Fluorimetric determination of lactate with immobilized lactate dehydrogenase

In measuring inter-phase distribution coefficients (k_{$\text{1}\text{11}$})it is often convenient, and sometimes necessary, to obtain all of the analytical data from one phase. A rigorous formula, applicable to both low and high k_{$\text{1}\text{11}$} values has been derived; measurements of the distribution of mercury, methylmercury and ⁶⁷Cu²⁺ between various pairs of phases with this formula gave k_{$\text{1}\text{11}$} values similar to those obtained from other approaches.     

The use of immobilized alcohol oxidase in the continuous flow determination of ethanol with an oxygen electrode

Immobilized alcohol oxidase was used in the determination of blood alcohol. The alcohol oxidase catalyzed the aerobic oxidation of ethanol and the oxygen concentration was monitored with an oxygen membrane electrode in a flow cell. The enzyme was immobilized either by covalent attachment via glutaraldehyde to the inside walls of nylon tubing, or by adsorption onto three separate controlled-pore glass support materials: TiO₂, SiO₂, or AL₂O₃. The supports were packed into 10 cm lengths of 3 mm i.d. glass tubing or 30 cm lengths of 5 mm i.d. nylon tubing. The five methods of immobilization were compared for stability and activity toward ethanol. Immobilization on silanized glass beads results in the highest activity and greatest stability of the reactor.     

Fluorimetric determination of zinc with pyrocatechin-1-aldehyde 2-benzothiazolylhydrazone

A rapid procedure for the spectrofluorimetric determination of 0.25 to 7.5 μg of zinc with PABTH, in a 50% ($\text{v}\text{v}$) DMSO-water medium at apparent pH 7.5 of Tris-HClO₄ is described (λ_ex = 425 nm, λ_ex = 490 nm, corrected). Interferences have been evaluated and the procedure applied to the determination of zinc in zinc-containing alloys.     

Spectrophotometric determination of ethanol in blood using a flow-injection system with an immobilized enzyme (alcohol dehydrogenase) reactor coupled to an on-line dialyser

The determination of ethanol in whole blood without pretreatment using spectrotometric detection with an immobilized enzyme reactor coupled to an on-line dialyser is described. Optimum working conditions are given. The effects of the injection volume and the flow-rate on the peak height using the dialyser were investigated. A good linear calibration graph over the ethanol concentration range 3–40 μg ml^?1 was observed; these concentrations correspond to 0.3?4.0 g of ethanol per 1000 g of whole blood prior to dilution. For comparison, whole blood samples were analysed by gas chromatography with direct injection. The effect of temperature on the peak height was also studied in a system without the dialyser.     

Heterocalixarenes. Part 2. Calix[m]uracil[n]benzimidazol-2(1H)-one[3] arenes: Synthesis and Binding Characteristics

The reactions of uracil/benzimidazol-2(1H)-one with 1,3-bis(bromomethyl)benzene provide respectively1 3-bis[(3-bromomethylbenzene)methyl]uracil/benzimidazol-2(1H)-onewhich on subsequent cyclization with1,3-bis[(uracil-1-yl/benzimidazol-2(1H)-one-1-yl)methyl]benzenederivatives provide respectively calix[m]uracil[n]benzimidazoI-2(1H)-one[3]arenes[m =3, n = 0 (9); m = 2, n = 1 (10);m = 1, n = 2 (11) and m = 0, n = 3 (12)]. The heterocalixarenes 9–12, both in liquid–liquid and solid–liquid extraction experiments, selectively extract ammonium picrates over the similarly sized K⁺ picrate. The selectivity is much more pronounced in the case of solid-liquid extractions. Both in L-L and S-L extractions, 10 exhibits the highest order oft-BuNH₃ ⁺/K⁺ selectivity.     

Stable free-radical complexing reagents in applications of electron spin resonance to the determination of metals: Part 2. Spin-labelled iminooxime [1]

Possibilities for the determination of metals by means of the intensity of the e.s.r. signal of the chelate-fonning reagent, spin-labelled iminomonoxime — 4-oximinomethyl-2,2,5,5-tetramethyl-3-imidazoline-1-oxyl, have been studied. The dissociation constant of the oxime group (pK^T_a = 9.36 ± 0.08) and the reagent partition constant in the chloroform—water system (log K_D = 0.80 ± 0.11) are reported. The reagent extracts copper, cobalt and nickel into chloroform. Copper is extracted as its CuA₂, chelate (log K_D = 0.91 ± 0.03; log K_ex = -3.35 ± 0.09; log β₂, = 15.8 ± 0.2). Several properties of the spin-labelled and conventional oximes are compared. It is confirmed that a radical-containing substituent produces a strong electron-acceptor effect. Unusual extractive and e.s.r.- spectroscopic behaviour of cobalt is indicated; an adduct of the spin-labelled chelate with atmospheric oxygen seems to be formed. Methods for the determination of cobalt and nickel based on the extraction with spin-labelled oxime into chloroform and subsequent separation of the excess of reagent on a chromatographic column are described. The detection limits are 3 × 10^-7 M for cobalt and 10^-6 M for nickel.     

25,26-Dialkoxycalix[4]arenes. Part 2: 1-Alkoxy-3-benzoyloxy route

Benzoylation of calix[4]arene monoalkyl ethers with benzoyl chloride yielded the corresponding 3-benzoates and/or 2,3-dibenzoates in different reaction conditions. A simple recrystallization process was able to isolate the 3-benzoates in good yield. In the presence of NaH as reaction base, the 1-alkoxy-3-benzoyloxycalixarenes were alkylated with active alkyl halides at proximal position and yielded the corresponding 1,2-dialkoxy derivatives. Basic hydrolysis of compounds afforded the expected 25,26-dialkoxycalix[4]arenes. For the less active alkyl halides, alkylation of 1-alkoxy-3-benzoyloxycalix[4]arenes afforded both the 1,2-dialkoxy derivatives and the benzoyl-migrated 1,3-dialkoxy derivatives. Only the highly symmetrical 1,3-diethoxy-2-benzoyloxycalix[4]arene was able to be isolated upon the deliberate recrystallization process. After basic hydrolysis of the dialkylated crude products, 25,26-dialkoxycalix[4]arenes were chromatographic separated.     

Synthesis of perfluorochemicals for use as blood substitutes. Part III. [1] electrochemical fluorination of quinolozidine, 4-methylquinolizidines and 4-trifluoromethylquinolizidines [2]

Electrochemical fluorination of quinolizidine gave F-quinolizidine in 16-23% isolated yields. 4-Methylquinolizidine was also fluorinated electrochemically to give the corresponding amine stereoisomers along with their fragmented and rearranged products in isolated yields of 28-34% and 2-3%, respectively. Introduction of an F-methyl group into the quinolizidine in place of the methyl group prior to electrochemical fluorination did not stabilize the 4-methyl quinolizidine structure and rather allowed the formation of F-quinolizidine to a greater extent. Oxidation of 4-(F-methyl)-F-quinolizidine with fuming sulfuric acid in the presence of a catalytic amount of HgSO₄ gave 6-oxo-4-(F-methyl)-F-quinolizidine in 60% yield, the F-nmr spectra of which gave further support to the structure of the starting material. The physical and spectral properties of these F-chemicals are described.     

[1,2]-Wittig rearrangement of acetals. Part 2: The influence of reaction conditions

Acetals of seven alcohols with (+)-camphor derived enantiomerically pure 7,8,8-trimethyl-4,7-methanobenzofuran-2-ol were subjected to different reaction conditions favorable for a [1,2]-Wittig rearrangement. Results with regard to conversion, yield and stereochemical course depending on the reaction conditions are discussed.     

Analysis of steroids: Part XXVIII. Kinetic method for the simultaneous determination of α- and β-ethynodioldiacetate [1]

The kinetics of the acid-catalyzed elimination reactions of α- and β-ethynodiol diacetate, leading to the spectrophotometrically active 17 α-ethynyl-3,5-oestradiene-17-ol acetate, are described. Under suitable conditions, the reaction rate of the α-epimer is about, five times greater than that of the β-epimer. Although acid-catalyzed isomerization takes place simultaneously and interferes with the elimination reaction, it is possible to determine not only the sum of the epimers but also their ratio if they are present in similar quantities.     

Fluorinations with complex metal fluorides. Part 6 [1] fluorination of yridine and related compounds with caesium tetrafluorocobaltate(III) [2]

Pyridine has been fluorinated over caesium tetrafluorocobaltate(III) (CsCo^IIIF₄) at 300–400°C to give a mixture of undecafluoro-N-methylpyrrolidine, bis(trifluoromethyl)amine, pentafluoropyridine and several polyfluoropyridines; the product composition depended to some extent on the geometry of the reactor. The fluorinations of pentafluoropyridine, piperidine and undecafluoropiperidine were also investigated.     

Fluorimetric kinetic studies and sub-muM determination of aluminium with 2-hydroxy-1-naphthaldehyde p-methoxybenzoylhydrazone

A fluorimetric study has been made of the kinetics of the reaction of 2-hydroxy-1-naphthaldehyde p-methoxybenzoylhydrazone with aluminium, and a rate equation and a possible kinetic scheme are proposed. Experimental conditions are defined under which 0.020-10.0muM aluminium can be determined with an average error of 3.7% and a coefficient of variation of about 4.6%.     

The mechanism of thermal eliminations. Part XXVIII [1]: Thermal decomposition of benzoylformic acid

The thermal decomposition of benzoylformic acid into carbon dioxide and benzaldehyde is a unimolecular first-order reaction which takes place according to the rate equation log k = 15.3–42,500/4.575 K. At 600 K the reaction takes place 18 times faster than the corresponding decomposition of pyruvic acid into carbon dioxide and acetaldehyde. This is consistent with the 4-center process proposed previously for pyruvic acid, in which a partial negative charge develops on the carbon of the α-carbonyl group in the transition state. Benzoylformic acid is considerably less reactive towards thermal elimination than is oxalic acid, which also accords with the proposed mechanism.     

Cyclic regeneration of nicotinamide adenine dinucleotide with immobilized enzymes: Flow-injection spectrofluorimetric determination of ethanol

Ethanol (0.05–0.5%) in water is determined by injection of a 20-μl sample into a solution of 1.5 × 10^?3 M NAD⁺ in pH 8.0 phosphate buffer flowing from a reservoir. The solution passes through a minicolumn of yeast alcohol dehydrogenase immobilized on controlled-pore glass (CPG). The NADH formed is monitored spectrofluorimetrically in the flow system, before reconversion to NAD⁺ in a minicolumn of glutamate dehydrogenase immobilized on CPG in the presence of glutarate and ammonium ions, also in the flowing solution. The solution then returns to the reservoir. The regeneration of NAD⁺ allows the same coenzyme solution to be used for 50 ethanol determinations daily for 4 days.     

Calix[4]quinones. Part 4: The ClO2 oxidation of calix[4]arene dialkyl ethers

Except for the special case of calix[4]arene diethyl ether 1, the chlorine dioxide oxidation of dialkyl ethers 2-5 yielded only the corresponding calix[4]diquinone dialkyl ethers 8-11. Chlorine dioxide oxidation of calix[4]arene diethyl ether 1 produced two isomeric products 6 and 7, which were stable enough to be isolated by column chromatography. However, a slow conformational interconversion between isomeric pair 6 and 7 was observed at room temperature, and the equilibrium was reached after 400 h at 18 °C with an amount of 5:3 in favor of syn-isomer.     

Flow-injection system for the fluorimetric determination of fructose with an immobilized mannitol dehydrogenase reactor

Immobilized mannitol dehydrogenase is used for the determination of D-fructose in a flow-injection system. The enzyme is immobilized on poly(vinyl alcohol) beads. The oxidation of NADH occurs simultaneously and the disappearance of NADH is measured fluorimetrically. The response is linearly related to fructose concentration in the range 6–600 μM; 30 samples per hour can be analysed. The immobilized enzyme retains over 80% of its initial activity after repetitive use for 2 months.