A METHOD FOR THE ESTIMATION OF CHLORIDES IN CHEESE 197 A METHOD FOR THE ESTIMATION OF CHLORIDES IN CHEESE. BY ELFREIDA C. V. CORNISH, M.Sc., AND JOHN GOLDING, F.I.C. (Rend at the ~Weeting, March 31, 1915.) THE usual method for the estimation of chlorine in cheese, by water extraction from the incinerated solid and subsequent titration with silver nitrate, using potassium chromate as indicator, was found to give unsatisfactory results unless the incineration was performed at a very low temperature.Carried out in this manner the experi- ments are very tedious, and attempts to hasten the process by raising the temperature were attended by considerable loss of chlorine, due to volatilisation of the chlorides present. The well-known method of incineradhg with lime gives satisfactory results, but is equally tedious.O’Sullivan in a paper (ANALYST, 1914, 39, 425) pointed out that chlorides are decomposed and chlorine lost on incinerating organic substances, especially in the presence of magnesium sulphate. The quantity of this latter compound present in a substance containing chlorides may be sufficient to cause the. total loss of the chlorides on incineration. We therefore determined to devise a method which should, if possible, be both more rapid and more accurate than those just mentioned.The method as first adopted was as follows : About 1 grm. of cheese was weighed on a chlorine-free filter-paper and placed in a Kjeldahl flask, and about 20 C.C. of strong, nitrogen-free sulphuric acid and a few small pieces of pumice added.These quantities were used because it was hoped that th6 ordinary Kjeldahl nitrogen determination might subsequently be carried out on the residue from the chlorine determination. A nitrogen bulb, containing a known volume of & silver nitrate and198 CORNISH AND GOLDING : some strong nitric acid, was inserted into the neck of the flask by means of a tightly fitting cork.The flask was clamped in an almost horizontal position and heated over an Argand burner. The hydrochloric acid formed by the action of strong sulphuric acid on the chlorides present in the cheese was driven over into the acid silver nitrate solution and precipitated as silver chloride. When the reaction was completed (determined by inserting another bulb with fresh solution) the silver chloride was filtered off, the precipitate washed till free from nitrate, and the washings added to the filtrate.Volhard's method of titration was used for the determination of the residue of the silver &rate, the result was subtracted from the original quantity, and the chlorine calculated as sodium chloride from the results so obtained. In this method the precipitated silver chloride must first be filtered off; the solution of silver nitrate is then titrated with potassium thiocyanate, using saturated iron alum as indicator.White silver thiocyanate is precipitated, and as soon as the reaction is oomplete brown iron thiocyanate is formed. The end-point is not readily determined in the presence of silver chloride, as a counter-reaction may take place, resulting in the formation of silver thiocyanate and ferric chloride, thus : Fe( CN S), + 3AgCl= FeCl, + 3AgCN S.For this reason it is necessary to filter off the silver chloride as already stated (Landw. V'rsuchsstat., 1914, 83, 309-316). About 1 C.C. of saturated iron alum is used as indicator, Preliminary experiments to determine the amount of nitric acid necessary to retain in solution all the salts other than silver chloride were made.Various solutions were made up containing 5, 20, 40, about 60 and 80 per cent. nitric acid and 10 per cent. water and the remainder & silver nitrate. I t was found that solu- tions containing 5 and 20 per cent. nitric acid gave results which were much too high, owing to the precipitation of carbonate and probably sulphate, while thoae containing 40 per cent. and above gave concordant results.It was decided that the solutions should always contain about 50 per cent. strong nitric acid. The apparatus first used, and already described, was not always satisfactory, as occasionally the silver nitrate solution sucked back into the Kjeldahl flask. I t was therefore decided to aspirate.This having been done, the next difficulty experienced was that of corks. It was necessary that the corks should fit perfectly, but their corrosion was rapid by reason of the oonstant exposure to strongly acid fumes. It was decided, therefore, to eliminate them altogether from the distillation flasks. This was done by inserting into the necks of the flasks the tubular end of a large soda-lime tube of such a size that the bulb just blocked the mouth of the flask.An extension of the narrow end of the tube dipped into the silver nitrate and nitric acid solution in a flask fitted with a rubber stopper with a second tube leading to the aspirator pump. It was decided later to add a second control wash-bottle between the first flask and the pump. Even with aspiration, however, it was found that the hydrochloric acid condensed in the bulb of the soda-lime tube.This was readily prevented by passingA METHOD FOR THE ESTIMATION OF CHLORIDES IN CHEESE 199 a Bunsen flame rapidly up and down the neck of the flask while the reaction was taking place. The hydrochloric acid was thus kept vaporised and its rate of absorption accelerated.Some difficulty was experienced in obtaining an absorption flask which was efficient and did not require an unnecessarily large quantity of silver nitrate and nitric aoid. The form finally adopted was that shown in the diagram. FIG. 1. Experiments using pure NaCl gave the following results : Amount taken. 0.246 0.1128 0.2362 0.184 0.1005 0.0879 0.0937 0.1174 0.1026 Amount found.0.2469 0.1139 0.2364 0-1865 0.0993 0-0886 0.0936 0.1170 0.1036 Control experiments were carried out with sulphuric acid and pumice, and also with pure casein, but no precipitation of silver chloride resulted in either case.200 CORNISH AND GOLDING : Further experiments with cheese were then carried out. The cheese used was a Stilton, showing brown discoloration, and it was thought that interesting results might be obtained by determining the chlorine in the brown as distinct from the white part.Chlorine, expressed as Sodium Chloride. Brown Part. White Part. (1) 2.519 per cent. (2) 2.468 ,, (2) 2.750 ,, (3) 2.432 ,, (3) 2.536 ,, (4) 2.584 ,, (1) 2.617 per cent. -- Maximum Error. Maximum Error. Average 2.473 ... k 1.6 per cent. Average 2-622 ... k 4.9 per cent.These results show no marked difference in the chlorine content of the discoloured and the white parts of the cheese, but the degree of accuracy which may be expected from this method may be seen from them. In order to save time in the duplicating of results, a battery of four flasks in series was fitted up, so that the aspiration of all four could be carried out simul- taneously from one pump.To prevent the condensation of hydrochloric acid in the necks of the flasks, a rectangular water-tank, provided with a cover and heated by a special burner, was used. Four round openings were made in each of the two long sides, those on the one side being lower than those on the other. These openings were joined up by slanting metallic cylinders soldered into the sides of the tank.The Kjeldahl flasks rested on a metal platform, and were heated by separate Argand burners. The neck of each flask passed through one of the slanting cylinders, and from the other side of the tank the soda-lime tubes were inserted into the necks of the flasks. The flasks were thus kept constantly heated by steam from the wate? in the tank. The absorption flasks and control wash-bottles were placed on adjustable wooden platforms.After the sulphuric acid (about 20 to 30 C.C. nitrogen-free) had been added to the Kjeldahl flask, and the neck inserted into the cylinder, connection was made with the absorption flask and control wash-bottle, and aspiration begun. The neck of the flask was gently rotated round the soda-lime tube until the contents of the flask were well mixed.I t was found advisable to add a little distilled water as well as the sulphuric acid, or to grind up the cheese, after weighing, with warm distilled water, and then wash into the flask before adding the sulphuric acid. The contents of the flasks having been well mixed by rotating, the flasks were heated, at first gently, then more strongly, until the contents boiled briskly.The length of time necessary to insure complete volatilisation of the hydrochloric acid depends naturally upon the amount of Eiubstance originally taken and the quantity of chlorides present. In no case was the time required found to exceed one and a half hours. After the removal of the first absorption flask, a second one was always connected with the Kjeldahl flask in order to make quite sure that all the hydrochloric acid had passed into the first.The Kjeldahl flasks were then removed to the fume chamber, the sulphuric acid reaction continued with subsequent addition of potassium sulphate and potassium permangenate, and the nitrogen determination completed by the ordinary Kjeldahl method.A METHOD FOR THE ESTIMATION OF CHLORIDES IN CHEESE 201 The following results were obtained, using the battery : STILTON CHEESE NO.c 50 (1ST SAMPLE), SHOWING BROWN DISCOLORATION. GI expressed as NaCI. White Part. Brown Part. (1) 1-821 per cent. (2) 1.825 ,, (2) 2.138 ,, (3) 1.802 ,, (3) 2.037 ,, (4) 1-814 ,, (4) 2.191 ,, (1) 2.209 per cent. -- Mrtximnm Error. -- Maximum Error. Average 1.815 ... +, 0.7 per cent. Average 2,144 ...+, 5 per cent. STILTON CHEESE NO. C 50 (2ND SAMPLE). CE expressed as NaCE. Brown Part. (1) 2.071 per cent. (2) 2.046 ,, (3) 2-053 ,, -- Maximnm Error. Average 2.057 ... +, 0.7 per cent. STILTON CHEESE No. 89, SHOWING BROWN DISCOLORATION. CE expressed as NaCE. Brown Part. (1) 2.610 per cent. (2) 2.771 ,, (3) 2.738 ,, (4) 2.732 ,, .., Maximnm Error. Average 2.713 ... * 3.8 per cent.CHEDDAR CHEESE. CI expressed as NaCE. (1) 1.574 per cent. (2) 1.553 ,, (3) 1.613 ,, Maximum Error. Average 1.580 ... & 2.1 per cent. In each case the cheese was prepared by passing through a mincer, and was then very carefully and thoroughly ground with a, pestle and mortar. Some experiments on butter gave the following results : CE expressed as NaCE.(1) 0.746 per cent. (2) 0,726 ,, (3) 0.759 ,, (4) 0.787 ,, Maximum Error. Average 0.7545 ... f 4.3 per cent.202 CORNISH AND GOLDING : The butter was heated by placing it in a flask in hot water till it melted, and was then well shaken until cool to insure thorough mixing. From the results obtained, the advantages of this method over that of incinerating and water extraction seem to be : (1) Greater accuracy and rapidity.There is no danger of loss of chlorides, and no necessity for lengthy and careful heating. (2) The same cheese residue, in the same flask and with the same sulphuric acid, may be used for the nitrogen estimation in the solid cheese by Kjeldahl’s method, part of the necessary heating having been carried out in the chlorine estimation.DAIRY RESEARCH LABORATORIES, UNIVERSITY COLLEGE, READING. DISCUSSION. The PRESIDENT, in inviting discussion, remarked that the method of ignition with alkali was perhaps not entirely free from error, particularly in the case of sub- stances-of which cheese was a notable example-which it was difficult to mix intimately with the lime or other alkali employed. I t was, moreover, a lengthy process, as the ignition must be effected at a low temperature, and this had to be followed by extraction with water, and possibly further ignition of the residue.The authors’ method, which evidently gave concordant results, appeared to have the advantage of being more speedy, and also of enabling the chlorine and the nitrogen to be estimated in one and the same operation.Possibly the authors might extend their experiments in the direction of meat extracts, in the case of which it would be a convenience to be able to determine the chlorine and the nitrogen together. The quantity of chlorine, however, was in that case much larger than in the case of cheese or butter. He should like to ask whether in the case of butter any trouble was experienced through bumping; and also-although this had no bearing on the actual subject of the paper-whether the authors could say what was the exact nature of the difference between the brown and the white portions of the cheese.Dr. VOELCKER remarked that, while it was undoubtedly valuable to have a reliable method for the estimation of chlorides in organic substances, this did not carry one much farther as regards the significance of chlorides in the case of such materials as cheese and butter.If, as the President had hinted, the presence of salt had some bearing upon the quality of cheese, and could indicate why some portions of a cheese were good and others inferior, the accurate estimation of the chlorides would be of importance. As it was, everyone who had to do with the making of cheese and butter knew that the use of salt in these was quite arbitrary, nor, unless where brine and not solid salt had been employed in butter-making, was there any likelihood of the salt being evenly distributed throughout a sample.Mr. E. M. HAWKINS, referring to the addition of water to the cheese, asked whether the quantity of water added was such that it might possibly affect the nitrogen determination.It was well known that in the Kjeldahl process the presence of an excessive quantity of water was liable to cause the results to be low. Mr. GOLDING, in reply, said that in the case of butter a little pumice was used,A METHOD FOR THE ESTIMATION OF CHLORIDES IN CHEESE 203 and no bumping had occurred. He desired to lay particular stress on the necessity for keeping the neck of the flask sufliciently warm. In reply to Dr.Voelcker, it was in the course of an endeavour to ascertain the cause of the discoloration referred to that this investigation was undertaken. The matter was an important one, for the occurrence of this ‘( yellow discoloration ” caused an annual loss of many thousands of pounds to Stilton cheese makers, since, when cheeses were so affected, their market value was greatly reduced, and they quickly deteriorated in quality and would not keep.It seemed that the discoloration was due to an organism producing tyrosinase, or some similar enzyme, which acted on the decomposition products of the casein. In his early work on the subject he had isolated such an organism, which blackened tyrosine media,.and many more had been isolated by Dr. R. Sten- house Williams, who has not yet published his results. While visiting the cheese factories he (Mr. Golding) had noticed that the cheeses were freer from this dis- coloration when but little salt was used than when the quantity of salt was large; but, as he had not been able to visit more than twenty-five or thirty factories, this evidence was not very weighty from a statistical point of view, although it appeared to have some scientific support, as was shown in a recent article in the Journal of the Board of Agricultum.It was thought that possibly accurate determination of the salt present in the cheese might throw some light on the matter. Uneven salting was, of course, very common ; the quantity of salt varied from about 7 to 13 ounces per cheese, and this was liable to be unevenly distributed; but if it appeared that the discoloration was regularly accompanied by the presence of an excessive quantity of salt, he thought that a step would have been made in the direction of providing a practical means for checking the fault. Now that the method of analysis had proved satisfactory, they proposed to make further estima- tions in the discoloured parts and in the white parts respectively. With regard to the addition of water, the quantity of water necessary to soften the cheese was very small, and was not enough to upset the nitrogen estimation. He had, however, made a good many estimations in which rather large quantities of water were used, and had never found this to cause any difficulty.