Topics in..
. Chemical Instrumentation
Edited by
5. 2. LEWIN, New York University, New York 3, N. Y.
These articles, most of which are to be contributed by guest authors, are intended to serve the readers of lhis JOURNAL by calling attention to new devebpmenls i n the t h e w , design, or auailability of chemical labmatoly inslrumenlation, oi by presenting useful insighls and explanations of topics that are of practical imporlance to those who use, or teach the use of, modern instrumentation and instrumental techniques.
XV. Instrumentation for Fluorometry. Part Two
reference phototube detector "H." The signal produced is amplified and applied to the recorder slidewire in such a. way aa to compensate automatically for muree ~ntensityfluctuations. The sample eompnrtment accepts solid samples, 1 cm square cells, or 1.9 cm cylindrical ceUs. The Perkin-Elmer Co. plans t o introduce a fluorescence attachment for the Hitachi P-E Model 139 speetrophotometer by June 1964. The first attachment t,o be offered will use s. filter excitation
the operation of the instrument as a fluorescence spectraphotometer.
.
Peter F. Lott.. De~artmentof Chemistry, University of Missouri ot Kansos City, Konsas City, Mo.
I n contrast to fluorescence spectrometers variable wavewhich have a conti~~uously leneth for the selection of excitation and fluorescence frequencies, same manuiscturers make it possible t o convert their spectrophotometers into fluororneten which will measure only the fluorescent spectrum of the compound. The fluarescenee attachment which provides the excitation light usually consists of the ext,ernal light source with filters for the selection of the appropriate excitation wavelength, s. sample holder and the optics necessary to reflect the fluorescent light into the munochronrstar of the speetraphot,ometer. The spectrophotometer then transform this fluorescent radiation into the fluorescent spectrum. In order to attain nmximum sensitivity, the photomultiplier of the spectrophotometer is used ;ls the light detector. The optical arrangement of the fluorescence attachment manufactured by Beckman Instruments, Inc. for their DU and DK spectrophot,ometersis shown in Figtire 16, the ~ttaehrnentis shown in Figure 17.
Figure 16. Optical diogrom Ruorereence anachment.
far
Beckman
The excitation light source is a General Electric Co. F4T5/BL low-pressure, phosphor-coated, mercury lsmp peaking a t
436 and 405 mfi The fluorescent spectrum is measured manually with the conventional DU instrument and recorded with the DK spectrophotometer. Also included with the fluorescence attachment is a sample holder for paper strips which serves t o measure the fluorescence directly on the paper after a, ehramatcgraphic separation. The Applied Physics Corp. also offers fluorescence attachments for their Cary
Figure 17. Beckman Ruorercence ottochment for DU ond DK rpecfrophotometerr.
Figure 18.
spectrophotometers. The optical path of the attachment for the Model 14 speetrophotometer is shown in Figure 18. A mercury are is used aa the excitation source and provisions are also made for correcting fluctuations in the intensity of the source. In operation, radiation from the mercury arc "A" passes through the aperture "B" and filters "C" and "D." The beam is then focused on the sample "G" by means of mirror "E." The fluorescent radiation ''I," from the sample, passes through the opening in the mirror "E," into the monochromator and then into the phototube detector. A small portion of the radiation from the mirror "En is reflected from the crystal quartz window "F" on t o the auxiliary
Hilger and Watts also offer a fluorescence attachment for the Hilger Uvispek spectrophotometer and the Unicam SF 500 spectrophotometer. These attachments differ from the previously described units in t h a t they do not use the monochromator of the spectrophotometer but convert the spectraphotometer into a filter fluorometer. The attachments are designed only to measure the amount of fluorescence and not fluorescent spectra. With both of these units a. tungsten lsmp is used as the light source and colored glass filters are used for limiting the excitation and fluorescent light. Operation of the Zeiss Model PMQ I1 spectrophotometer with a filter source has been mentioned previously.
Optical diagram for Cary Model
14 Rvorercence occertory.
Vol. 47, No. 6, June 7964
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A421
Chemical Ins trumentation Filter Fluorometers Historically, the early instruments used for fluorescence measurements employed a n external light source with a filter t,o obtain m~~nnehrumatia light for the ercitation of the sample and ;a second filter placed a t right angles t o the incident light path and the sample, t o screen out the exciting light and allow only the Hunrescent mdiat,ion t o pass on t o the detector. A simplified sketch of such a n instrument was shown in Figure 2. Although many modifieltt~ions have been made, many currently overed filter fluoramet,ers st,ill employ this same hasic design. The instruments vary as t o whether a photomultiplier or phototube detector is employed, and if some means is made t o compensate for light fluctuations. The cost of these fluornmeters ranges from several hundred dollars t o about $2500. Quite often these fluorometers are constructed for efficient fluorescence measurements of only a few compounds, and for routine use such as clinical analysis. A major factor is mer:hanical design for rapidity in operatinn Technicon Instruments offer s. Huorometer for use wit,h their Auto-Analyser for automated anal-. sis. Depending upon a number of faet,om, but particularly upon the light source intensity, a filter fluorometer may show a. greater sensitivit,y for t h e determinat,ion of trace quantities of a fluorescing sub-
A422 / Chemical Education
60
I
I
5.0 4.0
I
I
---- X
I
i
e m A r t 150 W
-H+Mtrtvry
rlrr 100 W
Figure 19. Spectrol charocterirticr of the 150-wan xenon orc and Pyrex-iacketed 10O.wm H.4 mercury lomp.
stance than does s. fluorescence speatrometer. Figure 19 compares the energy output of a, mercury lamp and xenon arc. The xenon arc produces essentially a continuous spectrum throughout the ultraviolet region. I n contrast, t h e mercury lamp spectrum is limited t o s. numher of lines. Should the esritntiun frequency of
the m o l e d e rorrespond t o one of bhr mercury lines, then the instrument PIIIploying this light source could shorv :L greater sensitivity, since the amount of fluorescence is directly proportit,nnl tu t,he intensity of the excitation sowee. Nerause of this factor, no true s i , i u ~ d : ~ ~ ~ l (Continzrd on page A434)
Chemical instrumentation exists for the comparison of the sensitivities of fluorometers. As they generally employ glass components, the uenal filter fluorometers are limited for excitation, and fluorescence measurements in the visible region. This would he the case with instruments employing Corning Glass Co. optical filters or Eastmsn Kodak Co. Wratten Filters. If the instrument is designed to illuminate the sample with a parallel beam of light, and sometimes when the exciting light source is
quite close to the sample, interference 6lters may be used quite advantageously in measuring the fluorescent light. Since these filters allow optical measurements bo be made with ementially monochromatic light, quite often they will screen out the fluorescence due to an impurity. To try to use interference filters in any fluorometer can cause difficulty. One of the problems is that the construction of the interference filter is such that it cannot be cut like ordinary glass filters to fit into the filter compartment of the i n s t ~ m e n t . Interference filters nmy be obtained from several manufacturers, in addition the Baird-Atomic Corp. (Cambridge, Mass.)
offers to supply the filters cut to any desired shape. characteristic of the conventional layout of components is the Coleman Model 12C Photofluorameter, an instrument which has been available for about 20 years. The optical path is shown in Figure 20. The fluorometer employs an H-3 mercury vepor lamp, test tube cuvettes, and plastic mounted glass filters. The fluorescent radiation is detected with a blue sensitive phototube, amplified with a linear amplifier, and the readout appears on a direct reading meter. If desired, an external galvanometer can be substituted for the meter to increase the sensitivity of the instrument. The Photovolt Model 540 fluorescence meter is a much more sensitive instrument of similar component arrangement. As shown in Figure 21, the instrument consists of two units, the Photovolt Model 520 M photomultiplier detector and their Model 54 fluorescence unit. The optical
Figure 21.
Photovolt Model 540 fluorercence
meter. Figure 20.
Optic01 path of Coleman Model 12C photofluorometer.
A424 / Chemicol Education
(Continued on page .44W
Chemical Instrumentation layout is shown in Figure 22. A variety of light sourcea can be used with the instrument, and as the condensor lens is made of quartz, UV excitation is possible. A number of filters, including interference filters, may be used with the instrument and the use of the photomultiplier (IF21 tube) permits high sensitivity det e r t ~ o nof fluorescenre in the ultraviolet resirm. n~~~~~
The Farrand Optical Co. Photoelectric fluorometer, Figure 23, employs a similar arrangement of components. I t is a high sensitivity, research-type insbrument. It. employs a photomultiplier detector, provision for using either a mercury or xenon arc source, quartz optics, con-
Figure 23. Farrand Optical fluorometer.
Co.
Photoelectric
ventiond or interference filters. Also offered as an accessory is a unit built into the instrument which permits the standardization of t,he instrument for the measurement of the fluorescence of two different materials or far decay studies. The Photovolt Corp. alao manufactures the Lumetron Fluorescence Meter, Model 402-EF. The instrument shows a, much lower sensitivity than the above instruments. It is of interest as it was one of the first fluorometers to compensate for fluctuations in the excitation source. Barrier layer cells are used as the fluorescence detertor without any electronic amplificstion. The optical path of the instrument is shawn in Figure 24. After passing through the condensinglens and excitation filter, the beam is split into two parts. One part of the light beam is used for the excitation of the sample, the fluorescence of which is detected with two barrier-layer photocells which me laterally arranged on both sides of the sample. The other part of the beam is deflected onto a third photocell which acts as the balance cell in the second arm of a bridge and thus compensates far light source fluctuations. The instrument employs a galvanometer for the detection of the null point; r e d out of the fluorescence is indicated by means of a slidewire potentiometer. Berkmsn Instruments, Inc. Ratio Fluorometer is shawn in Figure 25. The optical arrangement is shown in Figure 26. The fluorameter is a direct-reading, drruble-beam inst.rument which measures the ratio of the intensity of the sample and (Continued on page A428)
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Chemical Instrumentation reference beams. The source for the excitation of the sample is a low pressure lamp which is divided into two sections by n. light shield. Electrons flow from a common cathode alternately to the two anodes, one on the reference side and the other on the sample side. Since the lamp is of a diode type cmstruetion, it cyelcs in phase with the AC current. When one beam is on, the other is completely off. Thus the lamp supplies excitation radiation alternately t o the reference and sample cells, and in turn alternately to the photomultiplier. When the reference beanr impinges on the photo-
multiplier, the reference signal is amplified and fed back t o the input. I n the next cycle the sample signal passes onto the photomultiplier. The discriminator now passes this signal t o the meter circuit. By setting the reference solution equal to 100% on the meter, the sample signal a t the meter indicates the ratio of the sample to the reference. A zero control sets the aero reading on the m e k r in terms of the fluorescence genereted by a blank. A provision is also made for recording the fluorescent signal by simply plugging a recorder cable into recorder jacks at the rear of the instrument. The instrument has a rotary turret sample holder which slluws the sequential positioning of eight samples a t one "loading." A variety of
/
Mmwy kprlprlnmp
Figure
24.
Optia:ol
path
Lurnetron
fluorescence
meter
Model
402EF.
Figure 25.
Beckmon ratio Ruommeter.
filters is available as well as lamps peaking a t 253, 312, and 365 mrr. Vycor test tubes are recommended for use with excitation radiation below 325 ma. I n addition t o being able to measure liquid samples in test tubes, provisions are made for a continuous flow cell and s. paper strip holder for reading the fluorescence of samples which were prepared by ehramstographic separation. G. K. Turner Assaciatetes. Model 110 Fluorometer is a versatile instrument which can be used far conventional flumescence meaeurements of liquids, as well as continuous flow meaurements, pellet fluorescence measurements 23. in uranium analysis, and the measurement of the fluorescence on paper chromatograms or electrophoresis strips. Eleetronicdly snd optically the instrument is of the null balance design; the fluorescent output readings are measured on a d i d attached (Continued on page A4311
Save the time and trouble of investigating unknowns. Compare your spectrum for positive identification with a spectrum from one of these Sadtler collections: 22,000 Standard lnfrared Spectra (Pure Compounds) 10,000 Commercial lnfrared Spectra (19 groups of related Commercial Materials) 4,000 Standard Ultra Violet Spectra 2,000 Standard Near lnfrared Spectra 500 Standard Far lnfrared Spectra 250 Steroid Spectra Additional spectra are being readied for publication on a continuous basis.
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A428 / Chemiml Education
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amplifier to be used far the first stage of electronic r~mplifieation. The second elertronic stage is a phase sensitive detector whose output is eit,her positive or negative, depending upon whether there is an excess of light from the forward (sample) or rear light (reference) path. The output of the phase detector drives a meter amdifier which is in turn connected to a null meter. A balanced condition; i.e., equal light from the sample and from the
Chemical Instrumentation to a light cam which changes the light intensity of the reference beam. The fluommeter is basically an optical bridge. As shown in Figure 27, the optical bridge meamre8 the difference between the light emitted by the sample and that from a cnlihrated rear light path. A single p h o t e
I
I
I.1.rCa.lin.cd
Figure 26.
I.l.n"..6.l",in
Optical diogram of lhe ratio Ruororneler.
multiplier surrounded by a mechanical light interrupter sees alternately the light from the sample and the rear light path. Thus the photomultiplier output is an alternating current which permits a n AC
Vol. 41, No. 6, June 1964
/
rear light path, is indicated by the null position of the meter. The quantity of light in the rear path t o balance that from
A431
(Continued on page A436)
solvent blank. Because of the optical construction, errors due t o light source variations, such as aging of the U V
bridge Chemical llf~tt'~mt?lft~t;~lf
BLANK KNOB
PHOTOMULTlPLlER
LIGHT IrnEARUrnER
MOUNTNO BLOCK
-CITE LIGHT
/
ORWARD LlOHT PATH
Figure
COOLING FAN
Figure 27.
Optical design of
the Turner
Model 1 10 fluorometer.
the sample is indicated by the Fluorescence dial, which is connected t o the optical cam. Each of the d i s h 1 0 0 divisions comesponds t o the addition of an equal increment of light t o the rear light path by means of a n optical cam. Initially the light in the rear path may be adjusted with the blank control which sets the rear light path equal t o the fluorescence of the
source, or variations of the line voltage or frequency, are minimized. Dark current effects are also minimized as the photomultiplier sees only interrupted light, and the electronic circuit detects only the difference in light from the rear light path and the sample. The instrument is shown in Figure 28. No lenses are employed which has the advantage of
28.
Turner Model i
10 fluommeter.
permitting fluorescent applications which require excitation below 300 mp. For this purpose quarts cuvettes and an additional light source with excitation a t 245 mp, are available. The normal light source excites mainly a t 360 mp. The Turner Model 111 fluorometer is a self balancing modification of the Turner Model 1 1 0 fluorometer. The Model 111 is designed for use with recording equipment. The American Instrument Co., FluoraMieraphotometer Fig,,re 29, is a filter Ruorameter which incorporates the AMINCO photomultiplier 3,1icrophotometer as the detector of the radiation, The photomultiplier unit pro-
(Continued on page -4438)
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Chemical Instrumentation vides a dark-current bucking control, zero reference voltage for zeroing the meter, a continuous sensitivity adjustment over a range of 1000 to 1, and an outlet for recorder or oscilloscope attarhment. Built about the photomultiplier is the "saddle" whieh contains the optical partione of the fluorometer. The right side of the saddle contains prcvisions for using 6 different light sources, filter compartments for excitation and fluorescence filters, the sample holder and the photomultiplier tube. The left side p,.
.,.:.-
-,-.
---
I.'
Figure 29. American 1n.frument mtrophotometer.
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Chemicol Education
Co.,
fluoro.
of the saddle contains a regulated power supply offering constant voltage DC for the operation of a 4 w a t t General Eleetrio Co. Blacklite lamp peaking a t 360 mp or a General Electric Germicidal lamp peaking a t 255 mp. The other light sources offered include a GE Blue lamp with continuous emission from approximately 400 to 520 mp, a GE Green lamp covering the range from 520 t o 560 mp, and an %watt G E lamp covering the same range as the above bulbs but with increased energy. A tungsten source is also available. Offered with the instrument is a sample changer accessory of 20 euvette capacity which permits the rapid interchange of samples. Phosphoreseenee measurements may be made with a phosphorescence attachment whieh includes a special sample holder, a motor-driven rotary shutter, and a Dewar flask for operation a t liquid nitrogen temperatures. I n addition 8. bioloeirsl erowth attachtemperature and agitzted s t a aeleeted speed while fluorescence measurements are made. The fluorescence can be recorded to provide a permanent and continuous record of the biological growth. Provisions are also made for installat,ion of the fluoreseence unit a t a remote loastion should remote operation he desirable as, for example, if the sample is to bo kept in s. temperature-controlled roam. I n addition, the instrument may also be converted into 8. calorimeter or s.t,urbidimet,er.
Other instruments of American manufacture include the Jarrel-Ash Co., G-M Fluommeter. This instrument is shown in Figure 30. I t was originally designed as a reflectance flumameter for measuring the fluorescence of various ores, particularly uranium. Two 4 w a t t blacklight fluorescent lamps peaking a t 365 mp m e used for fluorescent excitation. A sample compartment ia d m weilable far measuring the fluorescence of liquids by measuring the amount of radiation coming directly from the exposed liquid surface.
Figure 30.
Jarrel-Alh, G-M Ruorometer.
Also manufactured, but perhaps less used a t the present time, are the Klett Fluorimeter manufactured by the Klett (Continued o n page .4440)
Chemical Instrumentation Manufacturing C . and the Model B Fluurarneter manufactured b,v Pfnlta and Uauer. Anrong foreign instruments whieh may he purchased through distributors in the United St,at,es, are the Hilger and Watts inst,ruments. The Hilger Spekker Fluorinreter, which is widely used in Europe and Great Britain, is based upon the Spckker Ahsorptinmeter. The fluororneter employs n photomult,iplier detector which is rmnerted into a null-balance elertronir riwuit. Kull balance is detected on a galvanometer. Conventional filtem are employed as well as a mercury excitation sowre. Also offered hy Hilger and Watts is their Fluorimeter, H 060. This high sensitivity instrument has a large cell compartment with facilities for accepting cells up to 10 cm in path length. The instrument employs a. photomultiplier detector, direct resd-out on B galvanometer sede, and a quartz envelope around the mercury an. lamp, permitting 1.T exritation.
Figure 31.
Hitochi, Ltd.. FPL-2 Ruomphotometer.
The Hitarhi, Ltd. F P G 2 Fluurophutometer has the conventiond optical component arrangement in whieh the photomultiplier detector is a t right angles t o the excitation beam. The instrument which is pictured in Figure 31, employs s. high-pressure mercury lamp as the excitation source, and conventional or interference filters for the selection of the excitation and fluorescent wavelengths. I t is made primarily for fluorescent radiation measurements in the visible region. The Jouan Co., Fluorometre Photoeleetrique, is a. high-sensitivity instrument with builtin compensation for light fluctuations. The instrument operates on the null-hdlance prinri~le, and the amount of fluorescence is measured by means of s. slidewire potentiometer. The unbalance is detected on an external galvanometer. A fluorescence attachment is also offered for the Eppendorf Photometer, manufactured by Netheler and Him, G.m.b.h. The attachment employs a. photomultiplier tube aa the receiver of the fluorescent radiation, uses a mercury lamp for excitation, and m e m e s the fluorescent radiation a t 45' t o the primary beam.
(Continued on page A 4 4 8
A440
/ Chemical Education
Chemical Instrumentation Addresses of Manufacturers
An nlphahet,iral listing of the addresses of the mnnufartnrers of these instruments folluws. Quite often, t,hese instruments are ulao sold by lahuratory supply houses. For thm0 instruments of foreign manufart,ure, the name of a li. S. dist,ributor is slsu inrluded where possible. Ameriran Instrument Cu., 80311 Georgia Ave., Silver Springs, Maryland Applied Physics Corp., 2724 Swl,ll Perk Rd., Monrovia, California Baird-Atomic, Int:., 33 llniversity Rd., Cambridge, Massachuset,ts 02138 Bausch & Lornb, Ine., 661011 Bauscl, St,., Ilwhester 2, Ii. Y. Beokman Instruments, Inr., 2Rllil Harbor Rlvd., Fullerton, California IYZD34 Coleman Instruments, Inr., 42 Madison Street,, Maywood, Illinois Farrand Optical Co., Inr., 4401 I h n x Blvd., New York, New York 10470 Hilger & W a l k , Ltd., 98 St. Pnnrl.ae Wag, Camden Rd., London, N. \V. 1, England. (I,.. S. Ilistributov, Engia 1Cquipment Co., 8135 Aust,in St,. Mort,on ( h v e Illinois 80053) Hit,xchi, Ltd., 4, l-rhome, Marunrnxhi, Chiyoda-ku, Tokyo, Japan. (Affiliated wit,h Perkin-Elmer Corp., Norwalk, Connertirut) Jamell-Ash Co., 590 Lincoln SL. \Irall,Imm, Messachusetts Jouan, 113 Bd St-Germain, Psris 6, France. (U. S. Dist,ributor, Brinkmsnn Instruments, Inc., Cantiague Rd., Westbury, N. Y.) Klett Mfg. Co., 179 East 87th St., S e w York 28, IT. Y. Set,heler & H i m , G.m.h.h., HamburyWellingshuettel, Germany. (11. S. l l s t,ribut,or, Brinkmann Instruments, Inc., Cantiague Rd., Westbury, New Ywk) Pwkin-Elmer Carp., 870 Main Avr., Forwalk, Connecticut Pfalti: & Bauer, 153 Waverly Plwe, S e w York 14, N. Y. Photovolt C o r p , 1115 Broadway, NPW York 10, N. Y. Schoeffel Instrument Co., 355 Hillsdde Ave., Hillsdale, New Jersey Terhnican Instruments Corp., ltesonreh Park, Chauncey, New York G. K. Turner, Assoc., 2.524 Pulgils Ave., Palo Alto, California Carl Zeiss, Inc., 444 Fift,h Ave., ITew York N. Y. 10018 The work leading t o this article was supported in part by Publia Health Service Research Grant GM-09792-02 from the Xationd Institutes of Health.
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