givc.11 solvent system. Since for both inetliods relative efficiency is a linear function of the observed variable (absorbance or channel ratio), only a few observations are necded once the appropriate conditions (wave length or discriminator sett,ings and high voltage) :ire determined for this linearity. It is necessary that solutions be counted a t that high voltage which gives thc m:tsimum count rate for the particuhr isotope in the uncolored solvent +,in. ;Is the voltage departs from t h k peak value, t’lie 1/A us. relative c>tficieric,ycurve departs from linearity m ( 1 exhibits a niasimuni. -4 serious dimdvantage, for lower .level counting. of the channel ratio met’hod, is t h a t since a low count rate is split into two rhnnncls thc count ratio of which is needed) a slight drift in the voltage of the middle discriminator will change the ratio grcatly. A combination of the usual iritmial standard technique wit’h the optical (or channel ratio) niethodoptical nietliod for background and intcmal standard method for isot’opeis quicker than eit,lier separately since fewer standard curves are required. ‘rlle data of Table I show that there is no single constant t’hat can be substracted from all count’s, sample and background. to make their quenching curves c>oincident. The isotope constant. varies with the isotope level; it is approximabcly numerically equal t o thc sample count in t’he same solvent systcm without added color, a number Ivhich is itsplf a n unknown quantity. S o practically useful application of this portion of the experiment is evident. ACCURACY AND PRECISION
The accuracy and precision of background adjustment by the optical mcthod depend on the counting time of thcl various solutions and on instru-
Table II.
Net Count Rate of Colored Solutions Containing C’* with Various Adjusted Backgrounds
Cnknown
Background 1 Background 2 Background 3 Recovery, %
260 270: 271 271 i 1 ITc 97.5
ment stability between time of determination of standard curves and time of sample counting. Inforniation about these factors is given by an experiment vAth bromocresol green in solutions containing toluene, ethyl alcohol, concentrated ammonium hydrolidc. PPO, and POPOP. Standard curves were determined for CI4 isotope and the corresponding background. Three unknowns all containing the bame amount of radioactivity but different amounts of color, Lvere prrpared and counted; three background., all of different color intensity and all different in color intensity from the unknowns, were prcpared and counted. All solutions were counted Fith channels 10.0 to 40.5 and 40.5 to 100. Unknowns and their backgrounds ere counted once for 10 minutes. Both isotope and background relative efficiency os. 1/A plots lvere linear over the whole range of color, and differed greatly in slope from each other. For H 3and its background, the standard curve plot of relative efficiency us. channel ratio was nearly a vertical line (slope 542 for H3) so that this procedure was not useful. d b sorbanees of all solutions were measured a t 400 mp. F e t count rates were calculated with each of the three backgrounds adjusted by the optical method to the color intensity of each of the three bamples. The results are shonn
3
2
1
288 280 294 287 ==! 1 4 7 103 2
-
283 276 292 284 =!= 1 6 c c 102 1
in Tablc 11. The precision for earh sample is good n-hatever background was used. The over-all accuracy is also good for the counting time employed. ACKNOWLEDGMENT
The author thanks C. N. Rice of this laboratory for valuable discussion and advice during this investigation. LITERATURE CITED
(1) Baillic, I,. -A,, “Determination of Liquid Scintillation Counting Efficiency by Pulse Height Shift,” submitted to Intern. J . I l p p l . Radiation and Isotopes.
(2) Davidson, J. D., “Liquid Scintillation Counting,’’ p. 95, Pergamon Press, S e w York, 1958. (3) Donicr. F. R., Hayes, F. S.,Nzcdeonics 18, 100 (1960). ( 3 ) Eisenherg, F., Jr., “Liquid Scintillation Counting.” p. 123, Pwgarnon Press, New l-ork, 1958. (5) Guinn, T’. P.,Ibid., p. 176. ( 0 ) Herberg, R. J., ASAL. C H E X 32, 42 (1960). ( i )Kinard, F. E., Rev. Sci. I n c f r . 2 8 , 293 (1957). (8) Passmann, J. M., Radin, S . S , Coouer. J. A. D.. AKAL. CHE3f. 28, 484 jl950:).
(9) Swank, R. K., “Liquid Scintillation Counting,” p. 29, Pergamon Press, Sew York, 1958. RECEIVEDfor review April 4, 1900. Accepted August 8, 1960.
Elimination of Anion Interferences in Flame Spectroscopy Use of (Ethylenedinitri1o)tetraacetic Acid A. C. WEST’ and
W. D. COOKE
Baker Laboratory, Cornel1 University, Ithoco, N. Y.
A method has been devised for eliminating anion interference in flame spectroscopy. (Ethylenedinitri1o)tetraacetic acid, when added to the solutions being analyzed, enhances emissivity and maintains a constant level of intensity which is independent of the anion present. Elimination of interferences of a variety of anions includ-
F -
L A M E SPECTROPHOTOMETRIC METHODS have been used for the deter-
ing phosphate and sulfate was found for calcium, magnesium, cobalt, copper, chromium, and manganese, some of the exDerimental variables in flame
mination of a \vide variety of metals a t low concentrations. Their general
spectroscopy have been examined and an instrument is described which has an absolute stability of 2% over a period of 2 months.
Present address, Department of Chemistry, iyilliams college, ~ ~ i l l i a m s t o , ~ m , Mass.
-
1
VOL. 32, NO. 1 1 , OCTOBER 1960
1471
applicability has been limited by the interferences of various anions. The emissivity of metals in a flame is strongly influenced b y the nature and concentration of such anions (1, 2, 11, 15-1 7 ) . Proposed techniques to eliminate this limitation include separation procedures ( 6 ) , ion exchange ( I d ) , and extraction techniques (5, 8). These methods are time-consuming and the manipulations involved introduce the danger of sample loss and contamination. Other methods not involving physical separation employ an internal standard (10, 15), the standard addition technique (4, 15), or elimination of interferences by buffering both standards and unknown with a large excess of the interfering species (15). However, these methods have disadvantages in that accuracy and sensitivity may be lowered, and a n exact knowledge of the sample composition and the nature of the interaction is usually necessary. Another technique for minimizing interferences which has been applied particularly to the determination of calcium involves the addition of a reagent which reacts preferentially with the interfering species during the evaporation process (7, 19). From an analysis of anion interferences made by Xlkemade and Voorhuis ( 1 ) i t appeared that it might be possible to eliminate them by complexing the metal with an anion which would be readily decomposed in the flame. The xork of TTirtschafter (18) showed that (ethylenedinitri1o)tetraacetic acid (EDTA) was a good choice and it was found to minimize or eliminate interferences caused by anions. Since flanie spectrophotometry is widely used for calcium determination and anion interferences are particularly troublesome, the most detailed study was made of thiq clement. EXPERIMENTAL
The basic apparatus used in this vork was the monochromator of a 13eckman D U spectrophotometer, slits fised throughout a t 0.0175 mm., and a Beckman hydrogen-oxygen flame attachment. The amplifier was removed from this instrument and replaced by an operational amplifier (Philbrick UPA-2) for increased stability. The amplified signal vias attenuated and recorded on a Leeds &I Korthrup Azar recorder. A low speed synchronous motor was connected to the wave-length spindle of the monochromator and permitted both forward and reverse scanning at a speed which varied from 2.3 A. per minute a t 2850 A to 8 A. per minute a t 4260 A. Two to 4 minutes were required per run. An TP 28 photomultiplier was used as the detector and was powered by a ,John Fluke 403 M regulated (0.1%) high 1472
ANALYTICAL CHEMISTRY
voltage supply which was set at 900 volts throughout this work. The hydrogen and oxygen supplies to the burner were regulated by manostat valves (Bellofram Corp.) and both the pressure and flow rate were monitored. The gas flow was measured to 1% with C-Mar Series 8000 flowmeters. Precision gages which could be read t o 0.01 and 0.005 p.s.i., respectively, were used t o indicate the oxygen and hydrogen pressures. Oxygen pressure was maintained at 10 p.s.i. Flow rates were 0.56 cubic foot per minute for hydrogen and from 0.11 t o 0.15 cubic foot per minute for oxygen. Control of solution feed to the burner is essential to eliminate the effect of viscosity changes among samples (9). The rate of solution feed was maintained a t 0.92 ml. per minute by a motor-driven hypodermic syringe. For rapid routine analyses a large number of syringes, one per sample, could be used. All solutions were prepared from ACS reagent grade chemicals; the calcium solutions from CaC03 which was dissolved in excess "03 and evaporated twice to dryness. Standard solutions of the other metals were prepared from the appropriate salts. Anions were added in the form of their free acids, and Al(N03)~was used to avoid the presence of extraneous anions. A 0.5M stock solution of the sodium salt of E D T A was prepared by dissolving either the disodium salt or the free acid in KaOH to give a solution of about p H 9, which mas filtered. The protein used was recrystallized bovine plasma albumin which contained considerable calcium, necessitating a correction of the calcium emission. All other solutions had negligible backgrounds with respect t o the six elements studied except for the 2852--4. sodium line from the EDTA which coincided with the magnesium line and had to be subtracted out. The wave lengths used are shown in Table I. All intensity values given are in arbitrary units. INS1 RUMENTAL STABILITY
T o achieve long term stability of peak intensities it is necessary to control closely the amplifier gain and photomultiplier dynode voltages as previously described. Variations in photomultiplier response caused no difficulty over 5 months. Precise control of solution feed and gas flow is also essential (3, 9, 12). The usual procedure for
Table I.
Elements and Emission Lines Studied
Element
Wave Length, A.
Ca
4226 3453 4254 3247 2852 3700 (band) 4203
co Cr
cu
Mg
?*In
controlling the pressure of the supply gases is inadequate for high precision work. Gradual encrustation of the burner tip can cause decreasing gas flow rates even though the pressure is kept constant. The flow rate is the more important parameter and determines the amount of heat generated by the flame as well as the velocity of the gases a t the burner orifice. The latter, in turn, controls the aton ization process and, in the case of suctior, feed, the sample flow rate. Even with a constant rate of solution flow the intenb'ty of calcium emission depends strongly on the oxygen flow rate and to a lessei extent on the hydrogen flow rate. This result is understandable since the flame is hydrogen-rich and the oxygen effects the atomization in the type of burner used. The long term stability of the instrument was determined from the reproducibility of the intensity of a standard calcium solution over 5 months. A total of 50 measurements was made and the standard deviation from their mean was =t4%. Over the last 2 months of this period, after the critical nature of the oxygen flow was fully realized, the value for 20 measurements was *3%. If these intensities were corrected for oxygen flow rates, the standard deviation was lowered to &2%. For a single day, the standard deviation of readings made a t the beginning and end of operation averaged i1%. Reproducibility of consecutive runs with the same solution was considerably better than 1%. It is believed that the major contribution to the observed variability was due to encrustation of the burner tip which affected aerosol formation and flame shape and temperature. The use of EDTA reduced encrustation due to slightly soluble salts and line intensities could be maintained constant for 12 minutes of continuous solution feed. The same solutions, without EDTA, exhibited a significant falloff of intensity over the same length of time. If a suction feed system had been used, this fall-off would have been further complicated by changes in sample feed rate. RESULTS A N D DISCUSSION
Preliminary experiments showed that EDTA was not only effective in eliminating anion interferences in calcium determination but also gave an intensity considerably enhanced over that obtained for Ca(SOa)* or any other calcium salt studied. This enhancement is not due to cation-cation interaction of calcium with sodium in the flame from the E D T A (10). The ammonium salt of E D T A produces a smaller enhancement and does not appear to be as effective in eliminating
to01
80
-
I-
42ot
0
;
I
I
I
1 I
I
I
1
I
I
2o
if40'
I
OO
.02
,MOLES .04 PER LITER.OS EDTA
.08
0.1
Figure 2. Effect of varying EDTA concentration on calcium emission with sulfate, phosphate, and aluminum interferences
A
0.0006M Ca(NO&
0
0.0006M Co(NO8)z 0.0006M Ca(N0a)z 0.0006M Ca(NO3)z
X
anion interferences. Figure 1 shows the degree of interference by both sulfate and phosphate on the emission of a Ca(NO8)z solution, with and without EDTA. The calcium concentration in all these solutions was 6 X lO-*M (24 p.p.m.) with 0.1M EDTA. For the elimination of anion interferences in the calcium system the optimum EDTA concentration was about 0.1M. The effect of this variable is shown in Figure 2. The calcium emission is independent of the anionic species in the presence of excess complexing agent. Since these curves are relatively flat in the region of O.1M EDTA, its exact concentration is not critical (if a spectral scanning technique is used). At an EDTA concentration of 0.5M the noise resulting from background radiation became intolerable and the calcium intensity increased only 3y0 above the value for 0.1M EDTA. This background radiation is decreased by using the ammonium salt of EDTA. The effect of pH on the calcium emission of solutions containing E D T A with and without phosphate was studied. No variation in intensity was noted over a pH range of 4 to 9 either with E D T A alone or with phosphate added. This was contrary to expectations since the stability of the calcium-EDTA complex varies considerably over this range. This fact is useful from a practical standpoint in that it eliminates the necessity for precise pH control. I t does, however, indicate that the mechanism of the anion interference elimination is not simple. At p H below 3 severe burner encrustation, probably by the free acid of EDTA, caused sharp decreases in intensity, accompanied by a rapid fall-off in oxygen flow. Aluminum exerts a strong depressing effect on the emission of calcium salts,
and a study was made to see whether E D T A would eliminate this interference. Figure 3 shows that the aluminum depression is not satisfactorily removed for any Al to Ca ratio greater than 0.5. Figure 2 shows the effect of E D T A on the calcium intensity when aluminum is present. The result of these experiments indicates t h a t the mechanism of the depressing effect proposed by Alkemade and Voorhuis (1) may be correct. They state that calcium emission is decreased because of the entrainment of calcium salts in the refractory aluminum oxide. Since flame spectrophotometry is
1vu 90
70
MOLES P E R L I T E R AI(NO,I,
Figure 3. Effect of aluminum on calcium emission with and without EDTA
0 0.0006M Ca(NOh X 0.0006M Ca(NO&
+ 0.1 M EDTA
0.002M HzSO4 +++ 0.00047M HsPO4 0.0006M AI(NO&
widely used in biochemical analyses, the effect of protein material on the emissivity of calcium was determined. An enhancement has been reported (6) but solution feed rate was not controlled. Figure 4 indicates an enhancement which is linear with protein concentration. At 5% protein this enhancement is 60%. The addition of 0.1M E D T A does not eliminate the interference, but it does decrease the magnitude of the enhancement to 30%. It is believed that the action of the protein is mainly physical rather than chemical in nature, producing a change in the size of the spray droplets or of the solid crystals formed after evaporation, and that the small amount of organic material involved does not appreciably affect the flame temperature and cause enhancement via this mechanism. To establish the applicability of the proposed procedure the ability of EDTA to remove anion interferences was tested for a number of other elements. Magnesium was chosen because of the pronounced effect of anions on its emissivity and the usefulness of flame spectrophotometry in its determination. E D T A removed the influence of anions on the magnesium intensities as efficiently as for calcium as indicated in Table 11. This result is rather surprising in view of the relatively high magnesium concentration which was used. I n the case of calcium a 160fold excess of E D T A was necessary to suppress interferences, while for magnesium a 10-fold excess sufficed. It is believed that this result follows from the higher solubility of the magnesium phosphate and sulfate, so that the E D T A competes more efficiently for the magnesium ion during the precipitation process. Table I1 shows that the atomic line a t 2852 A. yields a more precisely measurable intensity than the VOL. 32, NO. 11, OCTOBER 1960
1473
out EDTA, was determined for the emission of cobalt, copper, chromium, and manganese (Table 111). The depressing effect of sulfate and phosphate is generally less for these four elements than for calcium and magnesium. I n all cases, 0.1M EDTA eliminates the interference. The enhancement of the emission by EDTA is much less for all these elements than for calcium. ACKNOWLEDGMENT I I
I
1
2
3
I 4
The authors gratefully acknowledge a fellowship grant to one of them (A,C.W.) from the General Electric Foundation.
5
WEIGHT X P R O T E I N
Figure 4. Effect of protein on calcium emission with and without EDTA
A 0.0006M
0
Ca(N03)z
0.0006M CO(N03)z
+ 0.1 M EDTA
3700-A. band. This reflects the experimental observation of a higher noise level in the region of the band, probably due to increased background emission a t the longer wave length, and the necessity of using a higher amplifier gain to detect the magnesium.
Table II.
Effect
LITERATURE CITED
Because of the overlapping sodium line a t 2852 A. it would be preferable to work with the potassium E D T A salt. The ammonium salt appears to be much less satisfactory for magnesium than the sodium salt (IS). The effect of anions, with and with-
of Anion Interferences on Magnesium Emission with and without 0.1M EDTA
2852 A. Line Without With 29 31 0.01M Mg(N08)z 0.035M HC1O4 0.01hl Mg(N0a)z 30 31 0.02M HCI 0.01M M d N O i L 28 31 0.00785iM 0.01% hfg(NO8)z 24 30.5 0.01M H&04 0.01M hlg( N 0 3 ) ~ 24 30.5
++ + +
Table 111.
+ + + 0.0012.W Cr(S03)3 0 OLM HC1 + 0 0012M Cr(N03)8 0 00785M &PO4 + 0 0012Jf Cr(NO& 0 01M HzS04+ 0 0012M Cr(hO& 0,0006M MnClz 0.0035M HClO4 + 0.0006M MnC12 0,00785M &PO4 + 0,0006M hclz 0,OlM HISO, + 0.0006M MnClz
ANALYTICAL CHEMISTRY
25.5 12 5 12 5
22 20
24
Effect of Anion Interferences
0 0006144 C u ( S 0 3 ) ~ 0 Olhl HC1 0 0006M Cu(N03)z 0 00785M HjPO4 0 0006M cU(Yo3) 0,OlM HzSOa 0 000634 Cu(N0j)z
1474
3700 A. Band Without Kith 24 22 31 23 5
Without EDTA 15.5 13.5 13.5 40 5 41 39 40
25 25 22 5 22 5 64.5
62.5 54 56.5
Kith 0 . lhf EDTA 17.5 18.5 17.5 44 5 45
44 5 44 5 27 5 28 27
27 73.5 74
73 73
(1) Alkemade, C. T. J., Voorhuis, 31. H., 2.anal. Chem. 163.91 (1958). (2) Baker, G. L., Jdhnson, L.’ H., ANAL.
CHEY.26. 46.5 I1 Q54) (3) Baker, 31, 2036 (1959). (4)TBeukelman,T. E., Lord, S. S., Paper h o . 32, Pittsburgh Conference on A4nalyticalChemistry and Applied Spec. ___ 2 troscopy, Marrh -, 19.5q (5) Bryan, H. A., Dean, J. A., ANAL. CHEM.29,128i9 (1957). (6) Chen. P. E>,. Jr.. Toribara. T. Y.. Ibad., 25, 1642 (1953). ( 7 ) David, D. J., Analyst 84, 536 (1950). (8) Dean, J. A., Cain, C., Jr., A K A L . CHEM.29,530 (1957). (9) Foster, W. H., Jr., Hume, D. S Ibzd , 31,2028 (1999). (10) Ibad., p. 2033. (11) Fukushima, S., Mzkrochzm. Acta 1959, 596. (12) Fu-iva, K., Thiers, R. E , Vallee, B. L., Baker, M.R , ANAL.CHEU.31, 2039 (1959). (13) Grewling, H. T., private communication. (14) Hemingway, R. G., Analyst 81, 164 (1956). (15) Pietzka, G., Chun, H., dngew. Chern. 71,276 (1959). (16) Pungor, E., Hegedus, A. J., X z k r o cham. Acta 1960, 87. (17) Schuhknecht, IT., Schinkel, H., 2 . anal. Chem. 162,266 (1958). (18) Wirtschafter, J. D., Science 125, 603 ’ (1957). (19) Yofe, J., Finkelstein, R., A n a l . Chzm. Acta 19, 166 (1958). RECEIVEDfor review March 30, 1960. Accepted July 2, 1960. Division of Analytical Chemistry, 138th hleeting, ACS, Sew York, K.Y., September 1960. Supported in part by the Lnited States Air Force under contract Sumber AF 49( 638)-184 monitored by the Air Force Office of Scientific Research of the Air Research and Development Command.
