The cuvette

If pathlengths of 0.1 mm or less are required, it is probably best to use demountable cuvettes where the sample is dropped onto a quartz disc that is etched to a predefined depth and then another quartz disk is carefully placed on top. In this case sample recovery is very difficult, so the smaller volume of cylindrical cells makes them more attractive than rectangular ones. A simple cuvette holder for demormtable cylindrical UV cells may be created from an infrared cell holder and pieces of rubber (a mouse mat proves ideal) with holes drilled at the appropriate places to allow the holder assembly screws to pass through (Rgure 4). Any such cell holder must be located perpendicular to the light beam 

Cylindrical quaru short pathlength UV-vIsIble cuvette plates 

H ure 4 Celi holder for cylindrical quartz UV-vl ible short pathlength (less than 0.1 mm) cuvettes. 

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Kinetic measurements were performed employii UV-vis spectroscopy (Perkin Elmer "K2, X5 or 12 spectrophotometer) using quartz cuvettes of 1 cm pathlength at 25 0.1 C. Second-order rate constants of the reaction of methyl vinyl ketone (4.8) with cyclopentadiene (4.6) were determined from the pseudo-first-order rate constants obtained by followirg the absorption of 4.6 at 253-260 nm in the presence of an excess of 4.8. Typical concentrations were [4.8] = 18 mM and [4.6] = 0.1 mM. In order to ensure rapid dissolution of 4.6, this compound was added from a stock solution of 5.0 )j1 in 2.00 g of 1-propanol. In order to prevent evaporation of the extremely volatile 4.6, the cuvettes were filled almost completely and sealed carefully. The water used for the experiments with MeReOj was degassed by purging with argon for 0.5 hours prior to the measurements. All rate constants were reproducible to within 3%. 

In the process of performing a spectrophotometric determination of Ee, an analyst prepares a calibration curve using a single-beam spectrometer, such as a Spec-20. After preparing the calibration curve, the analyst drops the cuvette used for the method blank and the standards. The analyst acquires a new cuvette, measures the absorbance of the sample, and determines the %w/w Ee in the sample. Will the change in cuvette lead to a determinate error in the analysis Explain. 

Let us examine some batch results. In trials in which 5 mL of a dye solution was added by pipet (with pressure) to 10 mL of water in a 25-mL flask, which was shaken to mix (as determined visually), and the mixed solution was delivered into a 3-mL rectangular cuvette, it was found that = 3-5 s, 2-4 s, and /obs 3-5 s. This is characteristic of conventional batch operation. Simple modifications can reduce this dead time. Reaction vessels designed for photometric titrations - may be useful kinetic tools. For reactions that are followed spectrophotometrically this technique is valuable Make a flat button on the end of a 4-in. length of glass rod. Deliver 3 mL of reaction medium into the rectangular cuvette in the spectrophotometer cell compartment. Transfer 10-100 p.L of a reactant stock solution to the button on the rod. Lower this into the cuvette, mix the solution with a few rapid vertical movements of the rod, and begin recording the dead time will be 3-8 s. A commercial version of the stirrer is available. 

The instrument is occupied for 5 minutes per determination, including changing the solutions, rinsing the cuvettes, etc. 

Figure 9. Typical fluorescence signals obtained from a suspension of isolated rat cardiac myocytes after the application of maitotoxin (MTX). The arrow indicates the addition of MTX (10 g/mL), a detergent Emulgen 810 (1%), which frees all vesicular Ca , or EGTA (3.5 mM), a chelator that removes all free Ca in the cuvette. The intensity of Quin 2 fluorescence is expressed in arbitrary units. (Reproduced with permission from Ref. 20. Copyright 1987 Elsevier)...

Current instruments use complex and refined optical systems in order to radiate the cuvette with monochromatic light selected from the radiant spectrum of the light source. A radically different approach may be practical when tunable lasers become available at reasonable prices. 

Temperature Control. While it was well known that enzyme catalysis is a direct function of temperature, little attention was paid to its control in kinetic enzyme assays until the pioneer work of Schneider and Willis (11). These workers showed that the temperature compartment of the Beckman DU spectrophotometer varied widely as a function of room temperature and of the number of times the cuvet compartment was opened. Thus, while most authors have assumed that they were conducting their assay at room temperature (i.e., a nominal 25 ) direct measurements showed that the cuvette temperature was closer to 32 C. Schneider and Willis suggested that thermospacers, hollow plates adjacent to each side of the cuvette compartment through which water at a constant temperature is circulated, be used in order to standardize clinical enzyme assay temperatures. 

Fig. 2.14 The scheme of the cylindrical lens method for diffusion coefficient measurement (1) the source with the horizontal slit (2) the condenser supplying a handle of parallel beams (3) the cuvette with a refraction index gradient where the beams are deflected (4) the objective lens focusing the parallel beams to a single point (5) the optical member with an oblique slit and a cylindrical lens (6) the photosensitive material...

In the flocculated state for all suspensions, immediately after mixing, no transmission of light could be detected through the cuvette used and the sedimentation behaviour was different. Thus, instead of the parameters used for the pyrogenic silica systems we used the following characteristics for the precipitated silica mixtures to describe the flocculation and sedimentation behaviour ... 

A flour sample is weighed then mixed with water for a standard time and poured into a glass cuvette. The cuvette is inserted into the instrument, which moves into the measurement position and takes the reflectance reading. The results appear on a LED display and are printed out. The instrument is calibrated with an internal ceramic tile and standardised using a national standard flour. The normal range of flour grades is -5 to +18. 

As described for stopped flow experiments above, all commercially available SPR systems work under (pseudo) first-order conditions as well. This is realized either by a large excess of free ligand (in the large volume of the cuvette) compared with a nanoliter volume of the sensor layer [156] or by continuous replacement of free ligand in a flow injection system (e.g.,BIAcore [157]). 

The flow-through cuvette that is used for the four-channel sensor is made from Perspex and is 31-mm long and 7-mm wide. It has four flow chambers each with a volume of 1.2 pi (6 mm long and 3 mm wide), see Fig. 10.8. Each chamber of the cuvette has an inlet and outlet that are connected via a tubing system with the sampling reservoirs, which contain solutions to be monitored, and to the waste, respectively. Samples are flowed by means of a peristaltic pump (Ismatec... 

In practical situations the absorbance of a sample is determined by making two measurements, the first to determine 70 and the second to determine I. The determination of I0 is used to cancel a large number of experimental factors that could affect the result. When measuring I0 the sample container must closely match the unknown container in all ways except for the analyte content. The cuvettes should be a matched pair if a double beam instrument is used and the same cuvette can be used for both the blank and sample with a single beam instrument. The blank solution filling the cuvette should be identical to the solvent that the sample is dissolved in, except for the sample itself. If done correctly, the least-squares line for the calibration graph will come very close to the 0,0 point on the graph. 

Following the wavelength selection by the monochromator, the beam passes on to the sample compartment where the sample solution, held in the cuvette, is positioned in its path. The sample compartment is an enclosure with a lid that can be opened and closed in order to insert and remove the cuvette. When the lid is closed, the compartment should be relatively free of stray light, although this is not a requirement if a xenon arc lamp is used as the source. This is because of the high intensity of the xenon arc lamp. The cuvette is held snugly in a spring-loaded holder. 

A diode array is a series of several hundred photodiodes arranged in a linear array. Single-beam spectrophotometers have been invented that utilize a diode array as the detector. In this case, the cuvette is positioned between the source and the dispersing element. Then, following the dispersion of the fight, there is no exit slit. The spray of wavelengths created by the grating fall instead across the diode array,... 

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