3 Photometers and Spectrophotometers The basic principles of the construction of photometers and spectrophoto- meters is the same;i.e.they consist of a light source,monochromator or filter,cuvette compartment,detector and amplifier with an indicating device. Table 1.Filter combinations for isolating emission lines from metal-vapor discharge lampsac- cording to [1].The figures in the column 'filter combinations'refer to Tables 2 and 3 Element λinnm Filter Percentage For surpressing transmission the nea Filter-No. 163 16(36) 328/30/35 4+32+35 /53 0210030 4 456/59 468/8 2/81 2323201 1661666166 101510 16(36 668 050 257 +11 006 767/70 30+29 30+29 Cs 852-921 10 30+28 *Liquid filter No.36(see Table 3)can be used instead of NIR-filter 16. H.-H.Perkampus,UV-VIS Spectroscopy and Its Applications Springer-Verlag Berlin Heidelberg 1992
3 Photometers and Spectrophotometers The basic principles of the construction of photometers and spectrophotometers is the same; i.e. they consist of a light source, monochromator or filter, cuvette compartment, detector and amplifier with an indicating device. Table 1. Filter combinations for isolating emission lines from metal-vapor discharge lamps according to [1). The figures in the column 'filter combinations' refer to Tables 2 and 3 Element A in nm Filter Percentage For surpressing combinations transmission the near IR Filter-No. at room temp. and residual (approximate) red radiation Filter-No. Zn 308 4+32+33 5 16 (36)* Hg 313 4+34 35 16 (36) Cd 326 4+32+34 5 16 (36) Hg 334 4+32+35 10 16 (36) Zn 328/30/35 4+32+35 2 16 (36) TI 352153 2+ 10+32 10 16 (36) Hg 365 5+9+31 20 16 (36) TI 378 2+22 30 16 (36) Hg 404/07 1 +3+20+9 1 16 (36) Hg 435/36 10+17+6 4 16 (36) Cs 456159 9+22 40 16 (36) Cd 468/80 9+ 18 25 16 (36) Zn 468/72/81 9+ 18 25 16 (36) Cd 509 7+21 +8 20 16 (36) TI 535 14+ 19 35 16 (36) Hg 546 15+23+13+8 10 16 (36) Hg 577179 12+24+12 15 16 (36) He 588 12+25 + 12 10 16 (36) Na 589 12+25 + 12 10 16 (36) Zn 636 26 85 Ne 638-668 26 90 Cd 644 26 90 He 668 27 + 11 20 He 707 29 65 K 767170 30+29 25 Rb 780/95 30+29 25 Cs 794-921 30+29 10 Cs 852-921 30+28 1 * Liquid filter No. 36 (see Table 3) can be used instead of NIR-filter 16. H.-H. Perkampus, UV-VIS Spectroscopy and Its Applications © Springer-Verlag Berlin Heidelberg 1992
Photometers 11 3.1 Photometers In photometers,also called non-dispersive instruments,the monochroma tor is replaced by a set of filters by means of which specific spectral ranges can be selected from the continuum of a light source,eg.a tungsten halogen lamp in the visible region.Mercury-vapor high pressure lamps com source as the light source and fitted with an interference filter mo romatic be am.The number of spectral lines can be extended by using other metal vapor discharge lamps;for example a cad- Table 2.Glass filters in common use,from [2] Code Thickness No. mm 1 GG43 18 GG45 325 6- BG3 9012223 22 8910 BG12 122432 2526 28 RG100 35 3222222 +5 901 BG38 53 Table3.The most generally useful liquid filters Description Pathlength/mn of the cuvette) 32 Nickl-cobae 303g+86.5g 20 S04 16 mg 1314156 57g 0000 CS0,+5H20
Photometers 11 In photometers, also called non-dispersive instruments, the monochromator is replaced by a set of filters by means of which specific spectral ranges can be selected from the continuum of a light source, e.g. a tungstenhalogen lamp in the visible region. Mercury-vapor high pressure lamps combined with interference filters are often used so that mercury lill$s at 334, 365,404/407,435/436,546 and 577/579nm can be utilized. Photometers having a line source as the light source and fitted with an interference filter provide a more monochromatic beam. The numberbf spectral lines can be extended by using other metal-vapor discharge lamps; for example, a cadTable 2. Glass filters in common use, from [2) Filter Code Thickness Filter Code Thickness No. No. mm No. No. mm 1 V02 1 17 00435 4 2 V03 2 18 00455 3 3 V03 2 19 00475 2 4 V05 3 20 00385 5 5 VOll 2 21 00495 2 6 B03 2 22 00375 2 7 B07 1 23 00530 1 8 B08 2 24 00570 3 9 B012 2 25 00590 2 10 B012 4 26 R0610 2 11 K03 3 27 R0665 2 12 B018 2 28 R01000 2 13 B018 3 29 RON 9 2 14 B018 5 30 KOl 2 15 B020 5 31 W0360 1 16 B038 3 Table 3. The most generally useful liquid filters Filter Description Quantity/l Pathlength/mm No. H2O (inside dimension of the cuvette) 32 Nickel-cobalt sulphate 303 g+86.5 g 20 NiS04+CoS04 33 Picric acid 16mg 20 34 Potassium chromate 150mg 20 35 Nitric acid N/5 20 36 Copper sulphate 57 g 10 CuS04+ 5H20
2 Photometers and Spectrophotometers mium lamp supplies lines at 326,468/480,509 and 644 nm.A summary is given in Table 1 [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. 3.2 Spectrophotometers In a spectro photometer,no wadays 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 cu ette are placed one after another in the path of the light.The 100 point,previously setr a11 via the sli or by changing the amplification, is t day adjusted auto aticall in mo instrum which dis olay th hotometric result as per- tage ce n digital form. In a double-b or abs strument,the primary light beam is split and directed along two paths which traverse alternately the reference and measurement cuvette,which are approximately 10-15 cm apart.Thus,after both 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 these oingr non he beamine lmnc er on. trols this function.Figure 5 shows the optical sy em of a double-beam in- strument with a double (Perkin-Elmer Lambda 9). onen of photom pectro Here nd i mus ish ortan een inst ume s with nstruments with double monochromators.A dou the important advantage that the proportion of stray ight 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)
12 Photometers and Spectrophotometers mium lamp supplies lines at 326, 468/480, 509 and 644 nm. A summary is given in Thble 1 [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 substances (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. In a 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 instruments allow the continuous variation of the measurement wavelength over the whole spectral region. They are also called dispersive spectrometers. 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, i.e. the reference and measurement cuvette are placed one after another in the path of the light. The 100070 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 percentage transmittance or absorbance in digital form. In a 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 10-15 cm apart. Thus, after both beams have been refocused, light of varying intensity falls onto the detector and generates an alternating-voltage signal. This principle forms the basis of recording 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 controls this function. Figure 5 shows the optical system of a double-beam instrument with a double monochromator (Perkin-Elmer Lambda 9). The monochromator is the most important component of a spectrophotometer. 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 Monochr. 5 阳 ltiplier NIR regior nolographic gratings an quality of the gratings employed,the propor. ween 0.05 and 0.005%for single-grating monochromators. e equipment ers have introduced the no- tion of the "proportion of sca red ight"as a n index Thisterm refers to the intensity of the light leaving th ich wavelength in the immediate neighborhood of that of th edesired ly,it is also sometimes called "stray light see Sect.3.3. monochromators.the proportion of scattered light is lower by abou powers of ten.These figures are average values obtained from the manufac urer's literature.Holographic gratings provide a substantial improvement in stray light characteristics over ruled ones.See Sect.3.3 for the elimination deter ination of stray light. The advantage of a grating vis-a-vis a prism lies in the fact that a grating ows a a dispersion vhich is linear with wavelength.The correlation between the resol s and wa elength A in nm for the spec- po a tral bandwi is sho n in Tahle 4 Fot any other value of Av is obtai d by multiplying va es from hi b with the appropriate A.It can be seen that the ral band- ng po width increases from the U to the visible a constant or a constant resolving power the bandwidth decreases from UV V to visible
Spectrophotometers 13 113 DL ~amPie[omp.~rsampTe[omp·]j-l I II 119. I I Reteren,e R I i 10 : Sample II I II I II I II I s II I II II II II I I I I I Fig. 5. Optical layout of an UV-VIS-NIR spectrometer with a double monochromator (PerkinElmer Lambda 9) MI, M3, M4, M7 plane mirrors, M2, M5, M6, Ms, M9, MIO toroidal mirrors, SE entry slit monochromator I, SM centre slit = entry slit monochromator II, SA exit slit monochromator II. Detector: UV-VIS region photomultiplier NIR region PbS-detector holographic gratings for UV-VIS and NIR on a turntable Monochromators in use today are almost exclusively grating monochromators and, depending on the quality of the gratings employed, the proportion of scattered light lies between 0.05 and 0.005070 for single-grating monochromators. The equipment manufacturers have introduced the notion 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, AD. Erroneously, 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 manufacturer'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 v and wavelength A in nm for the spectral bandwidth LlA = 1 nm is shown in Table 4. For any other value of LlA, Ll v is obtained by multiplying values from this table with the appropriate LlA. It can be seen that the resolving power for a constant spectral bandwidth increases from the UV to the visible. Alternatively, for a constant resolving power the bandwidth decreases from UV to visible
Photometers and Spectrophotometers Table 4.Resolving powe 49 in VIS region for a spectral 入nml p【cm- [cm-' 200 50000 250 33333 600 16666 800 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:input of the recording ange recording in wavelength or wavenumber eat rec ording ove elec ted wavelen ath ra or at differ np and filter and time inte anges;I of the ist and nd,if n ary,o high de genera Goo ac prin r,e.g.printout for sets of mea ure nents;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 Ge ruments fitted with mic uters allow the recording of d 2nd has co increa ly t in applications since it can improve the sensitivity o 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
14 Photometers and Speetrophotometers Table 4. Resolving power LI ii in em -I in the UVVIS region for a spectral bandwidth of LlA = 1 nm A [nm] ii [em-I] Llii [em-I] 200 50000 250 300 33333 110 400 25000 62 500 20000 40 600 16666 28 700 14286 20 800 12500 16 With a few exceptions, all modern instruments are fitted with grating monochromators. 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 induding format selection with graph plotters; conversion of extinction values to concentrations; input of the recording 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 1 st and 2nd and, if necessary, of higher derivatives; generation of Good Laboratory Practice (GLP) protocols by the printer, e.g. printout of analysis data with sample identification for sets of measurements; calculation of difference spectra. The scope of application can be considerably extended by fitting supplementary modules and accessories, such as thermostated or temperatureramped cuvette changers controlled by a microprocessor, fluorescence attachments, 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 1st 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 recording spectrophotometers post manufacture, true double-wavelength spectroscopy 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