692 HART: THE LEAD CONTENT OF WHEATEN FLOUR [Vol. 76 The Lead Content of Wheaten Flour and its Determination BY H. V. HART A method for the estimation of lead in flour and other wheat products is described. The method involves dry ashing of the sample, preliminary separation of lead as the diethyldithiocarbainate complex and its determination absorptiometrically by the mixed colour technique. Destruction of organic matter by dry ashing under controlled conditions has been investigated and shown not to result in any significant loss of lead from flour. Quantities of lead of less than 1 p.p.m. were estimated with good duplica- tion of results. In the samples of flour examined, the normal range of lead was from 0.03 to 0.20p.p.m. (average 0.10). TRACES of lead in wheat and wheaten products hiave been reported by a number of investi- gators;lJJ the work described in this paper was undertaken to obtain figures for the lead content of commercially milled National flour.The amount of lead was not expected to exceed 1 p.p.m., and in the choice of a method of estimation dithizone offered much greater sensitivity than the sulphide procedure. The accurate application of the former method, however, is subject to certain precautions (Clifford and Wichmann4) and the method used in the final estimation of the lead was the mixed colour method of the Association of Official Agricultural Chemists’ “Methods of Analysis,” Sixth Edition, 1945, with certain adjustments of volume to give maximum sensitivity. A Committee of the Society of Public Analysts and Other Analytical Chemists has also been considering the same problem and details of a method they are examining (not yet published) were made available to us.As shown later, the two methods, which in fact follow similar lines, agree well. DESTRUCTION OF OKGANIC MATTER- Wet oxidation involves less risk of loss of lead, but it was found that the process resulted in the extraction of small but variable amounts of lead from the glass, which was a hard borosilicate type. Further, the blank on the reagents formed a large proportion of the total lead because of the limitation of the amount of sample taken. Dry ashing is generally considered to involve some loss of lead, and it was therefore necessary to carry out determinations of lead added to flour (as acetate or nitrate) in order to examine this point.Certain of the results are shown in Table I. Ashing was carried out in silica dishes, overnight, in a muffle a t a temperature not exceeding 500” C. For this range of lead, the recoveries were satisfactory and dry ashing was therefore adopted. Organic matter can be destroyed either by wet oxidation or by dry ashing. TABLE I EFFECT OF DRY ASHING ON LEAD CONTENT Initial lead content of flour, Added lead, p.p.m. p.p.m. 0.01 0.20 0.01 0.40 0.16 0.20 0.16 0.40 0.16 0.60 0.16 0.80 0.07 0.60 0.07 1.00 Total lead found, p.p.m. 0.19 0.44 0.35 0.53 0.74 0.95 0.67 1.06 Recovery, p.p.m. 0-18 0.43 0.19 0.37 0.58 0.79 0.60 0.99 Recovery, 90 108 95 93 97 99 100 99 Yo SEPARATION OF DIETHYLDITHIOCARBAMATE COMPLEX- In order to avoid precipitation due to the presence of calcium and magnesium phos- phates when the ash solution was made alkaline, a preliminary separation of lead and otherDec., 19511 AND ITS DETERMINATION 693 metals as the diethyldithiocarbamates was made from a slightly acid solution (Tompsetts).A chloroform solution of diethylammonium diethyldithiocarbamate was used (Strafford, Wyatt and Kershaw6) and the lead was readily extracted at a pH between 3 and 4. The subsequent wet oxidation of the carbamate residue was rapid and did not involve any measurable contamination from the glass. Filtration-Filtration at any stage of the estimation was avoided when it was found that the last traces of metals were not removed from filter-paper by dilute nitric acid or by a chloroform solution of dithizone.Silica-Solution of the ash in dilute hydrochloric acid usually resulted in a slightly opalescent liquid, probably because of the presence of traces of silica. By taking to dryness with hydrochloric acid and dehydrating at 100" C, the insoluble material became aggregated and the supernatant liquid cleared. Such treatment, however, always involved loss of lead, no doubt by adsorption on the silica. Recovery of this lead would involve treatment of the insoluble material with hydroffuoric acid. As the recovery of added lead was satisfactory, however, it was decided to ignore the slight opalescence of the ash solution. Bismuth-The method does not distinguish lead from bismuth, which forms an orange- coloured dithizonate, but the latter may be separated with dithizone at pH 3 (Sandell'). Several separations were carried out on the samples, but no evidence of interference by bismuth was found.An alternative method of separation is to extract the bismuth from a hydrochloric acid solution, which must be at least 2 N , with diethylammonium diethyldithiocarbamate solution (Strafford et al).6 METHOD REAGENTS- Nitric acid-AnalaR grade acid redistilled in an all-glass Pyrex still. Hydrochloric acid, diluted (1 + 1)-Mix equal volumes of AnalaR grade acid, sp.gr. 1.18, Kedistil in an all-glass Pyrex still and adjust the distillate to 5 iV. Sulphuric acid-AnalaR grade acid selected for its low lead content. Ammonium hydroxide, sp.gr. 0-880-We have found the AnalaR grade satisfactory. Ammonium citrate solution, 50 per cePzt.-Dissolve 500 g of citric acid in 300 ml of distilled water and carefully add about 450 ml of ammonium hydroxide, sp.gr.0-880, until the solution is neutral to thymol blue. Extract with successive quantities of dithizone in chloro- form until all traces of metal complexes are removed and the last extract is green. Finally decolorise by extraction with pure chloroform (Blaxter and Allcrofts) . Chloroform-Redistil from calcium hydroxide. Diethylammonium diethyldithiocarbamate solution-Prepared as described by Strafford Stock solzttion-Dilute 3.0 ml of redistilled diethylamine to 10.0 ml with chloroform and add slowly, with stirring, to 1.0 ml of redistilled carbon disulphide, previously diluted to 10.0 ml with chloroform. Cool and preserve in a dark-coloured glass-stoppered bottle.Extraction reagent-Dilute 5.0 ml of stock solution to 100 ml with chloroform. Prepare the stock solution once a week and the dilute solution daily. Dithizone-The commercial reagent was purified by the following method. and water. Filter. et aL6- Dissolve about 1 g in 100 ml of chloroform and filter off any insoluble material. Extract with four 100-ml portions of dilute ammonium hydroxide (1 ml of ammonium hydroxide, spgr. 0.880, added to 99 ml of water). Combine these extracts and precipitate the dithizone by adding a slight excess of sulphurous acid, containing about 5 per cent. of sulphur dioxide, as recom- mended by B a r n e ~ . ~ Extract the dithizone with two or three 20-ml portions of chloroform. Wash the combined extracts twice with water, and evaporate the chloroform gently.Dry the residue in a desiccator. Prepare a 0.1 per cent. stock solution in chloroform, and store away from light. Standard dithizone solzttion-Dilute the 0.1 per cent. stock solution to give a solution containing 4 mg per litre in chloroform. Nitric acid, 1 per cent.-As A.O.A.C. Methods of Analysis.lo Dilute 10 ml of fresh water- white nitric acid, sp.gr. 1.40, to 1 litre with distilled water. Boil off nitrous fumes from the re-distilled acid before making the dilution. To 100 ml of a 10 per cent. solution of recrystallised phosphate-free potassium cyanide in a 500-ml volumetric Store in a cool place away from light. Ammonia - cyanide mixture-As A.O.A.C. Methods of Analysis.1°694 HART: THE LEAD CONTENT OF WHEATEN FLOUR [Vol.76 flask add sufficient ammonium hydroxide to introduce 19.1 g of NH, and make to volume with distilled water. Standard lead solution-Prepare from AnalaR lead nitrate a solution containing 0.1 mg of lead per ml in 1 per cent. nitric acid. Potassium cyanide solution, 10 per cent.-Prepare a 50 per cent. w/v solution in water and extract lead with small portions of dilute dithizone in chloroform. Finally extract the excess of dithizone with chloroform and dilute the solution to 10 per cent. (Sandell,' p. 395). APPARATUS- Wherever possible apparatus should be made of Pyrex glass. Apparatus must be carefully cleaned with nitric acid; particular attention should be paid Reagents should be stored in Pyrex bottles. All apparatus should be kept for lead The first few results should be viewed with caution, in case lead has not been removed to ground-in stoppers and taps.estimation only. completely from the apparatus. PROCEDURE- Ash 25 g of flour in a 3-inch silica basin overnight (16 hours) in a muffle at a temperature of 450" to 500" C. If the residue is still black, :moisten it with the minimum of nitric acid, dry it at the entrance to the muffle and then complete the ashing in the interior for a few minutes. Add 5 ml of 5 N hydrochloric acid and 26 ml of water, and allow to simmer gently for 30 minutes. Cool the solution and transfer it to a separating funnel with water to bring the total volume to about 40ml. Add ammonium citrate solution to bring the pH between 3 and 4 (full yellow colour to thymol blue, olive to bromophenol blue).Extract with three 5-ml portions of diethylammonium diethyldithiocarbamate solution in chloroform. Run the extracts into a Pyrex micro-Kjeldahl flask, add 2 ml of diluted sulphuric acid (1 + 1) and evaporate the chloroform on a water-bath. Continue heating over a small flame and swirl the tube to dissolve all the residue until sulphur trioxide fumes are evolved. Continue heating to oxidise organic matter and drive off the excess of nitric acid. Cool slightly, add approximately 1 ml of water and evaporate to fuming again. Cool, dissolve the residue in a little water and then dilute to 15 ml. Add 2 ml of ammonium citrate solution and make alkaline to the blue colour of thymol blue with ammonium hydroxide, sp.gr. 0.880. Cool and add 1 ml of potassium cyanide solution.Transfer to a separating funnel and wash in with 5 to 10ml of water. Extract with 10-ml portions of a 10 mg per litre solution of dithizone in chloroform, shaking for about 1 minute. Two portions are sufficient if an excess of dithizone is present in the first extract. Run the chloroform layers carefully into a separating funnel. Shake the combined chloroform extracts with 15 ml of 1 per cent. nitric acid for 1 minute. Transfer the aqueous layer without any of the chloroform into a graduated flask (25 ml for each 10 pg of lead). Extract again with 10 ml of nitric acid. The first extract removes practically all the lead, the second acts as a washing. Measure 25 ml into a separating funnel, add 5 ml of ammonia - cyanide solution and 12.5 ml of standard dithizone solution from a burette.Dry the stem of the separator with filter-paper and run the chloroform layer carefully into a dry &inch test tube. Allow to stand for a few minutes, when any traces of water will collect on the side of the tube. Slight haziness of the solution may be removed by gentle warrning. The chloroform should show a mixed colour indicating a slight excess of dithizone. Measure the absorption with a Spekker absorptiometer at 510 mp in a 2-cm cell, using Ilford 604 filters and comparing with pure chloroform. The volume of standard dithizone used must be sufficient to fill the 2-cm cell and may be different from the 12.5 ml used above. Should the final colour be red only, indicating insufficient dithizone, then the nitric acid solution should be diluted and a fresh 25 ml treated as above.If all the solution has been used, then all the lead must be extracted again with the 10 mg per litre solution of dithizone in chloroform and the analysis repeated from that stage. Cool slightly and add 2 drops of nitric acid. Dilute to the mark with 1 per cent. nitric acid. Shake for 1 minute. This volume should be kept to a minimum.Dec., 19511 AND ITS DETERMINATION 696 Blank determinations must be made at the same time as each set of determinations, exactly the same procedure and quantities of reagents being used. The blanks under our conditions were normally from 0-3 to 0.5 pg of lead. Prepare a standard curve for the range 0 to 10 pg of lead by diluting appropriate quanti- ties of standard lead solution to 25 ml with 1 per cent.nitric acid, saturated with chloroform. Add 5 ml of ammonia - cyanide solution, 12.5 ml of standard dithizone solution and shake for 1 minute. The standard dithizone solution retains its strength fairly satisfactorily if stored in a Pyrex bottle in a dark place, but the standard curve should be checked periodically at a few points. RESULTS Measure the absorption of the solution as in the estimation. Results of estimations of lead on 24 commercially milled National flours of 81 per cent. extraction by this method and by the Society’s method are shown in Table 11. Sample C58 60 61 63 64 65 66 68 69 70 71 73 75 76 78 79 81 83 84 85 87 88 90 92 TABLE I1 ESTIMATION OF LEAD IN FLOUR Estimate by method described, p.p.m. 0.03, 0.04, 0.06 0.09, 0.10, 0.04, 0.06. 0.07 0.16, 0-20 0.14, 0.13 0.18, 0-24, 0.33, 0.32 0.12, 0.06, 0.08 0.07, 0.08 0-07, 0.06 0.14, 0.14 0.06, 0.08 0.10, 0.06 0.08, 0.19, 0.14 0.20, 0.20 0-06, 0.08 0.18, 0.18 0.10, 0.12 0.18, 0.15 0.08, 0.06 0.04, 0.02 0.08, 0.07 0.09, 0.08 0.08, 0-05 0.03, 0.06 Average 0.04 0.08 0.07 0.1 8 0.14 0.27 0.09 0.08 0.07 0.14 0.07 0.08 0.14 0.20 0.07 0.18 0.11 0.17 0.07 0.03 0.08 0.09 0.07 0.05 Estimate by Society’s method, p.p.m.0-04, 0.06 0-04, 0.07 0-06, 0.04 0-16, 0.20 0-22, 0.13 0.21, 0-30 0-06, 0.09 0-05, 0.06 0.08, 0.08 0-15, 0.14 0.08, 0.08 0.08, 0.07 0.13, 0.13 0-23 0-06, 0.12 0-16 0.10, 0.16 0-17, 0-20 0.03, 0.05 0.03, 0.04 0-04, 0-07 - - 0.04, 0.03 Average 0.05 0.06 0.05 0.18 0.18 0.26 0.08 0.06 0.08 0.15 0-08 0.08 0.13 0.23 0.09 0.16 0.13 0.19 0.04 0.04 0.06 - - 0.04 By the method described, 23 of the 24 samples are estimated to contain lead in the range 0.03 to 0-20 p.p.m., with an average of 0.10 p.p.m.The other sample (C 65), averaging 0.27 p.p.m., appeared to be unusual. Twenty-two of the samples were examined by the Society’s method and, again excluding sample C 65, the range of results is 0.04 to 0.23 p.p.m., the average being 0.10 p.p.m. Destruc- tion of organic material in this method was by ashing in the presence of magnesium nitrate, and the method was modified slightly for greater sensitivity. A t present calcium carbonate (Creta praeparata) is added to flour at the rate of 14 02. per 280 lb. of flour. Determinations on 8 different samples of commercial Creta praeparata indicated a lead content of 1 to 4 p.p.m.The B.P. limit for this substance is, however, 20 p.p.m., at which level it would account for 0-06 p.p.m. of lead in the flour. My thanks are due to Mr. C. L. Hinton (British Food Manufacturers’ Research Associa- tion) and Dr. H. H. Green (Ministry of Agriculture and Fisheries) for their advice in the early stages of this work. I am also indebted to Mr. G. Taylor, lately President of the Society of Public Analysts and Other Analytical Chemists, for kindly allowing me to see details of the method developed by the Society.696 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. HARRISON, LEES AND WOOD: THE ASSAY REFERENCES [Vol. 76 Kent, N. L., J . SOC. Chenz. Ind., 1942, 61, 183. Kehoe, R. A., Cholak, J., and Story, R. V., J . Nutr., 1940, 19, 588; 590. Monier-Williams, G. W., “Lead in Food,” Reports on Public Health and Medical Subjects, No. 88, Clifford, P. A., and Wichmann, H. J., J . Ass. 08. Agric. C h e w , 1936, 19, 130. Tompsett, S. L., Biochem. J., 1939, 33, 1231. Strafford, N., Wyatt, P. F., and Kershaw, F. G., Analyst, 1945, 70, 232. Sandell, E. B., “Colorimetric Determination of Traces of Metals,” Interscience Publishers Inc., Blaxter, I<. L., and Allcroft, R., Veterinary Laboratory, Ministry of Agriculture, Weybridge. Barnes, H., Analyst, 1947, 72, 469. Association of Official Agricultural Chemists, “Methods of Analysis,” Sixth Edition, 1945, p. 456. 1938. New York, 1950, p. 398. Private communication. RESEARCH ASSOCIATION OF BRITISH FLOUR-MILLERS CEREALS RESEARCH STATION ST. ALBANS, HERTS. June, 1961