Phase 2,4-dinitrofluorobenzene

Walle [100] carried out the derivatization after prior extraction of an aqueous sample (urine) with benzene. A 3-ml volume of the sample and 1 ml of an aqueous solution of an internal standard were mixed in a 50-ml centrifuge tube, 1 ml of 5 M K2C03 and 30 ml of benzene were added and the mixture was shaken for 10 min. After the centrifugation, the benzene phase was separated and 25 ml of the benzene solution of the amine were mixed with a five-fold excess of 2,4-dinitrofluorobenzene dissolved in 1 ml of benzene. After standing for 5 min at room temperature, the solution was heated at 60°C for 15 min, cooled and 2—5 pi were chromatographed. 

The effects of micelles on the rates of nucleophilic aromatic substitution reactions (equations 42a-42e) follow a similar pattern. The rate constant for reaction (42a) was unaffected by the presence of micellar sodium dodecyl sulfate, even though 2,4-dinitrofiuorobenzene partitions strongly in favor of the micelles and the free base of glycineamide is not appreciably solubilized. On the other hand, the rate constant for the reaction with glycylglycine (equation 42b) decreased by a factor of 3-6 in micellar sodium dodecyl sulfate solution and increased by a factor of ca. 15 in the presence of CTAB (Herries et al., 1964). Since the rate retardation in NaLS solutions was observed to parallel the partitioning of 2,4-dinitrofluorobenzene in favor of the micellar phase and the... 

To 25.0 ml of freshly voided urine is added 5 N NaOH with stirring until it is just alkaline to phenolphthalein paper. The solution is filtered through filter paper, and to 20.0 ml is added 5 ml of buffer (8.4 g NaHCOa dissolved in about 80 ml of H2O the pH is adjusted to 8.8 with 5 N NaOH and diluted to 100 ml with H2O). To this mixture is added 2.0 ml of freshly prepared 2,4-dinitrofluorobenzene (10% in ethanol w/v) and 40.0 ml of ethanol. Mix well and keep in a dark cabinet with occasional stirring for about an hour. The reaction mixture is cooled to room temperature and the pH adjusted with 5N NaOH to about 12.0. The mixture is transferred to a 100-ml separatory funnel and extracted three times with 10-ml portions of ether to remove excess reactant the ether phases are discarded. 

Aliphatic and aromatic nucleophilic substitution reactions are also subject to micellar effects, with results consistent with those in other reactions. In the reaction of alkyl halides with CN and S Oj in aqueous media, sodium dodecyl sulfate micelles decreased the second-order rate constants and dodecyltrimethylammonium bromide increased them (Winters, 1965 Bunton, 1968). The reactivity of methyl bromide in the cationic micellar phase was 30 to 50 times that in the bulk phase and was negligible in the anionic micellar phase a nonionic surfactant did not significantly affect the rate constant for n-pentyl bromide with S2O3-. Micellar effects on nucleophilic aromatic substitution reactions follow similar patterns. The reaction of 2, 4-dinitrochlorobenzene or 2, 4-dinitrofluorobenzene with hydroxide ion in aqueous media is catalyzed by cationic surfactants and retarded by sodium dodecyl sulfate (Bunton, 1968, 1969). Cetyltrimethylammonium bromide micelles increased the reactivity of dinitrofluorobenzene 59 times, whereas sodium dodecyl sulfate decreased it by a factor of 2.5 for dinitrochlorobenzene, the figures are 82 and 13 times, respectively. A POE nonionic surfactant had no effect. 

Sample preparation 2 mL Plasma + 100 ng tolbutamide + 500 jaL 1 M HCl + 8 mL chloroform, shake on a reciprocal shaker, shake for 10 min in a reciprocal shaker, centrifuge at 2000 g for 15 min. Remove 7 mL of the lower organic layer and evaporate it to dryness under a stream of nitrogen at 45°, reconstitute the residue in 100 (jlL 3 mg mL dinitrofluorobenzene in n-butyl acetate, heat at 120° for 30 min, evaporate to diyness under a stream of nitrogen at 60°, dissolve the residue in 100 p-L mobile phase, inject a 30-70 (jlL aliquot. (Recrystallize dinitrofluorobenzene from diethyl ether. Prepare solutions weekly, store at 4° in the dark.)... 

Sample preparation 5 g Ointment + 3 mL MeOH, heat at 55° for 5 min, vortex twice for 20 s, centrifuge at 2000 g for 2 min, discard the supernatant, repeat the MeOH wash twice more, add 30 mL chloroform, heat at 55°, vortex for 15 s, add 10 mL MeOH water 20 80, shake vigorously for 20 min, centrifuge at 1000 g for 3 min, remove the upper aqueous layer, repeat the MeOH/water extraction twice more. Combine the aqueous layers and make up to 50 mL with 20 mM pH 9.0 borate buffer. Remove a 10 mL aliquot and add it to 15 mL 150 mM 2,4-dinitrofluorobenzene in MeOH, heat at 100° for 45 min, cool, make up to 250 mL with mobile phase, inject an aliquot of the yellow organic layer. 

Binitrophenylatton Levy and Li [163] dissolve at least 0.2 pmole of the material in 3 ml of 0.05N aqueous KCl at 40° C and adjust the pH to 8 with 0.05 potassium hydroxide using an auto-titrator. About 0.1 ml of dinitrofluorobenzene is added and the solution energetically stirred in the dark at constant pH and temperature. The end of the reaction is indicated when alkali consumption ceases. The solution is then extracted three times with ether and the aqueous phase acidified in order to precipitate the dinitrophenyl derivative. The precipitate is centrifuged off, washed with water, acetone and ether and dried in a desiccator over P2O5. 

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