Instrument Designs for Molecular UV/Vis Absorption - Ultraviolet-Visible and Infrared Spectrophotometry

Instrumentation

Frequently an analyst must select, from several instruments of different design, the one instrument best suited for a particular analysis. In this section we examine some of the different types of instruments used for molecular absorption spec- troscopy, emphasizing their advantages and limitations. Methods of sample intro- duction are also covered in this section.

Instrument Designs for Molecular UV/Vis Absorption

The simplest instrument cur- rently used for molecular UV/Vis absorption is the filter photometer shown in Fig- ure 10.24, which uses an absorption or interference filter to isolate a band of radia- tion. The filter is placed between the source and sample to prevent the sample from decomposing when exposed to high-energy radiation. A filter photometer has a sin- gle optical path between the source and detector and is called a single-beam instru- ment. The instrument is calibrated to 0% T while using a shutter to block the source radiation from the detector. After removing the shutter, the instrument is calibrated to 100% T using an appropriate blank. The blank is then replaced with the sample, and its transmittance is measured. Since the source’s incident power and the sensitiv- ity of the detector vary with wavelength, the photometer must be recalibrated when- ever the filter is changed. In comparison with other spectroscopic instruments, pho- tometers have the advantage of being relatively inexpensive, rugged, and easy to maintain. Another advantage of a photometer is its portability, making it a useful in- strument for conducting spectroscopic analyses in the field. A disadvantage of a pho- tometer is that it cannot be used to obtain an absorption spectrum.

Instruments using monochromators for wavelength selection are called spectrometers. In absorbance spectroscopy, where the transmittance is a ratio of two radiant powers, the instrument is called a spectrophotometer. The simplest spectrophotometer is a single-beam instrument equipped with a fixed- wavelength monochromator, the block diagram for which is shown in Figure 10.25. Single-beam spectrophotometers are calibrated and used in the same manner as a photometer. One common example of a single-beam spectropho- tometer is the Spectronic-20 manufactured by Milton-Roy. The Spectronic-20 can be used from 340 to 625 nm (950 nm with a red-sensitive detector), and has a fixed effective bandwidth of 20 nm. Because its effective bandwidth is fairly large, this instrument is more appropriate for a quantitative analysis than for a qualitative analysis. Battery-powered, hand-held single-beam spectrophotome- ters are available, which are easily transported and can be used for on-site analy- ses. Other single-beam spectrophotometers are available with effective band- widths of 2–8 nm. Fixed-wavelength single-beam spectrophotometers are not practical for recording spectra since manually adjusting the wavelength and re- calibrating the spectrophotometer is awkward and time-consuming. In addition, the accuracy of a single-beam spectrophotometer is limited by the stability of its source and detector over time.

The limitations of fixed-wavelength, single-beam spectrophotometers are mini- mized by using the double-beam in-time spectrophotometer as shown in Figure 10.26. A chopper, similar to that shown in the insert, controls the radiation’s path, alternat- ing it between the sample, the blank, and a shutter. The signal processor uses the chop- per’s known speed of rotation to resolve the signal reaching the detector into that due to the transmission of the blank (P₀) and the sample (P_T). By including an opaque sur- face as a shutter it is possible to continuously adjust the 0% T response of the detector. The effective bandwidth of a double-beam spectrophotometer is controlled by means of adjustable slits at the entrance and exit of the monochromator. Effective band- widths of between 0.2 nm and 3.0 nm are common. A scanning monochromator al- lows for the automated recording of spectra. Double-beam instruments are more ver- satile than single-beam instruments, being useful for both quantitative and qualitative analyses; they are, however, more expensive.

The instrument designs considered thus far use a single detector and can only monitor one wavelength at a time. A linear photodiode array consists of multiple de- tectors, or channels, allowing an entire spectrum to be recorded in as little as 0.1 s. A block diagram for a typical multichannel spectrophotometer is shown in Figure 10.27. Source radiation passing through the sample is dispersed by a grating. The linear pho- todiode array is situated at the grating’s focal plane, with each diode recording the ra- diant power over a narrow range of wavelengths.

One advantage of a linear photodiode array is the speed of data acquisition, which makes it possible to collect several spectra for a single sample. Individual spec- tra are added and averaged to obtain the final spectrum. This process of signal aver- aging improves a spectrum’s signal-to-noise ratio. When a series of spectra is added, the sum of the signal at any point increases as (nS_x), where n is the number of spec- tra, and S_x is the signal for the spectrum’s x-th point. The propagation of noise, which is a random event, increases as ( Root of [nN_x]), where N_x is the noise level for the specctrum’s x-th point. The signal-to-noise ratio (S/N) at the x-th data point, there- fore, increases by a factor of  Rt[n]

where (S_x/N_x) is the signal-to-noise ratio for a single scan. The effect of signal averaging is shown in Figure 10.28. The spectrum in Figure 10.28a shows the total signal for a single scan. Although there is an apparent peak near the cen- ter of the spectrum, the level of background noise makes it difficult to mea- sure the peak’s signal. Figures 10.28b and Figure 10.28c demonstrate the im- provement in signal-to-noise ratio achieved by signal averaging. One disadvantage of a linear photodiode array is that the effective bandwidth per diode is roughly an order of magnitude larger than that obtainable with a high-quality monochromator.

The sample compartment for the instruments in Figures 10.24–10.27 provides a light-tight environment that prevents the loss of radiation, as well as the addition of stray radiation. Samples are normally in the liquid or solu- tion state and are placed in cells constructed with UV/Vis-transparent materi- als, such as quartz, glass, and plastic (Figure 10.29). Quartz or fused-silica cells are required when working at wavelengths of less than 300 nm where other materials show a significant absorption. The most common cell has a pathlength of 1 cm, although cells with shorter (>= 1 mm) and longer path- lengths (=< 10 cm) are available. Cells with a longer pathlength are useful for the analysis of very dilute solutions or for gaseous samples. The highest qual- ity cells are constructed in a rectangular shape, allowing the radiation to strike the cell at a 90° angle, where losses to reflection are minimal. These cells, which are usually available in matched pairs having identical optical proper- ties, are the cells of choice for double-beam instruments. Cylindrical test tubes are often used as a sample cell for simple, single-beam instruments, al- though differences in the cell’s pathlength and optical properties add an addi- tional source of error to the analysis.

In some circumstances it is desirable to monitor a system without physi- cally removing a sample for analysis. This is often the case, for example, with the on-line monitoring of industrial production lines or waste lines, for physi- ological monitoring, and for monitoring environmental systems. With the use of a fiber-optic probe it is possible to analyze samples in situ. A simple exam- ple of a remote-sensing, fiber-optic probe is shown in Figure 10.30a and con- sists of two bundles of fiber-optic cable. One bundle transmits radiation from

the source to the sample cell, which is designed to allow for the easy flow of sample through the cell. Radiation from the source passes through the solu- tion, where it is reflected back by a mirror. The second bundle of fiber-optic cable transmits the nonabsorbed radiation to the wavelength selector. In an alternative design (Figure 10.30b), the sample cell is a membrane containing a reagent phase capable of reacting with the analyte. 

When the analyte diffuses across the membrane, it reacts with the reagent phase, producing a product that ab- sorbs UV or visible radiation. Nonabsorbed radiation from the source is reflected or scattered back to the detector. Fiber-optic probes that show chemical selectivity are called optrodes.