Instrumentation for fluorometry. Part One

Topics in..

. Chemical Instrumentation

Edited by

S. 2. LEWIN, New York

University, N e w York 3, N. Y.

These articles, most of which are to be contributed by guest authors, are intended to serve the readers of this JOURNAL by calling attention to new developments i n the theory, design, or availability of chemical laboratary instmmmtolion, oi. by presenting useful insights and ezplanationa of topics that are of practical importance to those who use, or teach the use of, modern instrumentation and instrumalal techniques.

XV. Instrumentation for Fluorometry. Part One Peter F. Lott, Deporfment of Chemistry, University of Missouri of Konsos City, Mo.

Closely related to absorption spectroscopy is the phenomenon of phatolunlinesrence. Absorption spectrophotometry in the ultraviolet and visible region of tllr spprtrum uses energy in the farm of liglbt to raise molecules from their ground electronic level to higher electronic states; the difference in the intensity of the inrident and transmitted radiation is measured and used to ident,ify or determine tlw concentration of the substance present iu the light path. Since those moleculev which have absorbed light are in a higher electronic state, they must lose their excess energy to return back t o the ground stat". T h i ~mav be done either throueh n o -

excited molecule may slsa return tu tlir ground state by emitting light. If this is done, the substance is said to sliuw photolsrninescace. Should there be a measurable time delay of greater than about 10-0 seconds between the absorption and re-emission of radiation, then this photoluminescence is known as phosphorescence. However, if there is no messursble time delay between the absorption and emission of radiation, it is known as fEuoreseace. Measurement uf t , l k emitted radiation is done by instntments which is11 into the general classifi~at,ion of fluorometers. By appropriate measurements of this emitted radiation, s quditative identification of the substance can be made, as well as its concentration determined. When applirahle, fluorametric methods of analysis show a sensitivity approaching that of neutron activation. Thus, in normal spectrophotometry with cells of 1 cm. light path, there we few substances which can be measured a t concentrations oi 0.1 part per million. I n contrast, using simple fluorometers, it is not difficult

routinely to measure concentrations two orders of magnitude lower, such as 1 part per billion of quinine sulfate or vitamin US (riboflavin), and these are substances which itre not exceptionally fluorescent. Considering that the luminescence of mu,terial has been observed for about three renturies and that Stokes (I) identified the process of fluorescence in 1852, fluorometrie methods of analysis have been relatively neglected until recently. Most probably, this came about became of a lack of sophist,ieated instrumentation. General surveys of fluorescence may be found in several books, particularly those of Udenfried (93, Radley and Grant (31, and the section by West in "Techniques of Organic Chemistry" (4). Current analytical developments in fluorometry have been compiled by White (6) in the recent review issues of "Analytical chemist^."

Peter F. Lott obtoined his Ph.D. degree in 1956 from the University of Connecticut. He received his B.S. 11 9491 ond M S . I1950) from St. Lowrence University. Dr. Lon has worked or o research chemist far DuPont ond the Pure Carbon Co. He war on Armciate Profenor ot the Missouri School of

Miner and Metollurgy, and a t St. John's University in Jomoico, N. Y., where he tought groduate and undergraduate ondyt~ r .Lon recently joined icd chemistry. the staff of the Univerrity of M i m v r i a t Kansas City to arrume charge of the ondytical program. His research interests

vibrational level of this excited state by transferring the excess energy to other molecules through collisions as well as by partitioning the excess energy t o other poasible modes of vibration or rotztion of the excited molecule. Thus, if internal conversion has not taken place, the molecule now is in the ground state of the excited level. Far fluorescence to take place, F I R S T SINGLET LEVEL A

FIRST T R ~ P L F T LEVEL

Fundamental Mechanism

A more exact repre~cntation of the h n s i t i o n s responsible for fluorescence or plmphorescence may be att,ained from the simplified energy diagram shown in Figure 1,in which the verticnl distitnces reoresent G e r m diflerences between state; The absorption of light energy by a molecule mises the molecule from a vibrational level in the ground state to one of many vibrat.ionsl levels in the excited electronic level. If the spin of the electrons in the excited state remains the same as in the ground state, the molecule is excited to what i~ designated spectroscopically as a singlet level. Should an alteration in t h e spin take place, the molecule would either he excited to, or able to transfer into a triplet level. During the process of excitation, a number of higher vibrational levels of the excited state are populated. Molecules in it higher vibrations1 level of an excited state quickly return to the lowest

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GROUND ST*TE

Figure 1. Energy trander mechanisms involved in fluorescence, phorphorercence, and obsorption.

the molecule returns to the original eleetronic level by the emission of radis&iotion. Since the molecule has lost some energy in Vol. 41, No. 5, May 1964

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the higher level prior to this transition, the emitted light is of lower energy-i.e., longer wavelength-than the original,

to transitions between singlet levels. These bansitiona from the triplet to the singlet level a n usudly responsible for phosphoreseenee. Since there are many vibrational levels associated with any electronic state, the spectra observed in all three transitions-absorption, fluorescence,

EXCITATION FILTER

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U V SOURCE

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SAMPLE

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11 1

FLUORESCENCE

FILTER

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9 Figure 2.

Block diogromof the mmponenlsof o typical Ruorometer.

incident light. Transitions between a triplet and a singlet level are designated aa "forbidden transitions," which means thst they are improbable and only occur after an appreciable time delay compared

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Chemical Education

and phosphorescenc-are diffuse bands. In all tramitions only t h s t energy corresponding to the energy difference hetween states ( A E = hv) is either emitted or absorbed.

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Henee. the instrumentation reouired to produce und meawrr f l ~ ~ o r r a c cm w ~~ n~ prises :L ligl,r ~ K Wa mrnns , uf pruduvirrg monuel~rwurrtivr d i n t i m of just 1l.e rigl.1 frequency to excite the sample t o the higher energy state, and a second monochromator unit which would allow only the emitted fluorescent light to pass on to a detector. This is indicated in Figure 2. As excitation of molecules to higher electronio levels takes place in the ultraviolet region, the u s u d light sources for fluorometry must be rich in ultraviolet light such as mercury b m p s or xenon arcs. The monochromatic redistian is obtained either by means of filters or monochromators. The detector can be a photocell or photomultiplier and the readout may appear directly on a.meter dial, a recorder, or an oscilloscope. I n "building bloc!# the equipment for fluorometry is quite similsr to that employed for spectrophotometry. Mast commercial equipment for fluorometry is designed by the manufacturer to use the same components such as filters, photh cells, photomultipliers, monochromators, amplifiers, etc., as he employs in his spectrophotameters or colorimetors. Because these basic units have been described and reviewed in this aeries by Lewin (6), such components will not be described in this article. The great sensitivity of fluorometry comes about because the sensitivity is proportionsl not only t o the concentration of the sample and the length of the light path, but also to the intensity of the light source and the sensitivity of ~~~

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Chemical Instrumentation

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the photometer. Even though murh more intense light sources are used in fluornnretry, t h e major increase in sensitivity comes ahout because the detwtor i~ directly measuring the fluorescent mdi;~tion, which can be amplified much mow readily than is the case for the d i f l e r ~ n w or ratio between the incident and tmnsmitted radiation that is invnlved ill speetmphotometry. I n a series of samples, the innst roncnntrat,ed sample shows the largest anmonl, of fluorescence, and for small eoneentrstions the fluorescent intensity is gen~rally dirertly proportional to the concentr2ltion. Thus quantitative measurements arc made in the conventional manner: for example, by preparing a calibration rorvc (intensity of fluorescencevs. conrent,r:~;LIion) or by the method of standard addition. Once the right wavelengths of t,he excitntion and fluorescent radiation are known, fill.er instruments serve quite well for fluorometry. Using the same light, source and detector, filter instruments usunlly dlow grertter sensitivity than t.hasc indruments employing monochromators, ne there is less light intensity available for excit.ation with monochromator instruments. The filter instruments arc generdy called fluorometers or fluon,phot,ometers if they employ a photomoltiplier detector. Instruments with continuously variable wavelengths for hot11 excitation and emission, that is, instrumentswith two monochromatars, are callctl spectrofluorophotometers or flurwescencc spectrometers. In using the fluorescence spectrometer, the following procedure is ronmonl,v followed. Both the excitation and fluorescence manochromators are varied until t,he detector shows a fluorescence peak. T h e n the fluorescence monochromator is s e t s t t,his wavelength and t,he excitation nionorhromator is allowed to sran thr sample. This excitation spectrum shows a t which wavelengths abmrption of liplif. orrum. In theory, i t should he identical to the absorption spectrum of the crmmpound. The excitation monoehromxtor is then set a t the wavelength of mnrin~um shsorpt,ion, and the fluorescence mono-

Wavolmgth,

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Figure 3. Curve C ir the ercitotion spectrum of 2.1.3-nophtho-(2,3-r) selenadiarole; Curve D i. the fluorescence spectrum of the some [From Cukor, P., Wdrcyk, J., and Lott, P. F., And. Chim. Ado, 30 119641. -in press.

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1 Chemical Education

Chemical Instrumentation chromator is allowed to scan the sample to record the fluorescence spectrum, and to determine a t whieh wavelength maximum fluorescence oceun. A typical excitation and fluorescence spectrum, measured with a Farrand spectrofluorometer, is shown in Figure 3. Because these instruments generally measure the characteristics of the sample without s. continuous comparison to a. reference standard, the instrument has the same major drawbacks whieh appear in attempting to record absorption spectra with a single beam instrument (6). Thus, if the light source is unstahle and varies in intensity, it e m cause the appearance of false peaks in the spectrum. Equally as serious as the fluctuations in the light source is the variation of the sensit,ivity of the phototube with regard t o wavelength, as shown in Figure 4. For example, in

Figure 4. Wavelength sensitivity curve for typical photocell.

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recording the fluorescence spectrum with a 1P28 photomultiplier tube if the sample aotually has a strong pettk a t 500 mp, and a less intense peak a t 400 mp, because the tube shows a 50% higher output for 400 mp radiation, the 500 mp peak could appear to be the weaker one. Consequently, in addition t o producing a distorted spectrum, the sensitivity of an analytical method could be greatly deoreased by working a t the wrong wavelength. This drawbaok is well known and procedures for correcting spectra. have been reported (7-10).

Fluorescence Spectrometers Certain laboratory fluorescence spectrometers h w e been designed to overcome these distortion effects so as to allow the corrected spectrum t o be attained directly. To date, only two commercial units are offered for this purpose, the Perkin-Elmer Model 195, s linear energy spectrofluoraphotometer and the G. K. Turner Assoeit~tes Model 210 "Speetro" abaolute spectrofluorometer. The PerkinElmer instrument is pictured in Figure 5 and a. schemrttic diagram of the optics is given in Figure 6. The Perkin-Elmer (Continued on page A334)

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Figure 5. Perkin-Elmer Model 1 9 5 Spectrophotofluorirneter.

RECORDER

r=l :mi: U)NOC"ROMATOR

SAMPLE

Figure 6. Block diagram of optical components of Perkin-Elmer Model 195.

Model 108 monocl~rornators with fused silica prisms are employed fur both the excitation and fluorescence mono p m I c d lwlween 2lJtkROll mp. 'r > exle!nd ~ h t range , uf the inatrunwr~tto 1100 m u , the inrwchangeable grating on the fluorescence monochrometor may be replaced with one blazed a t 750 mp. Photomultipliers available for use with the instrument are the 1P21 or 1P28 detectors or an RCA type 7102 photomultiplier for usein t h e I R

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SPHERICAL

region, if the sample shows fluorescence above approximately 800 mw. A number of accessories are available for the instrument. These include a. number of temperature baths, including a liquid nitrogen bath for phosphorescence studies, a rotary turret oell holder which holds three samples and permits the sequentid positioning of samples into the excitation beam, s. wavelength calibration unit, and a polwising accessory which permits either horizontal or vertical polarization of the excitation or fluoresoenee beam. Aho offered is an automatic buret and reagent kit for EDTA titrations of edcium using t h e fluorornetric indicator Calcein. Either a conventional xenon arc source, or a xenon-mercury arc source may be used for excitation. The latter offen advantages for quantitative analysis, should the sample show excitation a t the same wavelength as one of the mercury lines. The instrument may &I80 be used to measure the fluorescence of solids and, interestingly, for shout 525 can be eon-

ELLIPSOIDAL

Figure

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Figwe 10a.

Farrand SpectroRuorometer.

XENON ARC LAMP Optisol Schematic, Farrand Spectrofluorometer.

lob.

verted into a, spectrophotometer, by purchasing an accessory adaptor plate. The cost of the instrument ranges from $4600 for the basic unit to over $10,000 depending upon the accessories which are purchased. Developed a t about the same time as the Aminco-Bowman unit is the F s r r m d Optical Co., Spectrofluorometer. The instrument is pictured in Figure 10. I t also employs two grating monochromators

(22S650 mw), a xenon arc lamp source for excitation, and a photomultiplier detector. The fluorescent output can be memured on a recorder, a n oscilloscope, or a meter for direct read-out. Instrument accessories include a solid sample holder, temperature-controlled sample compartments, a wavelength calibration unit and a transmission attachment for me*

(Contiwed on nooe A350)

Chemical lnstrumentation

Figure 1la.

Baird-Atomic, Model SF-1 Fluore-

scence Spectrophotometer.

Figvre 11 b. Opticol Schematic, Baird-Atomic Model SF-1 Fluorescence Spectrophotometer.

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1' Chemical Education

"ring al,slrption or twnsmissim. UPpending on accessories purchased, tlw price may range between 8:3100 h, approximately $5500. Baird-Atomic, Inr. has recently introdured (,heir model 81.'-1 fluorescwwe spertroph# are employed. Selection of the wavelengtlm can be done either manually or through motor-driven cams. The instrument is capahle of measuring the fluorescence of either solids or liquids and samples may be introrluced either from t,he tap or side. The fluoresvenre is again measured with a photo~nultipliertube and the rend-our can he obtained on a recorder, meter dial, or osrilloscope. Accessories include :* miwo~itmpleholder. The cost of the insl,nlment is nppnximately ~ 6 0 0 0 . Except for the Perkin-Elmer m d r l l!l5 instrument, t h e fluorescence spertrometers disrussed so far have employed grating manuchromators. Prism monor l m n ~ a t o r s , although they sliow better r e a h t i o n of light, particularly in the low wavelength region, have been less used fur fluoresrenre monochromators became of their larger inlierent light loss. For eqnslly int,ense light smrees, grating m~n,,rl~rrrmatw inslrument,s uaunllv slww :L higher sensit,ivity. Two flootwmmcc speet,rometers enrploping prium monoclinmmtors lmve heen int,rodured, and the ronstrwtion uf hoth of these inntrnments is founded upon an opt,ir.al 1 , r n d ~for wnvenienm in inurmling and inl.erc:lt:~nge u i wniponents.

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The Schoeffel Instrument Co. also offers

Chemical h ~ f r ~ m e n f a f i o n a flu0re~,encespectrometer designed about

their Model QPM-30 quartz prism mono-

Figure 12.

Corl Zeirr, Model PMQ 1 I nuommeter with two monochromotorr.

The Zeiss fluorescence spectrometer shown in Figure 12 is built around the Zeiss model PMQ I1 spectrophotometer by the addition of a fluorescence attachment, a second monochromator, and s. light source. The instrument can be scanned either manually or automatically, the read-out, which is nan-linear in microns on the wwelength axis, can be measured on a recorder, oscilloscope, or meter. Interchange of units can be done quite readily; for example, the excitation monochrorniltor can be replaced with a filter unit far higher light intensity for excitation a t a fixed wavelength. A 450-W xenon are lamp is used as the light source in contrast to the common use of 150-W xenon sources with grating instruments. The cost of the instrument with two monochromators is approximately 513,000.

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Chemicol Education

Figure 13. Schoeffel Instrument Fluorometer with recorder.

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Chemical Instrumentation chromator. The instrument covers the wavelength region from 200 to 700 mp. Tu compensate for the non-linear dim persion of the prism, a cam movement is incorporated in the monochromator to produce a linear wavelength scale, capable of being estimated to 1 mp. The unit, pictured in Figure 13, shows the instrument connected for recorder read-out. The instrument is built around standard eomponenb, uses a photomultiplier detector and 1511-W xenon arc source for excitation. The cost of the illustrated unit is approximately $6,000. The I'erkin-Elmer Corporation offers as an attachment, a xenon light source and munochromator, to convert their Model 350 Spertrophotometer into a conventional fluorescence spectrometer. As the spectmphotometer covers the UV, visihle and near I R wavelength regions, the attachment would permit the measurement of fluorescence over a wide wavelength range. The approximate price of the conversion unit is $4500.

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Figure 14. The Beckrnon DK Univerloi Spedrophotometer.

A fluorescence accessory is also available for the Beckman DK Universal Spertrophotometer, Figure 14. The optical diagram of the instrument is shown in Figure 14a, and the fluorescence accessory in Figure 14b. As s h a m in the optical diagram, the instrument mey be operated on a double beam principle. The excitation light, which may be either a hydrogen, tungsten, xenon, or mercury arc lamp, is focused into a prism monochromator for selection of the appropriate excitation radistion. This monochromstic radiation is then modulated and split into two beams by a rotating beam splitter to allow the light to pass in turn through the reference and sample mat,erial, so as to permit a measurement of the ratio of the fluorescent intensities. The light beam is recombined by a. second rotating mirror, and focused into the entrance slit of a grating monochromator for the dispersion of the fluorescent radiation. The dispersed radiation is then converted into an electrical impulse by a photomultiplier detector or a lead sulfide cell, amplified and fed into a recorder. The instrument may also be used for conventional "single beam" fluorescence measuremenb. I t is also possible to employ a stationary beam splitter and to have the light from each beam impinge upon a separate detector.

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Figure 140.

attachments may range from $26,000 to $32,000.

Optical diagram of the Beckman DKUniverral Spectraphotometer.

The main advantage of sueh double beam operation in whieh there is a. continuous comparison against a reference sample would appear to be in quantitative fluorescent measurements a t a fixed wavelength. For recording fluorescence spectre. tho disadvantage of sueh a made of operation would appear to be the difficulty of finding a reference material whieh has a. constmt and known quantum efficiency throughout the desired wavelength region and whieh absorbs either all or a known fraction of the excitation light with negligible absorption of the fluorescent radiation. Bv usine additional attachments the initrumeit may be used for measuring phosphorescence spectra or polarieed~uoreseencespectra. ~ ~ upon the accessories purchased, the cost of the spectrophotnrneter with fluorescence

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CONTROLS0 JACKET

REFERENCE PIT" MIRROR

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Figure~ 14b. Optical ~ d i w o ~m of the Beckman d R"orescenceoccertory~

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The Bausch and Lamb Optical Co. has exhibited a prototype fluorescence attachment, Figure 15, far their Model 505 epeetrophatometer. The instrument employs a monochromator for the selection of excitation radiation. A formal introduotion of the accessory attachment will be made probably before June, 1964. The price will be approximately $2250.

Bibliography (1) STOKES, G. C., Phil. Tram. Roy. Soc. London A142,463 (1852). (2) UDENPRIEND,S., "Flu~re.escence h s a y in Biology and Medicine," Academic Press, New York, 1962.

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(3) RADLEY,J. A., AND GR~NT,J., "Fluorescence Analysis in Ultraviolet Light,'' 5th ed. Van Nostrand, Princeton, New Jersey, 1954. (4) WEST, W., "Technique of Organic Chemistry, Vol. I X , Chemical Applications of Spectroscopy," Interscienee Publiahen, New York, 1956. (5) WHITE,C. E., Anal. Chem., 34, 81R (1962); 32,47R (1960); 30, 729R (1958). (6) LEWIN, S. Z., J . C h a . Ed., 37, A197, A271, A341, A401, A455, A507, A637, A705 (1960). (7) SPRINCE,H., A N D ROWLEY,G. R., Science 125, 28 (19.57). D. M., Science 125, 1242 (8) HERCULES, (1957). (9) WHITE,C. E., Ho, W.AND WEIMER, E. Q., Anal. Chem. 32, 438 (1960). (10) A R G A ~ R R., J., AND WHITE,C. E., Anal. Chem. 36, 368 (1964). (Part Two, conclusion of "Instrumentation Fluorometry," will appear in the June

ieaue.)

Instrumentation for fluorometry. Part One

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