REPORT FOR ANALYTICAL CHEMISTS
Development of Metallochromic Indicators b y H a r v e y Diehl D e p a r t m e n t o f C h e m i s t r y , Iowa S t a t e U n i v e r s i t y , A m e s , Iowa
T HE PUBPOSE OF THIS REPORT is to
review the work that has been done at Ames during the past eight een years on the EDTA titrations of calcium and magnesium and the metallochromic indicators used in such titrations. The narrative is presented in historical style, more or less as it happened ; the discourse may thus possibly prove of more in terest, particularly if the mistakes that were made and the difficulties that were encountered are not glossed over. Science does not proceed directly from A to Β as its formal record might lead the uninitiated to be lieve, and our scientific publica tions, written in the impersonal mood like telegrams, completely conceal from the public the devious path that is actually followed. I t ignores the blind alleys entered and discounts the frustrations experi enced. I t hides, too, the intellectu al satisfaction that the petty triumphs bring, these pleasures being all the more rewarding be cause of the difficulties. Yet, at its roots, science is a social activity of a very personal nature. At the risk, then, of violating the sacred principle of the impersonal, I will tell how we got into the EDTA work, or rather how we might have gotten into it six years earlier, and what wc have done about the indi cators Eriochrome Black T, Calmagite, Calcein, and DHAB. The EDTA Titration in America
During November of 1941, in connection with work for the Office 30 A
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of Scientific Research and Develop ment on the recovery of oxygen from air, I visited the F. C. Bersworth Laboratories in Framingham, Massachusetts, to purchase a large amount of ethylenediamine. The visit had an unexpected divi dend in that Mr. Frederick Bersworth had studied under the Swiss chemist Alfred Werner and was happy to have a perfectly en tranced listener to his stories about Werner and the early days of the coordination theory at Zurich. At the end of a fascinating day, Mr. Bersworth sent me home with sev eral pounds of a material which he called Versene, a substance he was proposing to market in New Eng land as a water softener, comment ing that it formed non-dissociated ions with the alkaline earth metals, that it was indeed an excellent water softener, and that it could even be used for the titrimetric de termination of calcium and magne sium. To a self-assured youngster, fresh from writing a long review paper (1) on the chelate ring com pounds, this appeared pretty un likely for it was well known that the alkaline earth metals simply did not form coordination com pounds in aqueous solution with ammonia and amines. Back in Ames, I made the cobalt compound of Bersworth's Versene, found that it did not take up oxygen reversibly, and retired the bottle of Ver sene to a back shelf. This was a mistake. Had I paid attention to what Mr. Bersworth was saying, the EDTA story might well have
started six years earlier. That it was a mistake became clearly evident in 1947 when Mr. Bersworth's persistence provided me with a second go at it. At the spring meeting of the American Chemical Society in Chicago, Mr. Bersworth learned with disgust how I had disposed of his earlier sample and threatened vaguely to do some thing about it. Sure enough, there soon appeared on the dock of the chemistry building a 55-gallon drum of a 30 per cent solution of the tetrasodium salt of ethylenediaminetetraacetic acid and our store keeper deposited the drum in the narrow aisle of my office; literally, I had to climb over it to get out. In desperation I tried the Versene at home that night, using it more than liberally with the dinner dishes. The results were spectacular: soap suds cascaded onto the floor. It was not long before my colleagues were using Versene to clean the ring off the bath tub, to remove lime from newly laid bricks, and to dis solve the shells off eggs. Quickly one of the local drug stores devel oped an advantage over its compet itors because it knew how to remove the milkstone from the glassware at its ice cream bar and had the only sparkling glassware in town. At the time we were making nu merous determinations of the hard ness of water for the local water conditioning people using the soap titration and the soda reagent titra tion. It occurred to Mr. Clifford Hach and me to use Bersworth's Versene as a titrating agent. This
DR. H A R V E Y D I E H L w a s p r e s e n t e d with the 1 9 6 6 Detroit Anachem A w a r d , a n d w a s c i t e d f o r his c o n t r i butions t o t h e p r o f e s s i o n of sanalytical c h e m i s t r y t h r o u g h his teaching, research, and service t o his s o c i e t y . T h i s R e p o r t is b a s e d o n Dr. Diehl's a d d r e s s g i v e n a t t h e t i m e o f his a c c e p t a n c e o f t h e a w a r d .
worked well; a bit of soap was added so that the end point was found by the same appearance of a permanent foam that marks the endpoint in the soap titration. This worked surprisingly well if the pH were controlled. The disap pearance of a turbidity of calcium oxalate worked even better and was quicker. Soon the chemists at the Ames and the Des Moines water works were using the method and we were about ready to submit a manuscript for publication when we found the Chemical Abstracts ab stract of the Biedermann and Schwarzenbach paper (2) of Chimia of April 1948. This paper de scribed the hardness titration with ethylenediaminetetra.acetate and also described a visual indicator for the titration, the indicator being of a new type, a metaLochromic indi cator—one in which a metal, usual ly the one being titrated, combines with the indicator and is pulled away from the indicator at the equivalent-point causing a color change. We secured Eriochrome Black T, the Biedermann and Schwarzen bach indicator, and found the red to blue color change more convenient than the soap or the calcium oxa late end point; we elaborated the details of the titration and, with mimeographed material, soon had numerous waterworks chemists throughout the Midwest using it successfully and enthusiastically. A manuscript describing the meth od was submitted during September of 1948, and despite a prompt and
favorable review, several difficul ties were encountered that con spired to delay publication of the paper (3). In the meantime, how ever, because of word-of-mouth communication among the water works chemists and the distribution of the mimeographed material, the method found wide adoption. In fact, it is probable that never in the history of analytical chemistry has a method been widely adopted so quickly as this one. I should add too, in fairness, that there has prob ably never been a method so poor as the soap titration it displaced. For various reasons I dropped the work with ethylenediaminetetraacetic acid as a titrating agent and went off to work on vitamin B 12 , another chelate-ring compound with a great deal of fascinating chemis try. After five years, I had ex hausted my ideas about the cobalt chemistry of B 1 2 and returned to ethylenediaminetetraactic acid. During these five years, changes had occurred in the hardness titra tion field. For one thing, Bersworth had attracted competitors in the manufacture of ethylenediaminetetraacetic acid and these people were expressing their resentment of our use of the term Versenate, a deriva tive of Bcrsworth's trade name, as a name for the hardness titration. This pressure on the editors of the journals to stop giving Bersworth free advertising resulted in replac ing the term Versenate by EDTA, a designation that caught on quickly and was almost universally adopt ed. One reputable journal persists
in using the more obscure but for mally correct Chemical Abstracts name ethylenedinitrilotetraacetic acid but the rest of the profession continues to use ethylenediaminetetraacetic acid or EDTA. (Editor's note:
ANALYTICAL CHEMISTRY now
permits use of ethylenediaminetetraacetic acid). During the five-year interval mentioned, a considerable corre spondence had developed, and the users of the EDTA titration asked for aid or for extensions of the method. These problems boiled down to three: the nature of the reaction of Eriochrome Black Τ with calcium and magnesium; the instability of stock solutions of Eri ochrome Black T; and the need for a method of differentiating calcium from magnesium. Fortunately during the succeeding years we were able to do something about each of these problems. Nature of the Reaction of Eriochrome Black Τ with Alkaline Earths
The first attack was an indirect one. There had been published in the American journals during 1953 and 1955 two papers reporting that the combining ratio of Eriochrome Black Τ with calcium (and also with magnesium) was two to one and three to one, contrary to the ratio of one to one reported by Schwarzenbach and Biedermann (4)- The stereochemistry involved as well as an enormous respect for Schwarzenbach as a chemist, develVOL. 39 NO 3, MARCH 1967
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REPORT FOR ANALYTICAL CHEMISTS
oped from a reading of his extensive work on the stability of chelatering compounds and from personal contact during two weeks he spent as a visiting lecturer at Ames, led me to suspect the results of these papers. With Mr. Frederick Lindstrom, I undertook to purify commercial Eriochrome Black T. Commercial Eriochrome Black Τ samples were found to vary in purity from 40 to 60 per cent and it proved difficult to separate the dye from the salt and other azo compounds which ac company this widely used dyestuff as manufactured commercially. We did finally succeed in obtaining Eriochrome Black Τ in crystalline form, actually as the dimethylammonium salt, the dimethylammonium ion having gotten in by the breakdown of the dimethylformamide used during the final recrystallization (5). Using the pure, crystalline material we found that the combining ratio was one to one only and obtained values for the formation constants almost exactly the same as those reported by Schwarzenbach and Biedermann. The error in the two American works arose from using the impure, commercial dyestuff in the method of continuous variations; Job states clearly in his original paper on the method of continuous variations that both ligand and metal must be absolutely pure. Pure chemicals are not required in the spectrophotometric method employed by Schwarzenbach and Biedermann based on measuring the absorbance of a series of solutions containing a
fixed amount of the dyestuff and varying the amount of magnesium out to a large excess ; there are vari ous ways of handling the data but all make the same assumption— that the concentration of the free magnesium ion is the same as that calculated from the total amount of magnesium added—an assumption that is satisfactory if the formation constant is fairly low (for Erio chrome Black T, log KMg = 5.75, log KCa = 3.72 at pH 10, ionic strength 0.100). Using the crystal line Eriochrome Black Τ we ob tained essentially the same values using the method of continuous var iations as obtained using the spectrophotometric method, handling the data in the latter method by what we call the log-ratio method (B). Our next study was an investiga tion of hydroxyazo compounds in general to determine just what fea tures of the structure are essential for reaction with calcium and mag nesium. Mr. John Ellingboe pre pared some thirty hydroxyazo com pounds, each selected to shed light on some aspect of the problem. Each compound was synthesized from carefully purified materials and subjected to purification until obtained crystalline. A definite answer was obtained (6). The nec essary and sufficient requirements for reaction with magnesium are simply the presence of two hydroxy groups, o- and o'- to the azo group. The nitro group is not essential, hy droxy groups placed other than in the ortho positions to the azo group are not necessary, and the sulfonic
Eriochrome Black Τ 32 A
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acid group is not essential although its presence gives the compounds greater solubility and superior sharpness and brilliance of color change. Of the compounds investi gated which did react with magne sium and calcium, the combining ratio was invariably one to one and the formation constant of the mag nesium derivative always apprecia bly greater than that of the calcium derivative. Two of the compounds gave color reactions with magnesi um but not with calcium; one of these compounds, o,o'-dihydroxyazobenzene, the simplest member of the series, was later developed as a reagent for the fluorometric deter mination of magnesium in the pres ence of calcium. A Stable Replacement for Eriochrome Black Τ
Armed with a knowledge of what is necessary for reaction with cal cium and magnesium, we next tackled the problem of finding a stable replacement for Eriochrome Black T. The guiding premise was that the presence in the molecule of both the nitro group, with oxidizing properties, and of the phenolic groups and the azo group, with re ducing properties, contributed an inherent instability which perhaps could be eliminated by excluding the nitro group from the molecule. After synthesizing some thirty com pounds, Mr. Frederick Lindstrom obtained one which had chemical and physical properties almost identical with those of Eriochrome Black Τ and which could be substi-
Calmagite See ACS Laboratory Guide for All Products/Sales OfficeCircle No. 75 an Readers' Service Card-
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tuted in working procedures for Eriochrome Black Τ without any change in the directions. We chris tened this indicator Calmagite (7). I t is actually somewhat sharper in color change than Eriochrome Black Τ and solutions of it undergo no deterioration on standing in light for years. The principal use of the EDTA titration of calcium plus magnesi um has, of course, been in the deter mination of water hardness. Here great accuracy is not demanded, be cause one or two parts per hundred are satisfactory for water plant control. The method, however, is inherently as good as any titrimetric method using volumetric glass ware and reasonable temperature control. Just how good it is was demonstrated by Mr. Loren C. McBride, using Calmagite as indi cator, by the analysis of NBS 88 Dolomite and the standard refer ence samples GFS 400 through GFS 419, Limestone and Dolomite, of the G. Frederick Smith Chemical Company ; the results are more pre cise than can be obtained by the classical gravimetric methods and are probably more reliable. The determination was made following dehydration and correction of the silica, but without removing the R 2 0 3 metals. The working details and the individual results are given in a brochure (8) of the G. Frederick Smith Chemical Company describ ing the samples and in a monograph (9) also published by the Smith company, on the EDTA titration. Calmagite has been the subject of more recent work. Lindstrom (10) has synthesized it in a very high de gree of purity and Ringbom (11) has utilized it for the direct spectrophotometric determination of mag nesium. Judging from the amount of Calmagite currently being sold, it has displaced Eriochrome Black Τ almost completely in routine work. An Indicator f o r Calcium in the Presence of Magnesium A solution to the problem of de termining calcium in the presence of magnesium by a direct titrimetric method actually preceded the EDTA-Eriochrome Black Τ titra tion of calcium plus magnesium. In the 1946 paper of Schwarzen-
bach, Biedermann, and Bangerter {12), which is also apparently the first description of the use of a metallochromic indicator," murexide is proposed as the indicator, the redviolet color of the calcium deriva tive changing to blue-violet as the
" Although not of an E D T A titration. The first published description of an E D T A titration is apparently the three preliminary announcements by Schwarzenbach: Schiveiz Chemiker-Ztg. u. Tech. lnd., 28, 181, 377 (1945) (no copies of which have apparently ever been received by a major library in the United States), and Helv. Clam. Ada., 29, 1338 (1946). Ethylenediaminetetraacetic acid first a p pears in the literature in German Patent 638,071 granted Nov. 9, 1936 to Fick and Ulrich and assigned to I. G. Farbenindustrie. I t s use as a water softener is described by Ender, Fette u. Seifen, 45, 144 (1938). I t is curious that Pfeiffer and Offerman, who described the copper and calcium derivatives in detail, Ber., 75B, 1 (1942); 76B, 847 (1943), did not see that the material could be used as a t i trating agent. I n a recent letter F . C. Bersworth confirmed that as early as 1938 he was adding a solution of Versene drop by drop to hard water containing a bit of soap to determine how much Versene should be added to a given water on a large scale to soften it. This is ob viously the E D T A titration, the end-point being detected by the first permanent foam. Dr. R. O. Bach, Director of R e search of the Lithium Corporation of America, Bessemer City, North Carolina, has related that as a student at the Uni versity of ZUrich he assisted Sehwarzenbach during t h e years 1941—42 in demon strating to t h e Zurich students the reac tions of the alkaline earth metals with nitrilotriacetic acid and ethylenediamine tetraacetic acid. Just how Schwarzenbach was led to these particular amino acids and to their reaction with t h e alkaline earths from his fundamental studies on the relation of structure to acid dissociation and chelating properties, how these compounds and reactions led in turn to the titration procedure sometime prior to 1945, and how he came to couple with the titration procedure the (almost ancient) observations of another Zurich chemist, C. Brenner, Helv. Chim. Acta., 3, 90 (1920), that calcium and magnesium yielded colored compounds with certain azo dyestuffs—can best be told b y Schwarzenbach himself. I n any case, there is no question as to the origin of the basic work on which t h e present body of analytical chemistry involving E D T A rests, for the large volume of work b y Schwarzenbach and his co-workers in cludes the fundamental measurements of acid dissociation and formation con stants of E D T A , the synthesis and similar studies on many ingenious, related aminopolycarboxylic acids, the fruitful adapta tion of metal-dyestuff compounds to endpoint detection, and the concept of con verting an acid-base indicator into a metal ion indicator by the introduction of chelating groups. This is scientific work of the highest order, which in overall i m pact and in general utility to the analyti cal chemist can be considered second only to the development of gas chromatogra phy. T h e work should bring to this brilliant Swiss chemist a Nobel Prize.
REPORT FOR ANALYTICAL CHEMISTS
calcium is pulled away from the murexide at the end point by the EDTA. The change is gradual and in the hands of most workers unsat isfactory, and a considerable num ber of papers have been published in recent years describing other in dicators for this titration. In prin ciple the problem is simply one of running up the pH to a value suffi ciently high to cause the formation of non-dissociated (or, for larger amounts) precipitated magnesium hydroxide; the calcium is then ti trated with EDTA. The problem is to find an indicator. Eriochrome Black Τ is not satisfactory because at high pH—12.5 to 13.5—it is red in color, its calcium derivative is red, and the change at the equiv alence point is not sufficiently marked to be satisfactory. The solution to the problem came from the development of a new class of indicators based on initial work by Schwarzenbach in which he introduced into ari acid-base in dicator the methyleneiminodiacetic acid group, —CH 2 —N(CH 2 — C0 2 H) 2 , (half of the EDTA mo lecule) thus converting the acidbase indicator into a r.ietallochromic indicator. The work on this ingeni ous concept is embodied in a series of papers describing the synthesis, which uses the Mannich condensa tion of a phenol (the indicator), formaldehyde, and iminodiacetic acid, and the properties of the che lating compounds formed, first from phenol itself (13), then from nitrophenol (14), and from phenolphthalein (15). Our own contribution was to in troduce the methyleneiminodiacetic acid group into fluorescein, con verting this fluorescent acid-base indicator into a fluorescent metal
ion indicator. Again the Mannich condensation was used. A yellow product was obtained which at high pH did not fluoresce, but it did fluoresce strongly when present with calcium. The disappearance of this green-yellow fluorescence (and color) serves well to mark the end point in the EDTA titration of calcium at high pH in the presence of magnesium. We called the ma terial Calcein (16). At the present writing (Fall of 1966) the bibliog raphy on Calcein numbers some eighty-five papers; the literature has been reviewed through June of 1963 in a monograph (9) published by the G. Frederick Smith Chemi cal Company. The indicator has been used for the determination of calcium in potable and industrial waters, blood serum and other bio logical fluids, silicate rocks, slags, phosphate rock, and lithium salts. The EDTA titration of calcium only using Calcein as indicator is inherently as good as any titrimetric method using volumetric glass ware and reasonable temperature control, as was demonstrated by Mr. R. C. Miller in the analysis of NBS 88 Dolomite and the reference samples of limestone and dolomite GFS 400 through GFS 419 (8). About twenty of the papers in the Calcein bibliography deal with the determination of calcium in blood serum. This large literature on a single determination shows not only how delighted clinical and biochem ists were to find a direct method for calcium, but is indicative also of underlying trouble. Clinical chem ists would like to determine the cal cium in blood serum on a sample of at most only a few hundred microli ters in volume, meaning that the ti tration must be made on only a few
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VOL.
39, NO. 3, MARCH 1967
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35 A
High
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