Light spectrophotometer

Which of the following can be achieved by using a visible-light spectrophotometer ... 

An ultraviolet-visible light spectrophotometer (Lambda 10, Perkin Elmer, Norwalk, CT) was used to measure the characteristic absorbance of the samples taken from the receptor half-cell. Using a calibration curve derived from known concentrations of the model dmgs, the concentration of each sample taken from the receptor half-cell could be determined. 

Some enzyme reactions can be studied colorimetrically when either the substrate or product can be converted chemically to a coloured product suitable for measurement in a u.v. or visible light spectrophotometer. In the case of alanine aminotransferase, the pyruvate formed in the reaction can be converted to pyruvate-2,4-dinitrophenylhydrazone by the addition of 2,4-dinitrophenylhydrazine (DNP). Addition of sodium hydroxide yields a product with an absorption maximum at 505 nm. Other examples of colorimetric procedures will be found in the last section. Colorimetric procedures are used for enzyme assays in the sampling mode, whereby samples of the reaction mixture are analysed at certain fixed times after starting the reaction. Graphs depicting the reaction rate must then be constructed by plotting amount of substrate transformed against time. 

As a result of these considerations, the primary difference between a spectrophotometer and a light-scattering photometer is the fact that the photodetector is mounted on an arm which pivots at the sample so that intensity measurements can be made at various angles. 

Gloss, or surface luster, is the property of a surface to reflect light specularly. It is associated with such phenomena as shininess, highlight, and reflected images. The gloss of paper is usually quantified with a spectrophotometer which measures light at a variety of angles of incidence and reflection. 

Measurement of Whiteness. The Ciba-Geigy Plastic White Scale is effective in the visual assessment of white effects (79), but the availabihty of this scale is limited. Most evaluations are carried out (ca 1993) by instmmental measurements, utilising the GIF chromaticity coordinates or the Hunter Uniform Color System (see Color). Spectrophotometers and colorimeters designed to measure fluorescent samples must have reversed optics, ie, the sample is illuminated by a polychromatic source and the reflected light passes through the analy2er to the detector. 

The goal of the basic infrared experiment is to determine changes in the intensity of a beam of infrared radiation as a function of wavelength or frequency (2.5-50 im or 4000—200 cm respectively) after it interacts with the sample. The centerpiece of most equipment configurations is the infrared spectrophotometer. Its function is to disperse the light from a broadband infrared source and to measure its intensity at each frequency. The ratio of the intensity before and after the light interacts with the sample is determined. The plot of this ratio versus frequency is the infrared spectrum. 

The scales of spectrophotometers are often calibrated to read directly in absorbances, and frequently also in percentage transmittance. It may be mentioned that for colorimetric measurements I0 is usually understood as the intensity of the light transmitted by the pure solvent, or the intensity of the light entering the solution /, is the intensity of the light emerging from the solution, or transmitted by the solution. It will be noted that ... 

When a spectrophotometer is used it is unnecessary to make comparison with solutions of known concentration. With such an instrument the intensity of the transmitted light or, better, the ratio I,/I0 (the transmittance) is found directly at a known thickness /. By varying / and c the validity of the Beer-Lambert Law, equation (9), can be tested and the value of may be evaluated. When the latter is known, the concentration cx of an unknown solution can be calculated from the formula ... 

The experimental technique is simple. The cell containing the solution to be titrated is placed in the light path of a spectrophotometer, a wavelength appropriate to the particular titration is selected, and the absorption is adjusted to some convenient value by means of the sensitivity and slit-width controls. A measured volume of the titrant is added to the stirred solution, and the absorbance is read again. This is repeated at several points before the end point and several more points after the end point. The latter is found graphically. 

A special titration cell is necessary which completely fills the cell compartment of the spectrophotometer. One shown in Fig. 17.24 can be made from 5 mm Perspex sheet, cemented together with special Perspex cement, and with dimensions suitable for the instrument to be used. Since Perspex is opaque to ultraviolet light, two openings are made in the cell to accommodate circular quartz windows 23 mm in diameter and 1.5 mm thick the windows are inserted in such a way that the beam of monochromatic light passes through their centres... 

Spectrophotometry. The instrument generally used for this basic type of measurement is the spectrophotometer. The data obtained, usually pictured in the form of a spectrophotometric curve, indicate the ability of the sample to transmit or reflect light of the various wave lengths. Various instruments are available which can be used to determine more or less complete spectrophotometric curves. 

Objective Evaluation of Color. In recent years a method has been devised and internationally adopted (International Commission on Illumination, I.C.I.) that makes possible objective specification of color in terms of equivalent stimuli. It provides a common language for description of the color of an object illuminated by a standard illuminant and viewed by a standard observer (H). Reflectance spectro-photometric curves, such as those described above, provide the necessary data. The results are expressed in one of two systems the tristimulus system in which the equivalent stimulus is a mixture of three standard primaries, or the heterogeneous-homogeneous system in which the equivalent stimulus is a mixture of light from a standard heterogeneous illuminant and a pure spectrum color (dominant wave-length-purity system). These systems provide a means of expressing the objective time-constant spectrophotometric results in numerical form, more suitable for tabulation and correlation studies. In the application to food work, the necessary experimental data have been obtained with spectrophotometers or certain photoelectric colorimeters. 

Colorimeter Also called color comparator or photoelectric color comparator. An instrument for matching colors with results about the same as those of visual inspection, but more consistent. Basically the sample is illuminated by light from the three primary color filters and scanned by an electronic detecting system. It is sometimes used in conjunction with a spectrophotometer, which is used for close control of color in production. 

The intensity of absorbed radiation. Sunlight or room lights may alter the rate of a reaction. Usually this effect is to be avoided unless the object is to study photochemical effects. The light level in an optical spectrometer that uses monochromatic light is not likely to cause problems, but if white light strikes the sample, as in a diode-array spectrophotometer, this is a possibility. 

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