The Spectrophotometers

Polychromatic light contains many wavelengths (literally, many colors ). 

CAUTION Ultraviolet radiation is harmful to the naked eye. Do not view an ultraviolet source without protection. 

Stepper motor adjusts monochromator exit slit width 

Grating optical element with closely spaced lines 

For each incident angle 0, there are diffraction angles at which a given wavelength will produce maximum constructive interference, as shown in Color Plate 19. 

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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 basic principle of most colorimetric measurements consists in comparing under well-defined conditions the colour produced by the substance in unknown amount with the same colour produced by a known amount of the material being determined. The quantitative comparison of these two solutions may, in general, be carried out by one or more of six methods. It is not essential to prepare a series of standards with the spectrophotometer the molar absorption coefficient can be calculated from one measurement of the absorbance or... 

No general rule can be given concerning the strength of the solution to be prepared, as this will depend upon the spectrophotometer used for the study. Usually a 0.01-0.001 M solution is sufficiently concentrated for the highest absorbances, and other concentrations are prepared by dilution. The concentrations should be selected such that the absorbance lies between about 0.3 and 1.5. 

Procedure, (a) Determination of molar absorption coefficients and verification of additivity of absorbances. The molar absorption coefficients must be determined for the particular set of cells and the spectrophotometer employed. For the present purpose we may write ... 

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... 

Procedure. Place 80 mL of the arsenic/antimony solution in the titration cell of the spectrophotometer. Titrate with standard bromate/bromide solution at 326 nm taking an absorbance reading at least every 0.2 mL. From the curve obtained calculate the concentration of arsenic and antimony in the solution. 

Procedure. Charge the titration cell (Fig. 17.24) with 10.00 mL of the copper ion solution, 20 mL of the acetate buffer (pH = 2.2), and about 120mL of water. Position the cell in the spectrophotometer and set the wavelength scale at 745 nm. Adjust the slit width so that the reading on the absorbance scale is zero. Stir the solution and titrate with the standard EDTA record the absorbance every 0.50 mL until the value is about 0.20 and subsequently every 0.20 mL. Continue the titration until about 1.0 mL after the end point the latter occurs when the absorbance readings become fairly constant. Plot absorbance against mL of titrant added the intersection of the two straight lines (see Fig. 17.23 C) is the end point. 

Procedure. Transfer 10.00mL of the iron(III) solution to the titration cell (Fig. 17.24), add about 10mL of the buffer solution of pH = 4.0 and about 120mL of water the pH of the resulting solution should be 1.7-2.3. Insert the titration cell into the spectrophotometer immerse the stirrer and the tip of the 5mL microburette (graduated in 0.02 mL) in the solution. Switch on the... 

Detection of the infrared signal is, of course, of prime importance. A range of detectors is available for this purpose, the type used in any particular instrument depending upon the type and quality of the spectrophotometer. 

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. 

About 5 ml of sample is withdrawn for every 4-6 hours. The absorbance reading of the sample at 580 nm was measured using a Hitachi U-2000 spectrophotometer. The sample is filtered in a vacuum through Whatman filter paper with a pore size of 2.5 pin and diameter of 47 mm. The dry weight of cells is measured to monitoring microbial cell population and cell density. A plot of optical density reading from the spectrophotometer against cell dry... 

In the spectrophotometer, the crystal may be placed between source and sample, in which case it acts as a monochromator. Or, it may be inserted between sample and detector, where it acts as an analyzer of the transmitted beam. 

There are available also several kits for the assay of calcium, in 10 or 20 microliter samples by chelate formation colorimetrically or fluorimetrically. (Pierce Chem. Co., Rockford, 111.). These are read either with the spectrophotometer or by spectrofluorometry. In our experience, while these systems can be used for approximate results, the plot of concentration versus reading curves are rather flat and only an approximation of the values can be obtained. This may be very important late at night or at times when the atomic absorption machine is down, but if the atomic absorption instrument is available it should be used in preference to these procedures. 

The recent availability of low cost microprocessors has brought the possibility of on-line control of all elements of the spectrophotometer, plus on-line data reduction into the realm of practicality. 

In liquid medium, the thiobarbuturic acid test was used to determine polygalacturonase and pectate lyase activity (Sherwood, 1965). 1 ml of the crude enzyme preparation was added to 2 ml of 0.5 N HCl in a test tube. 4 ml of 0.01 M thiobarbuturic acid, dissolved in distilled water, were added. The tubes were heated in a boiling water for Ih and centrifuged. The absorption of the supernatant was determined in the spectrophotometer over the range 480-580 nm. Reaction mixtures without enzyme, which showed no reaction with thiobarbuturic acid, were used as controls. 

Pectolytic activity was also studied in batch reactors, following the reaction progress in thermostated quartz cuvettes. The reaction medium (3 cm ) was prepared with 1.5 g/L pectin in the standard buffer and 0.063 mg of enzyme. The absorbance of the reaction mixture against the substrate blank was continuously recorded at the spectrophotometer (Perkin Elmer Lambda 2, USA). Typical reaction time was 15 minutes, but initial reaction rates were estimated considering only the absorbances recorded during the first 200 seconds, range of totally linear response. 

Shell Chemical Company), exhibits a maximum at 300 nm, corresponding to that of the model chromophore anisole. The fluorescence intensity decreases monotonically with increasing concentration of 2,4-dihydroxybenzophenone (DHB) and, furthermore, decreases with time on continued excitation (274 nm) in the spectrophotometer. The fluorescence loss with time may be resolved into two exponential decays. Initially, a relatively rapid fluorescence loss is observed within 20 sec, followed by a slower loss. Loss constants for the initial (k ) and secondary (kj) exponential decays for 1.5 ym films (on glass slides) containing varying concentrations of DHB are provided in Table I (entries 1-3). The initial loss constants are seen to decrease more markedly with increasing DHB concentration than the secondary constants. 

Instrument standardization, v - a procedure for standardizing the response of multiple instruments such that a common multivariate model is applicable for measurements conducted across these instruments, the standardization being accomplished via adjustment of the spectrophotometer hardware or via mathematical treatment of one or a series of collected spectra. 

Optical background, n - the spectrum of radiation incident on a sample under test, typically obtained by measuring the radiation transmitted through or reflected from the spectrophotometer when no sample is present, or when an optically thin or non-absorbing standard material is present. 

Optical reference filter, n - an optical filter or other device which can be inserted into the optical path in the spectrophotometer or probe producing an absorption spectrum which is known to be constant over time such that it can be used in place of a check or test sample in a performance test. 

This layer is then analysed directly by internal reflectance infra-red spectroscopy. Since there is no handling of the sample, contamination is reduced to a minimum. However, only infra-red spectral analysis is possible with this system since the material absorbed on the germanium prism is always a mixture of compounds, and since the spectrophotometer used for the production of the spectra is not a high-precision unit, the information coming from this technique is limited. While identification of specific compounds is not usually possible, changes in spectra, which can be related to the time of day, season, or to singular events, can be observed. 

The term calibration is often used when in fact what is meant is verification . Calibration of an instrument or a piece of equipment (e.g. glassware) involves making a comparison of a measured quantity against a reference value. For example, to calibrate a spectrophotometer response, the appropriate reference material is selected and the spectrophotometer response to it, under the specified conditions, is measured. Then, the measured value is compared to the value quoted in the literature. Either a correction is made to the results from subsequent measurements or an adjustment is made to the instrument. 

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