Stopped spectrophotometer

Stopped flow mixing of organic and aqueous phases is an excellent way to produce dispersion within a few milliseconds. The specific interfacial area of the dispersion can become as high as 700 cm and the interfacial reaction in the dispersed system can be measured by a photodiode array spectrophotometer. A drawback of this method is the limitation of a measurable time, although it depends on the viscosity. After 200 ms, the dispersion system starts to separate, even in a rather viscous solvent like a dodecane. Therefore, rather fast interfacial reactions such as diffusion-rate-limiting reactions are preferable systems to be measured. 

The reaction of Ru(III) chelate with diimine is about 99 times more efficient than that of Ru(III) with hydrazine. Computer-simulated chemiluminescence time curves based on the kinetic data of the above reaction scheme exactly matched light-intensity time curves recorded in a stopped-flow spectrophotometer 166h At high hydrazine concentra-... 

SCN-complex - mono- (Rh(lll) and 7.4 (Cr(IlI)) and mechanisms respectively spectral cell in stopped-flow or spectrophotometer ... 

A number of stopped-flow systems are commercially available. Three of the most used are manufactured by Atago Bussan (formerly Union Giken), Japan Dionex (formerly Durrum), USA and Hi-Tech Scientific, UK. These also manufacture rapid scan spectrophotometers, multimixer, temperature-jump and flash photolysis equipment. 

Any advanced absorbance/fluorescence spectrophotometer designed for routine acquistion of absorption or emission on the subsecond time scale. The basic goal is to obtain a series of complete UV/visible or fluorescence spectra as a function of time, usually after samples are mixed in a stopped-flow device. Such data help the investigator to infer the most likely structures of transient intermediates whose electronic spectra or fluorescence spectra can be determined by deconvoluting the spectra with appropriate reaction kinetic simulation software or by some other global analysis method (Fig. 1). 

For measurements at temperatures other than ambient, cells with double walls, which can be thermostatted, are also available commercially. If measurements are required at temperatures between ca. —5°C and room temperature, the sample compartment of the spectrometer can be flushed with dry air or nitrogen to reduce condensation on the cell windows. Below ca. — 5 °C the windows can be covered with a thin polythene film, but measurements below —25 °C are very troublesome. The problems associated with low temperature spectroscopic measurements were solved by enclosing the cell in an air-tight box fitted with glass windows (Dadley and Evans, 1967). The box was so designed that it fitted into the spectrophotometer and the air inside the box was dried with phosphoric oxide which, it is claimed, stopped condensation even at temperatures as low as — 60 °C glass windows could be used because only absorptions above 380 nm were of interest. 

HjO]/[AOT] ratios influenced the equilibrium sizes of CdS particles generated rates of CdS growth, determined in a stopped-flow spectrophotometer, were consistent with the rate-determining intermicellar exchange of solubilizates... 

Stop the reaction with 250 pi 1 N NaOH. Read the extinction in a spectrophotometer in microcuvettes at 515 nm. The molar extinction coefficient of p-nitrocat-echol at 515 nm is 12,400/Mol/cm. 

The rate at which liquid crystalline bile salt monoolein aggregates were dispersed by bile salt solutions was determined by Quentin Gibson (Cornell University, Ithaca, N. Y.) using a rapid mixing stop-flow spectrophotometer designed in his laboratory (4). 

Electrophoretic mobilities of the quartz particles in cobalt (II) perchlorate solutions were determined with a calibrated Zeta-Meter apparatus. Coagulation sedimentation behavior was followed using a stop-flow type apparatus. The dispersion is pumped in a closed loop from an equilibration vessel through an optical cell located in the sample compartment of a recording spectrophotometer. From the optical densitytime curve obtained from the time the pump is switched off, the turbidity index (in arbitrary units) is obtained as the slope of the curve at zero time. 

The thematic approach to isolating the deacylation step is to generate the acylen-zyme in situ in the stopped-flow spectrophotometer by mixing a substrate that acylates very rapidly with an excess or stoichiometric amount of the enzyme. The acylenzyme is formed in a rapid step that consumes all the substrate. This is then followed by relatively slow hydrolysis under single-turnover conditions. For example, acetyl-L-phenylalanine p-nitrophenyl ester may be mixed with chy-motrypsin in a stopped-flow spectrophotometer in which the enzyme is acylated in the dead time. The subsequent deacylation may be monitored by the binding of proflavin to the free enzyme as it is produced in the reaction.8... 

There are also nonthematic methods that allow the formation of acylenzymes under conditions where they are stable, so that they can be stored in a syringe in a stopped-flow spectrophotometer. For example, it is possible to synthesize certain nonspecific acylenzymes and store them at low pH.9 12 When they are restored to high pH, they are found to deacylate at the rate expected from the steady state kinetics. This approach has been extended to cover specific acylenzymes. When acyl-L-tryptophan derivatives are incubated with chymotrypsin at pH 3 to 4, the acylenzyme accumulates. The solution may then be pH-jumped by mixing it with a concentrated high-pH buffer in the stopped-flow spectrophotometer.1314 The deacylation rate has been measured by the proflavin displacement method and by using furylacrylolyl compounds. 

Two points should be noted (1) Because the rate constants are pseudo-unimolecular, there is a concentration dependence, so ka and koff may be resolved without the amplitude factor. (2) There is a lower limit to 1/r that is, 1/t cannot be less than koS. This sets a limit on the measurement of these rate constants. A good stopped-flow spectrophotometer can cope only with rate constants of 1000 s 1 or less, and many enzyme-substrate dissociation constants are faster than this. 

The hydrolysis of amides in the presence of acceptor nucleophiles gives the same product ratios as those found for the hydrolysis of the methyl ester (Ac-Phe-OCH3) under the same conditions (Table 7.4). Furthermore, these product ratios are the same as those expected from direct rate measurements of the attack of the nucleophiles on Ac-Phe-chy-motrypsin, generated in situ in the stopped-flow spectrophotometer (Table 7.5). 

Measurements of reactions that occur in less than a few seconds require special techniques to speed up the mixing of the enzyme and substrate. One way to achieve this is to place solutions containing the enzyme and the substrate in two separate syringes. A pneumatic device then is used to inject the contents of both syringes rapidly into a common chamber that resides in a spectrophotometer for measuring the course of the reaction (fig. 7.5). Such an apparatus is referred to as a stopped-flow device because the flow stops abruptly when the movement of the pneumatic driver is arrested. In this type of apparatus it is possible to make kinetic measurements within about 1 ms after mixing of enzyme and substrate. 

Spectrophotometer any suitable microplate reader able to measure absorbance at the appropriate wavelength. For TMB, this is 450 nm, having stopped the reaction with 0.5 M H2S04. 

For qualitative screening, a visual comparison of color with standards can be made. For semiquantitative determination, however, a spectrophotometer should be used to read absorbance to plot a calibration standard curve. The color should be read as soon as possible because it becomes unstable after 30 min. The required period for incubation varies from substance to substance but can range from 5 to 10 min to 1 or 2 hours. In certain analysis, the immunochemical reaction may require quenching after a specific amount of time. The reaction can be stopped by adding an acid, such as 1 N HC1, which turns the blue to yellow. The intensity of yellow too can be measured to determine the analyte concentration in the sample. 

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. 

A Multi-Microcomputer Controlled, Vidicon, Stopped-Flow Spectrophotometer... 

Me had a number of goals in mind when we set out to design a new vidicon rapid scanning stopped-flow spectrophotometer. 

We wanted to design an instrument which was highly automated so that it would be simple to use. Versatility in the vidicon spectrophotometer was also desired so that it could be used in a variety of applications in addition to stopped-flow kinetics. 

The design of the instrument will be discussed primarily on the block diagram level. Details on the construction of the vidicon spectrophotometer (19), the multi-microcomputer system (20) and the stopped-flow mixing system (20) are presented elsewhere. 

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