12 _Photometers and Spectrophotometers ‘mium lamp supplies lines at 326, 468/480, 509 and 644 nm. A summary is ‘given in Table | [1]. Details of the filters are given in Tables 2 and 3 [2]; see also [3]. Photometers are used in the photometric determination of single sub- stances (see below). In recent years, they have also been employed in the clinical-chemical and biochemical fields, as well as in special equipment developed for the analysis of waste gases. Ina spectrophotometer, nowadays more usually termed a spectrometer, the ‘measuring light is split up (dispersed) into its constituent wavelengths by a prism or grating monochromator. With a deuterium lamp for the UV region and a tungsten (tungsten-halogen) lamp for the VIS region, these in- struments allow the continuous variation of the measurement wavelength over the whole spectral region. They are also called dispersive spectro- meters. Most instruments cover the range 190 to 900.nm [4]. ‘We differentiate between single-beam and double-beam instruments. Single-beam instruments generally operate on the substitution principle, ie. the reference and measurement cuvette are placed one after another in the path of the light. The 100% point, previously set manually via the slit or by changing the amplification, is today adjusted automatically in most instruments which normally display the spectrophotometric result as per- centage transmittance or absorbance in digital form. Ina double-beam instrument, the primary light beam is split and directed along two paths which traverse alternately the reference and measurement cuvette, which are approximately 10~ 15cm apart. Thus, after hoth beams have been refocused, light of varying intensity falls onto the detector and generates an alternating-voltage signal. This principle forms the basis of re- cording spectrophotometers. In the case of fixed beam-splitting elements, the alternating direction of the light through the two cuvettes must be made by means of a chopper. In the case of a rotating sector mirror, the beam-splitting element itself con- trols this function. Figure 5 shows the optical system of a double-beam in- strument with a double monochromator (Perkin-Elmer Lambda 9). ‘The monochromator is the most important component of a spectro- photometer. Here we must distinguish between instruments with single ‘monochromators and instruments with double monochromators. A double ‘monochromator has the important advantage that the proportion of stray light is very small. Stray light is light from another spectral region which is superimposed on the ‘useful light’ of the spectral region which is selected for measurement. It can distort the required measurement considerably (see Sect. 3.3).Spectrophotometers 13 Fig. 5. Optical layout of an UVVIS-NIR spectrometer with a double monochromator (Perkin- Emer Lambda 9) My, M;, My, My plane mirrors, My, Ms, Ma, My, Mp, My toroidal mirror, SE entry st monochromator 1, SM centre sit entry slit monochromator II, SA exit sit ‘monochromator IL. Detector: UVIS region photomultiplier NIR region PbS-detezor holographic gratings for UVAVIS and NIR on a turntable ‘Monochromators in use today are almost exclusively grating monochro- mators and, depending on the quality of the gratings employed, the propor- tion of scattered light lies between 0.05 and 0.005% for single-grating monochromators. The equipment manufacturers have introduced the no- tion of the “proportion of scattered light” as an index of quality. This term refers to the intensity of the light leaving the exit slit which has a wavelength in the immediate neighborhood of that of the desired light, 2p. Erroneous- ly, it is also sometimes called “stray light”, see Sect.3.3. For double monochromators, the proportion of scattered light is lower by about two powers of ten. These figures are average values obtained from the manufac- turer’s literature, Holographic gratings provide a substantial improvement in stray light characteristics over ruled ones. See Sect, 3.3 for the elimination or determination of stray light. ‘The advantage of a grating vis-a-vis a prism lies in the fact that a grating shows a dispersion which is linear with wavelength. The correlation between the resolving power in wavenumbers # and wavelength 2 in nm for the spec- tral bandwidth 44 = 1 nm is shown in ‘able 4, For any other value of 42, AV is obtained by multiplying values from this table with the appropriate ‘Al. It can be seen that the resolving power for a constant spectral band- ‘width increases from the UV to the visible. Alternatively, for a constant resolving power the bandwidth decreases from UV to visible.14 _Photometers and Spectrophotometers ‘Table. Resolving power 7 in em~! in the UV- VIS region for a spectral bandwidth of 2 = {nm A fom) * 45 fem 200 250 300 110 ‘400 @ 500 0 600 28 10 20 30 16 With a few exceptions, all modern instruments are fitted with grating mo- nochromators. With fully automated instruments, depending on the sophistication of the software, the following functions can be recalled or continuously monitored by a microcomputer: Base-line correction; conversion of analog to digital data; recorder, printer or plotter control including format selection with graph plotters; conversion of extinction values to concentrations; range; recording in wavelength or wavenumber; repeat-recording over selected wavelength ranges or at different wavelength and time intervals; lamp and filter changes; formation of the ist and 2nd and, if necessary, of higher derivatives; generation of Good Laboratory Practice (GLP) pro- tocols by the printer, eg. printout of analysis data with sample identific tion for sets of measurements; calculation of difference spectra. The scope of application can be considerably extended by fitting sup- plementary modules and accessories, such as thermostated or temperature- ramped cuvette changers controlled by a microprocessor, fluorescence at- tachments, accessories for diffuse and specular reflection spectroscopy, and for enzyme kinetics, sipper systems for repeated measurements, gel scanners and chromatographic attachments. Several newer techniques such as derivative spectroscopy [5], see Sect. 5.1, and dual- or double-wavelength spectroscopy [6], see Sect. 5.2 have recently gained in importance. Generally, instruments fitted with microcomputers allow the recording of Ist and 2nd order derivative spectra. This method has come increasingly to the fore in analytical applications since it can improve the sensitivity of detection considerably. Whilst devices for derivative spectroscopy can be fitted to many record- ing spectrophotometers post manufacture, true double-wavelength spectro- scopy requires a special instrument whose most important components are two optically identical monochromators. However, double-wavelength spectroscopy can also be pursued with a ‘microcomputer-controlled spectrophotometer by entering the extinctionSpectrophotometers 15 value at a specific wavelength A, into the memory and by retrieving it from the memory for comparison with the extinction values at wavelengths Aa, Several manufacturers have taken advantage of this possibility in software. Diode-array spectrometers are an interesting new development pioneered by Hewlet-Packard. Instead of a monochromator, a polychromator is used in which the dispersed light of a continuum source is brought to a focus in ‘one plane, the focal plane of the instrument. This method of operation cor- responds to the classical method where a photographic plate, which “recorded” the whole spectrum directly, was fitted in the focal plane of a spectrograph. Today a silicon-diode array detector is used, instead of a photographic plate, and this allows direct, fast electronic processing of the spectral information stored for a short time in one row of 256 or 512 diodes (Channels), The components are not arranged as in a conventional spec- trophotometer, in particular the cuvettes are mounted between the light source and the entry slit of the polychromator. Figure 6 shows the optical layout of the Perkin-Elmer Lambda-Array- LUVNIS Spectrometer 3840, The light of a deuterium lamp is focused on to the sample by the plane (flat) and toroidal mirrors (Mz and M3) and, after traversing the sample, arrives via mirror M, and another plane mirror at the entry sit of the polychromator. Here the continuum of the light source is directed onto a grating via Ms. Finally the light, now spectrally dispers- 4, falls via mirror Mg onto the photodiode array (PDA) detector with 512 photodiodes. Two gratings are mounted on a turntable. The first grating (100 tnes/mm) is used for the fast acquisition of a survey spectrum in the 200-900 nm region (theoretical resolution 1.5m, effective resolution ca. 451m, ie, the survey mode). The second grating (600 lines/mm) is used a high-performance mode in which itis rotated in 7 steps to achieve a high resolution (~0.25 or =0.75nm). In this mode, spectral regions of ca. 100nm arrive at the diode array, ie. the whole region of measurement is divided into 7 sections. Depending on the width of the selected 4 region, the time for measuring a whole spectrum is approximately 816s. The deuterium lamp (type D 802 or D 902, Heraeus Original Hanau) transmits light along the optical axis and, as shown in Fig. 6, this allows the introdue- tion of the beam of a tungsten lamp into the light path, The W and source filter wheels are used to cut out one lamp when measuring with the other. {All functions (filter change, shutter, rotation of grating, PDA read-out, col- lection of data, etc) are controlled by the Perkin-Elmer PC 7500. Hewlett-Packard have developed a similar instrument, model HP 8452. lips and Milton Roy also market diode array spectrometers. PI‘The Stray Light Error__17 334 General Observations Since the vast majority of UV.VIS spectrophotometers are still fitted with a single monochromator, errors can occur due to monochromator stray light at the limits of the monochromator transmission (eg. 4<220 nm 345000 cm™"); particularly in the case of small transmission values. By stray light we mean light of other wavelengths which is superimposed upon the useful light. Often mistakenly called scattered light, itis caused by scattering at the optical surfaces in the monochromator. If a monochromator is set to wavelength Ap and if the slitwidth cor- responds to the effective bandwidth 4, the useful light lies in the region between Ag—AA and At Ad. ® We shall call this the useful-light region. In ideal circumstances, a mono- c’chromator should only transmit in the useful light region with the transmis- sion decreasing linearly from Ap in both directions. However, due to stray light there is a certain transmission by the monochromator of light outside the useful light region. Although its intensity is small (order of magnitude 10°) it can still be significant since the radiation detector sums the stray light over the whole wavelength region to which it is sensitive and in which the light source emits radiation. ‘The proportion of the photoelectric current given out by the detector and reaching the display, which is due to stray light, is the critical measure of the stray light effect when making spectrophotometric measurements. For that reason, we understand by the term proportion of stray light the ratio of the photoelectric current arising from stray light to the total photoelectric current. Although the proportion of the photoelectric current ising from the stray light is generally low (<0.1%), it can cause signifi- cant problems if the photoelectric current due to the useful light becomes relatively small. In practice, this occurs in the following cases: 4. The useful light can be weakened by absorption occurring in the light path whilst the stray light is hardly diminished. This occurs particularly below 230.nm because: ) The optical elements in the light path (envelope of the hydrogen lamp, source mirror, lenses, mirrors, prisms or gratings in the monochromator, lenses in the sample changer, multiplier envelope) absorb increasingly swith decreasing wavelength. In addition, there is the effect of the pres, ence of absorbing deposits on the accessible optical surfaces (envelope of the hydrogen lamp, lenses in the sample changer, cuvettes). Further- more, atmospheric oxygen which is present in the optical path of the in- strument absorbs at wavelengths below 200 nm.