Chemical Instrumentation Edited by GALEN W. EWING, Seton Hall University, So. Orange, N. J. 07079 These articles are intended to serve the readers o f n n s JOURNAL by calling attention to new deuelopmenls i n the Iheory, design, or availability of chemical laboratory instrumentation, or by presenting useful insights and ezplanations of topics that are of practieal imporlance lo those who use, or leach the use of, modern instrumentation and instrumental techniques. The editor inmles correspondence from prospective conlributors.
LXXVII. Instrumentation for Fluorescence and Phosphorescence (Concluded) Peter F. Loti, Chemistry Department, University of Missouri-Kansas Citv, Kansas C ~ t v ,Missouri 64110 a n d Robert J. Hurtubise. Pfizer Inc., Terre Haute, Indiana 47808 Commercial Instruments Commercial speetmfluomphotometers which allow the scanning of excitation and emission spectra, and filter fluommeters have been previously described in this series 111). Certain of these instruments are still offered commercially, and some models have been imomved while keeoinr" their usme basic design. Accordingly, only tuu cunven[~onali~rrctrurluorophotometers and onr fi1tt.r Iluorometer are desvr~bedin this article. Phosphorescence accessories and pulse instrumentation are also described.
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11 shows an optical diagram for the Model MPF-3 fluoresceme spectrophotometer. An earlier instrument in the same MPF series; the MPF-4 instrument employs essentially the same Rhodamine B type of spectral correction system. As can be seen in Figure 11, the instrument employs two grating monochmmston for excitation and emission, respectively. A portion of the radiation from the xenon lamp is reflected hy a beam splitter onto a detector photocell. The source light then illuminates the sample (in the sample compartment) and the emitted fluorescent radiation is detect-
Fluorescence Instrumentation
The spectrofluorophotomete~~ offered by the Perkin-Elmer Corooratian are reoreientatwc respectively of the conwntional simple spevtn~fluurophutumete~j and more complex insrrumrntation required to correct for non-constant source intensity and non-constant detector response throughout the ultraviolet-visible spectral region. An example of the first type is the Model 204 Fluorescence Speetrophotometer (shown in Figure 8) whose functional layout is shown in Figure 9. The system is designed to pmvide qualitative spectral emission and excitation data as well as quantitative fluorescence measurements with good sensitivity, stability and convenience. It consists of a 150-watt xenon source, grating monochromators for both excitation and emission, with a sample compartment in between, a photomultiplier detector to provide the signal for s 5-in. meter readout or a recorder. The bandpass of both monochromators is fixed at 10 nm. The sample compartment accommodates a holder for four standard cells. A number of accessories including micro cells, flow cells, temperature control and test tube holder, and Technican interface aecessories are optionally available. The Model MPF-4 fluorescence speetmphotometer is pictured in Figure 10. Figure
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Figure 8. Perkin-Elmer Model 204 cence Spectraphotometer.
Fluores-
ed in turn by a second detector photocell. A difference amplifier measures the ratio of the difference in intensity between the sample and the reference circuits. Correction for the excitation soectrum is made by rerurdmg the ratio of the znrnplc drtrrtor output signal to that ul a reference dctertor which monrtors the emnsiion of a quantum counter. The quantum counter consists of a concentrated solution of Rhodamine B in s triangular shaped cell. The quantum counter will absorb all the ineident radiation and convert it into a pmportional number of emitted quanta of fluorescent radiation. Rhodamine B is used as a quantum counter because it has the pmperty of maintaining a constant ratio of quanta absorbed from 2W to 600 nm to quanta emitted a t 630 nm. The fluorescent radiation from Rhodamine B is passed through a 630 nm filter, onto a detector.
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Figure 10. Perkin-Elmer MPF-4 Spectrophotometer.
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Fluorescence
The detected signal is fed to the reference amplifier, and the sample fluorescence is directed through the emission monoehromator to the sample photomultiplier. The amplified sample signal is electronically ratioed to the reference signal and recorded. A 25-tap pre-programmed potentiometer is coupled to the wavelength drive of the excitation monoehmmator to alter the sample amplifier gain to compensate for the non-constant reflectance of the beam splitter and for energy changes due to polarization from the monochromator. When the excitation system is properly adjusted, the excitation spectrum of a sample of
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ELECTRICAL CONNECTION OPTICI\L PATH
YONOCHROMATOR
LIMP SOURCE
SHUTTER
VOLTbGE POWER
AMPLIFIER
RECORDER OUTPUT
DlGlTIL OUTPUT
SENSICV~Y CONTROL
Figure 9.
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b
I00 % ADJUST
b
ZERO ADJUST
Functional layout of the Perkin-Elmer Model 204 Fluorescence Spectrophotometer.
EXCITATION MARKER SWITCW
CLUTCH
EXClTATlON WAVELENGTH C A Y
I
AYPLIFIER
SIGNAL
W O N M A R I E R SIGNAL
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t r
XENON LAMP POWER SUPPLY (INCLUDING STARTER I
E X C I T A T I O N MARKER SIGNAL
Figure 11. Mechanical and electrical system diagram of the Perkin-Elmer MPF-3 Fluorescence
Rhdsmine B run against the Rhodamine B reference cell will give a response of relative intensity versus wavelength flat to within +5%. For correction of emission spectra, the emission monochromator is treated separately. A flat response curve for the detector output is obtained by preadjustment of another 25-tap ~otentiometercoupled to the emission monochromator. One way to preadjust the emission system is to use a tungsten filament lamp that has heen calibrated against a National Bureau of Standards spectral radiance reference lamp. The output of the lamp is measured directly through the emission monochromator. The 25-tap potentiometer coupled to the emission monochromator is adjusted to match the calibrated values of the tungsten lamp. Another way to preadjust the emission system is to ahtain the spectrum of the xenon source by scanning the excitation monochromator with a concen-
trated solution of Rhodamine B in the sample position. This gives the true relative intensity versus wavelength of the source emission because the Rhodamine B acts as a detector and emits fluorescent radiation of 630 nm which is directly proportional to the quantum intensity of exciting light. To adjust the corrected emission system, both the exeitation and emission monochromators are scanned synchronously using the Rhodamine B sample, and the trimming potentiometers of the emission corrector are adjusted so that the spectrum obtained is identical to that of the xenon source. At wavelengths beyond 600 nm, the corrected emission system is adjusted using a calibrated National Bureau of Standards lamp. The Model MPF-4 uses an AC optical and electrical system to provide high stability and automatic gain control which serves in conjunction with the Rhodamine B reference detector to automatically adjust dynode
voltage to maintain constant amplifier gain regardless of the variations in source energy. This provides better pen response in the short wavelengths (200 to 240 nm) where source energy is relatively law, and it also improves near infrared performance where detector response is relatively low (700 to 850 nm). The monochromators use 1200 lines per mm gratings which permit a wavelength range from 200 to 1200 nm. Optional detectors may be used to obtain fluorescence data from 850 to 1200 nm. In using the MPF-4, one first adjusts the excitation monochromator to an appropriate wavelength that is absorbed by the sample. The emission monochromator is allowed to scan and the emission spectrum is recorded. If the emission monochromator is set to a fluorescence wavelength for the sample and the exeitation monochromator is allowed to scan, the excitation spectrum can he recorded. From these spectra, one obtains the optimum operating conditions. Once these conditions are obtained, one can then prepare conventional calibration curves by plotting the fluorescence intensity versus concentration and then use the calibration curves for quantitative analysis. A phosphorescence accessory is available for the MPF-4 that permits recording of excitation and phosphorescence spectra, measurement of average life of phosphorescence, and recording of low'temperature fluorescence. The accessory includes a sample holder assembly and an amplifier. The sample holder includes a "ratatingcan chopper" (Figure 3) that permits periodic excitation of the sample and periodic out-of-phase measurement of phosphorescence. This allows the measurement of the phosphorescent signal without interference from scattered light and short-lived fluorescence. A Dewar flask filled with liquid nitrogen is incorporated into the sample compartment to permit luminescence measurements a t reduced temperatures. Phosphorescence accessories are also available for various other commercial instruments, such as the Aminco-Bowman spectrophotofluorometer, the Baird-Atamic Fluorispee, and the Farrand MK-1 Spectrofluorometer, which will be described. The accessories available from Famand allow measurement af phosphorescence and enhanced fluorescence at reduced temperatures. For measurement of luminescent signals at low temperatures, two cryogenic accessories are available. With both accessories, the samples are cooled by means of an immersion probe. With one accessory, a refrigerant such as liquid nitrogen is used to cool the sample to 77°K. The other accessory can cool samples in the temperature range from -15°C to -125"C, and utilizes a mechanical cryogenic unit operated by compressed air. The same air is also used as the refrigerant. Other accessories are a rotary chopper which is used to obtain phosphorescence spectral data and a single pulse phosphorescence shutter which provides fast cutoff of the excitation light and allows phosphorescence decay times down to two milliseconds to be ohserved and recarded. The Farrand MK-1 instrument is shown in Figure 12. There are other means of obtaining corrected spectra, such as the use of a flat response thermal detector rather than the (Cont~nuedon pageA360) Volume 51, Number 7, July 1974
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Chemical Instrumentation Rhadamine B system. With any corrected fluorescence spectrophatometer advantages obtained include the following (12): (a) Problems associated with non-constant source energy and non-constant detector response are minimized and data can be compared from instrument t o instrument. (h) Probably the most important use is the capability of obtaining a corrected excitation spectrum in place of a n ahsorption spectrum when solution concentrations are below the absorption threshold for conventional spectrophotometers a t concentrations perhaps 1000 times more dilute. For many compounds the corrected excitation spectrum and absorption spectrum are identical. (c) Corrected emission spectra can he used t o determine fluorescence quantum efficiencies of pure materials. (d) The presence of impurities, moleeular associations, tautomerism, and transfer of electronic energy among molecules can he detected by the dissimilarity between absorption and excitation spectra or the non-constancy of the quantum efficiency as a function of wavelength (13). These advantages are enhanced because of the great sensitivity of fluorescence methodology. For example, it is very often possible to measure impurities which would not he reflected in the absorption spectrum.
Filter Instruments Filter instruments are commonly used for routine quantitative measurements. If flow cells are incorporated into the sample
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Figure 12. Farrand M K - I Spsctrofluarometer with law fernperature accessories.
compartment, they can he incorporated into continuous flow instrumentation such as the Technicon "AutoAnalyzer." Wheeler and Lott recently incorporated a fluorometer a s a detector in a HPLC system for the determination of Se(IV), nitrite, and nitrate a t the part-per-billion level (14). The spectra for this selenium compound are shown in Figure 5. Generally filter fluorometers offer the advantage of lower cost, convenience far repetitive mea-
surements and, particularly if the sample excitation wavelength is a t an emission line of the light source, perhaps higher sensitivity than obtained with fluorescence spectrophotometers since there may he less light loss through simple filters compared to monochromators. The Farrand Ratio Fluommeter-2, with digital meter, is shown in Figure 13 and the optical diagram in Figure 14. The main optical path of the instrument is es-
Figure 13. Farrand Ratio Fluorometer-2.
sentially the same as a conventional single-beam filter fluommeter. A high-intensity mercury vapor lamp is used as the excitation source. The Farrand instrument features all-quartz optics (because of their W transmittance): lenses A, B, and C collimate and condense the source radiation to the sample; lenses D and E collect, collimate, and focus the emission radiation to the photomultiplier detector. Either optical elass or narrow-hand interference filtern are rrnpltryed fur ercnatwn and em,,a i m wnvrlen~?h iaolatton. Thc apenure disc scnes a, a s l i t to w n f r o l t h e i n f m s i t y ofradiation to the sample. The ratio system greatly reduces longterm signal drift caused by changes in the Light source. The optical path of the reference beam is shown in the right side of the diagram (Figure 14). A portion of the lamp radiation passes through the primary optical system and is attenuated by means of the reference aperture disc before reaching the reference photomultiplier tube. The electrical signal from the reference photomultiplier and the sample signal from the sample photomultiplier are fed into a solid-state electronic divider which perioms the computation of ratioing the sample-to-reference signals. Matched lowdrift photomultiplier tubes contribute to long-term stability. The ratioed signal can be read directly on the meter or on any strip-chart recorder. Either transmission or interference filters are employed for wavelength isolation.
lnstrumentatlon for Measurement of Luminescence Lifetimes As generally the decay of normal fluorescence is several nanoseconds, special techniques are needed to measurelhe fluorescent lifetime of a molecule. These include stroboscopic and phase-shift techniques. High-speed flash tubes that generate subnanosecond Light pulses are used in the stroboscopic method. Such flash lamp systems are available from TRW Instruments, El Segundo, Calif., and Xenon Corp., Medford, Mass. (3). This technique usually employs a gated amplifier for detection af the fluorescent signal. The technique is described in more detail along with pulse source phosphorimetry. Muller et al. (201 have described a fluarameter utilizing a phase-shift technique in which the exciting radiation is modulated up to 27 MHz. A portion of the madulated source radiation is reflected from a quartz plate onto a reference photamultiplier and the remainder excites the sample. Fluorescence from the sample is detected by a second photomultiplier and the phase shift between the fluorescent (Continued on page A362) Volume 51. Number 7, July 1974
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MERCURY -VAPOR LAMP
Chemical instrumentation and reflected light is measured electmnieally. The fluorescent Lifetime can then be determined knowing the phase shift and source modulation frequency (21). Time resolved phosphorimetry whereby the concentration of two similar phosphorescent molecules can be determined by recording the logarithm of the phwphoreseence signal from the mixture versus time after the termination of excitation was mentioned earlier. However, with present day commercial instrumentation this technique cannot he used for molecules with relatively short phosphorescent lifetimes, e.g.
