal activity can be checked by Caenorhabditis elegans survival (108). Spores of 32 fungal species were investigated in the TLC detection of carbendazim, thiabendazole, imazalil, captafol, folpet, and chlorothalonil (104).
D. Fluorgenic Detection
Fluorescence gives numerous possibilities in the detection of pesticides. One possibility is the well-known fluorescence quenching, which can be observed on TLC plates impregnated with a fluorescent indicator sensitized for UV 254 and/or 366 nm. A second possibility is much more specific for pesticides. Some compounds exhibit native fluorescence or can be easily transformed into such compounds. For example, carbaryl produces, after alkaline hydrolysis, a-naphtolate anion, which is strongly fluorescent. Its residues can be measured by in situ spectrofluorimetry (66).
A third possibility is the very sensitive and selective enzyme inhibition method as mentioned in Section V.B. This method uses TLC plates without fluorescent indicators, in contrast to fluorescence quenching. The whole TLC plate becomes fluorescent after enzymatic hydrolysis of the substrate, and the nonfluorescent inhibition zones of the pesticides appear on a fluorescent background. This method is of high selectivity and sensitivity (see Section V.B.).
The fluorogenic labeling of pesticides is the last, most interesting possibility for detection and densitometric determination. Earlier, 2,3-dichloro-4,5-dicyano-1,4-benzoquinone [DDQ (XXII), Fig. 8] was applied in fluorescent detection of pesticides. The detection limit was reported as 0.05 (ig/spot (172). 4-chloro-7-nitrobenzo-2,1,3-oxadiazole (NBD-C1) (XXIII, Fig. 8) was another compound used for fluorogenic labeling. N-methyl- and //^-dimethyl carbamates were coupled with this reagent after hydrolysis, and the liberated amino part of these pesticides gave a fluorescent derivative zone on a nonfluorescent background with nanogram/spot sensitivity (173).
Dansyl chloride (iV^-dimethylamino-a-naphthalene-5-sulfonyl chloride) (XXIV, Fig. 8) was used as a derivatizing agent for the TLC detection of herbicides and insecticides such as carbaryl, metacrate, alon, etc. After coupling with dansyl chloride, the pesticides were separated and tris(2-hydroxyethyl)amine or paraffin oil was sprayed on the plates to stabilize the fluorescence. The excitation maximum was at 320 nm and that of the emission was at 500-550 nm (174). Dansyl chloride reacts not only with amino groups but also compounds that hydrolyze to anilines. Forty-nine such compounds were detected on TLC plates by dansylation (175). Earlier the hydrolysis of pesticides was performed in a tube, and the products of the hydrolysis were spotted on the plate and chromatographed. According to a newer method, the hydrolysis of the pesticide metoxuron can be carried out
XXII XXIII XXIV
Figure 8 XXII, DDQ; XXIII, NBD-CI; and XXIV, dansyl chloride.
in situ on the plates after development. These treated TLC plates were sprayed with dansyl chloride and evaluated by densitometry (176).
E. Liquid Crystals as TLC Detectors
Liquid crystal detectors were developed for the visualization of pesticides. Compounds having liquid crystalline properties around room temperature were incorporated into a porous foil, which was pressed onto the developed plates for several minutes. The compounds on the TLC plates were transferred into the foil where the liquid crystalline structure was disturbed by the pesticides incorporated. Light transmittance changed in the spots of pesticides, which were measured between crossed polarizers to give quantitative evaluation of the spot areas (177).
F. Laser-Pyrolysis Scanning (LPS)
A new method for the quantitative determination of pesticides is laser-pyrolysis scanning (LPS). No color reaction or visualization procedure is necessary; simply the TLC plates are placed in a chamber after development and irradiated with an infrared laser to produce a high temperature at the location of the spot. The analyte is swept by a carrier gas to a gas chromatograph and analyzed by an appropriate sensitive GC detection method.
The pesticides p,p'-DDT and methoxychlor were analyzed by this technique. These two pesticides were separated on a TLC plate containing no binder by hexane-methanol (99:1) mobile phase. Their values were 0.6 and 0.1, respectively. A CO2 laser was focused onto the TLC plate, and the pesticides were swept to the detector. The limit of detection was 20 ng for methoxychlor and 50 ng for pj?'-DDT. The response was linear from 50 ng to 2.5 (ig for methoxychlor, and from 200 ng to 2.5 [Ag for p,p'-DDT.
This quantification method requires very careful cleaning of the TLC support before use. The thoroughly-cleaned plates should be stored under nitrogen atmosphere until use (178).