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
- 4
4 r
P
w
- 2
u
-
mperature. H o w v e r , this may he corrected by lowering the freezing point of the dioxane scintillator hy incorporat'ing snmc aqueous solution count,ing samplc, 01' 1)y mixing with half or equal volunic of the toluene scintillator, drpentling upon the solvent polarity d e s i r d . Kihh an option t o use a volume of 10 or 15 ml. of liquid scintillator in the counting vial! and using hyaniine hydroxide to solubilize proteins and to absorb carbon-14 dioxide (14), a homogeneous counting solution was obtained in most of the samples encountered. The multiplier phototubes of the liquid scintillation spectrometer were operated at -8" C. to reduce the background noise due to thermionic emission of the phototubes. The temperature of the refrigerated unit was raised on occasions when the use of the dioxane scintillator alone made such a change necessary.
RESULTS AND DISCUSSION
Quenching Correction. Kerr et al. (11) reported that the counting efficiency decreases as an exponential function of the sample concentration of a n unlabeled quenching compound. This relationship has, however, not been applied to the counting of labeled samples that may quench. For correcting the effect of quenching in the liquid scintillation counting of some sulfur-35 labeled compounds, Peng (16) derived the following equation: N = SoCexp. ( - q C )
(1)
where N = the observed counts, SO = specific activity of the sample in the absence of quenching, C = sample concentration, and q = quenching constant. The quenching constant is equal to o.693/c1,2, where CllZ is the sample concentration that will reduce the counts to half of its initial value by quenching. This equation may be used for samples containing other weak beta emitting radionuclides. If the ratio of N / C is known as the apparent specific activity, Sa, of the sample a t concentration C, then Equation l may be espressed as: S , = So esp. ( -qC)
(2)
This equation gives a linear semilogarithmic plot which may be extrapolated to zero sample concentration to afford the specific activity of the sample in the absence of quenching. The validity of the equation for correcting fluorescence quenching in biological samples may be illustrated with the counting data of Vaughan et al. (18) on tritiated protein samples dissolved in hyamine solution. These investigators used the internal standard method. Their data were plotted in Figure 1 according to Equation 2. The extrapolated value for the specific activity of the protein sample (curve p ) in the absence of quenching is 2385 c./m./mg., which is 6.2% lower than the average (calculated by the present author) of the values given by Vaughan (2512 c. 'm.,hg.), and the
extrapolated value for the internal standard (curve s) is 16,000 as against the reported activity of 16,500 c./m. These observed discrepancies may be attributed either to an improper m eighting of the individual observations in obtaining the average of the reported values and, or to an altered counting geometry after the addition of the internal atandard which increases the total sample volume. The above extrapolation method has been u ~ c din this laboratory to measure the specific activity of some labeled organic compounds and medicinals, such as acetyl-C14 derivatives of mol phine, tetramethylene-1 ,1-C214 (or 2,3C214)bismethane sulfonate (Busulfan). tritiated berberine, bilirubin, Nyleran, morphine, etc. The radioactivity of these compounds was determined by liquid scintillation counting after dissolving them in suitable solvents. The fluorescence quenching due to either the solvent or the sample solute, or both, including the effect of any impurities which may be accidentally present, mas corrected for as described above. I n the case of carbon-14-labeled Myleran. the value for specific activity obtained by extrapolation was almost identical within the experimental error to that obtained by absolute beta counting using a large, thin, end-window Geiger-Mueller tube. I n precision determination by liquid wintillation counting such as d w r i b e d above, the extrapolation method is inherentll- more accurate and dependable than the internal standard method. The former method makes use of many observations a t varying sample concentrations in plotting and extrapolating, thus alloning the result of each observation to be properly weighted relative t o others and, in addition, the counting samples are not disturbed once they have been prepared; counting \$ ith an internal standard involving t n o operations on each sample pospesses none of these advantages. This method of estrapolation to zero sample concentration may also be applied to routine counting of samples. The quenching property, or C1t2, is dependent upon the composition of the sample medium. If this remains unchanged, the samples may be counted a t any one concentration and from the count observed it is possible to correct to zt'ro quenching. An alternate is to use t\i o different concentrations of a sample in a ratio of 2 to 1 and obtain the activity in the absence of quenching by extrapolation. I n this manner, providing that the counting efficiency for the radionuclide is known, the activity of a sample can be readily measured in terms of absolute units without the use of an internal standard. I n practice, biological specimens such as urine, etc., may be counted in the VOL. 32, NO. 10, SEPTEMBER 1960
1293
IO!
5
IO'
I \
J
5
I-
?
I-
0
4
lo'
5
IO2 SAMPLE ADDED, ML.
Figure 2. Activity vs. added volumes of quencher
Table I.
C,,, Values of Some Substituted T h i 0 ~ r e a s - S ~ ~
Compounds, RHS-CS*-SHR' R R' Phenyl H Phenyl Phenyl p-Bromophenyl Phenyl o-Ethoxyphenyl Phenyl p-Ethoxyphenyl Phenyl p-hlethoxyphenyl Phenyl 2-P yrid yl Phenyl o-Tolyl I'hcnyl p-Tolyl Phenyl
c,,,, Mg./ 10 M1. Liquid Scintillator Calcd, ~ o u l l d 36.7 36 19.0 20
2 3 . 9 21 . 7 27.1 28
27.4 29.0 28.8 29.5 30.3 28.0
36.6 34 21.8 22
Table II. Half-Quenching Concentration of Mixed Quenchers
Cl,?] All./l0 111.Llql1i(1 scintillator e t h j l alcohol (v./v ) Calcd. Found 0 77 0 68 2/98 5/95 0 312 0 ,312 10/90 0 177 0 17tj 15/85 0 12 0 12
Quenching llixtiirea I' heny1 is0t hio cyma t e /
Ethyl alcohol/\$ater
( v /v.) 80/20 60/40 10/60 20/80
4'K3 3 5
291 2.48
1 3 3 2
8 4 0 ci
Mixture prepared by pipetting first component into a volumetric flask and diluting to volume with second component. 5% added to liquid scintillator. a
manner described and the results conveniently expressed in terms of microcuries per liter. This method is also valuable in personnel monitoring programs, especially in the case of urinalysis of those n h o work with carbon-l4--labeled and tritiated compounds. The effect of varying the counting sample geometry on the rounting rate whm the total solution volume mas increased by the added wmple is shown in Figure 2. The slightly concave upward curve, A , waq given by a series of samples prtparcd by adding varying amounts of a qucmlher to 10 ml. of liquid scintillation solution containing carbon-14. Curve B , which is more linear than A . waq obtained from the samc series of saniplcs after the differences in solution volume \\ere compensated for by the addition of more liquid scintillator. It became apparent from Figure 2 that for correcting fluoresrencc quenching by Equation 2, a constant counting geometry for the counting samples must be maintained and only a proportionately sniall amount of the sample solute should be introduced into the liquid scintillation solution. Quenching US. c,,,. Differentiation of Equation l gives
and
c,,,
= l/y = 144
c,
A
ANALYTICAL CHEMISTRY
.
N
0
.5
qK'>)I
TAP N O
(4)
t
t
t
t
5
6
7
E
10
9
VOLTAGE,
12
I1 XlO'
13
VOLTS
Figure 3. Half-quenching concentration of THO v5. high voltage supply to multiplier phototube
previously defined, and the numeric:d subscript indicates compounds 1 and 2 . lly letting C equal C, C2, Equation 4 becomcbs
+
where C1'C and C2/C represent concentration fractions of quenchem 1 and 2 in the mixture. The validity of Equation 5 was established by using niistures of phenyl isothiocyanate and ethyl alcohol. The observed and calculated quenching constants of these mixtures are given in Table 11. A more general form of Equation 5 is So
The symbols have the sanic nicaning a < 1294
-I I 1.0
2
Increasing the sample concentration of the labeled quenching cnompound in solution not only incrrases the activity (the counts observcd). but also increases the degrec. of fluorescence quenching. When the rate of incrcase of activity balances that of quenching, a peak in the plot of concrntration us. activity is reached whcrc the sample concentration yields thc ma\iinum counts and is represented as C,,,,,. Table I shows the observed and the calculated C,,, values of somc qulfur35-labeled compounds. Tlir observed C,,,, valuw were obtained from different samples of each compound with specific activities varying appro\imntcly from two to fivefold. Thc ronsiitrncy of the value of C,, or C1 I in thc cowbe of changing specifics avtivity of the sample (16) verifies tho mnstancy of the q value in Equation 2 for snmpleq a t varying levels of activity. Quenching Effect of Mixtures. The quenching of fluoiescence is dependent upon the concentration of the quenchel. I n a mixture of quenchers, thc effect of quenching is euponentially additivr; thus S , = Soe\p [ - ( q l C ,
1.5
=
So exp. [--iz?ic,)C]
Quenching Constant us. Pulse Amplification and Particle Energy. Although the quenching constants or half-quenching concentrations (C,12) of some compounds have been reported ( 1 1 15) thcir rnlue has since been found to vary with certain characteristics of the counting instrumrnt. Quenching diminishes thc light output from the scinbillation solution and decreases the pulse height of the input signal to the lincar amplificr. To offsct the effect of quencahing, counting may be performed :it a higher voltage supply t'o t,he multiplicr phototube ( I S ) . The height of the signal pulse above the threshold of the pulse-height selector or a b o w t h r bias setting for the acccpt:inw by t h r scdcr is the factor n.hic.11 is charnctcrietic of each counting instrunirnt, anti it plays a part in dettrmining the magnitude of t h r quenching constant or C,.?. Qurnching decreases the p,ulw amplitude but so long as it remains :Ihove thc threshold, the pulse will bc counted. thcwby making th hctnetm the taps 11as approxinxttely 80 volth antl a tl! ofold change ( i f :ittenwition cauwd a shift of the I~alniict~point by m v voltage tap. I n this particular inhtrunicnt uscd. tritium peaked at thc voltagr tap 30.6 .ind attenuation tap KO. 1 : c:irbon-14 or sulfur-35, 3 and 1 : (.;ilc~iuin-45, 2 and I ; chloriric-36. 1 :inti 2 ; phosphorus-32, 3 and 16, rcspcdively. -4ssuming that the pulw heights arc' inomparable in magnitude a t the balnnw points, it follows that thc half-qur>nching concentration of a qucnrher should IIC thri sanic 13 ith thcw radionuclidcs. 'l'hr Clip values of a strong quencher, phenyl isothiocyanate, in liquid scintillation solutions containing carbon-14. d f u r - 3 5 , calcium-45, chlorine-36, or phosphorus-32 w r e idmtical within
Table 111.
Counts/50 Min. /Channel Width, VoltsC ____.
Sampleb ~-
Settings __ Material 10-50 10- m Dark viald 36 3Z 6 . i 60 f 1 0 . 8 Blank sample well 162 f 9 . 0 231 f 11.5 Empty vial 230 f 15.6 330 f 18.0 Ethyl alcohole 382 f 19.5 549 f 25.8 Ethyl acetate 363 f 15.2 535 f 25.7 Toluenc 389- f 24.4 619 f 26.3 Phenyl isothiocynnate " 320 f 13.8 468 f 15.6 I)io>ane' 316 =t18.1 504 f 26.4 PPO in toluene 638 3Z 26.8 1879 f 49.3 P1'0-POPOP-toluene 591 f 26.8 1853 f 46.6 Plexiglass vial 383 f 40.9 480 f 43.9 Solid plexiglass vial 470 f 37.6 681 f 4 0 . i C l 4
'
(;labs vial sprayed inside and outside with black paint. Ten milliliters of solution used in counting. Froxcn at -8' C.
the cxpcrimental error. The Cli2value for tritium is lower probably o\ving t o t,he small light pulses given by this isotope in the sc~intillationsolution. In t,liia ronnwtion, it may be ment,ioned that Cl.2 also varies with thc composition of the liquid scintillatioil solution bcing usrd. For example. in it solution medium containing 10 nil. of liquid sciihllator 1. the C1,2i d u c of phenyl isothiocyannte \vas 32.7 mg.. \vhilr in a mixture of 5 nil. each oi scintillatore I and 2 this vduct incw:iscd to 68 m g . This discrepancy iii:i!. bo csplnincd on the basis of partid rwtoration of the quenchd light hy tho j m s m r e of n a p h t h a h e in thc niised srintillation solution (4. 6, 7 ) . Quenching of Background. 'l'hv background c*ount in liquid s h ~ t i l l a tiori rounting was alw studicstl in tlic, light of fluorescence qutmc.hing. I k rausc rounting samples cwitaining strong qucwhers niay yivltl l c m c s r . caounts than thosc tqwctctl lor 1iac.kground alone. it was of intcwst to asctrtain thc composition of thc backsround and to det,ei,ininc w h a t fraction of i t ]nay be quenched i n thih manner. T:ible 111 shoxvs the, Ivsults of the study. 'The background rount iu liquitl svintillation counting niay a t
s35
P32
5
Figure 4. Particle energy vs. high volta g e a t balance point
0.0 I
I
0.05
I
I
0.1
E
MAX,,
0.5
M.E.V.
... . .
~~~
c136
It
... . .
4y-y
c045
-
H 3 Settings __ 10-50 10- m 125 f 14.9 245 f 38.8 881 f 3T.4 1636 f 51.1 1895 f 61.0 2747 f 76.9 2066 f 4 i , l 3155 f 50.9 2033 =k 56.9 3079 =t 72.3 2420 f 64.2 3622 i 92.0 1722 i 43.5 2614 f 42.3 2130 f 51.3 3140 rt 60.6 2382 f 89.7 4823 f 142.3 2407 =k 73.8 4837 f 94.2
a Counting performed on new automatic Tri-Carb liquid scintillation spectrometer of R. J. Havel, Cardiovascular Research Institute, University of California Hospital. ') Samplev dark-adapted a t 0" C. overnight before counting. No attempt made to remove dibsolved oxygen in sample. Average of more than 20 individual counts taken uninterrupted. Std. dev. = ____
l o l l c'4
Background Countings"
I
I
I
2
4
following sources: chancc coincidencei.e., two noise signals, each from a multiplicr phototube, can arrive a t t'he electronic gating circuit within its resolving time to be passed as a coincidence cvent antl counted; :L reciprocal light sensitization of the photocathodr of on(' multiplier phototube by scintillations cmittcd by the glass envelope of t'he other mult,iplier phototube in the 'Lhrad-on":irr:tngcnic,nt in the spectronicxtcr; radiation from potassium40 i n the glass minting vial; (:herenkov rarli,ition---i.c,.. thc, cllwtromngnetic padiation tmiitttd by :I chargcd partide moving through :i niedium a t :i speed escwding that of light in the medium ; vomiic. radiation : and radiation due to cnvironmrntwi ratlioartivity. Contribution of thew sourrce to the background c*ount ni:iy b(, ()valuated by a scritv of sainplw listcstl in l'able 111. In l'abltl 111. s:iriiplv 1 (thc dark vial) g : i \ ~thv c,ounts t l u c h to vliancc coincideiic*c: sampl(~2. thv canbirird effect of chaiicc, ewincidcncc. and light smsitization of thr p1iotoc~:ithodes; and sarnplc 3, thc supc.rirnt,(iR('(i c 4 f i b c t of chancr and r:idi:ition from potascoinridc~ncc~ sium-40 i n glass. Tht, light, sensitization of tlic photot,ubcs i s not c w h a t e d in this ('as(>. l l o s t likely, its effcct is small sinw iiiscrt>ion of a raniple rvill obstruct tho dirc3c.t vicwing of t h r photocathodes of c,acah othcr. S:implcs 1, 5 , 6, 7 , anti S \Y(W rounting vials fillcd. rc~sptvtiwl\-,\\.it11 10 i d . of ethyl alc~)hol,ethyl :ic.c,tatcs, phwyl isothiocyanatcb (a w r y strong qucwhcr), toluencl. and frozcw dioianc~; t h e incrc.asc i n rounts in thwc. saiiiplcs as romparcd to eaniplc 3 was attrihuttd to (?herenko\. ratliotion. S:implc*s 9 antl I O mnVOL. 32, NO. 10, SEPTEMBER 1960
1295
tained liquid scintillators and yielded normal background counts. The inrrease in counts in these two samples over the previous ones was due to cosmic radiation and environmental radioactivity. Samples 11 and 12 were fabricated from optical plastics to differentiate the relative importance of the effects of potassium-40 and Cherenkov radiation in glass. However, from the large number of counts observed in these samples as compared with sample 3, i t can be seen that the plastic constitutes a copious source of Cherenkov radiation, possibly due to a greater optical transmission of light in the short wave-length region. This observation agrees with that of Arnold ( I ) . By assuming the normal background counts as unity, the per cent contribution from the above sources may be computed from the results shown in Table 111. Thus, for counting a t optimum settings for carbon-14. the contributions from chance coincidcnce, potassium40 (light sensitization of photocathodes), and Cherenkov radiation were approhimately 6, 31 (20), and 25% in the 10-to-50 volt channd and 3.2, 14.6 (9.21, and 12% in the 10-toinfinite volt channel, respectively. At optimum settings for tritium these values became 5, 74 (32), and 9% in the lower channel and 5, 52 (29), and 8% in the upper channel, respectively.
It may be noted that increasing the high voltage supply increases the sensitivity of the multiplier phototube, thereby resulting in a more efficient detection of potassium-40 in glass. The background counts of a liquid scintillation solution may now be considered t o be composed of a hard component and a soft component fraction. The hard component consists of the counts usually observed in solution media without an organic scintillator present and, for this reason, these counts are not fluorescence quenchable. I n contrast, the soft component is affected by quenching; however, the extent to which this ocrurs depends largely upon the concentration and the quality of the quencher. Only under extremely adverse quenching conditions can the soft coniponmt be completely eliminated.
(6) Hayes, F. S.,Ott, D. G., Kerr, V. X., hucleonics 14, No. 1, 42 (1956). (7) Hayes, F. N., Ott, D. G., Kerr, V. K., Rogers, B. S.,Ibid., 13, No 12, 38 (1955). ( 8 ) Kallmann, H., Furst, M., in “Liquid Scintillation Counting,” C. G. Bell, Jr., F. N. Hayes, eds.. p. 3. l’ergamon, New York, 1958. (9) Kallmann, H., Furst, 1\1 , Phys. Rev. 79.857 11950). (10) jbid.,‘81, 853 (1951). (11) Iierr, V. S . , Hayes, F. S . , Ott, D. G.. Intern. J . A.nw1. Radia/ion and
LITERATURE CITED
6 ) Swank, R. K., “Liquid Scintillation Counting,” C. C;, Bell, Jr., F. N. HayeE, edp., p. 93, Pergamon, ?;em- York, 1058. 7 ) Sn-ank, R . K . .l’ircleonics 12. S o . 3. 14 11954).
2 ) Lingham, iV. €I:, Eversole, W. J., Hayes, F. S . , Trujillo, T. T., J . Lab. Clin. X e d . 47, 819 (1956). 3 ) Packard, L. E., in “Liquid Scintillation Counting,’’ (7. G. Bell, Jr., F. N. Hayes,, eds., p. 50, Prrgamon, S e w York, 1058. 4) Paaeman, J. It.,Rntiin. S . S., Coouer. J. -4. I).. . ~ X . A L . C H m i . 28.
484 -( 1956). 5 ) Peng, C. T., in ,‘Liquid Scintillation Counting,” C. G , Bell, Jr., F. S . Hayes, e&., p. 108. Pergamon, Xcw York,
1958.
(1) Arnold, ,J. R., in “Liquid Scintillation Counting,” C. G. Bell, Jr., F. ?J. Hayes, eds., p. 129, I’ergamon, Xew York, 1958. (2) Arnold, J. It., Science 119, 155 (1954).
(3) Birks, J. B., “Scintillation Counters,” p. 62, McGraw-Hill, New York, 1953. (4) Furst, M., Kallmann, H., Brown, F. H., i4’z~cleonics 1 3 , No. 4, 58 (1955). (5) Guinn, V. l’., “Liquid Scintillation Counting,” C. G. Bell, Jr., F. N. Hayes, eds., p. 170, I’tlrgamon, S e w York, 19.58.
5) T’aughan, M.,Steinberg, D., Logan, J., Srzence 126,446 1957).
RECEIVED for review December 21, 1959. Accepted June 20, 1960. Work aided by grants from U.S. Public Health Service (C-3630) a n d .4merican Canwr Society (T-431,
Extraction and Flame Spectrophotometric Determination of Vanadium CORNELIUS M. STANDER African Metals Corp. Lid., P . 0. Box 66, Meyerton, Transvaal, Union of South Africa
b A flame spectrophotometric method that is sufficiently sensitive to determine vanadium in a variety of materials has been developed. The method consists of separation of vanadium from interfering elements by extraction with N-nitrosophenylhydroxylamine plus ethyl acetate, followed by aspiration of the organic phase directly into the flame and measurement of the emissivity of vanadium by using the band at 550 mp. With a slit width of 0.065 mm., the sensitivity is 1 p g . per ml. per scale division T ) . The working curve is linear between 0 and 100 pg. per ml. of vanadium. The interference of metals that are extracted with the vanadium has been examined and methods for their separation have been devised.
(%
1296
ANALYTICAL CHEMISTRY
A
s PART of a study on the flame photometric determination of metals belonging to groups IV and V of the periodic table, a method for the determination of vanadium has been developed. Burriel-RIarti, Ramirez-MuBoz, and Asuncion-Omarrementeria (2) in their study of the emission characteristics of various metals in the flame reported that the response of vanadium in the flame was small and that i t was affected by large amounts of iron. The dissociation energy of vanadium oxide in the flame is given as 5.5 e.v. by Gaydon (5) and as about 6.4 e.v. by Mahanati ( 8 ) and Herzberg ( 7 ) . As a result of this high dissociation energy only a very small fraction of the vanadium oxide molecules present will be dissociated into vanadium atoms a t the
temperatilie of the oxyacetylene flame. Line spectra emitted by neutral or ionized vanadium atoms are therefore absent from the flame spectrum. The flame spectrum consists of a series of bands associated with a continuous emission starting at approximately 400 mp and extending into the infrared region. Bands that may be used for analysis occur in the region from 500 to 580 mp. I n the present investigation the bands occurring a t 529 and 550 mp were used (6). Many metals interfere in the flame photometric determination of vanadium, However, the interference of a large number of these metals can be obviated by extracting the vanadium with ethyl acetate plus S-nitrosophenylhydroxylamine (cupferron) from dl-
