Micro-EDTA Titration of Calcium Magnssium I nterference J. CH. VAN SCHOUWENBURG laboratory of Soils and Fertilizers, Agricultural State University, Wageningen, and lnstitute for Soil Fertility, Groningen, The Netherlands )The negative interference of magnesium in the EDTA titration of calcium when murexide is used as an indicator is probably caused by the adsorption and inactivation of calcium murexide by the magnesium hydroxide precipitate. This adsorbed dye-bound calcium is very slowly or not at all released. If Carbocel is used as a protective colloid, no hydroxide precipitates. This calcium determination with the proposed microtitration procedure is applicable to systems containing low calcium and high magnesium levels.
K
( 4 ) describe the interference of magnesium with the (ethylenedinitri1o)tetraacetic acid (EDTA) titration of calcium when murexide is used as an indicator. If more magnesium is present, less calcium will be recovered; if a t a given magnesium and calcium level more murexide is used, the negative magnesium interference will be diminished; with a lower calcium level the negative magnesium interference will be less pronounced. Kenny and Cohn point out, referring to the work of Diehl and Ellingboe (29, that if calcein is used there will be no negative magnesium interference. Contrary to this, Socolar and Salach (11) using calcein report a negative interference of magnesium the moment a magnesium hydroxide precipitate becomes visible; lowering the p H to 11.8 causes the magnesium interference to become positive-that is, more calcium will be recovered than is actually present. This paper proposes an explanation for these facts and describes a method for microtitration of calcium in the presence of large amounts of magnesium. It seems reasonable to expect a slight positive magnesium interference with the EDTA titration of calcium because the formation constants of both metals differ by a factor of 100 only. log KC.-EDTA= 10.6, log KYI-EDTA = 8.7. This positive magnesium interference actually is found when the azo dyes Eriochrome Blue SE and Eriochrome Blue Black R (Calcon) are being used (Table I) ENNY and Cohn
If instead of EDTA, ethylene glycol bis-(b-aminoethyl ether)-N,N’-tetraacetic acid (EGTA) is used as the titrant, the magnesium interference is not important, as shown by Ringbom et al. (IO). However, these authors used a different indicating system Zn-EDTA). The formation (Zincon constants of the calcium- and magnesium-EGTA complexes are, respectively, 11 and 5.4. Lowering the p H to 11.8 brings about a positive magnesium interference (4). At this p H less magnesium hydroxide will be formed, and the free magnesium ions are able to compete with the calcium ions for the EDTA. I n addition to this positive magnesium interference, which undoubtedly influences the titration when murexide is used, there seems to exist a superimposed negative influence of magnesium. Several authors (1, 8, If) attribute this negative influence to the coprecipitation of calcium during the formation of magnesium hydroxide. This, however, can hardly be the case, because neither Eriochrome Blue SE nor Eriochrome Blue Black R shows this phenomenon (Table I). Magnesium hydroxide adsorbs organic dyes and, presumably, also the metal indicators used with the EDTA titration. Calcon shows this phenomenon very distinctly: The solution is already hlue while the magnesium hydroxide precipitate is still colored red. I t seems probable that not only the murexide but also the calcium murexide will be adsorbed by the magnesium hydroxide, rendering the calcium thus adsorbed more or less inactive because the adsorbed calcium murexide releases its calcium very slowly or not a t all. Such a slow subsequent delivery of adsorbed dye-bound calcium is, although to a lesser extent, known from the experiments of Lott and Cheng (6, 7 ) working with Calcon. The assumption of the simultaneous adsorption of murexide and calcium murexide explains all the facts mentioned by Kenny and Cohn. Adsorption of calcium murexide by the magnesium hydroxide and the impeded subsequent delivery of its calcium are responsible for the negative magnesium
+
Table 1. Titration of 1000 y of Calcium in Presence of Increasing Amounts of Magnesium
Mg Added, y 0
12 36 60 120 600
1200
Calcium Recovered, % ’ EdoSE Erio R 100
100
101 101 102 103 104 105
102 103
~ . .
...
104 105
107
influence; if more murexide is used, relatively less calcium murexide will be adsorbed, which implies a decrease of the negative interference of magnesium; for the same reason, less calcium will diminish the negative magnesium interference; and the different behavior of the various indicators seems understandable in view of their structural formulas. Lott and Cheng (6, 7) repress the adsorption phenomena of the magnesium hydroxide by adding either gelatin or poly(viny1 alcohol). This author tried to reproduce their results using Wacker 28/20 as the poly(viny1 alcohol) instead of the Elvanol (Du Pont) used by Lott and Cheng. Perhaps because of this modification it proved impossible to reproduce their results. The magnesium, hydroxide precipitate still existed and produced a sluggish end point caused by the subsequent delivery of calcium from the adsorbed calciumCalcon complex. I n this laboratory Carbocel (sodium carboxymethylcellulose from Nijma, Nijmegen, The Netherlands) is used as a protective colloid for the EDTA microdetermination of calcium. By adding Carbocel the formation of a visible magnesium hydroxide precipitate is prevented. The consequences are obvious: When murexide is used, the negative magnesium influence is no longer encountered. If, however, the azo dyes are used, a vrry strong positive magnesium interference ciisues. Because the formation of magnesium hydroxide precipitate is prevented, the magnesium ions, by whatever cause, apparently will be in a position to VOL 32, NO. 6, MAY 1960
709
Sodium hydroxide solution (LY) Murexide (Llerck), 25 mg. of murexide per 100 ml. of distilled a a t e r . Several other qualities gave too high results because of impurities. This seems to be the reason why Kibrick et al. ( 5 ) found a high blank with their microtitration of calcium. The murexide solution has to be renewed everv other day. EDTA solution (10-3~Y)standardized against calcium chloride (calcium carbEnate hydrochloric acid). I
Table II.
Replicate Determinations of Calcium
Calcium.
Y
1.20 1.11; 1.11 1.1; Ll3 1.24 1.28 1.19 1, 2 3 1 ,24 1.2ti 1.19 1.2x Meail Std. dev., s Coeff. of v u
12.0 1.19 1.24 1.26 1.19 1.23
11.8 11.8 11.9 12.2 12.0 12.0
1.19 1.16 1.13 1.23 1.23
11.9 .11.8 12.2 12.2 12.0 11.6
11.8 12.0 11.9 11.7 12.0
12.2 12.2 12.1 12.2 12.4 12.0 0.20 1.65%
1.20 0.044 3.G7%
+
METHOD
influence the color of the azo dye indicator. This latter difficulty is not encountered when murexide is used, because murexide is not as sensitive for magnesium ions as the azo dyes. Wtli this in mind an attempt was made to develop a micro-EDTA titration of calciuni. Because of the micro quantities involved it was necessary t o use a colorimetric detector (Vitatron, Amsterdam). Use of Carbocel was necessary because the titrator reacted unfavorably on the magnesium hydroxide turbidities. Because of the Carbocel, the use of the azo dyes was eliminated and murexide was chosen as an indicator. The difficulty in observing
Table 111.
the end point of the murexide titration is of no concern with a good titrator. Interference of iron, nickel, cobalt, copper, zinc, manganese, and aluminum is, according to Pfibil ( 9 ) , readily suppressed by the addition of potassium cyanide and triethanolamine. However, i t is not advisahle to use reducing agents in this connection. Although ascorbic acid (3, 8) and hydroxylamine hydrochloric acid (8) aid the masking of iron and the heavy metals, manganese, if present, will be reduced. In this case it is not masked by the triethanolamine and will be titrated by the EDTA, resulting in too high calcium recoveries.
Interference of Some Elements with Titration of Calcium
Calcium,
y
1.2
Ion .hided, -, 200 Fe Xi
12
Mn hfn Mg
120 50 100 1200
Pb A1 Po4
20 100
12.0
Ca found, Y
Mg
Ion Added,
Ca found, y
Y
1.17 1.19
200
Fe
11.9
1.32 1.39 1.24 1.39 1.32" 1.32 1.26"
40 100
Mn
lln
12.4 12.8
hIg Pb A1 POI
12.3 12.3 12.2 12,4a 12.3
60
50 200 2400 5000
so,
The titration is slow because of drifting of the galvanometer needle. Readings were taken after t h p drifting had stopped. 0
REAGENTS
Table IV. Titration of Calcium with Increasing Amounts of a Mixture Of Interfering Metals
Calcium, 1.2
Mixture atlded,
nil.
0.2 0.5 1.0 2 0
710
e
Ca
found, y 1.34 1.37 L47 1 45
y
12.0 Llisture
added, ml. 0.2 0.5 1.u 2,0
ANALYTICAL CHEMISTRY
Ca found, 12.3 12.3 11.9 11.7
y
Triethanolamine. Dilute 25 ml. to a total volume of 100 ml, with distilled water. Potassium cyanide solution. Dissolve 3.2 grams of potassium cyanide in 100 ml. of distilled water. The potassium cyanide used in this laboratory contained impurities which caused high results. Therefore 3 ml. of, lO+N E D T A are added to the solution. Carbocel (0.5%) in distilled water. T o each 100 ml. of the solution are added 5 ml. of 10-3N EDTA.
To a 1- to 6-ml. neutral aliquot in a hard-glass test tube (Thermax, Leerdam, The Netherlands) add successively: 0.2 ml. of triethanolamine solution, 2 drops of potassium cyanide solution, 0.2 ml. of Carbocel solution, and 0.4 ml. of sodium hydroxide. Stir after each addition. Wait half an hour to give the masking agents enough time to react. Then add 0.1 ml. of murexide solution and make u p t o 8-ml. total volume with distilled water. Titrate with a Vitatron titrator to a colorimetric end point with 10-sN EDTA. A standardized addition of murexide and a constant total volume of 8 ml. give a n end point at about the same galvanometer reading. All the glassware has to be hard glass, cleaned with 4N nitric acid and distilled water. It is not advisable to use polyethylene containers for the storage of reagents. Stabilization of the indicator with a reducing agent is not allowed, in view of the manganese interference. All the reagents must be added separately. Attempts to combine the reagents prior to their addition decreased the reproducibility. Table I1 gives tIvo examplcs of replicate determinations. Standard deviations and coefficients of variation were calculated for two calcium levels. It is important to restandardize every time a new reagent is used. Because of impurities, the stsndard curve may be shifted parallel to its former course. I n case a new EDTA solution is used, the slope of the standard curve might change. INTERFERENCES
For a microtitration as proposed, mere dilution is sufficient tjo reduce the concentration of interfering metals below the critical level. Because dilution introduces a n extra error, the use of a micropipet for taking an aliquot of the sample under consideration is preferred. The interferences (Table 111) prove that calcium in wet-digested plant material as well as in soil extracts can be titrated without difficulty. Phosphate will not interfere, even without a chemical separation. The only elements which might cause errors are zinc and manganese, but thcsc clcmcnts are not expected to cauw
trouble in plant and soil extractions, hccause there always will be an excess of calcium in comparison.’ There still exists a slight positive magnesium interference. Only with another titrant (EGTA) and perhaps anothw indicator (calcein) could this intcrferrnce possibly be prevented. To see the cffect of combining interfering metals. a mixture was composed which contained 20 p.p.m. of iron, 5 p.p.m. of nickel. j p.p.n-,, of cobalt, 10 P.pam* Of zinc, P*P,lll*Of coppers *50 p.p.m. of manganese. 120 p.p.in. of
magnwium, 20 p.p.m. of aluminum, and 10 p.p,m. of lead. T o either 1.2 or 12.0 Y of calcium were added increasing amounts of this mixture solution (Table TV). LITERATURE CITED
( l& ) Bond, D’J 1954, 1236.M., Znd. (London)
(2) Diehl, H., Ellingboe, J. L., ANAL. CHEM.28,882 (1956). (3) Flaschkai H.,I-’iischel~R.9 2. anal. Chem. 143,330 (1954). ( 4 ) Kenny, A. D., Cohn, V, H., AXAL. CHEM.30,1366(1058).
( 5 ) Kibrick A. c., R O B & 51, Rogers, ~ 3 ~ ; , g ~ ~ EzPtl. ~ ~ cB‘o‘* -‘fed. 81, (6) Lett,, p, F., Cheng, K L,, C/ientist Analyst 46,30(1957). (7) Zbid., 48, 13 (1959) (8) Malmstadt, H. V., Hsdjijoannou, T.P., Anal. Chim. Acta 19, 563 (1958). (9) Pfibil, R. “Komplexometrie,” p. 74, Chemapol, krague, 1954. (10) Ringbom, A,, Pensar, G., Wanninen, E., Anal. Chim. Acta 19,525 (1058). ( 1 1 ) Socolar, s, J,, Salach, J, I,, CHEM.31,473 (1959).
RECEIVED for review Sovernber 5, 1959. Accepted Febrnary 20, 1960.
Determination of Free Magnesium Ions in Body Fluids Improved Methods for Free Calcium Ions, Total Calcium, and Total Magnesium MACKENZIE WALSER Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore 5, Md.
b A study is reported of all of the pertinent variables concerning the use of the metal-indicating dyes, Eriochrome Black T and acid ammonium purpurate (murexide), to determine free ion concentrations of magnesium and calcium, respectively. The color change on adding dye to a proteinfree sample, measured spectrophotometrically, yields an accurate estimate of the free ion concentrations, without changing them appreciably in the process of measurement. It i s shown theoretically and experimentally that, at constant pH and ionic strength, reciprocal absorbance is a linear function of reciprocal metal ion concentration. Heavy metals must b e removed prior to analysis for precise results. No interference occurs from other cations in body fluids. Dissociation constants of calcium murexide and magnesium-Eriochrome were determined a t varying pH and ionic strength. The variation with pH conformed to theoretical expectations. Improved complexometric methods are also presented for determining total calcium and total magnesium in body fluids. Using these methods, the recovery of added magnesium and calcium from plasma freed of these metals was complete, even in the presence of added phosphate.
S
and associates reported that certain dyestuffs change color in the presence of trace amounts of calcium or magnesium (18, 19). The CHWARZENBACH
spectrophotometric characteristics of these dyes and their metal compleses were analyzed in detail. The two best known dyes, murexide (acid ammonium purpurate) and Eriochrome Black T, are now widely employed as end point indicators in complexometric titrations of calcium and magnesium, respectively (17). Subsequently Raaflaub (15 ) showed that murexide can also be employed to determine the concentration of free calcium ions in solution. If the amount of dye added is small in relation to the metal ion concentration, the shift in equilibrium due to the dye itself is negligible. Raaflaub applied this method to the determination of free calcium ions in ‘spinal fluid. Others have subsequently applied i t to milk and plasma ultrafiltrates (12,16,20, 22)As employed by these authors, the method involves adding dye t o ultrafiltrate and comparing the observed absorbance t o a standard curve derived from solutions with varying calcium content whose composition otherwise resembles that of the unknown sample as closely as possible. Although the shape of the standard curve varies with p H and ionic strength (16), this variation has not been analyzed in the physiological range of these variables. The effect of the various ionic species present in plasma or milk ultrafiltrate on the reaction of calcium with murexide has not been studied, with the exception of magnesium ions. The results of calcium ion determinations in normal plasma ultrafiltrate (12, 16) have con-
firmed the c1assic:d studies of 1IcLc:rn and Hastirigs ( 1 4 ) using the frog heart. Both techniques iiidirate that all but ct small portion of the nonprotein-bound calcium present in plasma is free and ionized. Definitive studies of magnesium ion concentration in body fluids have not been reported. Christianson, Jenness, and Coulton (6) employed a n ion exchange technique to estimate approximately the magnesium ion concentration in milk. This procedure requires a sample volume of several liters. I n this report, the use of metal-indieating dyes to determine free cation concentrations is re-examined. Linear relationships exist between reciprocal absorbance and reciprocal metal ion concentration both for calcium in the presence of murexide, and for magnesium in the presence of Eriochrome Black T. The intercepts of these straight lines permit evaluation of the respective indicator constants and absorptivities.’ The chief difficulties in applying these dyes t o the determination of free metal ion concentrations in body fluids are the dependence of the reactions upon pH, necessitating the handling of biological samples without exposure to air, and the interference caused by traces of heavy metals, for which both dyes have great affinity (1-3). These difficulties have been overcome by the addition of a n organic amine (tris) as a buffer and by removing traces of heavy metals prior to analysis. With these modifications, the two methods VOL. 32, NO. 6, MAY-1960
71 1
