ANALYTICAL CHEMISTRY
1130 be reported elsewhere. -411 the thiophene derivatives were considered t o have a purity of at least 99%. Samples of 2,3,5-trimethylthiopheneand 2,3,4,5-tetramethylthiophene were obtained from F. F. Nord of Fordhani University. F. G. Bordwell of Sorthivestern University supplied samples of thiacyclohexane and 2-methylthiacyclopentane. The 2- and 3-tert-butylthiophene were obtained by precision distillation of a so-called “pure” 2-tert-butylthiophene. The “2tert-butylthiophene,” “2-tert-amylthiophene,” and “2,5-di-tertbutylthiophene” used originally ( 3 ) were found to be mixtures, after the disclosure of Appleby et al. ( 2 ) that alkylation of thiophene yielded mistures of isomers. Most of the compounds of Table I1 were Eastman reagent grade products.
ACKNOWLEDGMENT
The author wishes to espress his appreciation to F. P. Richter for advice and encouragement in the course of this work. LITERATURE C I T E n
(1) .4nderson, H. D., and Hammick, D. L., J . Chem. SOC., 1950,
1089. (2) .4ppleby, W. G., et nl., J . -41)~.C h e m SOC.,69, 1552 (1950). (3) Hartough, H. D., A h . 4 ~CHEY., . 20, 860 (1948). (4) Hartough, H. D., unpublished data. (5) Keswani, R., and Freiser, H., J . -4m.Chem. SOC.,71, 218 (1949). (6) Sedgwick, X. V., “The Chemical Elements and Their Compounds,” pp. 450-2, London, Oxford University Press, 1950. RECEIYED January 13, 1951.
Improved Photoelectric Fluorometer W. C. ALFORD AND J. H. DANIEL Experimental Biology and Medicine Institute, National Institutes of Health, Bethesda 14. Md. Rapid expansion of the field of fluorometric analysis has emphasized the need for a photoelectric fluorometer which displays the sensitivity, stability, and flexibility required for quantitative measurements. .4s no commercially available instrument meets these requirements fully, an instrument was designed. It employs vacuum-type phototubes and an electrometer tube in a circuit which permits measurements of fluorescence, by a null-point method, over an un-
T
H E use of fluorescent reagents in analytical chemistrj- has become increasingly common during the past seventeen years. They were used first as simple qualitative reagents for various inorganic and biological materials, but in recent years many precise quantitative methods have been developed for a wide variety of substances. S o attempt is made here to review this field, as t v o comprehensive reviews of fluorometric analysis have been published recently ( 7 , 8). Instrumentation has tended t o lag behind the development of analytical methods, so that often the worker has been handicapped by lack of an instrument that would meet his needs. Visual comparison methods have been used ( 3 ) , and various, more or less successful, attempts have been made to adapt spectrophotometers to fluorometric measurements ( 4 ) . At present, there are a t least ten or twelve instruments on the market which have been designed for photofluorometric work, and several others have been described ( 7 , 8 ) . Practical experience has shown, honever, that most of these suffer from one or more of the following objections : lack of stability, inadequate sensitivity, insufficient flexibility to meet widely varying conditions, or excessive cost. The fluorometer dekrihed below overcomes these difficulties to a large extent. DESCRIPTION OF INSTRUMENT
The need for sensitivity and stability dictates the use of a vacuum-type phototube as the light-receiving element. The phototube emissive surface is selected from commercially available types to give greatest response in the spectral region in which fluorescence is expected. For wave lengths above 5000 rl., a n RCA 919 tube (spectral response S-1) was used; for wave lengths below 5000 A,, a Cetron-CE-99 (class Q, spectral response S4, Continental Electric Co., Geneva, Ill.) was used. The extremely high impedance and comparative independence of current on voltage of the phototube make it ideally suited to work into a n electrometer-tube current amplifier with its as-
usually wide range of intensit). The basic fluorometer is powdered by dry cell batteries and is characterized by a very low power consumption, which tends to promote excellent stability. The circuit is such that full advantage m,ay be taken of the ultimate sensitivity of the phototube surface. This fluorometer should permit more accurate analyses by existing methods and facilitate research in the development of new methods.
wciated high input impedance ( 2 , 5 ) . This type of rlectiicnl amplification was chosen in preference to an electron multiplier because it appears to be more flexible, reliable, and simple, while taking full advantage of the ultimate Sensitivity of the phototube. The Victoreen electrometer tube VS-41.4 xith its Ion filament curient requirement is well suited to the circuit shown in Figuie 1. This is essentially the DuBridge-Brown tvpe circuit ( 2 ) . The functioning of the circuit may be explained briefly as followe: With a 10-ma. current through filament F , no light incident on phototube T , and the vaiiable taps of potentiometers PI and Pz set a t ground potential, Pa is adjusted to give no deflection of the galvanometer. Current induced by light falling on the phototube then will produce a voltage change (the IR drop across the high resistance connected to S,) on grid G,, which causes the electron currents to plate P and grid GI to change in opposite directions, thus giving a deflection of the galvanometer. The galvanometer may be returned to zero by applying an equal but opposite voltage change to G? by means of P, (or PI). Thus the setting of Pz becomes a measure of the light intensity on the phototube, with the advantages associated a i t h a null point method of measurement. The function of thr remaining components is described below. STRUCTURAL DETAILS
The instrument described here, and shown in Figure 2, was assembled from equipment that was immediately at hand. Thus, its design is not necessarily that which would provide maximum convenience to the operator. HoTvever, it has given trouble-free and entirely satisfactory service for over two years, during which time only one new set of 1.5-volt batteries has been installed. The various parts of the fluorometer, esclusive of the 1.5volt batteries, E l , E2, and. EB, the ultraviolet source, and the galvanometer are housed in a light-tight wcoden bos (13 X 13 X 6 inches) n-hich is metal-lined to furnish the necessary electrostatic shielding of the grid lead, G , , of the electrometer tube. The box is painted inside and out with a nonfluorescent,,
V O L U M E 23, NO. 8, A U G U S T 1 9 5 1
1131 sketch is largely self-explanatory but it is desirable t o keep the light path as short as possible in order to obtain maximum sensitivity-for example, 'the phototube should be not more than 1 inch from t,he cuvette.
The filter holder is made of black sheet brass with si 1-inch-square window in the center. It is spring-loaded, as shown, to hold the filter8 snugly and a t the same time permit emy insertion of any desired filter combination. Felt and metal washers under the springs serve to pre20,000 7,500 vent leakage of light. A shutter is not needed between the cuvette and phototube, as. such tubes are not subject to fatigue and the galvanometer is protected against sudden surges of current by microswitch Sa. Thus, the beam of ultraviolet light falls on the surface of the fluorescent solution from above. The resultant fluorescent light, picked up a t right angles to the path af the exciting beam, passes through an appropriate filter system and then strikes the active surface of the phototuhe. Figure 1. Circuit Diaeram of Photoelectric Fluorometer T h e basic filter system for most work consists of a. Corning glass filter Eo. 5874, mounted in the lamp housing to isolate t.he 3657 A. mercury line, and a Corning filter KO.3060 between the cuvette and phototube to stop scattered ultraviolet radiation. Additional filters are installed 8.6 needed to isolate the wave length of fluorescent light that is to he measured. The phototube must be shielded from the light emitted by the filament of the electrometer tube. The galvanometer used for most work is a small Leeds & Northrup portable pointer type with a sensitivity of 1 micraampere per millimeter scale division. A more sensitive galvanometer with variable sensitivity (Figure 1)is advantageous when measuring very low intensity fluorescence. In such 6888, the galvanometer should preferably be of the high resistance type (500 ohms or more) rather than the low resistance t ". w e (25 ohm8 or less) as is used M nth thermocouples. OPERA'ITONAL PROCEDURE
Relative measurements
-
-..-. ...
a null paint method by mea
Figure 2.
Photoelectric Fluorometer
ale units t.he volt-
COLEMAN U.V. LAMP
( W D U-II-M
~~
~.
flat black paint. A separate wooden box, fitted with a p probriate binding posts, holds batteries E l , &, and Ea. As may be seen in Figure 2, all operating controls are mounted on the metal top of the box. The actual layout of these is unimportant,, except t h a t the most frequently used controls ( P , , Pa, and P a )should be in a convenient location. The source of ultraviolet radiation 18 a Coleman Model U-11M high pressure mercury vapor lamp (AH-4) with constant voltage transformer. I n keeping with its design, it is mounted on the top of the instrument at the right rear corner. It is held in place by three pins in the lamp housing, which fit into holes bored in the top of the case. Leakage of light is prevented by a black felt gasket cemented to the under side of t.he lamp housing. A flat strip of sheet metal (4 X 9 inches) with a hole 1.5 inches square 3 inches from one end Serve8 as a sliding shutter between the lamp and the cuvette compartment. Guide pins in the top of the case and slots in the metal strip assure accurate positioning of the shutter. The cuvette compartment is located immediately beneath the ultraviolet lamp and measures 4 X 6 inches. It is constructed of wood and Masonite and is completely walled off from the rest of the osse, so that no mattered ultraviolet light can reach the phototube. Samples a t r e inserted through 8 light-tight side door and are held by a two-place cuvette holder (Coleman No. 11-103, Figure 2) which slides on parallel brass rods mounted on a wooden platform of appropriate height. A slat in the base of t.he cuvette holder and a pin in one of the brass rods permit accurate placement of either cuvette in the light path. Coleman No. 11-121 rectangular fused glass cuvettes (25 ml.) are used.
A cross-sectional diagram of the optical system, a8 seen from the door of the cuvette compartment, is shown in Figure 3. This
- _.. ... . -...-
i I I
RII J
.
C
b 4
FILTER
WOODE RI
nrL
Figure 3. Diagram of Optical System of Fluorometer
ANALYTICAL CHEMISTRY
1132 age required t o balance that produced across a high resistance by the phototube current. This phototube current is, of course, strictly proportional to the intensity of the fluorescent light. The instrument is calibrated in the usual manner-it., the fluorescence of a graduated series of known standard solutions is measured to determine the relationship beta een fluorescent intensity and concentiation. Measurement of the fluorescence of a similarly prepared unknown sample then gives directly its concentration. The ultraviolet lamp is turned on, the filament circuit is closed by means of SI,and meter -11,is adjusted to 10 ma. with potentiomete'r P,. After a warm-up period of 10 to 15 minutes all other swit,ches (except microsv-itch 86) are closed. The shut,ter between the ultraviolet lamp and the instrument is closed (giviug "dark conditions"), and with PI and Pp set a t ground potential (zero scale), P, is adjusted until no galvanometer deflection is noted \Then S6 is closed. Final adjustment is made with Ss set for maximum galvanometer response (position 3). Pa is left in this "balanced" position and the instrument is ready for use. A reagent blank, a standard solution, and an unknown are prepared simultaneously in such manner that they are identical except for concentration of the fluorescent substance. After the appropriat,e st,anding period, the reagent blank and st.andard solution are placed in the cuvette holder, and the blank is poritioned in front of the phototube with the fluorometer set a t medium sensitivity-Le., selector switch Sd set on the 1On-ohm resistor, and S3 in the No. 1 position. The galvanometer microswitch, 86, is closed, and any deflection of the galvanometer ir balanced out by adjustment of P I . PI is'left in this position and the standard solution is moved into place. The linear potentis is ometer, P,, is set a t any desired scale reading, say 100, S closed, and the galvanometer is nulled by adjustment of Pa. By this procedure, the instrument has been set so that, on P 2 , the blank reads zero, and the standard solution reads 100. The standard is replaced by the unknown, and the galvanometer is zeroed by adjustment of P,. The scale reading of P? is now a measure of the concentration of the unknown relative to that of the standard. If', for example, the unknown gives a reading of 70 compared to a standard which reads 100, the unknown contains TOY0 as much fluorescent material as the standard.
Table 1. Test of Fluorometer with AluminumPontochrome Blue Black R. Fluorescence .lIuiiinnm, 7/25 Mi, 0 2.0 4.0 6.0 8.0 10.0
~
Hun 1 0 20 39 60 80 100
Scale ~Readings ~ _
_ _ _ _ Run 2 0 21 40 60 81 100
This simple procedure is applicable when it has been found that fluorescent intensit!. is proportional to concentration (linear calibration curve). If this is not the case, concentration may he determined by reference to appropriate calibration curves. However, because of such variables as temperature; battery strength, ultraviolet light intensity, fxtc., it is necessary to calibrate the instrument with solutions of known concentration immediately before measuring the fluorescence of an unknown. This technique is required for fluorometric methods in general. Thc cuvettes must be carefully matched, especially when measuring fluorescence of low intensity. \Then perfect matching cannot lie arhieved, the same cuvette must be used for all solutions. The arbitrary instrument settings of S3 and S4 given in the above procedure are applioablr only when the fluorescent iiitensity is in a cert.ain rather narrow range, and are changed to meet varying conditions of concentration and fluorescent intensity. As a given light intensity is measured by balancing the potential drop due to a constant current through the high resistance connected to S4,the response of the fluorometer to any given light signal is proportional t,o the magnitude of this resistance. It is thus
necessary to select the appropriate resistor and' galvanometer sensitivity to give the desired response. I n this respect the instrument offers unusual flexibility. Consideration of the circuit diagram (Figure I ) will show that galvanometer response to a given photo current-Le., sensitivity-can be varied t)v a factor of approximately 10,000. This represent5 the difference tietiieen the settings: 8 4 on the lO*-ohm resistoi with Sjin thr KO. 1 position and Saon the 101"-ohm resistor with S,in the Xo. 3 position. Such flexibility is desirable because the fluorescent intensity of different substances varies over a very a i d e range and also because it allows the operator considerable latitude in selecting a suitable concentration range.
Table 11.
0.0 0 .5 1.0 1.5 2.0 2.5 3.0 3,3 4.0 4.5 5.0
Relation of Zirconium Concentration to Scale Readings
0 8 19 29 39 51 60 71 80 92 1.00
n n i.0
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10 0
0 9 20 31 40 48 60 70 79 90 100
n o 2 0 4 0 6.0 8 0 10 0 12 0 14 0 16.0 18 0 20 0
0 10 19 30 38 49 61 71 80 90 100
0 5 10 1.5 20 25 30
35 40 45 30
0 0 0 0 0 0 0 0 0 0 0
0 11 21 29 40 51 61 70 79 89 100
"Curve" is used here t o represent a set of calibration d a t a * ,\Iicrograms per 25 ml. of solution.
Flexibility is also provided by the resistors, which may be paralleled across P1 by means of SI to reduce the potential across Pz,and thus permit setting of the instrument to give any desired scale reading (100 in the example given above) on any appropriate fluorescent standard. Each successive position on S3 (1 to 4) approximately doubles the scale reading of a given fluorescent solution. The need for such a system is obvious when it is considered that the high resistors, &, vary from each other by factors of 10, and that, with S3in any given position, the potential across Pz can be varied only by a factor of roughly 3 by adjustment of P,. This ability to set the instrument to give a large scale reading for any given fluorescence serves to increase the percentage accuracy of scale readings and is a convenience to the operator in establishing calibration curves. REPRESENTATIVE DATA
The completed instrument was first tested by using it for the determination of aluminum by the Pontochrome Blue Black R method under the optimum condit,ions given by Weissler and White (6). Typical calibration data are shown in Table I. The instrument was also used in the development of a new fluorometric method for zirconium ( I ) . Table I1 gives the results obtained on a series of calibration runs covering different ranges of zirconium concentration. This table illustrates one of the desirable features of the instrument-that for any usable concentration range (of fluorescent material) the percentage accuracy of a particular scale reading is constant. For example, the percentage accuracy in the determination of 10 micrograms of zirconium dioxide (scale reading 21 of curve 4)is the sanie as that of the l-microgram sample (scale reading 19, curve 1). It is also apparent that the absolute error is approximately constant over the entire range of scale readings, although percentage accuracy is best at the higher scale readings. Measurements were also made on fluorescein and quinine sulfate solutions, and in both cases scale readings were a linear function of concentration. In order to demonstrate the sensitivity and range of the fluorometer, solutions of quinine sulfate were prepared, in 0.1 S sulfuric acid, to contain, respectively, 20, 10, 1, 0.1, 0.01, and
V O L U M E 23, NO. 8, A U G U S T 1 9 5 1
A Wratten '2-5 grlatin filter solut,ions was used (between to isolate cuvette the blue and fluorescent phototube),light Twentyof the
0.001 microgram of the salt per milliliter.
five milliliters of each solution Rere measured in the fluorometer, using 25 nil. of 0.1 LV sulfuric acid as a reagent blank in each case. The galvanometer ueed had a sensitivity of 0.05 microampere per millimeter scale division. Results are shown in Tiihle 111. Inst,runiental settings arc givrln to illustrate the various combinations which may be empl(i>-edto permit mrasurement of a given fluorescent solution.
1133
complpte at)sence of "drift" untler "(iark" coll~~itiona at est sensitivity the need for rehalancing srldom occurs. The data given in Tables 1 and 11 were oht'ainrd with no rebalancing of the circuit after the initial settings had been made. T h a t stability is to expected is eviderlt \vheIl it is that the electrollle t r r tube is operated with grid re6iston c*onsiderably smallrr than those which can be used with this t u l w The manufacturers state thut the tuhe itself has an rxtrrnal Irttkage resistance of l O I 5 ohms anti that resistances up to lo'? ohms can he successfully used in cii,ruits of the type describrd.
DlSCUSSIOS
~____ Table 111.
Awmiing a judicious choice of light filters, and the absence of I d a g e curi,eiits (discussed below), the accuracy of measurements tlrpendP primarily on t,he linearity of potentiometer P 2 (General Radio Type, 301-.4 is satisfactory) and the stability of the ultraviolet source. St,ability of the electronic amplifier becomes a fartor only if a vcr?- sensitive galvanometer is used for measuring estrrmely ivrak fluorei;crnce. This type of instability can he minin i i z d as follows:
-4s thcs filament currriit is varied betn-ren 9 and 13 init. (rebalancing with P, it' the galvanometer goes off-scale), the galvanometrr deflection will pass through a masimum (or niinimum). The filimient current is thrii yet a t the value giving this masinium -i.e., where the rate of change of deflection a-ith filament currrnt is zero-and the ealvanometer zeroed with PA. If this filament current is not in thr larige 9.5 to 11.0 ma., values of I?? in the range 400 to 600 ohms, and, if necesxar~.,of Rl iIi the range 150 to 400 ohnii; can he ti,ird until the deflection maslnium (or minimum) t1oc.s occur :it a filanic,nt current in the neighhorhood of thr r;ttcd value of 10 ma. \\lth the tuhe used, arid the \-slues of I?, ;ml I?? .shown iii Figure 1 , thiv "compensated" condition x a s obtained :it a filanient current of 10.0 ma. Howcvrr, for t h r rangr of fluoresctxnt intensities normally eiicoun\vi13 uscd "uricoml~rn trrrti, thr fuoi~omett~r. t o :300 ohlils ;11itl R, ('(IUaI to 450 0 l i 1 i i ~ ( ~~Iectronit~tcr u w b y the ni o thr galvunonirtcr with Should it lie found imi /',) :I tiiff'rrriit value of I?, .-\i ii safeguard against a grounticd guaid ring i.- provided. turns of fine, barc wire wound around 1 with .4quadag to ensurr good electrical T h r surface of the glass envelope lwtweeii ode is kept c~lranarid free of dirt and gt'raw.
\\.it11 such precautioiiP, Irialage currrlits have causcd trouble oiilv under extremelv humid conditions. and it has not heen n w rspary to use a dericcant in the fluorometer case undrr normal ~ ~leakagr~ ~ arr, evidrncrcl l ~ \,.eather corlditiolls, ~ l)y (1) a decrravd rcyonsc. of the galvanometer to changes in P I 01' P 2 for the higher rtssistances of S,, and ( 2 ) for a given light signalj a lack of proportionality of galvanometrr respoiwe 01' nulliiig voltage to the VHIUP of the rrsistor connrctrd to S4-i.e., thi, respoiis~or nulling voltage, for the 10lO-ohniresistor will IIC, less th:in trll tinips that for thc 109-ohm i.esistor. If such symptoms iiidirat(' the iiecd for
desiccant, a nonliqupfyiny type (such than cslciuln chloride or pklosphorus Iwiitoxidt~)should hr U P P ~ . Stability in a n instrument of this kind is an easily observed quality, hut one that is not readily demonstrated with experinimtal data. However, a t any low or medium sensitivity setting thra insti.ument descrikd above is characterized by an almost g(,~ or calcium
:I
Sensitivity and Range of Fluorotneter il'or iiiiinine sulfate solritionzi
Qiiinine Sulfate, -, 2.5111. 0.023 0.2.5 2 .5 2 .j 2 .i 23.0 25 0 z.50. 0 .loo. 0
Grid Resistor, Ohnib.
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of
s4
10'0
10'0 10-0
Position of X I 1 1
in Series w i t h P? 100 100 I00 100
3 8 3
3
100
6 22
1
100
,123
4
4
s
10 y 109
73
I'ii-ition of ,S6 B ,3 :i
10'
108 108 108
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,
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Scale Reading 7 ii3 59
80 ( a p p r o y . ) 100 0 3 $1 0 46
___
-____
Viidrr normal working conditions, any lavk of reproducihility of iiir:i,~ui'c'inent,s can be attributed mostly to the ultraviolet soui'rr'. The intensity of the rxriting radiation varies with line voltage and the constant voltage regulator? urually employed do not provide perfect control. Chaiigiiig Iiattery strength over any ,shoi.t period of tinir. is negligible and, in fai(.t. thr oprrating life of thr batteries used in this vircuit m:t!. I)c expected to approach tli& shelf life.
SU313IARY
Aine\v photofluoronwter, rmployiiig a vacuum-type phototulle arid a \-ictoreeen electrometer tube, has been developed. Aleasuwments are made by a null point nitlthod in n.hich the fluorescriice of :in unknon-n solutio11 is rompared to that of a standard solution. The design of the instrument offer.+dPfinite advantages y . convrnience of of st:il)ilit:., sensitivity, flrsil)ility, a c c u ~ ~ a r and operation ovrr coninierrially availulllr instruments. The instrument :IF dewrihed may be rpadily cotiutructrd at a matrrial cost roiisiderahly helon that of any conimrrcially available fluoromrtrr. Current price lists iiidirate a total parts cost of slightly over $100, esclu>iveof ultmviolrt lamp and galvanometer. L I l E R 4 T U R E CITED
~ IT.~C., Shapil.0, ~ ~IJ.,2nd TThite,
(1) -Iliord,
,
c. E..
AS-AL.C H E Y . ,
23, 1149 (1951).
( 2 ) DuBridge, L. (;3)
.L,and Brown, H., Rei. Sci. I n s t r u n w ~ t s ,4, 6 3 2 (1933). Fletcher, 11,H,, hit^, c, E,, and Sheftel, 11, s,, rSD, CHEY.,-%~YAL.ED., 18, 179 (1946).
(4)Ihid., p. 206. (5) Gabus, G. H., and Pool, 11. L.,Re?. S c i . I r i s f r f m e n t s , 8, 196
(1937). (6) Weissler, A , and V-hite, C. E., ISD. Est. CHEW 18, 530 (1946). (7) Xhite, C . E., A12-I.. CHEJI.,21, 104 (1949). (8) Ibi'd.. 22, 69 (1950).
R
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