Analyst, February, 1967, Vol. 92, pp. 83-90 83 The Component of Commercial Titan Yellow Most Reactive towards Magnesium: Its Isolation and Use in Determining Magnesium in Silicate Minerals BY H. G. C. KING AND G. PRUDEN (Rothamsted Experimental Station, Harpanden, Herts.) The component of Titan yellow most reactive towards magnesium has been isolated from the acetone-extracted dye by adsorption chromatography on Sephadex G-10. I t is so reactive that i t can be used at a concentration as small as 0.008 per cent., a t which concentration the response is linear between 20 and 15Op.g of magnesium. Values found for the magnesium content of silicate minerals agree with those obtained by spectrographic and gravimetric methods. TITAN yellow, variously described in the literature as Clayton yellow, Thiazole yellow, Acridingelb 5G and Azidingelb 5G, was first used by Kolthoff1y2 to determine magnesium.The chemical constitution of the dye, which is synthesised by coupling a molecule of a sulphonic acid of dehydrothio-9-toluidine with a molecule of its diazonium salt, is usually given as- H 3 C y - J q 5 3 N=N-HN /(---3&UCH3 ’,4 3 / 6L5’ - \N / -t S03Na Na03S t N (The positions of the sulphonate groups are uncertain.) There are inconsistencies in the published formulation of Titan yellow, particularIy in the positions of the sulphonate groups; some authors state that it differs from Clayton y e l l ~ w , ~ whereas others* find no specific differences between any of the compounds named above, At least one manufacturer uses the names Titan yellow and Clayton yellow synonymously.* Samples of commercial Titan yellow, Clayton yellow and Thiazole yellow were first shown to differ in their reactivity towards magnesium by Mikkelsen and Toth,B who found that Thiazole yellow was the most active, the inference being that the dyes tested were of different composition. Bradfield5 analysed four samples of Titan yellow and found variable, and often large, amounts of sodium chloride (55 per cent.for one). He demonstrated by paper chromatography that Titan yellow was a mixture, and separated a contaminant that did not react with magnesium, and under ultraviolet light, gave the blue - violet fluorescence characteristic of a dehydrothio-p-toluidine sulphonate. Recently Hall, Gray and Flynn’ confirmed the complexity of fourteen commercial Titan yellow samples by using paper and thin-layer chromatography, and showed spectrophotometrically that, at the wavelength of maximum absorption, the optical densities of equal concentrations of the dyes have a wide range of values.They also isolated the active component on a micro scale by thin-layer chromatography. The presence of variable amounts of sodium chloride in Titan yellow is largely responsible for the observed differences in optical densities, and perhaps explains earIier inconsistent results in magnesium determination because the amount of magnesium t o be determined may sometimes have exceeded the reactivity of Titan yellow. Some workers have attempted * Kolthoff1p2 and Hirschfelder and Searles3 show formulae for Titan yellow inconsistent with the chemical consitutions described in their texts.Kalthoff also describes the dye as a derivative of dehydro- thio-p-toluidine sulphonate. Bradfield5 gives the formula as a 3’-disulphonate, as does Sandell,O although he describes i t as a 2’-disulphonate; Hall et a1.’ describe Titan yellow as a 2’-disulphonate.84 KING AND PRUDEN: THE COMPONENT OF COMMERCIAL [Artalyst, vol. 92 to overcome the variability of Titan yellow by using the reagent at a standard optical density. However, this is unsatisfactory, because the criterion of reactivity is not the optical density of the mixture of components of different reactivity, but of the proportion of the most reactive component in the dye. We find that the colours of alkaline blanks of the magnesium-reacting components differ in optical density, and that the blank of the most active component has the least colour.The use of the most active component thus ensures greatest sensitivity in determining magnesium. By the use of the dextran gel Sephadex G-10 we have prepared enough of the active component for several hundred magnesium determinations. The blue - violet fluorescing contaminant was largely removed by extracting the dye with acetone, and the remaining components were separated on Sephadex G-10, by using the gel not as a molecular sieve, but by making use of its property of adsorbing differentially the main components with water, followed by aqueous acetone, as eluent. This procedure has enabled us to assess the relative amounts of the active components in a commercial sample of Titan yellow and to prepare amounts of a standard, reproducible fraction that has been used for the routine determination of magnesium in silicate minerals.EXPERIMENTAL Eastman Kodak’s Titan yellow, P4454, a grade also studied by Mikkelsen and Toth,* was used for the detailed examination and preparation of the most active component, It contained 374 per cent. of sodium chloride. A sample of Hopkin and Williams’ Titan yellow (Clayton yellow) containing 42.0 per cent. of sodium chloride was examined by paper chromatography only. SPECTROPHOTOMETRY- Ultraviolet and visible absorption spectra of Titan yellow and its fractions, at concen- trations of 1.5 to 2.5 mg in 100 ml of water, were measured with a Unicam SP500 spectro- photometer, with quartz cells of light path 1 cm.The spectrum of an aqueous solution of commercial Titan yellow between 200 and 500 mp showed two maxima, one at 325 mp (Eke% = 152) and the other at between 405 and 410 mp (Elc$ = 348). Because the optical densities of the maxima vary inversely with the amount of sodium chloride mesent. the molecular extinction coefficient of Titan yellow cannot be obtained directly. Bradfieldg found the maxima to be 330 mp gave only the higher wavelength maximum, as 405 mp. I 200 300 400 500 Wavelength, mu Fig. 1. Ultraviolet and visible absorption spectrum of Eastman Kodak’s Titan yellow, P4454 (2.1 mg in 100 ml of water) and 405 mp, and Hall et aZ.7 PAPER CHROMATOGRAPHY- The descending method was used with Whatman No. 1 paper and aqueous ethanol, 80 per cent., as solvent.(This simple solvent gives as good resolution of the components as more complex combinations of butanol, ethanol, ammonia solution or acetic acid, although none of the solvent mixtures examined gave satisfactory resolution of the components between the origin and the head of the most active component [R, about 0.36 in aqueous ethanol].) Fluorescent areas were marked out under ultraviolet light (366 mp, Wood’s glass filter).February, 19671 TITAN YELLOW MOST REACTIVE TOWARDS MAGNESIUM 85 Papers were dipped in aqueous magnesium sulphate solution (2 per cent.), suspended in a current of air until almost dry, and dipped in alcoholic sodium hydroxide solution (2 per cent.). The relative activities of the reactive areas should be assessed as soon as possible, because the less active components begin to fade within 25 minutes.Paper chromatography did not reveal some of the minor fluorescent compounds described by Hall et aZ.,' who used thin-layer chromatography, but confirmed the presence of the main components. TABLE I PAPER CHROMATOGRAPHIC PROPERTIES OF COMPONENTS OF TITAN YELLOW Component RF Colour in daylight Colour in ultraviolet light . . 0.32 (head) Yellow Yellow 1 (= Band 1) . . 0-00 (tail) 0.20 (tail) 2 (= Band2) . . . . 0.36 (head) Bright yellow 1~e110~ 3 . . , . .. . . 0.49 Colourless Bright blue - violet 4 .. . . . . . . 0.40 Colourless Faint violet 5 . . . . . . . . 0.00 Pale yellow Yellow Only bands 1 and 2 reacted with magnesium to give the characteristic red colour in With band 1 the colour began to fade in 25 minutes, the presence of sodium hydroxide.but the colour obtained with band 2 was stable for 45 minutes. PRELIMINARY FRACTIONATION OF TITAN YELLOW- Titan yellow was extracted with acetone in a Soxhlet apparatus. The solvent was removed from the extract by vacuum distillation and the extracted Titan yellow dried in air. The acetone extract, 6.3 per cent. of the starting material, consisted of a yellow coni- ponent (25 per cent.) whose chromatographic behaviour was similar to band 1, and the colour- less component 3 (75 per cent.). The latter was isolated by passing an aqueous solution of the acetone-soluble extract down a column of Solka Floc (powdered cellulose) with 6 per cent. acetic acid as eluent. The yellow component was immobile in this system.SMALL-SCALE SEPARATION OF COMPONENTS OF EXTRACTED TITAN YELLOW ON SEPHADEX G-10 Twenty grams of Sephadex G-10 (Pharmacia Ltd., Uppsala), particle size 40 to 12Op, were allowed to swell overnight in 0.1 M sodium chloride solution. The supernatant liquid was then poured off and the gel was stirred with three successive 100-ml portions of water, decanting any fine material after each stirring. The gel was poured as a slurry into a glass chromatographic column, 25 x 2.25 cm diameter, having at its lower end, as a support for the gel, a sintered-glass plate of porosity 3, i.e., pore size less than 40 p. (For the large-scale preparation of the most active fraction of Titan yellow 75 g of Sephadex G-10 and a column, 32 x 3.25 cm diameter, were used.) When the gel had settled, a filter-paper disc was placed on its surface and the column was washed with water until free from sodium chloride.The water in the prepared column was allowed to drain to the level of the filter-paper disc, then 2.5 ml of an aqueous solution containing 2.5 mg of acetone-extracted Titan yellow 0 Wavelength, mu Fig. 2 . Ultraviolet and visible absorption spectra of fractions of Titan yellow isolatcd by gel filtration and chromatography on powdered cellu- lose: A, band 1 ; B, band 2 ; and C, blue - violet fluorescing component86 KING AND PRUDEN: THE COMPONENT OF COMMERICAL [Analyst, VOl. 92 was introduced. The column was eluted with water at a flow-rate of about 1 ml per minute. Sodium chloride was eluted first, followed by the first yellow band (band 1).The solvent was then changed to 50 per cent. aqueous acetone, and the column washed until the effluent was colourless (band 2). The acetone was distilled from the band 2 effluent, and band 1 and band 2 solutions were diluted to 100 ml with water for spectrophotometric examination. The acetone-insoluble fraction, fractionated on the small Sephadex column, gave the ratio 1/0.86 for the optical density of band 1 (salt-free) to that of band 2. The optical densities of bands 1 and 2 were 51.0 per cent. and 45.3 per cent., respectively, of the optical density of the unfractionated Titan yellow, measured at 410 mp. The remaining 3.7 per cent. of the absorption at 410 mp is accounted for by the acetone-soluble fraction, and a trace of yellow material held at the top of the Sephadex column.Fig. 2 shows the ultraviolet and visible absorption spectra of the Titan yellow fractions. DETERMINATION OF THE REACTIVITY WITH MAGNESIUM OF TITAN YELLOW FRACTIONS- For these measurements band 1 and band 2 fractions were prepared in larger amounts 2.s described below. Fifteen ml of standard magnesium chloride solution (equivalent to 150 pg of magnesium) were added to the stabilising colloid mixed reagent solution,1° followed by 5 ml of a solution of the Titan yellow fraction under examination and 5ml of aqueous, 30 per cent. sodium hydroxide. Concentrations of Titan yellow fractions ranging from 0.02 per cent., by steps of 0.001 per cent., to a final concentration of 0.001 per cent. were used. The volume was made up to 100 ml with water and the optical density of the solutions measured at 545 mp, with glass cells of light path 4 cm, against a blank of the corresponding Titan yellow fraction treated with sodium hydroxide.The point at which magnesium was in excess of Titan yellow was found by plotting the optical density of the latter against its concentration. The optical density at 545 mp of each blank was also measured against water and again the optical density was plotted against concentration of the Titan yellow fraction. Titan yellow fraction, per cent. Titan yellow fraction, per cent, Fig. 3. Reactivity of Titan yellow fractions Fig. 4. Variation of optical density of re- with 150 pg of magnesium: A, band 1 ; B, band agent blank concentration of Titan yellow : A, 2 ; C, acetone-soluble fraction ; and D, unfraction- band 1 ; B, band 2 ; C, acetone-soluble fraction; ated Titan yellow and D, unfractionated Titan yellow Figs.3 and 4 show the least concentration of each Titan yellow fraction reacting with magnesium chloride - sodium hydroxide ; and with sodium hydroxide alone. The relative activity of the fractions is measured by the minimum concentration of each that reacts with the given amount of magnesium. The difference in the reactivity of bands 1 and 2 is apparent from the curves. METHOD Fractionation of Titan yellow-Extract 1.0 g of Titan yellow with AnalaR acetone in a Soxhlet apparatus for a total of 12 hours. Dry the extracted dyestuff in air, and dissolve 0.5 g of it in 25.0 ml of water. Introduce the aqueous solution into the top of the Sephadex column and allow the liquid to drain to the surface of the gel, applying a little suction if necessary.Elute the column with water to wash out sodium chloride and the less activeFebruary, 19671 TITAN YELLOW MOST REACTIVE TOWARDS MAGNESIUM 87 component (band 1). When the effluent is colourless, change the eluent to 50 per cent. aqueous acetone and wash out the more active component (band 2). Distil the solvent under vacuum and dry the residue over phosphorus pentoxide. ANALYSIS OF SILICATE MINERALS The solution of the silicate mineral to be examined is prepared by Meyrowitz's method,lo with the acidity adjusted to be about 1.2 N with respect to sulphuric acid. REAGENTS- Acid blank solution-About 1.2 N sulphuric acid.Standard magnesium solution-Dissolve 500 mg of pure magnesium ribbon in 15 ml of 6 N hydrochloric acid. Evaporate off excess of the acid, dissolve the residual magnesium chloride in water and make up the solution to 500ml. Ten ml of this solution, diluted to 1 litre gives a working standard containing 1Opg of magnesium per ml. Complexing solution-Dissolve 16 g of potassium cyanide in 100 ml of water. Add 100 ml of triethanolamine, and mix the solution. Sodium hydroxide solution, 30 per cent.-Dissolve 60 g of sodium hydroxide pellets in about 50 ml of water. Dilute the solution to slightly less than 200 ml. Cool, dilute to 200 ml and transfer the solution to a polythene bottle. Titan yellow reagent solution, 0.008 per cent.-Dissolve 8 mg of Titan yellow band 2 in 100 ml of water.Mixed reagent s0lutio.n-Add 100 mg of poly(viny1 alcohol) to 150 ml of water in a 600-ml beaker. Place the beaker on a hot-plate and heat gently with constant stirring until the temperature of the solution is 60" C. Heat at 60" to 70" C, with constant stirring until the solution is clear. Add, with mixing after each addition, 300 ml of water, 5 ml of 9 N sulphuric acid, 750 mg of hydrated aluminium nitrate, Al(NO,),.SH,O, and 20 g of hydroxyl- ammonium chloride. Cool the solution and dilute it to 500 ml. Filter with a fine filter-paper. DETERMINATION OF MAGNESIUM- Dilute to 250ml with water and mix well. Transfer 5 ml of acid blank solution to a 100-ml calibrated flask. Transfer 2, 4, 6, 8, 10, 12 and 14ml of working standard magnesium solution (20 to 140 pg of magnesium) to seven 100-ml calibrated flasks.Add to the flasks containing the standard magnesium solution, 5 ml of acid blank solution. Transfer to the 100-ml calibrated flasks 2 to 5 ml (containing no more than 140 pg of magnesium) of the sulphuric acid solution of the mineral (about 1.2 N). Add to each of the flasks enough acid blank so that the volume of sample solution taken together with the added amount of acid blank is 5 ml. Introduce 5 ml of the mixed reagent, 45 ml of water and 2 ml of complexing solution to each flask, mixing after each addition. Add to the first of the flasks 5 ml of Titan yellow reagent solution. Immediately add 5 ml of 30 per cent. sodium hydroxide, swirling the contents of the flask. Dilute the solution to the mark and mix.Follow this sequence systematically through the 10 Wavelength, mp Fig. 5. Visible absorption spectra: A, Titan yellow band 2 - magnesium complex against water as blank; B, reagent blank against water blank; and C, Titan yellow band 2 - magnesium complex against reagent blank88 [AnaZyst, Vol. 92 whole series of flasks. Allow the solutions to stand for at least 20minutes. Determine the optical density at 545mp, relative to the reagent blank, with 4-cm absorption cells. The wavelength at which the Titan yellow - magnesium complex is measured has been reported variously as 530 mp,497 545 mpl0 and 550 r n p 5 We find that when the coloured complex is set against the blank, with the optimum concentration of Titan yellow band 2 found above, the wavelength of maximum absorption is at 545mp.However, this is not the wavelength of maximum absorption with water as blank, but is the wavelength that gives the greatest difference between the optical densities of the complex and the blank, i.e., the wavelength at which the method has the greatest sensitivity (Fig. 5 ) . KING A-ND PRUDEN: THE COMPOYENT OF COMMERCIAL Table I1 shows results for some typical silicate minerals. TABLE I1 MAGNESIUM CONTENT (AS MAGNESIUM OXIDE) OF SILICATE MINERALS Mineral Granite (G-1) . . . . .. .. Diabase (W-1) . . . . .. .. Granite (G-2) .. .. .. Peridotite (PCC-1) . . . . . . Dunite (DTS-1) . . . . . . .. Basalt (BCR-1) . . .. .. . . Granodiorite (GSP-1) . . .. . . Andesite (AGV-1) . . .. . . Biotite quartz monzonite, Keller Peak, San Bernarclino Mts., California Biotite quartz monzonite, Rattlesnake Mt., Pluton, San Bernardino Mts., California .. . . . . . . Biotite granodiorite, Mt. Edna, San Jacinto Mts., California . . . . Woodson Mt., granodiorite, Jarupa Hills, Riverside County, California .. Lakeview Mt., tonalite, Lakeview Mts., Homeland, California . . . . La Sierra, tonalite, La Sierra, California Standard sample, sulphide ore-1 . . Granite (GH) . . . . . . .. Granite (GA) . . . . .. .. Basalt (BR) . . . . . . . . . . BY Titan yellow 0.41 6.70 0-73 1-48 43.55 49-09 3.46 0.96 0.46 1.09 1.25 0.41 3.27 1.86 3-85 0.03 0-88 12.61 Spectrographic Gravimetric 0.35 - 6.58 - 0.76 - 1.53 - 43.4 to 44.6 43.86 48.2 to 49.7 49.40 3.48 - 0.93 - 0-52* 1*09* 1-25* 0-48* 344* 1-89* 3.71 t o 4.49'3 0.0313 0*9513 1 2*6013 * Secondary chemical standards determined by X-ray methods. Results supplied by Prof.A. I<. Baird, Pomona College, Claremont, California, U.S.,4. DISCUSSION Although the structure of Titan yellow is uncertain, it seems that the commercial dye is essentially a mixture of two compounds that react differently with magnesium; in the sample studied these two components account for at least 90 per cent. of the total active material. The amounts of these components vary from sample to sample, and sodium chloride is always a major impurity. Standardisation of Titan yellow is made difficult by the fact that all of the components are water-soluble. Removing sodium chloride and the blue - violet fluorescing compound from different samples of Titan yellow gives products of different reactivity with magnesium, although the spectra and optical densities at the wave- lengths of maximum absorption are closely related. The difference in activity is shown in the greater colour developed by the less active component (band 1) in the presence of alkali.With a fixed amount of magnesium and different concentrations of dyestuff components, the optimum concentration of the more active fraction (band 2) is about one-third of that of our unfractionated Titan yellow. At this concentration the intensity of the colour of the blank is minimal, whereas the colour of the magnesium complex develops strongly. With unfractionated Titan yellow, interference caused by the blue - violet fluorescing component (about 5 per cent. of the starting material in our sample) is negligible.Bradfields considers that this compound is the 7-sulphonate of dehydrothio-P-toluidine, some of which remains unreacted during the synthesis of Titan yellow, but this is at variance with Bradfield's own formulation of the dye as a 3'-disulphonate. We have confirmed Bradfield's finding that Titan yellow can be degraded to a blue - violet fluorescing compound that exhibits theFebruary, 19671 TITAN YELLOW MOST REACTIVE TOWARDS MAGNESIUM 89 L Weight of magnesium, pg Fig. 6. Calibration for magnesium in silicate materials same behaviour on paper chromatograms and to ultraviolet light as the contaminant seen on paper chromatograms of the unfractionated dye. Bradfield degraded Titan yellow with an acid - sodium dithionite mixture; we degraded the band 2 component with 2 N sulphuric acid, neutralising the hydrolysate with sodium hydrogen carbonate.It is unlikely that such treatment would cause the sulphonate groups to migrate, and the fluorescent compound is probably a 3’- or 2’-sulphonate of dehydrothio-P-toluidine. The ultraviolet spectrum of the compound has a maximum at 332 mp (E:,2 = 93), significantly different from the shorter wavelength maximum of Titan yellow. We do not attribute the shorter wavelength maximum of Titan yellow to contamination by the blue - violet fluorescing compound (contrast Brad- fieldg) but to the contribution of the chromophoric groups of the active components to the spectrum of the dye. Traces of the fluorescent compound are found after fractionation in bands 1 and 2, possibly because of slight hydrolysis on the Sephadex column, but the ratio of the 405 to 410 mp maximum to that at 320 to 325 mp is almost constant in unfractionated Titan yellow band 1 and band 2, with values of 2-30, 2.47 and 2-43, respectively.Paper chromatography has confirmed that acetone removes all but a trace of the blue -violet fluorescing compound from Titan yellow, but this does not increase the ratio of Titan yellow maxima given above. By using model compounds we have confirmed that Sephadex G-10 has a molecular weight exclusion limit of 700. It would be expected that Titan yellow (estimated molecular weight 696) would also be excluded within the void volume of the column. However, the active components of Titan yellow are adsorbed differentially whereas the sodium chloride, behaving as expected with Kd = 1, appears when a volume of water equal to the void volume (V,) and the column internal volume (Vi) has passed through the gel.Presumably the behaviour of bands 1 and 2 indicates a structural difference, which, in view of the similarity of their spectra, may be no more than a difference in the positions of the sulphonate groups. The evidence does not allow us to say whether or not bands 1 and 2, which are mobile in water and in aqueous acetone, respectively, are single compounds or mixtures of isomers. An improved solvent system for paper chromatography would probably resolve this question. Bands 1 and 2 do not separate into further components when passed down the Sephadex column a second time.The most active Titan yellow fraction (band 2) gives excellent results in the determination of magnesium in silicate minerals. With Meyrowitz’slO procedure, in which the samples are prepared at a constant acidity, it is unnecessary to buffer the solution when developing the Titan yellow - magnesium complex’ because the final alkalinity is the same for each sample, pH 13.2. As such small amounts of sample are needed for the determination, inter- ference by other metals is usually negligible in the analysis of silicate minerals. By using the active fraction at a concentration of 0-008 per cent. instead of the usual 0.02 per cent., the calibration can be extended down to 20 pg of magnesium, while the high upper level of 150pg is maintained. For the determination of magnesium in other materials, e.g., soil, the Titan yellow method may need to be modified, see, e g ., Hall et aL7 With the technique described above, a reproducible, highly active fraction, salt-free and relatively uncontaminated by other less active components, can be isolated from com- mercial Titan yellow. The sodium chloride content of the starting material and the amount90 KING AND PRUDEN of less active material present may vary within wide limits; in our sample the amount of the most active fraction, recovered quantitatively from the Sephadex column, was 28.5 per cent. of the original dyestuff. We thank Dr. Francis J. Flanagan, United States Geological Survey, Washington, D.C., Professor A. K. Baird, Pomona College, Claremont, California, and Dr. K. Govindaraju, Centre de Recherches Pktrographiques et Geochimiques, Nancy - Vandoeuvre, France, for samples of analysed silicate minerals. REFERENCES 1. 2. 3. 4. 5. 8. 7 . 8. 9. 10. 11. 12. 13. Kolthoff, I. M., Biochem. Z., 1927, 185, 344. -, Chem. Weekbl., 1927, 24, 254. Hirschfelder, A. D., and Searles, E., J . Bid. Chem., 1934, 104, 635. van Wesemael, J. Ch., Analytica Chirn. Acta, 1961, 25, 238. Bradfield, E. G., Analyst, 1960, 85, 666. Sandell, E. B., “Colorimetric Determination of Traces of Metals,” Third Edition, Interscience Publishers Ltd., London, 1959, p. 591. Hall, R. J., Gray, G. A., and Flynn, L. R., Analyst, 1966, 91, 102. Mikkelsen, D. S., and Toth, S. J., J . Amev. SOC. Agron., 1947, 39, 165. Bradfield, E. G., Analytica Chim. A d a , 1962, 27, 262. Meyrowitz, R., Amer. Minev, 1964, 49, 769. Fleischer, M., Geochim. Cosmochim. Actn, 1965, 29, 1263. Webber, G. R., Ibid., 1965, 29, 229. Roubault, M., de la Roche, H., and Govindaraju, K., Sciences Terve, 1962-1963, 9, 339. Received J u l y 19th, 1966