of acetophenone and benzaldchydc introduced an error of 7.7%. The presence of acetophenone causes higher deviations than does benzophenone. Since the methyl carbonyl group has weak absorption closr to the 2.21-micron band, the overlapping could introduce error in locating the base line. The results were not imljroved when the ratio of intensities betn-ern 2.210 and 2.185 mirrons n as used for measuring the absorbances ( 1 4 ) . DETERMINATION OF NITROBENZALDEHYDES
The near-infrarotl y)ectra of nitro1)c~iizaldeIiydrsare very siniihr. Honv\.cr, the oonibination hand. of o-nitroI)cmzaldeliytlc was a t 2.176 microns, n hile for m- and p-nitrobcIizaldeh\-dc it \vas a t 2.203 microns. Thih tliffcrcwe in position makw possiblc the detwmination of a nii.\tiir.e of (ither the ortho and pnra isomers or the ortho l hulimpti\-itics and nwt:i isomers. ‘
at each wave length were measured (Table 111), and from the measured absorbances the concentration of each component mas calculated by simultaneous equations. ,2 good agreement b e t m e n the actual and calculated amount was obtained (Table IV) . The spectra of the nieta and the para isomers are almost identical. Only the weak bands a t 2.474 and 2.266 microns seemed to he characteristic of the nieta and t h r para isomer. respectively. The authors tried to utilize these two bands for dcterniining these two isomers, but the deviations \$ere too large to be of practical significance.
ACKNOWLEDGMENT
The authors thank C. E. Irdand for helpful suggestions. and Leon Mandcll for the XhlR nnalj-sis of tlcwterohrnzaldehyde.
LITERATURE CITED
(1) Crisler, It. O., Burrill, A. hl.. .4x.41,. CHEW31,2055 (1959).
(2) Fowler, I,., Mitchell, R. S.,Ihid., 27,1688 (1955). (3) Goddu, R. F., Ibid., 29,1790 ( 1 9 5 i ) . (4) Ibid., 30, 2009 (1958). (5) Goddu, R. F., Delker, I). .4.,Zbid., 30,2013 (1058). ( G ) Gorden, B. E., mopat, F., Jr., 13urham, H. D., Jones, L. C., J r . , Ibitl., 23, 1754 (1951). ( 7 ) Holman. R. T., Edmontlmn. 1’. 1i.. Ibid., 28, 1533 (lobe). ( 8 ) Holman, R. T., Sickell, C‘., I’rivc>tt, 0 . S.,lcdmondson, 1’. It., J . i l t r 1 . Oil Chemists’ SOC.35, 422 (1958). (9) Kaye, W., Spectrochirn. .4cfn 6, 281 (1054). (10) Pinchas, s., ASAL. C m X . 27, 2 (1955). ( I 1 ) Ibid., 29, 334 (195i). (12) Whekel, K., Roberson, T\-. E , Krell, hf. \V.,Ibad , 30, 1954 (1958). (13) Wiberg, K. \T .. J 4m. C ~ P , , ,Yo( I 76,5372 (19a4). (14) Willard, 11. H., Merritt, Jr , 1, I, , Dean. J. A.. “Instrumental hfpthotls of Analysis,” p, 14i, 3rd ed., J7:m SOPtrand, l’rinreton, S. J., 1958. RECEIVED for review Frbrnary 18. 1!)GO. Acceptrd June 3, 1960 ~
Determination of Lanthanum, Cerium, Praseodymium, and Neodymium as Maior Components by X-Ray Emission Spectroscopy DAVID R. MANEVAL and HAROLD L. LOVELL Department of Mineral Preparation, College o f Mineral Industries, Pennsylvania State University, University Park, Pa.
b The advantages of the fused disk technique in x-ray emission spectroscopy suggest its application to analyses of the rare earths. Improvements in accuracy, convenience, speed, and economy result from this process. Standard disks for cerium, lanthanum, praseodymium, and neodymium are prepared by fusion with sodium borate and are employed for daily calibration. The analyses of four samples of widely varying composition show a With mean error of less than 5%. these four elements, interference from other rare earth oxides is generally insignificant.
N
o
m i p m TECHNIQUE^ is available for the mrt-chemical dctcrniination of Irznthanuni, prascotlyiiiium, or niwdymium Spectrographic :tnalysis of t h r rare carths is ni:ide diffic lilt by t h r compleuity of tclc hniquc antl t h r lot ation of cyanogen band. i n the region of tlic rarc earth RU Iinc’-. -\]though :ipplictl in our laboratoriei t o minerals or rarc carth coniplc\cs (of n idely
varying nature), the use of a constant matrix permits applications t o most any type of rare earth-containing substance with lanthanum, cerium, praseodymium, or neodymium as a major component). I n very unusual circumstances a preliminary oxalatc separation ma>- be helpful to reniovc interferences or achieve concentration. The procedure set forth hert‘ >in can be used for the determination of lant,hanum, ccrium, praseodymium, anti ncodymium with sufficient accuracj. for most separation or caontrol purposes. The three simpk Qrocc.dnl(,s--u-eighing, disk preparation, antl intensity tletcrniination-can be readily accomplishcd by a technician. Also, spectrochemical analysrs have heen chnractcristically npplicd to low concrntration ranges. ‘The errors rrlatcd to self-ahsorption, excitation vwiablcs, hackground effects:. and densit’ometry of intensc lines tend t’o hc morc prwalent at high concentrations arid arc additive to the uncontrollablc variables charac,teristic of photographic emission spectroscopy.
Q u a n t i t h w x-ray fluorescencc prowdures art’ widely used (9, 12, 15) foi, the an:tIysis of minerals because of thi. simplicity of standardizat,ion, specvl of analysis (clapsed time). infrcqwiit interfcwnw between components. and :tccurnry at’tainablc a t high concwitrations. Early in the Manhattan District Project, Clarke (3) investigatcd the possibilities of x-ray analysis of thc: rare earths, while in 1955 Dunn ( 5 ) studied the quantitative aspects ~ n i ploying :tn internal standard but without cmrections rclated to particii1:ir systems. Romans (11) used a comparison-type proccdurc based on synthctic 1nixturt.s. Salmon and Blacklrdgc (12) have rcportcstl a semiqumtitn t i w method utilizing otnpirical caorrections. Rewntly Lytlc, h t s f o r d , a n d Hvllw (7) as w 1 1 as Lytlc and H w l y (8) deseribcd :L potvtler-t>ype x-ray fluor(+ cence analysis of bastnaesite :tnd high purity rarc cnrth oxides. Standartiization rtwiltcd from s y n t h h c ~niiililw packed into a plastic holder. ’Tho authors found several disadv:int:igcs VOL. 32, NO. 10, SEPTEMBER 1960
1289
to this approach. The costliiiwb of pure rare earth oxides used as standards, the repeated packing of thc powders for making the calibration curves, and the danger of contamination of thc powdered sample are major drawback hiatrix effects can bc large in packed powders requiring uniformly small partid e size to obtain reproducible s-raj intensities. Such small particles ran be only partially attninerl by tcdiouq grinding and screcning. Claisse (1, 2 ) introduced a fusion technique in \\ hich the sample is dissolved in a flux of light dements, suclr as sodium boratv ghss. In addition to Claisse, Sherrnnn ( I S , 14) among others lias dealt with the theoretical considerations of dilution techniques. E\cellent results have been reported for boric acid-lithium carbonate fusions (6, 16) in emission spectroscopy. The fused standards may be retained for reme with no changc in character. The possibility of yuantitativc, analysis of the. rare cwths l ) j x-ray fluorcs-
Table 1.
.inalytical Element 1,nnthaniim
cencv using the "borax disk" technique was invwtigated. A proportional relation mists between measured-line intensity antl weight fraction. Deviations from the proportionality h a w been classificd :wording to t h e e principal causw : (1) absorption and enhancement efftd,s, ( 2 ) heberogeneity effects, and (3) instability, including drifts and fluctuations in the equipment. Tlic, h a s disk trdinique niiniinizes the first antl sc.cond effects and t'he repetition OT stanclardization during each day's operation al1rviatc.s thc third by cva1u:itiug slope a r i d thc practical linearity of tlic working curves. Thc rcinaiiiing signifi~antctifficdty (in raw ) results froin inadrquatv instruintwtal rcwlution. PROCEDURE
Choice of Analytical Lines. T h e conimonly e n i p l o y d analyzing crystal, lithium fluorictc., is hasically illadeqrr:itti foi, imolviiig lines of s e ~ t ' r a l
Elements Possibly Interfering with Determination of Lanthanum, Cerium, Praseodymium, and Neodymium ( I 0)
l'eak hiiglc.. IkgreeR 28 $2 85 Lnl
Ba I'whible _ _ _ _ Interference line Breadth, Angle, Relative Degrees 28 degrees 'LO Element intensity 1 00 84.41 Hf I,@ 30
0.04
0.81
0 .94
83 57
1211
LB5
:30
8 3 23 82 43 81.60 80 85 80.86 80.25 78,04 77.75 69. 74 09.57
S d LL. Hf I&:!
30 60
'hi
'I'h
Idyl 12; 1
6!),47 68 4:i 67.37 67.23 6 6 . 48'. ii6.75 ii6.48 (iG.18 64 i!) 6 4 . 22 iiY . 5 I (it%. 48
40 40 40
30 40
20 60
80
30 30 30 30 60 30
60 100
30
40 100 60
prnseodymium. b Although this line lies I)ryond the f 2 H twig('. it :ippnr"11tly h:is an twhancenient effect, on thr l'r line nt 68.Z3". Table 11. Analytical Lines and Operating Conditions I'enk Backgroliritl Sollrr Slit .\ngle, A4nglr, Voltage,. Ciirreiit, Width, lhnent I)rgrera %e l k y r e r s %e Kv Ma Inch 50 25 0 010 80 85 82 85 Lanthanum 50 25 0 010 80 US 78 90 Cerium Pmseodymiuni 68 28 77 20 50 50 0 005 ti 010 50 25 6 2 00 65 O(i Neodymium a
'I'ypical v:tlur.i 1):imI o i l o\id(b :it conc~entrut~on of 25( 1)) wrlght of mnplr
1290
ANALYTICAL CHEMISTRY
Set
Intensity, CPSn 3 78 06 9 25 (i
11
88
of t,hc rare earth near-neighbors ill both the K and L sprctra ( 5 ) . The boras disk techniqur outlined here docs not modify this sit'uation, but permits quantitative deterniinat'ioii of elements which possess lines without significant interfcrencc. The resolution obtainablt. is rrlated to slit widbh, which controls line breadth, and to dispersion. which is ti&vnined 11y the analyzing Dumi ( 5 ) rlaims that most lines 0.5" :Lpart :tnd very strong lilies 0.75" s p i t girt, no dr~twtahlcintwference. 1-sing this criteria. he lists no interferenw for La L a ] . Ce L a l , Pr @I, or Sd L p I . Cullitj- (4) notes that an :dequat,e resolut'ion rsists if no other line orcurs within =t2 line breadths (dcsignatd hcrrafter as +2B) as iiieasurctl hnlfnay bctwecn pcak and hackground intmsitics in tcmns of dcgrces 2 theta. ('oncentration should not alter this prrniisc. Table I list's possil~le interftwncc within this criterion of thc choscn lines considering thosc lines of thca i'aic cxrths, scandium, yttrium, hafnium, zirconium, and thoiium \vhich ha\.(, rclxtive intcnsities grcatcr than 20. l'hosc elements n-host. lincs lich \vithin thcL iI3 range could caonstitutv major intcrfcrence. If ticenied necessary. thorium, hafnium, zirconiumi, and many other possible interferencw c*ould be rcmovrd by a preliminary oxalat'c srparation. The Ce lines I432 a t 66.48' and L/36 a t 69.02", although occurring beyond the 1 2 B range. apparmtly h a w an additive effect on tlit. Pr line :it ti8.23". A sc.rirs of cq)thriments s h o w d that for every 10% CeOs content prescnt, the cvaluated yo Pr6011 would be 0.107, too high. This correction factor lias b w n applied to :ill of thc Pr6OI1 values. The La ,562 line a t 69.74" appears to linve no effect on this Pr lint). On this hisis, analytical lines were chosen and standard curves were constructed for lanthanum, rerium, praseodymium. and neodymium. Background linrs \ v t w rhosen as near to thv tlnalyticwl line :is practical, with due consideration of possible interferenco from other rlrnic,nts. The analytical lines (angle 20) art' given in Table I1 with typical net int,t.nsity values. Preparation of Disks. The prepnration of the b o r a s glass disks is siniplc. .A 0.10-gram port,ion of sample anti 10.0 grams of fused, ground XCS rcyigrnt grade sodium borate are mixed in a platinum crucible. T h e fusion is made over a Meker burner. Swirling thc crucible is not essent'ial but permits R homogeneous melt to be obtained niorr rapidly. The> matcrials are heatetl from 8 t o 15 minutes until the solution appears rlear. h b h l e s should appear only on th(. sides of t,he crucible a t this point. Thc f i i e c ~ lmaterial is quickly poured onto :i
preheated (400' C.) :duniinum slnb with one quick motion. An asbestos board cover for the aluminum slab, which is removed while casting, aids in temperature control. After 10 t o 20 minutes' cooling on the aluminum slab, the disk is transferred to the asbcst'os board and permitted t o cool slowly. Careful casting procedure is necessary to avoid cracking by sudden temperature change. If a disk does crack, because of unequal cooling, i t may simply be recast. The top of each disk is identified with a laboratory number. The excess borax glass remaining in the platinum crucible can be conveniently removed by fusing with a small amount of potassium pyroeulfatc and leaching with hot water. Instrumental Conditions. All mwsurements were made on a General Electric XRD-5 iiiiit with flow proportional counter A 4952F and count'er tube S o . 4SPG. The operating volt'age \\-as 1.8 kv. with a gas mixture of 90% argon and 10% methane. The flow rate \\-as 2.0 inm. per minute. -4 lithium fluoride analyzing crystal was used in an air path. Because the instrument is stable, yielding reproducible data a t 25 ma. and emits sufficient radiation with a 0.010-inch slit, these conditions n-ere chosen. However, to evaluate praseodj.mium, i t was necessary to employ the higher resolution obtained n i t h a 0.005-inch slit. The desired radiation intensity necessitated using 50 ma. in this case. The operating conditions are shown in Table 11. The x-ray intensity for each element \vas nieasurcd thrre times a t the emitt,ed line peak and three times at the background location. The average counts per 40 seconds a t the peak minus t'he average counts per 40 seconds for the background \vas used as the net peak height or intensity. -4standard curvc of net intensity tis. per cent concentration was made each day, using the standard rare earth disks. The standardization on rach day of opera t 1011 ' ry, inasmuch as the instru:ed with many tliffercnt s of slits, cwintcri., :tiid\-xiiio.sph~r(~s.From tlw v:ilucs for n - c i ~wad ~ clircvtl>from the standard ( ~ T X X Preparation of Calibration Curves. I o prl:J?:lr(' tlir :inalytic.al working r ~ i r wstandard ~ disks ~ e r pprc'p:irc~l contaiiiing know1 aniounts of t h e givcBn olcmrmt. tTnknon-n samplrs and purc i ' a r ~ c'nrt'lis should 1)t. ignit,cd lirforc ivcighing and disk ~)rcparation, becauw of thr, liygrosccipic, natiii,cb of some of the rare rwth 0xidc.s. 1,:111thanuiii osidc will slon.1~-convert t o t h r hytlroxidc~ and absorb cai.bon dioxidv. thus disrupting stoichiometric, values. Frwhly igiiitcd pare eart,h oxide iuis diluted with pottt+s flint or sotliuiii borate to iiiakc up thv 0.l-grani suniplc of the desirod conctntration anti then fused \vit'li 10 granis of sodium borate. For cx:iniplc, a t1irc.c-point calibration line may bc> detrrmined by using 100, -r ( a , xiid 50% c-erium oxitle samples. The 100(rc t l i 4 \vould cwntain 0.1 gr:im ?
>
Table Ill.
Rare Earth Compound Lanthanum oxide Crriuni oxide
X0.n
70
43 44 45 68 43
21.3 5.3 28.5 12.4 42 0 5 3 56.5 2'; 9 5 2 5 3 4 5 3 1 20.1 5.3 10.5 9.9
44
Praseodymium oxide Srodyrniurn oxide
Analytical Results
Rare ___ _Earth Oxide Arith. mean Disk Known,
45 63 43
44 45 63 4:3 44
45
u3 Disk 43. Lindsay Code X50. Disk 63. Monazite sand B-10.
forlrld, 7G Std. Dev 0 45 21.4 5.6 0 34 28.9 0 47 0 53 13 6 0 65 42 . 3 0 41 5.3 0 63 56.3 0 43 27.8 0 Iti 5.2 0 1:< 6.2 0 21 4.8 0 21 3.1 19.9 0.22 5.7 0.37
Error, + + + + +
0 5 5 7 1 1 9 T 0 7 0 0 - 0 4 -0-1 0 0 +17 0 + 6 i 0 0 - 0.1 7.5 1. o +l4 1
+ +
10.7 0.3'3 11.3 0 43 I>i& 44. Spex Mix. Disk 45. Synthetic mixtriw.
of cwiuni oxide, the 7 5 7 , disk \voultl contain 0.075 grain of cerium oxidc arid 0.025 gram of diluent, and the 50% disk would contain 0.050 gram of cerium oxide and 0.050 grain of diluent. Potter's flint, which contains 99+% silica. is convenient :is 21 diluent; because of their lon atomic numbrrs, thc silicon and oxygen do not interfere. The rare eart,h oxidcs uscd in prcpa,ring the analytical working (wrvc \\-ere purchased from Research Cheniicds, Inc., as assaying 99.87, or better. Curves were plot'ted of n-right per cent rare earth oxide z's. net intcnsit,\- of the analytical lint for lant8hununi, cvriurn! Iirascwiymiiim, nnd ncorl!-niium osidcs. ANALYTICAL RESULTS
'l'lie analytji(vilrosults of four standard sninplcs are I)rtwnt'ed in '1':iIilc 111. These saniplcs r q m w i i t a nidc variation of concentration m d of c>lcinerits prrswt . One standxrd suniplt, \vas rare earth oxitlt,, Code 330 (Lindsay Chemical Co.). In addition t'o the data for t'hiq s m i p l ~suppliccl by tho manufacturer. it had hwn :innlyzed s~~ecitrochcniicully.I t contsincd samarium. gadolinium, yttrium, yttc.rbium. and tlysprosiuni in addition to lanthanum, ccriuni. prnscodyniium. arid neodymium. T h t swond sample w:is rare carth Spes 11is (Spes Industi Inc.). I t contained 5.28% of c w h of the 14 rare ositles in acltlit~ionto s c ~ n dium ouiclc and yttriuni osidrx. The third saniplc! was a synt'hctic haatnaesite-like mixture conipountlwl by the authors from Resvarcli Cli(mirals, Inc., research grade lanthanum o d e , cerium oxide. praseodymium oxide, and neodymium oxide. The fourth sample and its spectrographic analysis were provided by Heavy Minerals, In(,. This sample was a inonazitc, sand ant1 containcd ytt'riuni, tlmriiiiu. and a11 tlie rare earths.
Each sample ~ : t b analyzed on 10 different days. Iluring this periotl the instrument was used t)y others who varied conditions so that each analysk also incorporated rvproduvibility of instrument adjustment for voltage, current, analyzing crystal, 28, and recording. The per cent error was calculated by dividing t h e differenw between the accepted value and tht, arithmetic mean by the accepted valuv and niultiplying the quotient by 100. The percentage error generally increases n ith decreasing lanthanum praseodymium, and iieodyiniuni contrnt. The accuracy as indicated by thv per cent error is considered satisfactor! for lanthanum, cerium, and neodymium The praseodyniiuni values are acceptable aftcr t h c rerium correction. Thv accuracy does not vary appreciabli from Iiiulticomponciit sainples 43-41 and sample 45 which contains onlj four rare earths. 'This suggests littlc or no rare earth clciiirnt interferenLv at the ana1ytic:il lincs c~hovn. DISCUSSION
Thi. p r ~ c c d u r cN :I< ticsigned for industrial control purposes, not for the most precise analyticd work, but it shows promise that furtlicr refinrments may waluatc the vuisting limitations, thus increasing tlie accuracy atid iniproving prccisiori suffiriently for finer work. The standard aiialyticd curve's arc linear over tlic concentration range considered. Routine iamples have bern satisfactorily analyzc~l, with rerium and lanthanum contents approaching those of the pure oxides. The niiriimuni ction have not been dctermined, but samples nithin the range of the standard s:iniplt,s are most typical of the routine samplcs for which the VOL. 32, NO. 10, SEPTEMBER 1960
1291
procedure was designed. If necrssary, lower limits might be obtained by removing air from the beam path ( 8 ) . The number of rare earths which may bc readily determined by x-ray fluorescwwe may be increased with the availability of higher dispersion. The use of second-order rare earth L lines with lithium fluoride, or topaz as a n analyzing crystal should be helpful but will sacrifice intensity. The d sparing of topaz is smaller than lithium fluoride and Khen used as the analyzing ciystal, the resulting spectra would bc spread over a wider rangt. This drould niakc more “interference-free” 1int.s available. Lines posscssing interference from only one element may be employed utilizing appropriate corrections. This interference should be from an rlcnient which can nom be determined quantitatively. ACKNOWLEDGMENT
This work was done wliile D. R.
Maneval held a fellowship sponsored by the Molybdenum Corp. of America. H. A. McKinstry, Mineral Constitution Laboratory, The Pennsylvania State University, contributed significant suggestions during the coursr of thew studies. LITERATURE CITED
F..,VoreZco Rewtr. 4. 3 11957). ( Z j Claisse, k., Provinc‘e of Quebed,
(1) Claisw.
Dept. of llines, R.P. 327 (1956). (3) Clarke, 0. I,.,Wagner, W. F., Carley, D. JV., Univ. Illinois, 6 Eori 71, Chemistry Task Force VII, 1947. 14) Cullity, B. D., “Elements of X-Ray Diffraction.” I). 410. A4ddison-Weslev. Reading, Mass., 1956. (5) Dunn, H. W., Oak Ridge Natl. Lab., ORNL-1917, Contract my-7405-eng-26 (1955). (6) Hasler, hl. F., Barley, F., Spectrographers :Vews Letter 5 , 2 (1952). (7) Lytle, F. W., Botsford, J. I., Heller, H. A,, U.R. Bur. MincR, Rept. Invest. 5378 ( 1957).
\I.., Heady, H. H., Ax.41,. 31,809 (19%). (9) Parrish, K.,>Yorelco Reptr. 3, 24 (1956). (10) Powers, 11 C., “X-Ray Fluorescent Spectromet er Conversion Tables, ’ ’ Philips Electronics, Mount Vernon, IT.Y., 3957. (11) Romans, P A., AIlIE, Pacific Korthwest Regional Conference, Spokane, JTash., 1955. (12) Salmon, 11. L., Blackledye, J. P., ,Vorelco Reptr. 3, 68 (1956). (13) Sherman, J , A S T M Spec. Tech Publ. 157. 27-:33 13957). ( 1 4 ) Sherman, J , Sprrtrochim. .4cta 7 , 283 (1955). (15) Sun, S. C., ;\SAL,. CSEM. 31, 1322 (1959). (16) Tingle, IT. H., hlstocha, C. K., Ibid.,30, 49-1 (1958). (8) Lytle, F. CHEM.
RECEIVEDfor reviex July 14, 1959. Accepted June 15, 1960. hleeting in Miniature, Central Pennsylvania Section, SCS, March 14, 1959. Contribution 58-124, College of Mineral Industries, The Pennsylvania State University, University Park. 1’s.
Quenching of Fluorescence in Liquid Scintillation Counting of Labeled Organic Compounds C. T. PENG Radioactivity Research Center and School of Pharmacy, University of California Medical Center, San Francisco, Calif.
b A method is reported for correcting fluorescence quenching in liquid scintillation counting without the use of an internal standard. The quenching constant or half-quenching concentration, which expresses the quenching property of a compound, was found to vary with instrumental amplificcation of the pulse signal and particle energy. The nonquenchable fraction of the background count was also analyzed.
I
x THE USE of liquid scintillation counting for assaying radioactive biological samples or labcled organic compounds containing weak beta r m i b ters such as carbon-14, su1fu1-35, antl tritium, the efficiency of the method is greatly impaired by the cffcct of fluorescence quenching which may lw caused by the presence of these compounds in the scintillation solution. I n theory, the energy dissipated by the beta particle is transfrrred molecularly through the solvent to the organic. scintillator which becomes c w i t t b d and thrn de-excites by the emission of photons (8-10). The photons are emitted as fluorescence antl ran bc detected by s multiplier phototube and counted. Whcn impurities and nonfluorcscent
1292
ANALYTICAL CHEMISTRY
materials are prcqent in solution, these substances may also be excited by the radiation energy from the emitted beta particles. HONever, the excited molecules of these substances return to the ground state by radiationless transition without roncomittant emission of photons (3); the eyxnditure of beta radiation energy in this manner results in a decrease of the maximum fluorescence yield and directly rrduces the counting efficiency from a given radioactive source in a scintillation solution. The quenching property of many classes of organic, compounds has been reported by Kerr, Hayes, and Ott (If) and Guinn ( 5 ) . In general the fluorescence quenching in liquid scintillation cbouiiting can be rorrected by recounting the sample after the addition of an internal standard solution of knon n activity and adjusting the, obscrved counts of the ~ a m p l vpiopoi tionatel! dnothcr method according t o tliii lo-. of corrrcting for qurnc~hing l o s v i without the, i i w of n n iiitcmal standaid is by c~\tiapolation of thv obscrved count< :it difft3i cmt sample concentrations t o obtain t l i v true spec3ic. activity of the s:tmplv (16) This report ii con( tmicLcl Iiitli tlw aj)plic~itionof thc.
latter method for assaying labeled biological samples and studying the quenching property of a mixture oi quenchers. The relationship of quenching to certain characteristics of thv counting instrument and the effect oi quenching on background are also considerrd. METHOD
An automatic two-channel liquid scintillation spectrometer (Packard Instrument Co.. La Grange, Ill.) ( I S ) was set with the lower channel scanning IO-to-50 volt pulses and the uppri. channel 10-to-infinite volt pulses. The arhitrary setting of the two cha,nnel~ for scanning pulses of different amplitudes was found desirable inasmuch as a chaiigc, in the i.elative counting efficieiic*y hc,tn.ccn the channels signifies either, that, an unsuspected quenching may lie taking place, or t,liat the bslanccb point (2) of the voltage characteristic, ciii’vc of the spectrometer has shifted. At ant1 w a r the balance point, fluctuations in voltage do not cause significant changes in the counting rate. ,411 the samples were counted to a statistical accuracy of approximately & lyc at the respective balance point found for each particular isotope. The balance point was determined by counting
