Spectrophotometer

light source halogen lamps are commonly used for the visible region of the spectrum, and hydrogen or deuterium discharge lamps for the ultraviolet region. 

Monochromator almost all equipment presently used is fitted with grating monochromators prism monochromators are only found in old apparatus. Simple equipment, which usually means a single beam apparatus, is often fitted with a single stage monochromator. Higher specification equipment uses double monochromators, and here the beam is usually split for simulta- 

Light from the monochromator is directed alternately through the sample and reference cuvettes, and detection is with a single photomultiplier. 

Steam from the flask is directed on to the inside walls of the cuvette where it condenses and is collected in the beaker. The external walls of the cuvette can be rinsed with water or ethanol. 

White light is passed alternately through the diode array, sample and referencecuvettes. The light is then 

Cavity ring-down spectrum of 3 mbar of C02, which is similar to the concentration in human breath. [From E. R. Crosson, K. N. Ricci, B. A. Richman, F. C. Chilese, T. G. Owano, R. A. Provencal, 

Glasser, A. A. Kachanov, B. A. Paldus, T. G. Spence, and R. N. Zare, Stable Isotope Ratios Using Cavity Ring-Down Spectroscopy Determination of, C/I2C for Carbon Dioxide In Human Breath,  

Spectrum (c) shows absorbance measured for C02(g) with the natural mixture of 98.9% l2C and 1.1% l3C. Peaks arise from transitions between rotational levels of two vibrational states. The spectral region was chosen to include a strong absorption of l3C02 and a weak absorption of l2C02, so that the intensities of the isotopic peaks are similar. Each point in the spectrum was obtained by varying the laser frequency. 

The areas of the l3C02 and l2C02 peaks from human breath were used to determine whether a patient was infected with Helicobacter pylori, a bacterium that causes ulcers. After ingesting l3C-urea, H. pylori converts l3C-urea into 13C02, which appears in the patient s breath. The ratio l3C/l2C in the breath of an infected person increases by 1-5%, whereas the ratio, 3C/I2C from an uninfected person is constant to within 0.1%. 

A single-beam spectrophotometer is inconvenient because the sample and reference must be placed alternately in the beam. For measurements at multiple wavelengths, the reference must be run at each wavelength. A single-beam instrument is poorly suited to measuring 

In principle, any detection system which could be adapted for flow-through detection may be used as detectors for FIA. However, some detectors are inherently more suitable than others in the interfacing, and therefore are used more frequently in FI systems. These include the spectrophotometer (visible and UV), atomic absorption and ICP spectrometer, chemiluminescence and various electrochemical detectors, and will be discussed here in more detail. 

Visible and UV spectrophotometers are by far the most frequently used type of detectors in FI systems. This is also true for FI separation systems. Provided the light source intensity is strong enough, a conventional batch spectrophotometer can easily be converted into a flow-through spectrophotometer by substituting the conventional cuvette with a flow-through cell. 

A most often used flow-cell configuration, which may be furnished with either glass or quanz windows, is shown in Fig. 2.12. Flow cells of 18 /il capacity in the light path (1.5 mm diameter. 10 mm long) are commonly used. The inlet and outlet conduits of the cell are so arranged that the stream is fed in from the bonom of the cell and leaves from the top to facilitate the release of accidentally introduced air bubbles. Care should be taken not to reverse the flow directions through the cell, in order to avoid the trapping of air bubbles in it. 

In some applications, separation and detection systems are integrated by packing the flow cell with panially transparent sorbent material. The analyte may be first collected on the sorbent, transformed in situ into a detectable species, and deteaed in the cell. This technique, termed sorbent absorptiometry, will be discussed in Chapter 4. 

A frequent trouble encountered in spectrophotometric readouts is the generation of spurious peaks due to differences in refractive properties of the sample and car-rier/reagent. In case of a substantial difference in the refiractive index, the parabolic interfaces at the two ends of the sample zone create convex and concave lens effects on 

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Willey R R 1976 Fourier transform infrared spectrophotometer for transmittance and diffuse reflectance measurements Appl. Spectrosc. 30 593-601... 

All the cations of Group I produce a characteristic colour in a flame (lithium, red sodium, yellow potassium, violet rubidium, dark red caesium, blue). The test may be applied quantitatively by atomising an aqueous solution containing Group I cations into a flame and determining the intensities of emission over the visible spectrum with a spectrophotometer Jlame photometry). 

Measurements were performed employing a Perkin Elmer X2, 5 or 12 UV-Vis spectrophotometer at 25 O.r- C. Equilibrium constants were determined by measuring the extinction coefficient at a suitable wavelength of the partially complexed dienophile (y,.hs) as a function of the concentration of... 

Kinetic experiments were performed on a Perkin Elmer 12, 15, or 12 spectrophotometer following methods described in Chapter 2. Values for k. , given in Tables 3.1 and 3.2 were calculated using equation A8, derived in Appendix 2.1 in Chapter 2. 

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%. 

To test a spectrophotometer for its accuracy, a solution of 60.06 ppm K2Cr207 in 5.0 mM H2SO4 is prepared and analyzed. This solution has a known absorbance of 0.640 at 350.0 nm in a 1.0-cm cell when using 5.0 mM H2SO4 as a reagent blank. Several aliquots of the solution are analyzed with the following results... 

The accuracy of a spectrophotometer can be checked by measuring absorbances for a series of standard dichromate solutions that can be obtained in sealed cuvettes from the National institute of Standards and Technology. Absorbances are measured at 257 nm and compared with the accepted values. The results obtained when testing a newly purchased spectrophotometer are shown here. Determine if the tested spectrophotometer is accurate at a = 0.05. 

Block diagram for a single-beam fixed-wavelength spectrophotometer with photo of a typical instrument. 

Finally, values of sx are directly proportional to transmittance for indeterminate errors due to fluctuations in source intensity and for uncertainty in positioning the sample cell within the spectrometer. The latter is of particular importance since the optical properties of any sample cell are not uniform. As a result, repositioning the sample cell may lead to a change in the intensity of transmitted radiation. As shown by curve C in Figure 10.35, the effect of this source of indeterminate error is only important at low absorbances. This source of indeterminate errors is usually the limiting factor for high-quality UV/Vis spectrophotometers when the absorbance is relatively small. 

Atomic absorption spectrophotometers (Figure 10.37) are designed using either the single-beam or double-beam optics described earlier for molecular absorption spectrophotometers (see Figures 10.25 and 10.26). There are, however, several important differences that are considered in this section. 

Atomization The most important difference between a spectrophotometer for atomic absorption and one for molecular absorption is the need to convert the analyte into a free atom. The process of converting an analyte in solid, liquid, or solution form to a free gaseous atom is called atomization. In most cases the sample containing the analyte undergoes some form of sample preparation that leaves the analyte in an organic or aqueous solution. For this reason, only the introduction of solution samples is considered in this text. Two general methods of atomization are used flame atomization and electrothermal atomization. A few elements are atomized using other methods. 

When the identity of the matrix interference is unknown, or when it is impossible to adjust the flame to eliminate the interference, then other means must be used to compensate for the background interference. Several methods have been developed to compensate for matrix interferences, and most atomic absorption spectrophotometers include one or more of these methods. 

Other methods of background correction have been developed, including Zee-man effect background correction and Smith-Iiieffje background correction, both of which are included in some commercially available atomic absorption spectrophotometers. Further details about these methods can be found in several of the suggested readings listed at the end of the chapter. 

When using a spectrophotometer for which the precision of absorbance measurements is limited by the uncertainty of reading %T, the analysis of highly absorbing solutions can lead to an unacceptable level of indeterminate errors. Consider the analysis of a sample for which the molar absorptivity is... 

Spectrophotometer Works, /. Chem. Educ. 1979, 56, 681-684. Consult the following sources for more information about reflectance techniques for IR spectroscopy. 

Two common detectors, which also are independent instruments, are Fourier transform infrared spectrophotometers (FT-IR) and mass spectrometers (MS). In GC-FT-IR, effluent from the column flows through an optical cell constructed... 

Write directives outlining good measurement practices for (a) a buret, (b) a pH meter, and (c) a spectrophotometer. 

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