Sample preparation stopped-flow

The luminol reaction has also been used for the CL determination of organic substances such as penicillins [32] and tartrate ion [30] in pharmaceutical preparations by their inhibitory effect on the luminol-iodine and luminol-periodate-manganese(II)-TEA system, respectively. As can be seen from Table 1, the results were quite satisfactory. In the indirect determination of penicillins by their inhibitory effect on the luminol-iodine system, the stopped-flow technique improves the accuracy and precision of the analytical information obtained, and also the sample throughput [32], Thus, in only 2-3 s one can obtain the whole CL signal-versus-time profile and calculate the three measured parameters formation and... 

Radical Generation. The ESR spectrometer, flow system, and general procedure have been described (46). The apparatus was calibrated with freshly prepared diphenyl picrylhydrazyl (DPPH) solutions. The peroxy radical concentrations were determined by double integration of derivative spectra. A standard coal sample in the dual cavity allowed corrections to be made for changes in cavity Q. The rates of decay of the less reactive radicals were determined by stopped-flow techniques with manually or electrically operated valves. The decay was recorded... 

Hi-Tech Scientific Limited (Salisbury, England) recently introduced a stopped-flow (SF-51) instrument with conductivity detection that uses a five-mixer aging block that gives preparative quench aging times in the range of 1.0 ms to >10 s (Fig. 4.16). Preparative quench and stopped-flow experiments can be performed under total thermostatted, anaerobic, and chemically inert conditions. The entire stopped-flow package consists of the sample handling unit, a spectrophotometer, and a data processor based on the Apple lie. 

Because stopped-flow techniques are widely used with optical detection, samples should be prepared in solution and produce detectable signal changes after mixing into the cell. In some situations, if the reaction of some samples is very rapid and complete within the dead time of the stopped-flow instruments, the majority and indeed the entire kinetic time course may be lost. Selected adjustment of concentration, solution conditions, temperature, and so on, may be able to slow the reaction into an accessible time range, but this is not always possible or desirable. Such systems are not amenable to the stopped-flow technique. In general, other techniques will have to be used, and these will be... 

Sample preparation is critical to the success of any spectrophotometric experiment whether using absorbance or fluorescence detection systems. Even the rapid mixing of two samples of buffer in a sensitive instrument can result in what appears to be a reaction curve. This may be caused by simple effects such as the rapid compression and decompression of an air bubble in the flow path, the nuxing of two solutions at different temperatures or the effect of the stopping process upon small dust particles present in the solution. Therefore it is essential that solutions be prepared thoroughly before use. Sometimes it is necessary to degas buffers to remove dissolved air that may otherwise come out of solution following rapid decompression of the reaction solutions. In practice, it is... 

Sample Preparation. Cobalt catalysts were prepared by subliming Co2(C0)g into the pores of dehydrated NaX zeolite in a vacuum line at pressures of 1 x 10- f torr. Argon was flowed over the metal loaded zeolite sample at a pressure of 0.3 torr. A microwave plasma was induced with a static gun and the decomposition of the metal carbonyl precursor occurred for two hours. After total decomposition of the metal carbonyl which can be determined by the color of the plasma, the argon flow was stopped and the sample was sealed off by closing the Teflon stopcocks at both ends of the reactor. The sample was then brought into a drybox and loaded into catalytic reactors or holders for spectroscopic experiments. Further details of this procedure can be found elsewhere (11, 25). Iron samples were prepared in a similar fashion except ferrocene was used as a metal precursor. 

Using a computer-controlled FIA system, certain technical arrangements can be implemented in order to raise the sensitivity of the assays. Stopped flow procedures where the sample is kept in contact with the immobilized enzyme preparation for a longer time than the passage takes is one such arrangement being used [37]. Reversal of flow rate in order to improve mixing in the enzyme... 

The AV data of Fig. 5.1 that are satisfactorily accounted for by Eqs (5.5)-(5.8) are fewer in number than the anomalous cases of Table 5.1. This is a rather unsatisfactory situation, even though most of the anomalies can be explained away - indeed, deviations from the predictions of Eqs (5.5)-(5.8) can often provide important mechanistic information. More AV data are clearly desirable, but the prospects for further successful experiments are poor. The measurements of AV summarized in Fig. 5.1 and Table 5.1 were obtained at high pressures by radiochemical tracer methods for the slowest reactions [12, 17, 25], NMR linebroadening techniques for the faster cases [11, 13, 15, 19-22, 34], and stopped-flow circular dichroism [13, 14, 18] for moderately rapid reactions of reactants that could be prepared as resolved enantiomers. There are, however, many self-exchange reactions that are inaccessible to these techniques. For example, rates of electron transfer in couples where both reactants have unpaired electrons generally cannot be studied by NMR methods, while other couples that undergo electron transfer at intermediate rates may not be resolvable into optical isomers or be amenable to radiochemical sampling procedures under pressure. 

Methods of monitoring aldehydes in food and pharmaceutical samples have also been reported. A semiautomatic method by a stopped-flow flow injection analysis technique was successful in identifying the presence of furfural and 5-hydroxymethyl-2-furfuraldehyde in several commercial pharmaceutical preparations and food samples [398], The analysis is based on the reaction of the aldehydes with 2-thiobarbituric acid, with determination of the derivatives. 

A rotating enzyme-immobilized reactor and a flat pH electrode were incorporated into a sealed cell for use under continuous-flow/stopped-flow (SF) operation for the rapid determination of penicillins G and V in tablets and injectables [50]. A co-immobilization in a rotating bioreactor and amperometric detector resulted in a sensitive system for determination of succinylcholine and acetylcholine in pharmaceutical preparations [51]. A tandem system incorporating two rotating bioreactors into a continuous-flow/SF sample/reagentprocessing setup was apphed for the determination of alkaline phosphatase activity in serum samples [52]. By functional combination of the SF and flow-injection analysis (FIA), an automated micro apparatus was constructed resulting in significant reduction of the injection volumes of enzyme and substrate [53]. SF/continuous flow methods were apphed to acquire kinetic information also [54, 55]. 

Takeuchi et al. published a mechanized assay of serum cholinesterase by specific colorimetric detection of the released acid [40]. The cholinesterase reaction was carried out on a thermostatted rack at 30° C with a reaction mixture of serum (10 pL), 50mM barbitone-HCl assay buffer (pH 8.2 140 pL), and 12.5 mM acetylcholine solution (50 pL). The solutions were prepared by programmed needle actions, and a sample blank was also prepared. The reaction was stopped after 9.7 min by injection of the mixture into a flow injection analysis system to determine the quantity of acetic acid formed. The carrier stream (water, at 0.5mL/min) was merged with a stream (0.5mL/min) of 20 mM 2-nitrophenylhydrazine hydrochloride in 0.2 M HC1 and a stream (0.5 mL/min) of 50 mM 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride in ethanol containing 4% of pyridine. The sample was injected into this mixture (pH 4.5), passed through a reaction coil (10 m x 0.5mm) at 60°C, 1.5M NaOH was added, and, after passing through a second reaction coil (lm x 0.5 mm) at 60°C, the absorbance was measured at 540 nm. 

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