Cuvettes, spectrophotometric

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 solubility was determined spectrophotometrically on a Shimadzu UV160A using the reference quartz cuvette filled with pure vegetable oil under testing and the sample cuvette filled with the solution of C60 or C70 in a given vegetable oil. 

B 7. A solution of purified DNA gave in the spectrophotometric assay an A16a of 0.35 when measured in a 1-cm quartz cuvette. What is the concentration of the DNA in /xg/mL ... 

For the spectrophotometric method, there is no sample workup, allowing one to run -4 assays/hr. This can be increased to -16 to 100 or more samples/hr depending on equipment features and automation, such as multiple cuvette holders/changers and 96-well microplate readers. For the colorimetric procedure, sample workup requires -10 min/subsample, but several samples can be batch processed simulta-... 

For CD and CT determinations (see Basic Protocol 1), quartz cuvettes must be used, as glass ones will absorb UV radiation. A 1-cm-path-length quartz cuvette is most often employed for spectrophotometric detection however, quartz cuvettes with shorter path lengths (0.01 to 0.5 cm) are available. The equation relating CD value to absorbance assumes that the cuvette has a path length of 1 cm when cuvettes of shorter path length are used, the... 

Disposable plastic cuvettes are more convenient to use in spectrophotometric detection however, plastic melts after extended exposure to strong acids. In addition, for more linearity and accuracy in the measurements, many important parameters must be considered. It is advantageous to use cuvettes with path lengths of (0.01 to 0.5 cm), because they use a smaller volume of sample and often give more reproducible results. 

Spectrophotometric readings should be carried out immediately after the solution has been placed in the cuvette to avoid evaporation of the solvent or degradation. 

From this equation, we see that the changes of attenuation of the initial beam are equally affected by the changes of the optical path length and/or by the changes of the concentration. In a normal spectrophotometric experiment, the optical path L is constant and defined by the spacing of the transparent cuvette windows. A similarly well-defined relationship often does not exist in optical sensors. 

In principle, any property of a reacting system which changes as the reaction proceeds may be monitored in order to accumulate the experimental data which lead to determination of the various kinetics parameters (rate law, rate constants, kinetic isotope effects, etc.). In practice, some methods are much more widely used than others, and UV-vis spectropho-tometric techniques are amongst these. Often, it is sufficient simply to record continuously the absorbance at a fixed wavelength of a reaction mixture in a thermostatted cuvette the required instrumentation is inexpensive and only a basic level of experimental skill is required. In contrast, instrumentation required to study very fast reactions spectrophotometrically is demanding both of resources and experimental skill, and likely to remain the preserve of relatively few dedicated expert users. 

By inserting either an interference filter or a colored filter, it is possible to select a more or less extended region of the spectrum likewise, by adding an optical fiber it is possible to direct the beam where desired. This set-up best exploits the characteristics of these powerful lamps, and offers an excellent choice for the irradiation of small surfaces. Consequently, spectrophotometric cuvettes or cylindrical cuvettes are used for the irradiation, which involves small volumes. Such restrictions, as well as the high price and short lifetime of the lamp and its accessories, favors the use of these arcs for kinetics studies and quantum yield measurements, rather than for preparative photochemistry. 

Colorimetric or spectrophotometric analysis pipet, buret, test tubes and/or cuvettes, spectrophotometer, buret support stand... 

Table 1. The effect of 1 mM NaF + 20 pM A1C13 on the acetylcholinesterase activity (AChE) in freshly prepared intact RBC and in hemolysate of patients with AD (mean age 72.5 5.1 years), age-matched healthy controls (AM-HS) (72.1 1.6 years), and the group of young healthy subjects (YS) (35.9 8.5 years). Whole venous blood samples were drawn from each subject after overnight fasting., always at 07 30 AM. Red blood cells (RBC) were isolated from the blood of patients with AD, AM-HS, and YS by centrifugation [68], RBC AChE activity was evaluated in intact freshly prepared RBC or hemolyzate following the spectrophotometric method [45] with modifications. Buffer was Tris-HCl, pH 7.5 in the solution of 154 mmol L 1 NaCl, acetylthiocholine iodide was a substrate. Measurement of enzymatic activity was performed in fluorimeter polystyrene cuvettes for 3 min (UV/VIS spectrophotometer Shimadzu, Japan). The effects of 1 mmol L-1 NaF in the presence of 20 pmol L 1 A1C13 were measured. Data are expressed in percentage of the AChE activity in the absence of aluminum and fluoride ions. No differences between the AChE activity were found between the investigated groups...

Two procedures have been adopted to prevent oxidation of excess unreacted iodide by molecular oxygen. Either unreacted iodide is com-plexed with cadmium (Buege and Aust, 1978), or spectrophotometric measurements are made in air-free stoppered cuvettes and development of I3 is followed continuously. The second procedure allows reliable detection of the reaction end point and identifies any oxygen contamination (Hicks and Gebicki, 1979), but it is not applicable to routine measurements of many samples as the time taken for complete reduction of the hydroperoxide is prohibitive. Methods are therefore described which are based only on the procedure utilizing cadmium. 

The minimum eoncentration of an element (in moles per litre) that can be determined by spectrophotometric methods may be caleulated from the expression A = eel. If one assumes that the minimum measurable absorbance of a solution is A = 0.02, with a cuvette width 1 = 2 em, and for a moderately sensitive speetrophotometric method, e = 1-10", the respective concentration will be ... 

Notes. 1. On account of the high concentration of HCI in the sample solution the cuvettes should be suitably covered before being placed in the spectrophotometric apparatus. 

A Beckman DU spectrophotometer was used for all measurements. The slit width was chosen so that a half-intensity band width of 1.6 nm was obtained in the region X = 650-590 nm, 0.6 nm in the region X = 590-A60 nm, and 0.15 nm in the region of the Soret bands. The wavelength of the instrument had been checked with the aid of the mercury emission spectrum. Measurements at X > 590 nm were made in a layer thickness of 0.0993 cm, at X < 590 nm in a layer thickness of 0.0128 cm. The latter was obtained by inserting a plane parallel glass plate into the 0.0993-cm cuvette (Fig. 13). The layer thickness of the cuvette had been determined spectrophotometrically using HiCN solutions and a 1.000-cm cuvette as a reference. 

The different interactions of radiation with the flowing sample in relation to the different spectrophotometric techniques are presented below. Basic aspects are dealt with in relation to a static cuvette, whereas those aspects more related to flow analysis are discussed in relation to a flowthrough cuvette. 

Figure 5.19. Progress in miniaturization of spectrophotometric flowthrough cells, (a) Hellma cuvette, which fits into most commercial photometers, b) Z-cell, where A are the transparent windows B, Teflon made body C, cell house, and CH, the inlet channel, (c) A fiber optic reflectance cell for optosensing at active surfaces [848], where CH is the channel of a microconduit, R is the chemically active reflecting material, OF is the optical fiber.

To a metapyrocatechase solution in 0.05Af potassium phosphate buffer, pH 7.5 (approximately 2.5 ml.), which was equilibrated with air 5 /xmoles of catechol were added in a closed system. The reactions were carried out in a cuvette with a 0.5-cm. light path at 24 °C. The cuvette was filled with reaction mixture and covered with a glass plate to avoid contact with air. The product formed was directly determined spectrophotometrically at 430 m/x. 

Spectrophotometric pJCa values. .. were determined. .. using 1 cm cuvettes at 25 0.5 °C. The experimental results showed that tire three analytes were fully protonated and deprotonated at about pH... 

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