Patrick G. McCormick Marquette University Milwaukee, Wisconsin 53233
Titration of Calcium and in
Milk with EDTA
i n the undergraduate analytical chemistry laboratory, complexometnc titrations employing EDTA are invariably encountered. In those experiments where samples other than typical commercial unknowns are used, the determination most commonly run seems to be water hardness. Interest seems greater in those instances where the student collects his own sample, but the analysis of calcium and magnesium in water presents little challenge to him except in cases of rather complicated waters, and such exciting samples are not always readily available. A sample which we have found to he interesting and somewhat challenging to students is milk. The titration of calcium and magnesium in milk and dairy products is seriously complicated by the presence of proteins and orthophosphate ions, which precipitate calcium and magnesium a t the high p H required for titration with EDTA. These interferences can be removed by precipitation with potassium metastannate ( 1 , 21, yielding a clear filtrate in which calcium and magnesium may be determined. This filtration is time consuming, and the quantity of sample must be corrected for the volume occupied by the precipitate in the volumetric flask (1, 2). Another means of remaving phosphate ions is anion exchange (>5), but application of this technique to milk requires prior removal of protein by precipitation or ashing. Elution of the ion exchange column is also time consuming, and yields a very dilute sample for titration, leading to indistinct end points. Kamal has shown ( 6 ) that addition of excess NazEDTA to a neutral or slightly acidic solution containing calcium, magnesium, and phosphate ions causes chelation of calci-' um and magnesium, preventing their precipitation with phosphate when t h e solution is made alkaline. By backtitrating the excess EDTA a t pH 10, the EDTA complexed with the sum of calcium and magnesium can be calculated. If the titration is performed a t p H 12 or bigher, magnesium is released from its complex with EDTA and precipitates with phosphate and/or hydroxide. The back-titration in this instance determines t h e EDTA complexed with calcium alone. These data, together with a blank determination, allow calculation of both calcium and magnesium in the sample. Kamal ( 6 ) applied this method to milk a n d found no interference from the proteins present. Ntailianas and Whitney (7) employed essentially the same idea to milk using a single indicator, Calcein, with similar findings! Since beginning students often ex~eriencedifficulties with fluorescent indicators, the experiment is based primarily upon the method of Kamal ( 6 ) . It should be noted, in light of the above discussion, t h a t this is only one possible approach to the determination of calcium and magnesium in milk, and represents, possibly, the simplest method. More challenging approaches are certainly available. Experimental Reagents
Standard Magnesium Solution. Dissolve 0.2 g (accurately weighed) of reagent grade magnesium turnings in the minimum 136 /Journal of Chemical Education
amount of dilute HCI (about 3 ml of 6 M and dilute to 1 I. This solution contains about 0.2 mg Mg per ml. Standard Calcium Solution. Dissolve 2.5 g (accurately weighed) of previously dried reagent grade calcium carbonate in dilute HCI, and dilute to 1 I. This solution contains about 1 mn. Caper ml. EDTA Solution. Dissolve 15 g of reagent grade disodium EDTA in water and dilute to 1 1. The solution will be standardized against the standard Mg and Ca solutians. The titer of this solution is about 1.6 mg Ca per ml and just under 1 mg Mg per ml. Ammonia Buffer, pH 10. Dissolve 67.5 g of ammonium chloride in 200 ml distilled water, add 570 ml of reagent grade concentrated ammonium hydroxide, and dilute to I I. Basefor High pH Titrotion. 0.5 NKOH or NaOH. Mupnrrium I n d ~ o t u r . Calmngitc; either a dr). preparatmn (Mnllinrkrodt Chemical Worksj. or n O.U5% solutlm in water of the w r p material !(;. F. Sm:th Chemrcal C o . , . ~hleiumIndicator Hydroxy Naphthol Blue: dry preparations are available from Mallinckrodt Chemical Works and Fisher Seientific Ca. Procedure
Calcium. Pipet 5 ml of milk into a 125-ml erlenmeyer flask; pipet 10 ml of the EDTA solution into the flask, and mix thoroughly. Add 15 ml of 0.5 N KOH to the sample to adjust the pH to about 13, and add 200-300 mg calcium indicator. The color of the solution will change from white to light blue. The excess EDTA is back-titrated with standard calcium solution until the color changes from blue to violet. Dilution with water should be avoided as much as possible. The volume of EDTA reacted with calcium is determined from the difference between this titration and a similar titration where water is substituted for the milk. The amount of calcium in the sample is calculated by multiplying the volume of EDTA complexed with calcium by the calcium titer of the EDTA, which can be determined from the blank titration. Magnesium. Pipet 5 ml of milk and 10 ml of EDTA into a 125-ml erlenmeyer flask and mix thoroughly. Add 10 ml of ammonia buffer and 200 mg of magnesium indicator (or an appropriate volume of indicator solution). The color of the solution will change from white to light hlue. To minimize loss of ammonia, do not add the buffer and indicator until just before titrating. Titrate the sample with standard magnesium solution until the color changes from hlue to red-violet, avoiding dilution with water. From the difference between this titration and a blank titration, run with water instead of milk, determine the volume of EDTA complexed with the sum of calcium and magnesium. Subtract the volume of EDTA which was found to be eompleaed with calcium in the previous experiment, and multiply the result by the magnesium titer of the EDTA to obtain the amount of magnesium in the sample. Discussion
In the calcium determination, adjustment of the pH with KOH instead of a buffer was found to be easier, and is a reasonably common practice (1, 2, 7). Buffers have and often use cyanide as one of been used by others the buffering compounds. It was the desire to avoid use of cyanide in the student laboratory which led to the use of hydroxide alone. Other buffer materials are certainly' available, but are not really any better buffers a t high p H than is cyanide. The choice of indicator materials was governed by experience and ease of student use. Calmagite has given us
(a,
very good results as a general substitute for Eriochrome Black T, and was suhstituted in this experiment. The desirable qualities of Hydroxy Naphthol Blue include its stability and the similarity of the color change to that of Calmagite. Students have no difficulty recognizing this change, while some of them do have troubles with Calcein, which is best used with specialized lighting. Of course. Calcein can he suhstituted in the calcium determination (@, and has been successfully used for determination of hoth calcium and mamesium in milk (7). A prohleni which must he acknowledged is the fact that the end point color change appears somewhat different in titrations of milk and hlank titrations, due to the opaque background provided by the milk, the hlank solution remaining clear. This fact was also recognized by Kamal (6) who minimized the difficulty by noting that a sharp change in color is observed at the end point in either case. This may he true for the trained eye, hut students are generally less than enthusiastic about the sharpness of color changes experienced with metalochromic indicators, and any differences between titrated solutions which affect the color of the indicator just make the situation worse. In a desire to ease this difficulty, we sought ways of making the solutions similar in appearance. It has already been noted that problems arise in clarifying the milk sample, so we tried, instead, making the hlank opaque. One material which seemed particularly appropriate for this task was non-dairy coffee creamer. These products, especially the liquid versions, simulate the physical character.istics of milk very well. The listed ingredients on several hrands led us to believe that they were entirely free of materials which would complex with EDTA, rendering them ideal for the desired purpose. To our surprise, titrations using 5 ml of liquid coffee creamer required about 1.5 ml less titrant (magnesium standard solution) than those using water in place of milk. Thinking this was due to differences in appearance of the end points several titrations were done with differing amounts of creamer. A linear relationship was found between the titration volume and amount of creamer used. The titration required about 0.3 ml less titrant per ml of coffee creamer taken than did a water hlank, indicating that the creamer was
complexing with EDTA. This was found t o he true'for two hrands of liquid coffee creamer, and four hrands of the dry powdered product, used in water solution in the concentration recommended as a suhstitute for cream. Emission spectrographic analysis of three hrands of coffee creamer identified the presence of hoth calcium and iron, two ions which could cause the observed difficulty. The addition of cyanide to the buffer caused only a slight improvement, however, indicating that the major interfering substance is probably calcium. This ion could he present as an impurity in the sodium caseinate used in preparation of these products, assuming the source is cowb milk in which casein probably exists as the calcium salt (8). The only method which can he suggested for successful use of non-dairy coffee creamer to make the blank resemble the sample is to add the creamer to the EDTA reagent. Any reduction in concentration caused by the creamer would not he noticed since the reagent is standardized. On the other hand, the coffee creamer makes an excellent sample for the student seeking - an extra challenge. We did not use anvthing to make the hlank opaque, hut found that the end point color change in the sample took just a little getting used to. Titrating to a more definite violet than usual seemed to give better results. We did find that end point colors tended to darken somewhat on standing, so comparison of an end point with that from a previous titration is not recommended. The wide varietv of wssihle samples (homoeenized milk, skim milk, d;y milk', ice cream, &c.) a i d v&iations available in terms of indicators and techniques make the titration of calcium and magnesium in milk a very versatile and interesting student experiment. Literature Cited (11 Lin& E.R..Anulysf,81.179 (19581. (21 Bird, E.W., Wsbcr, J., Cox, C. P., and Chen, T. C., J DoiwSei., 44,1036(1961). 131 Jennes,R.. A m 1 Chem.,25.966(19531. 141 Mason. A. C.,Anolyst, 77.529(19521. 151 Gehrke, C. W., Aff.pmng,H.E.,andLoe,Y.C.,Ainl. Chsm.,26,1944119541. (61 Kama1.T. H.,JAgr Food Chem..8,156(19Ml. 171 Ntailianas. H.A . and Whitney,R.McL.,J D a b S C L 47.19119bll. . (81 Cohn. E. J., and Hendry, J. L.. "Organic Syntheses." eoll. val. n. John Wiley 8 Som, Ine.,NewYork, 1943.p 120.
Volume 50,Number 2, February 1973 / 137
