be revealed easily by any other technique. ACKNOWLEDGMENT
The author acknowledges with gratitude the patient help of L. P. Adda in obtaining the infrared spectra. The Sic14 purification was done by C. E. Shoemaker. REFERENCES ( 1 ) Beachell, H. C., liatlafsky, B., J . Chem. Phys. 27, 182 (1957). (2) Beattie, I. R., hlcQuillan, G., J . Chem. SOC.1962, 2072. (3) Bethke, G. W., M'ilson, M. K., J . Chem. Phys. 26, 1107 (1957). (4) Clabaugh, W. S., e ! al., J . Res. Nat2. Bur. Standards 55, 261 (1955). ( 5 ) Crawford, Y.A., et al., J . Chem. Phys. 37, 2377 (1962). (6) Curl, R. F., Jr., Pitzer, K. S., J . Am. Chem. SOC.80. 2371 11958). (7) Delwaulle, I f . L.; et bl., J . Phys. Radium 15, 206 (1954).
(8) Dove, M. F. A., et al., Spectrochim. Acta 1962,267. (9) Drake, J. E., Jolly, W. L., J . Chem. Soc. 1962. 2807. (10) Gibian, T. G., McKinney, D. S., J . Am. Chem. SOC.73,1431 (1951). (11) Griffiths, J. E., J . Chem. Phys. 38, 2879 (1963). (12) Hawkins, J. A , , et al., Ibid., 21, 1122 (1953). 113) Hawkins. IT. J.. Camenter. D. R.. ' J. Chem. Phis. 23, i700 (i955). ' (14) Jam, G. J., Mikawa, Y., Bull. Chem. SOC.Japan 34,1495 (1961). (15) Johannesen, R. B., et al., J . Res. Natl. Bur. Standards 53, 197 (1954). (16) Kriegsmann, H., et al., 2. Chem. 1, 346 (1961). (17) Lindeman, L. P., Wilson, &I. K., Spectrochim. Acta 1957, 47. (18) SfcConaghie, V. M., Nielsen, H. H., J . Chem. Phus. 21.1836 119531. (19) McKean,"D. C., Schatz, P: N., Ibid., 24,316 (1956). (20) Meal, J., Wilson, M. K., Ibid., p. 385. (21) Moszynska, B., Bull. Acad. Polon. Sci. 1111) 5. 819 (1957) fC.A.. 52. 3515~1. (22) Kixon, E. R., J . Phys. Chem. 60, 1054 (1956). I
.
~
I
.
I
I
(23) Overend, J., Scherer, J. R., J . Chem. Phys. 32, 1297 (1960). (24) Reimert, L. J., Rand, PIT. J., J . Electrochem. Soc., in press. (25) Robinson, C. C., et al., Proc. Roy. Soc. (London)A269,492 (1962). ( 2 6 ) Schindlbauer, H., Steminger, E., Monatsch. 92,868 (1962). (27) Schumb, W. C., Stevens, A. J., J . Am. Chem. SOC.72, 3178 (1950). (28) Smith, A. L., J . Chem. Phys. 21,1997 (19.53). (29) Straley, J. W., et al., Phys. Rev. 62, 161 (1942). (30) Theuerer, H. C., J . Electrochem. SOC. 107,29 (1960). 1311 Tindal. C. H.. et al.., Phus. " Rev. 62. ' i b i (1942). (32) Tsekhovol'skaya, D. I., et al., Zavodsk. Lab. 25,300 (1959). (33) Tsekhovol'skaya, D. I., Zavaritskaya, T. A., Zh. Analit. Khim. 16, 623 (1961). (34) Zavaritskaya, T. A . , Titan i Ego Splavy, Akad. A'auk S S S R , Inst. illel. 1961, 195. (35) Zavaritskaya, T. A., Zevakin, I. &4., Zhur. Prikl. Khim. 34, 2783 (1961). \ - - - - I -
RECEIVEDfor review July 18, 1963. Accepted September 10, 1963.
Sensitive land Selective Spectrophotometric Reaction for Determination of Trace Amounts of Calcium MANOLITA HERRERO-LANCINA' and T. S. WEST2 Chemistry Department,, The University of Birmingham, Birmingham 7 5, England
b The reagent Calcichrome, cyclotris-7-( 1 -azo-8-hydro.~ynaphthalene-3, 6-disulfonic acid), provides a method for the spectrophotometry of calcium down to the 0.1-p1.p.m. level. At p H 12 and 61 5 mp the procedure has a molar absorptivity of 7600. The method may b e applied in the presence of the other alkaline earths-e.g., 5000 bg. of Ba+-with some reduction in sensitivity. Several hundred micrograms of metal!; such as AI, Pb, Zn, Co, Hg, and Cd do not interfere under specified conalitions, but M g +* and interfere. The color system develops within I5 to 20 minutes and maintains an unchanged absorbance for more than 24 hours. The reagent solution is also stable.
A
many rrethods have been proposed for the spectrophotometric determination of cdcium, in general the majority are subject to interference from other ions, and are rather unstable or not very sensitive vhen compared to spectrophotometric pi*ocedures for the determination of othei. metals. Sandell ( 7 ) has recently reviewed and discussed these methods. The oxalate procedure is a very indirect one and is subject to rather restrictive conditions. It is dependent on initial complete separation LTHOUGH
of calcium oxalate with minimum coprecipitation of oxalate ion and i t involves a filtration or centrifugation step. The chloroanilate method is less restrictive, but also involves filtration. The most advantageous procedures so far devised appear to be those based on murexide (ammonium purpurate), phthalein complexan (2,6-xylenolphthalein-a, a'-bisiminodiacetic acid), and glyoxal bis(2-hydroxyanil). The murexide method appears to be applicable in the range 1 to 3 p.p.m. of calcium, but a high concentration of reagent must be used to ensure quantitative formation of the calcium complex. Unfortunately, the reagent is unstable and ea. 50% decomposition occurs over 4 hours a t room temperature a t the p H of determination. Practically all heavy metals interfere and the tolerance for strontium and barium is 1 and 5 p.p.m., respectively. Magnesium also forms a color and >400 p.p.m. of sulfate interfere. The results obtained by the method appear to be somewhat lacking in reproducibility, though a more detailed and reliable method has been reported (3, 5 ) . The phthalein complexan method is also nonselective and the color system is unstable, so that the absorption must be measured immediately under carefully prescribed conditions.
Undoubtedly the best reagent to date appears to be glyoxal bis(2-hydroxyanil). According to Williams and VVilson (9), the method based on this reagent operates in the range 0.5 t o 10 pg. per ml. The color system also lacks stability, but when extracted into chloroform it remains unchanged for 15 minutes. Centrifuging of precipitate is required, but removal of liquid is avoided by extraction with chloroform and clarification of the extract by centrifuging. The color of the extract obeys Beer's law in the range up to 10 pg. (0.5 to 10 p.p.m.) and the calibration curve appears to be fairly reproducible, though it must be checked whenever used. The information about interferences is limited. Ten times the concentration of magnesium, or one tenth the amount of strontium or of iron, does not interfere with the determination of 210 pg. of calcium. Strontium gives a color with the reagent, but the addition of carbonate is apparently successful for amounts I: 1 to 10 ratio. Earlier papers on the use of the reagent (4) state that in the presence of carbonate ion, cobalt and nickel give red precipitates with the reagent, and On study leave from the University of Zaragosa, Zaragoza, Spain. 2 Present address, Imperial College, Universit of London, South Kensington, London, g.W.7. VOL. 35, NO. 13, DECEMBER 1963
2131
;Ir4 a
$0.8 9
T
0.6
0.4
0 '2
400
rnl
500
600
700
Figure 1. Absorption spectra of Calcichrome and alkaline earth complexes A.
E.
+
copper and cadmium form colored compleses; only the cadmium complex is extracted into chloroform. A highly selective colorimetric reagent for calcium, cyclotris-7-( 1-azo-8-hydrosynaphthalene-3,6-disulfonic acid) (Calcichrome), has been synthesized and examined by Close and West ( 2 ) . It was reported that, qualitatively a t pH 12 to 13, of some 37 cations esamined only calcium produced a soluble colored product with the reagent. Magnesium, strontium, and barium did not produce colors. The reagent was used at pH 12 for the complesometric titration of calcium in the presence of soluble barium with trans-1,2-diaminocyclohexane-X,Ar,X ',N'-tetraacetic acid as titrant. I n addition. i t mas used as a spot test a t a dilution limit of 1 to 1,000,000. The interference of heavy metal hydroxides, which tend to adsorb reagent, was prevented by addition of sulfide ion. At this time, the esact structure of the reagent is not finally decided, but it is thought to be:
Work is now in progress to establish the exact identity of the molecule by synthetic and degradative techniques, and will be reported in a subsequent communication. The unusual selectivity of this hydroxyazo compound may, according to our present information, be attributed to the cyclic arrangement of the o-hydroxyl groups 2132
+
10 ml. of 1 O-% Calcium complex. 10 ml. of 1 O-3M Ca+2 10 ml. of p H 1 2 buffer per 100 ml., 4-cm. cuvettes Calcichrome. Same as A, but without Ca+2
ANALYTICAL CHEMISTRY
Calcichrome
which, with the azo nitrogens, form a clathrate network or chelate cage into which only ions of a certain size may fit. Thus, within the alkaline earth group, while chemical affinity doubtless has its role, there can be little doubt that the decisive factor which allows calcium to react, but not barium and strontium, is the ionic radii of the ions concerned. For n = 6 these are Ca+2,0.99A.; Sr+2,1.12A.; andBa+2, 1.34 A. Beyond this group, other ions which might fit into the reagent are bound as anionic compleses or are insoluble a t p H > 12 (8). Several other ions react with the reagent a t p H 10, but the reaction is restricted to calcium a t p H 2 12. I n view of the need for a sensitive and selective reagent capable of producing stable colors 15-ithcalcium, i t was decided to apply the above reagent to its spectrophotometric determination. EXPERIMENTAL
Reagents. 10-3M Calcichrome, 0.5916 gram of t h e sodium salt per liter of distilled water. The solid dissolves readily with efficient stirring and is stable in solution. The reagent was prepared as described ( 2 ) . It is available commercially from Burdick-Jackson Laboratories, 1953 South Harvey St., Muskegon, Mich. l o - 4 x Calcium, prepared by dilution of a 10-lllf calcium solution. [The latter was prepared by dissolving 10.009 grams of CaC03 (AR) (analytical reagent grade) in the minimum amount of hydrochloric acid (iiR) and diluting with distilled lvater to 1 liter.] This solution contains 4 pg. of C a T 2per nil. p H 12 Buffer, 3.002 grams of glycine (AR) 2.34 grams of KaC1 (-1R) in 500 ml. of water. This solution is then mixed with 3.0 grams of r a O H dissolved in 500 ml. of diqtilled water.
+
When used as prescribed, this buffer produced color solutions with a final p H of 12.0. It is essential that the p H be maintained a t 12 and consequently the buffer and its performance should be checked periodically with a p H meter. Solutions for Examination of Interferences. Barium, 1.5 X 10-3M, prepared from Johnson Matthey Specpure barium carbonate. This solution contains 200 pg. of Ba+* per ml. Strontium, 2.3 X 10-3V, prepared from Specpure strontium carbonate. This solution contains 200 pg. of Sr+2 per ml. Other Cations. 10-3Jf solutions of zinc sulfate, cadmium sulfate, copper sulfate, magnesium sulfate, aluminum sulfate, lead nitrate, mercury(I1) nitrate, and cobalt nitrate. Anion Solutions. potassium cyanide (1%) in water, 1% sodium sulfide in water, etc. Apparatus. Unicam SP. 600 spectrophotometer and Hilger & \f7atts Uvispek with 4-cm. cuvettes. The spectra s h o n n in Figures 1 and 2 were measured on a Hilger Ultrascan automatic recording spectrophotometer in 4-cm. cells. Procedure. ClLIBRATION C U R V E , 10 to 70 pg. of Ca+*. Aliquots (2.5 to 17.5 ml.) of 10-4M calcium solution were pipetted into 100-ml. standard flasks plus 10 ml. of p H 12 buffer qolution, followed by 10 ml. of 10-3AlI Calcichrome reagent solution. The contents of the flasks mere then diluted to 100 ml. d blank solution containing the standard amounts of buffer and reagent only n as prepared simultaneously and all Tolutions Jvere allowed to stand for 1 hour. The absorption of the blank solution vias then measured in turn against zero absorbance set on each of the calcium-containing solutions in 4-em. cuvettes a t 615 mp in the spectrophotometer. The calibration curve is a straight line from 10 to 70 pg. of Ca+2 with the absorbance ranging from 0.075 to 0.535. CSLIBRATION CURVE, 2 to 10 pg. O f Ca+2 Aliquots of 10-43f calcium solution containing from 2 to 10 pg. of Ca+* were treated in 25-ml. flasks with 2 ml. of pH 12 buffer and 2 ml. of 10-3M Calcichrome and diluted to 25 ml. The solutions were then allowed to stand for 1 hour and measured a t 615 mM in 4-cm. cuvettes as described above. The absorbance ranges from 0.075 to 0.290. DETERMINATIOK OF UNKI~OWN CALCIUM SOLUTIOSG.The procedure appropriate to the ranges 2 to 10 or 10 to 70 pg. of calcium is used. When other metals are known to be present, the calibration curve should be drawn up with an approximately equivalent amount of that element present in the blank and calibration solutions. If the metal is one such as Co, Cd, or Cu, which is likely to precipitate out a t pH 12, sufficient K C N should be added before the buffer and reagent throughout the procedure. It is also appropriate to check the pH of the solutions for rise of p H when appreciable amounts of cyanide are used.
L 10 40 0
mP
500
600
50
30
70
.
/4g. co
700
Fiaure 3. Calibration curves " Figure 2. Absorption spectra of Calcichrome and alkaline earth A. to 7o pg. of Ca+2 by recommended procedure a t 6 , m p complexes 6. 10 to 7 0 pg. of Ca+2 b y recommended procedure a t 5 1 0 m p A. 6.
C.
D.
Calcium complex as in Figure 1 A Calcichrome strontiuln. 2000 pg. of Sri2 10 mi. of 1 0-3M Calcichrome 10 ml. of p H 1 2 bufFer per 100 rnl., 4-cm. cuvettes Calcichrome. As in 6, but without Sr barium. As in B, but with 2000 pg. of Bat2 in place of Sr t 2 Calcichrome
+
+
+
C.
D.
10 to 70 pg. of Caf2 in presence of 4 7 0 pg. of
a t 61 5 mp (reagent blank) Like c, but measured against reagent blank containing 470 pg. of A l t 3
+
Calibration curves n ith more poqitive slope and a wider range may be obtained by using larger amcunts of reagent solution-e.g., 15 or 20 ml. of 10-3,u Calcichrome. The procedure mentioned here is designed for the average small spectrophotometer used in routine laboratories-the Unicam S.P.600 is sufficiently qensitive to cole with 15 mi. of 10-3X Calcichrome per 100 ml., but not with 20 ml. Where KCN is used it must be added before the buffer and reagent. It is also important to add buffer before reagent n hen the method is applied generally. If the spectrophotometer w e d operates its check control via slit adjustment rather than electronically, a slightly sigmoid-shaped calibration curve may be obtaincd because of the slightly different 1%-idtliof the spectrum scanned over the range of measurements. K e ourselve, have not observed thi- phenomenon. DiSCUSSlON AbID RESULTS
The optimum p H conditions for applying the reagent to .;he determination of calcium in pure solution were first established by adding varying amounts of sodium hydroxide to a solution of calcium and the dyest iff and measuring the pH with a pH mt3ter following absorption measurements at the viavelength of maximum alxorption. These studies revealed that the maximum sensitivity was obtained at pH 12. When the method p;as placed on a routine basis subsequently, it was found preferable to replace the sodium hydroxide solution n ith a glycine-sodium hydroside buffer a t p l l 12, since varia-
tions in pH obtained with sodium hydroxide alone affected the reproducibility of the method from day to day. The wavelength of maximum absorption of the calcium complex of the reagent lies a t 520 to 530 mp a t p H 12 and a t 540 mp at p H 11, while that of the reagent is a t 615 and 610 mp, respectively. From the data obtained (Figure 1) i t is obvious that the formation of t'he complex may be measured positively a t 510 mp or three to four times more sensitively at 615 mp by folloviing the decrease in the absorption band due to the reagent. The calcium complex also exhibits a n interne peak in the ultraviolet region a t 299 mp (pH 12) or 300 mp (pH l l ) , while the reagent shows a strong band a t 310 mp at both pH values. However, no advantage may be gained by measuring the complex in this region because of the high background absorpbion. To obtain positive readings for the calibration curve a t 615 mp t'he normal measurement process of spectrophotometry was reversed-Le., the blank solution was measured against zero absorbance set with the test solution in each case. The stability of the color sy;t em was then examined by preparing a solution of the calcium complex under normal conditions of determination, and nieasuring its absorption against the reagent solution over a period of several hours, taking due precaution against absorption of carbon dioxide. This study re7;ealed that the color system had developed quantitatively within 15 to 20 minutes and thereafter maintained an unchanged absorption for 24 hours. For the sake of uniformity, the color system was always allowed to stand for
-
1 hour in all subsequent determinations, although it is apparent that the time factor is not critical. Solut,ions allowed to stand in open cuvettes absorb atmospheric carbon dioxide and decrease in pH and in absorbance accordingly. This effect becomes apparent' with open cuvettes after 1.5 to 20 minutes. Initial experiment3 v-ere carried out with 10-3-11 solutions of calcium and Calcichrome, thus obtaining calibration curves with 1-cni. cuvettes in the range u p to 400 pg. of Ca-2. Subsequently, similar experiments n-ere performed with 10-4M solution- is e615,p = T600. or eS,Ornp = 4500. According to the terminology of Saiidell ( C ) , this represents a -ensitivity index of 0.005pg of Ca+' per q.cm. a t 615 mp, or 0.009 pg. of Ca+* per >q. rm. at, 510 nip (Figure 3). The reagent -elution was -talde and the calihation curvei n-ere reproducible from day to day and with different^ preparations of reagent solution. Effect of Foreign Ions. Since i t had h w n established qualit'atively ( 2 ) that a wide range of foreign cations do not form colored products with the reagent or int,erfere in any way with the VOL. 35,
NO. 13,
DECEMBER 1 9 6 3
0
2133
calcium chelate except in some cases where precipitation occurs, only a few typical cations were examined in these studies: aluminum, cadmium, cobalt, copper, lead, mercury(II), zinc, barium, strontium, and magnesium. In these experiments with the first-mentioned seven metals, the effect of the addition of 400 to 600 pg. of each was tested on the recovery of 30 to 50 pg. of calcium. Potassium cyanide was added when Cd, Co, Cu, and Hg(I1) were examined to prevent their precipitation, but none was necessary where aluminum, zinc, and lead were involved, since these are soluble a t p H 12. The results obtained in all cases were almost identical, but the absorbance was lower than the value predicted by the calibration curve. When a blank solution containing the foreign ion, but no calcium, was prepared and employed in the usual way, the straight-line graphs obtained were identical in all cases and passed through the origin (Figure 3). At 615 mp in the presence of these ions the molar absorptivity was reduced from e6lJmp = 7600 to 4700. It was apparent from these results that while none of these ions interfered by producing colored complexes with the reagent, or by preventing color formation with calcium when present in 10- to 20-fold amounts, they did reduce the sensitivity of the color reaction. The curves for calcium obtained in their presence were virtually identical in all cases when measured against a blank containing the same amount of the foreign ion. A similar effect is obtained by adding varying amounts of an indifferent univalent-univalent electrolyte such as sodium chloride. Figure 4 shows the effect produced by determining 40 pg. of calcium against a reagent blank in the presence of 0 to 100 mg. of sodium chloride in a final volume of 100 ml. This measurement was made a t 510 mp, corresponding to direct formation of the red calcium complex, rather than a t 615 mp as for analytical measurements, and with 15 ml. of 10-311f reagent solution instead of the usual 10 ml. The value of €510 m p decreases from 5200 in a medium of ionic strength p = 0.01 to 2800 a t p 0.03. Accordingly, when calcium is to be determined in the presence of any of the above metals (and presumably many others), a roughly similar amount should be incorporated in the blank and standard solutions used to prepare the calibration curve to compensate for this electrolyte effect. As will be seen from Figure 4,the amount used is not very critical. An alternative procedure could probably be devised by incorporating indifferent electrolyte in the buffer or reagent solutions. Finally, where cyanide addition is necessary--e.g., in the presence of Cu, Co, or Cd, where the ions must be held 2 134
ANALYTICAL CHEMISTRY
I
20
Figure 4.
Effect
40
mg.bci
in solution-it should be added before the reagent and buffer. The effect of barium and strontium is of particular interest, since the determination of trace amounts of calcium in their presence is of the utmost importance. I n this instance the amount of these metals added was extended from 200 to 5000 pg. This spectrophotometric study showed that at 615 mp, and to a lesser extent a t 510 mp, these ions give a slight positive reaction, but the effect of 200,2000, and 5000 pg. is by no means proportionate. I t is indeeddoubtful that a colored complex is formed with barium or strontium, and even so, the compounds formed have a nearnegligible molar absorptivity a t any known wavelength. Figure 2 shows the absorption spectra of the reagent in the presence of 2000 pg. of Sr and Ba and 400 pg. of calcium. This graph illustrates the nonreactive depressant action of barium and the slight positive effect of strontium when present in large amounts. The over-all effect of the large amount of strontium is to broaden out the reagent band a t 530 to 630 mp. Figure 5 shows the calibration curves obtained for calcium in the range 10 to 70 pg. when 2000 pg. of Ba+2 or were present, and when measurement was made against a blank containing a similar amount of the appropriate ion. The barium curve is closely similar to that obtained with
PLCO
40
60
Figure 5. Calibration curves in presence of barium and strontium A. 6.
C.
Calcium b y recommended procedure (reagent blank) a t 615 mp a t p H 12, 4-cm. cuvettes Like A, in presence of 2000 pg. of Ba t z (reagent f 2000 pg. of Bo+* blank) like A in presence of 2000 pg. of Srtz 2000 pg. of Sr" blank) (reagent
+
80
100
of added electrolyte on sensitivity of color reaction
+
40 fig, of C a f P 15 ml. of 1 O-s M Calcichrome per 100 ml., 4-cm. cuvettes a t 51 0 mp
zo
60
+ 10
ml. of p H 12 buffer
+
mg. of N a C l
400 to 600 pg. of the previous metals, but that for strontium shows some divergence. This is not apparently due to the calcium content of the strontium carbonate used (1 p.p.m. of Ca), since the barium carbonate had a higher calcium content (2 p.p.m. of Ca). However, these graphs show clearly that it is possible to determine calcium in the presence of large amounts of barium or strontium without resort to separation, extraction, or masking. The addition of large amounts of magnesium is detrimental, however. When magnesium is incorporated in the blank, a straight-line graph through the origin is obtained, but its gradient is low and its analytical value is probably very small. There is little doubt that the magnesium interferes by coprecipitating calcium and probably Calcichrome also. Small amounts of M g f Z ions-e.g., 25 pg.-caused high results, but the correct C a f Z recovery was obtained when similar amounts of Mg+2 were incorporated in the blank. Finally, the effect of a few selected anions was examined. Calibration curves for 10 to 70 pg. of calcium were obtained in the presence of 4800 pg. of SOk-', 1300 pg. of S-', 3300 pg. of F-, and 5300 pg. of CN-, as described for the cations. The same electrolyte effect was noted, and when similar amounts were incorporated in the blank solution, the results were virtually identical in all cases, and with those obtained previously with Al, Pb, Cd, Cu, etc. For example, the curve for calcium in the presence of 3300 pg. of F- is exactly superimposed on the curve obtained with 2000 pg. of Ba+*. Studies with 4700 pg. of PO4+ yielded considerable interference. -4straight-line calibration curve was obtained, but the gradient is too low to be of any analytical significance. Small amounts of phosphate (
