Spectrophotometer components

This method compensates for the spectral inhomogeneities of the spectrophotometer components and for contributions from substrates or solvents. It doesn t... 

Optical Applications. Vitreous siUca is ideal for many optical appHcations because of its excellent ultraviolet transmission, resistance to radiation darkening, optical polishing properties, and physical and chemical stabiUty. It is used for prisms, lenses, cells, wiadows, and other optical components where ultraviolet transmission is critical. Cuvettes used ia scatter and spectrophotometer cells are manufactured from fused siUca and fused quart2 because of the transmissive properties and high purity (222). 

Mounting electrodes in a bioreactor is costly, and there is an additional contamination risk for sensitive cell cultures. Some other sensors of prac ticai importance are those for dissolved oxygen and for dissolved carbon dioxide. The analysis of gas exiting from a bioreactor with an infrared unit that detects carbon dioxide or a paramagnetic unit that detects oxygen (after carbon dioxide removal) has been replaced by mass spec trophotometry. Gas chromatographic procedures coupled with a mass spectrophotometer will detect 1 the volatile components. 

Detection is also frequently a key issue in polymer analysis, so much so that a section below is devoted to detectors. Only two detectors, the ultra-violet-visible spectrophotometer (UV-VIS) and the differential refractive index (DRI), are commonly in use as concentration-sensitive detectors in GPC. Many of the common polymer solvents absorb in the UV, so UV detection is the exception rather than the rule. Refractive index detectors have improved markedly in the last decade, but the limit of detection remains a common problem. Also, it is quite common that one component may have a positive RI response, while a second has a zero or negative response. This can be particularly problematic in co-polymer analysis. Although such problems can often be solved by changing or blending solvents, a third detector, the evaporative light-scattering detector, has found some favor. 

A modern spectrophotometer (UV/VIS, NIR, mid-IR) consists of a number of essential components source optical bench (mirror, filter, grating, Fourier transform, diode array, IRED, AOTF) sample holder detector (PDA, CCD) amplifier computer control. Important experimental parameters are the optical resolution (the minimum difference in wavelength that can be separated by the spectrometer) and the width of the light beam entering the spectrometer (the fixed entrance slit or fibre core). Modern echelle spectral analysers record simultaneously from UV to NIR. 

FIGURE 23.5 Effect of feeding captive male ring-necked pheasant (Ph. colchicus) young a high- or low-protein feed for the first three weeks of life on the expression of wattle coloration (mean+SE) at 20 (open circles) and 40 (filled circles) weeks of age. Coloration was determined using a principal components analysis (PCA) of tristimulus scores (hue, saturation, and brightness) obtained with a Colortron II reflectance spectrophotometer. 

When using a spectrophotometer for a colorimetric analysis, both the 0% and 100% transmittance (oo and 0 absorbance) readings must be set. Once the instrument has warmed up, with the light beam blocked and with nothing in the sample compartment, the readout is set to 0% transmittance (oo abs.). Again, this measurement is done to set / in the absorbance equation shown earlier. A blank, a solution containing all the components used in the analysis except the analyte being measured, is placed in a cuvette, placed in the sample... 

An HPLC detector is often a modified spectrophotometer equipped with a small flow cell, which monitors the concentration (or mass) of eluting sample components. A number of detectors used in HPLC are discussed below. Most applications utilize absorbance detectors such as UV/Vis or... 

The system used by these workers consisted of a Microtek 220 gas chromatograph and a Perkin-Elmer 403 atomic absorption spectrophotometer. These instruments were connected by means of stainless steel tubing (2mm o.d.) connected from the column outlet of the gas chromatograph to the silica furnace of the a.a.s. (Fig. 13.2). A four-way valve was installed between the carrier gas inlet and the column injection port so that a sample trap could be mounted, and the sample could be swept into the gas chromatographic column by the carrier gas. The recorder (lOmV) was equipped with an electronic integrator to measure the peak areas, and was simultaneously actuated with the sample introduction so that the retention time of each component could be used for identification of peaks. 

Draw a block diagram showing all the components of a basic spectrophotometer. 

Discuss troubleshooting procedures for spectrophotometers for 1) failure of electrical components, 2) unexpectedly high absorbance readings, and 3) an unexpected peak in an absorption spectrum. 

The function of the HPLC detector is to examine the solution that elutes from the column and output an electronic signal proportional to the concentrations of individual components present there. In Chapter 12, we discussed a number of detector designs that serve this same purpose for gas chromatography. The design of the HPLC detectors, however, is more conventional in the sense that components present in a liquid solution can be determined with conventional instruments, including spectrophotometers, fluorometers, and refractometers. 

FIGURE 13.8 The HPLC variable-wavelength UV detector—a UV spectrophotometer with a flow cell. A peak appears when a mixture component that absorbs the set wavelength elutes from the column. 

The various components of a double-monochromator infrared spectrophotometer shown in Figure 22.3 are as follows below ... 

The optical path of a double-beam atomic absorption spectrophotometer is depicted in Figure 26.2. The various essential components comprising the optical arrangement in Figure 26.2 are enumerated after the figure. 

Consider now multivariate data, e.g. measurements at many wavelengths instead of only one, say kinetics followed by a diode-array spectrophotometer. Assume the instrument records the spectra at 1024 wavelengths. Compared with monovariate data (single wavelength), there is a dramatic increase in the number of parameters to be fitted. In addition to the rate constant, there are now 1024 molar absorptivities for each reacting component that need to be fitted. The algorithm devised so far cannot cope with that number of parameters. 

Instruments for studying UV-visible absorbance, i.e., spectrophotometers are available in many biomedical laboratories and most investigators are familiar with the basic components ... 

Peak purity tests are used to demonstrate that an observed chromatographic peak is attributable to a single component. Mass spectrometry is the most sensitive and accurate technique to use for peak purity evaluation because of the specific information derived from the analysis. However, a good number of HPLC methods use mobile phase conditions that are incompatible with mass spectrometry detection. In this case, PDA spectrophotometers using peak purity algorithms may be used to support the specificity of the method. Almost all commercially available diode array detectors are equipped with proprietary software that will perform these calculations. Although this technique is more universal in application to HPLC methods, the data provided is neither particularly... 

Experimental methods for determining 0 are well documented (2). These experiments are conveniently carried out and require only a method of producing reasonably narrow-bandwidth radiation, a method of measuring the flux of that radiation per unit area, and a UV-visible spectrophotometer. The quantum efficiency of typical diazonaphthoquinone sensitizers of the type that are used in the formulation of positive photoresists ranges from 0.2 to 0.3, whereas the quantum efficiency of the bis-arylazide sensitizers used in the formulation of two-component negative photoresists, ranges from 0.5 to 1.0. 

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