248 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION* BY C. L. HINTON F.I.C. FOREWORD.-At the time the agreed Jam Standards were introduced the desirability of extending the methods for determining the fruit-content of jams was discussed by a small committee of the chemists engaged in this industry. Arising out of these discussions the late L. K. Boseley suggested that the quantity of lead precipitate formed when a lead acetate solution was added to a solution of a jam, might afford a useful indication of the amount of fruit in the sample and as preliminary tests showed that there appeared to be some value in the method it was decided that the Research Association should investigate it. This investigation has been carried on during the three past fruit seasons in order to put the process on a satisfactory basis and to obtain suitable data for calculating the percentage of fruit in the jam.It will be seen from what follows that the method will not by itself indicate the percentage of fruit in a jam with any more certainty than the other methods already employed; it may be particularly useful in the analysis of jams made from a mixture of fruits the acids of which differ i.e. one fruit containing citric acid and the other malic. It is also sometimes useful in indicating whether a fruit juice or a commercial pectin has been used in making the jam. By its use it is possible to place the various fruits in three classes i.e. those containing mainly (i) citric acid (ii) malic acid (iii) lactic acid or an acid having similar lead-precipitating properties.Mr. C. L. Hinton has been responsible for the carrying out of this investigation, and has drafted the report which follows. The Council of the Association has given permission for the publication of any work of this description which may be of assistance to analysts in maintaining the Jam Standards. T. MACARA BOSELEY’S METHoD.-The method proposed by Boseley as mentioned in the foreword was to dissolve 50grms. of jam add 100 ml. of 2 per cent. lead acetate solution make up to 500 ml. and filter off the lead precipitate together with the insoluble matter of the fruit; 100 ml. of the filtrate were heated to boiling and the unprecipitated lead was titrated with approximately 1 per cent.ammonium molybdate solution with tannic acid as an outside indicator. A blank titration was also made on 20 ml. of the lead solution diluted with water. The difference was called the “lead number” of the jam. When the molybdate solution was adjusted in strength to be equivalent volume for volume to the 2 per cent. lead solution the “lead number” represented the number of ml. of 2 per cent. lead acetate solution completely precipitated by 10grms. of jam. Essentially this is the procedure still used though various refinements have been made which will be described later. The “lead number,” though empirical, * Communicated by the British Association of Research for the Cocoa Chocolate Sugar Confectionery and Jam Trades HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 249 is quite a convenient mode of expressing results and as will be seen is readily linked up with the chemistry of the precipitation.THE NATURE OF THE LEAD PREcrPrTATE.-Preliminary experiments on strawberry and raspberry jams of known composition soon showed that the “lead number” was not as Boseley supposed a measure of any constituents peculiar to the jam fruits but was directly related to the amount of acid both free and in the form of salts in the jam. In further experiments it was found that malic acid gave no precipitate whilst citric and tartaric acids gave “lead numbers,” on 0.05 grm. of acid of 6.7 and 6.1 respectively. A simple calculation shows that for the complete precipitation of 0.05 grm. of these acids as normal lead salts 6.8 and 6.3 ml.of 2 per cent. lead acetate solution would be required. Thus practically within the accuracy of the titration (which is not greater than about 0.1 to 0.2 ml.) these acids seemed to be completely precipitated. The behaviour of malic acid seemed likely to account for the fact noticed by Boseley that pomace extracts etc. (in which most of the acid presumably would be malic) lacked the lead-precipitating constituents of the jam fruits. However, further experiments described later showed that the matter was by no means so simple. the acidity of the solution from which the precipitation was made was varied it was found that in more strongly acid solutions (e.g. by adding acetic acid to the jam solution to bring it down to a @= of 2*5) the end-point of the titration of the excess of lead was upset; but addition of sodium hydroxide to give a p of 6.7 caused a slight increase in the “lead number,” whilst complete neutralisation of the jam before adding the lead acetate raised the “lead number” about 1 ml.Finally addition of excess of sodium hydroxide had as its main result the precipitation of some or all of the excess of lead as hydroxide. In a series of precipitations of citric acid solution first brought to varying @= (from 2.5 to 8.0) by addition of sodium hydroxide it was found that the lead equivalent for 0.08 grm. of citric acid increased gradually over the range mentioned from 11.0 ml. to 12.2 ml. even the lowest figure being rather higher than the theoretical. At the same time the p values of the filtrates (tested colorimetrically) were different from those of the original solutions; they were all now compressed into the range 4.4 to 5.6.In view of this tendency to high results an attempt was next made to keep down the j5 by means of additions of acetic acid. Mixtures of citric acid and sodium hydroxide (from j5* 2.5 to 8.0 with constant amounts of citric acid) were treated with moderate and relatively large amounts of acetic acid before addition of the lead acetate. The lead equivalents expressed on 0.1 grm. of citric acid and also the j5 values are shown in Table I. Since the theoretical figure for the lead equivalent of 0.1 grm. citric acid should be 13*55ml. the results showed that an excess of acetic acid prevented complete precipitation while a lack of it tended to permit the precipitation of too much lead.In view of these experiments it was decided to adopt a modified IMPROVEMENT OF THE LEAD ACETATE REAGENT.-h experiments in whic 250 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION reagent prepared by incorporating acetic acid in the lead acetate solution in proportion corresponding with the 2-ml. additions above. Thus the new reagent was composed of :-Lead acetate crystals 20 grms. ; glacial acetic acid 2-5 ems. per litre. TABLE I CONTROL OF LEAD CITRATE PRECIPITATION WITH ACETIC ACID 2N acetic acid added (per 500 ml. of mixture) 0 ml. 2 ml. 20 ml. && & Lead equiv. pH (before 2 per cent. Citrate addition of p lead PE Lead P H Lead mixture acetic acid filtrate acetate filtrate equiv.filtrate equiv. I 2.5 4.4 13-8 3.1 13-5 3.6 12.8 I1 5 . 0 5.2 14-3 4 . 9 13.7 3 . 9 12.8 I11 8 . 0 5 - 6 15.2 5 . 0 13.8 4 . 0 12.9 DEPENDENCE OF RESULTS ON TOTAL AMOUNT OF PRECIPITABLE AcID.-In tests with the improved reagent it was found that the amount of lead precipitated per unit of acid varied slightly but definitely according to the total amount of citric acid present; or what is almost the same thing according to the total mount of lead precipitated. Thus when amounts of citric acid from 0.2 grm. to 0.6 grm. (per 100 ml. of lead solution) were used in the test the lead equivalents were slightly higher than expected for the smaller amounts of acid, and slightly lower for larger amounts. For the intermediate amounts the precipitation agreed with the theoretical values.Table I1 gives the actual figures. In preparing the mixtures for precipitation sodium hydroxide was added to neutralise about one-third of the citric acid so as to correspond with conditions in fruit extracts. TABLE I1 DEVIATION FROM CORRECT LEAD PRECIPITATION WITH TOTAL AMOUNT PRECIPITATED Citric acid used (per 100 ml. lead solution) ml. 0.20 0-30 0-40 0.50 0-60 Calculated titration diif erence ml. 5.4 5.1 10-85 13.55 16.25 Titration difference found Difference ml. ml. 5 . 6 +Om2 s - 3 +om2 10.9 +0-05 13.55 0 15.9 -0.35 A few experiments on varying quantities of fruit extracts showed as was anticipated that discrepancies were even greater than with citric acid solutions.The results are given in Table 111 where the lead equivalents for the varying amounts of fruit extract taken have been calculated in the last column t o a common basis of 10 grms. to correspond with the fixed 10 grms. of Boseley's original method HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 251 TABLE I11 VARIATION OF LEAD NUMBER OF FRUITS WITH AMOUNT OF FRUIT EXTRACT TAKEN Amt. of 50 per cent. extract represented ml. Raspberry 6 8 10 12 6 8 10 12 Blackcurrant . . I 3 4 5 Fruit in titration Gooseberry 4 9 Lead titration diff. ml. 7 . 7 10.0 12-0 14-3 5.1 7 . 3 9.5 11.6 13.7 6 . 7 9 - 6 12.6 15- 1 Lead number per 10 grms. of fruit ml. 25.7 25.0 24.0 23.8 25.6 24.3 23.8 23.2 22.8 67.0 64.0 62.5 60.4 In view of these results it seemed necessary to study further the conditions governing the precipitation of the pure fruit acids themselves both separately and in admixture and thus establish conditions for correct precipitation when applied to fruit and jam solutions.found more convenient to work with solutions of twice the concentration of those previously used i.e. with 250ml. instead of 500 ml. and with 50ml. of filtrate for titration instead of the former 100 ml. In further tests on the precipitation of varying quantities of lead citrate under these conditions a mono-sodium citrate was used (the total citric-content of which had been determined by titration of free acidity and alkalinity of ash). As before there was a high result compared with theory for the smallest amount of citrate precipitated and a low result for the largest amount (Table IV).For the intermediate amount with a lead titration difference of 12 ml. the correct precipitation figure was obtained. CONDITIONS FOR SATISFACTORY PRECIPITATION OF LEAD CITRATE.-It WaS TABLE IV DEVIATION FROM CORRECT LEAD PRECIPITATION FOR DIFFERENT AMOUNTS OF CITRATE Monosodium citrate used Calculated Titration diff. (per 100 ml. lead solution) titration diff. found Difference grm. ml. ml. ml. 0.30 8.0 8 . 2 +0.2 0.45 12.0 12.0 0 0.60 16.0 15.8 -0.2 These figures are similar to those of Table 11 except that the deficiency of precipitation at 16 ml. was not so great. This may have been due to the fact that the doubling of the concentration had had some effect.However it seemed that correct results were obtainable with citric acid if the total precipitation were such that a lead titration difference of about 12 to 14ml. was obtained. BEHAVIOUR OF MALIC ACID IN PRECIPITATION OF MIXTURES.-h preliminary experiments on the precipitation by lead of citric acid when mixed with mali 252 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION acid (so as to simulate the conditions in fruit juices) a co-precipitation of part of the malic acid was found about 40 to 50 per cent. of the theoretical amount of lead being thus precipitated by the malic acid; but the amount was dependent, to an appreciable extent on the total amount of malic acid present as well as on the total amount of lead precipitated.For a more complete investigation mono-sodium citrate and malic acid of analysed composition were used and sodium hydroxide was added to the mixtures to keep the 9 in the ordinary range for fruits viz. about 3.5. In the first series the citric acid was kept constant and the amount of malic was varied. In calculating the amount of precipitation due to the malic acid the effect due to the citric was deducted with a suitable smallallowance for the deviations from correct precipitation already established for that acid. In Table V the effect of. the malic acid has been calculated as a percentage of the full amount theoretically precipit able. TABLE V PRECIPITATION OF MALIC ACID IN ADMIXTURE WITH CITRIC ACID CITRIC ACID CONSTANT Per 100 ml.of lead solution I 1 Amt. of mono-sodium citrate Malic acid Grm. GrlIl. 0.35 0.104 99 0.208 n 0-312 Y Y 0.416 97 0.520 Lead Amt. due to Theor. for Per cent. titration diff. malic acid complete pptn. precipitated 10.9 1.5 2-94 51 12- 15 2 - 8 5.89 48 13 36 4.0 8-83 45 14.15 4 - 8 11.76 41 14.95 5-66 14.72 38 ml. ml. ml. From these results it appeared that the precipitation of the malic acid was relatively less when the total amount of malic (or of malic + citric) was increased; or looking at it in another way there was less precipitation of the malic acid as the amount of excess of lead acetate available for its precipitation was reduced. A second series was tried in which the malic acid was kept constant but the citrate was varied.Again in calculating the amount of precipitation due to the malic acid allowance was made for the slight variation in the citric acid effect (Table VI). TABLE VI PRECIPITATION OF MALIC ACID IN ADMIXTURE WITH CITRIC ACID MALIC ACID CONSTANT Per 100 ml. of lead solution L t . of monu- Lead Amt. due to Theor. for Per cent. sodium citrate Malic acid titration dif€. malic acid complete pptn. precipitated Grm. Grm . ml. ml. ml. 0.20 0.298 8.85 3-35 8.43 40 0.30 9Y 11-75 3-75 Y Y 45 0.40 97 14-35 3-75 99 45 0.50 Y Y 16.4 3.2 w 38 Under these c.onditions the malic acid precipitation again varied reaching a maximum when the lead titration difference was from 12 to 14ml. or unde HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 253 about the same conditions (as regards excess of lead) as for the correct precipitation of citric acid.In a third series the total amount of precipitation was kept approximately constant (by varying the proportions of citrate and malic atid) at about this point of correct citric and maximum malic precipitation (Table VII). TABLE VII PRECIPITATION OF MALIC ACID IN ADMIXTURE WITH CITRIC ACID TOTAL LEAD PRECIPITATION CONSTANT Per 100 ml. of lead solution Amt. of mono-sodium citrate Grm. 0.40 0.35 0-30 0.25 0.20 0.15 Malic acid Grm. 0.178 0.296 0.415 0.543 0.661 0.780 Lead titration diff. ml. 13-2 13.2 13.2 13-15 13.0 12.8 Amt. due to malic acid ml. 2-55 3 . 9 5 . 2 6 - 5 7 - 7 8.8 Theor. for complete pptn.ml. 5-04 8.38 11-75 15.36 18-71 22-08 Per cent. precipitated 51 46 44 42 41 40 This series showed again the effect of increasing quantities of malic acid as in Table V so that this effect is manifested over and above the effect noted from Table VI since it occurs when all the lead precipitations are at what should be the point of maximum malic acid precipitation. The practical outcome of these experiments was that if the quantity of malic and citric acids were kept within suitable limits the malic acid precipitated could be taken as a fixed percentage. In particular if the malic acid used in the test (per 100 ml. of lead solution) amounted to at least 0.3 grm. and the citric acid was from about 0.2 grm. to 0.3 grm. about 40 to 45 per cent.of the malic acid was precipitated. A mean figure of 43 per cent. can be taken t o cover all such cases correctly within the limits of experimental error. Subject to these restrictions we have a means of determining the proportion of malic acid in a mixture of the two acids (or their salts). For this purpose it is convenient to reckon all the acid as citric acid (this total acid is ascertainable directly from titrations of free acidity and of ash when salts are present). Then the theoretical amount of 2 per cent. lead acetate solution equivalent to each 0.1 grm. of acid (as citric) is 13.55 ml. But whilst all the citric acid is precipitated, only 43 per cent. of the malic acid accompanies it. Thus if T be the grms. of total acid present in the solution used for the test; D the lead titration difference (i.e.on one-fifth of T); and M the grms. of malic acid (reckoned as citric) contained in the T grms. of total acid, D =? [ i M x - 43 +z(T 1 - M ) ] 0.1 100 = 27.1 [T - 0.57 MI, whence M = 1.75 T - 0.065 D . . . . (I) 264 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION To take an illustration suppose the total of the two acids in a solution taken for the test is 0.75 grm. reckoned as citric and that the lead titration difference is 12.8 ml. then and the citric acid would be M = 1.75 x 0.75 - 0-065 x 1243 = 0.48 grm. 0.75 - 0.48 = 0.27 grm. 67 70 The actual malic acid as malic would be 0.48 x - = 0.46 grm. It will be noted that the quantityof each of the acids conforms to the restrictions previously set out.In that case, in order to obtain a more accurate result the determination should be repeated, with suitable additions of either citric or malic acid as the case may require and, if-necessary also an accompanying alteration in the amount of the mixture taken for the test. The titration difference may f a l l outside the range of 12 to 14 ml. required for correct precipitation of the citric acid. In that case a small correction based on the data of Tables I1 and IV may be applied before calculating the proportions of malic and citric acids. It may sometimes happen that this is not so. These corrections are given in Table VIII. TABLE VIII CORRECTIONS TO BE APPLIED TO LEAD TITRATION DIFFERENCES Lead titration diff. ml. 8 .5 - 9 9-1-11 11.1-14 14.1- 15 16-1-16 Correction ml. -0.2 -0.1 0 +o. 1 + 0 * 2 Titration values outside the range shown here are less reliable and when obtained a fresh determination on an altered quantity of original solution should be made. method elaborated for pure acids to fruit extracts it was thought desirable to examine the behaviour of pectin when precipitated by lead. A solution of orange pectin containing 0.82 per cent. of pectin as calcium pectate was used. The free and combined acidity of the pectin expressed on the calcium pectate basis was equivalent to 13.6 per cent. as citric acid. Thus for theoretical precipitation of the pectin as the lead salt 100 ml. of pectin solution would require 13.55 x 0.136 x 0.82 ml. = 15-1 ml. of 2 per cent.lead solution. In a first experiment in which 100 ml. of the pectin solution were treated alone by the usual procedure the titration difference was 2.9 ml. This was as usual on one-fifth of the total volume so that the theoretical lead equivalent should have been "*' - = 3-02 ml. Within ordinary limits therefore the precipitation result was in agreement with the theoretical value. In a subsequent experiment 125ml. of pectin solution were mixed with a known amount of citric acid solution (partly neutralised with sodium hydroxide BEHAVIOUR OF PECTIN I N THE LEAD PRECIPITATION.-Before applying the HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 255 to correspond with conditions in a fruit juice). The titration difference was 12.8 ml.whilst a control test of the citric acid solution without the pectin gave 10*6ml. or a difference of 2.2 ml. due to the pectin. The theoretical lead 1 -25 equivalent should have been 15.1 x - = 343 ml. so that in presence of citric 5 acid the precipitation of lead by the pectin was reduced considerably. The amounts of pectin used above were four or five times as large as would be present in the test as carried out on an ordinary fruit or jam but in order to prevent any possible interference and also to improve the filtration which tended to be sluggish in the presence of the pectin a preliminary treatment with acetone was introduced into the procedure to remove the pectin. A fruit or jam extract was made up to double its volume with acetone being meanwhile swirled round to avoid entangling too much air and then filtered with precautions against evaporation.A suitable amount of this pectin-free filtrate was taken for the test, and after removal of the acetone by evaporation the solution was cooled and the lead test proceeded with as usual. The free and combined acidity of the fruit were also determined on the pectin-free filtrate so that all results should be on the same basis. possible (though doubtful) presence of small amounts of phosphates in fruit extracts or jams known amounts of a solution of acid potassium phosphate (KH,POA were added to citric acid solutions and the mixtures were treated by the usual lead process. In Table IX the quantities taken and lead titration differences found are shown; and the effect due to the phosphate obtained by deducting that of the citric acid is compared with the theoretical amount required for the complete precipitation of triplumbic phosphate [Pb,(POp)J.EFFECT OF PHOSPHATES ON LEAD NUMBER.-In view Of the effect Of the TABLE IX PRECIPITATION OF LEAD BY PHOSPHATE IN ADMIXTURE WITH CITRIC ACID Citric acid Lead Amt. due to Theoretical Grm. Grm. ml. ml. ml. 0 . 1 0 0.30 1 5 . 9 13.3 12.54 0.20 0-20 13.7 8 . 3 8.36 0 . 3 0 0.10 12.2 4 . 1 4.18 taken KH,PO taken titration diff. phosphate for phosphate The precipitation was close to the theoretical value. It should be noted that any phosphate present in a fruit extract would behave in the determination of free and combined fruit acids as though it were the salt of a dibasic acid, e.g.KH(HPO,) whereas the lead salt precipitated is the tribasic salt. Thus any part of the apparent “total fruit acid” which may be present as phosphate would appear t o precipitate 3/2 times the amount of lead that would be precipitated by its equivalent of citric acid. Thus whilst 0.1 grm. of citric acid (equivalent weight = 70) has a 2 per cent. lead acetate equivalent of 13-55 ml. 0.1 grm. of phosphoric acid (virtual equivalent in fruit analyses = 49) has a lead equivalent o 266 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION The phosphoric acid in the ash of a large number of samples of fruit has been found to be usually between 0.03 and 0-06 per cent. of the fruit. Since the lead number of the fruit by Boseley’s scheme is expressed on 10 grms.of sample that portion of it due to this phosphoric acid would be 29 x O*Oo3 - - 0-9 to 1.8 ml if it were present as phosphate in the fruit extract. This however is doubtful at least as regards the whole of it; so that the effect of any phosphate in this way would not be great and certainly very much less than the natural variations in lead number from one sample of fruit to another. BEHAVIOUR OF OTHER ACIDS I N THE LEAD PRECIPITATION.-Although the evidence discussed later indicates that citric and malic acids are the principal acids occurring in jam fruits at least in quantity sufficient to affect significantly the amount of lead precipitate the effect of a few other acids has been studied. Tartaric Acid.-This is of course a prominent acid in the grape but does not occur to any extent in most jam fruits.A few experiments with a solution of cream of tartar of known composition showed that this acid was precipitated as neutral lead tartrate when submitted to the standard (‘ lead number” process (see Table X). TABLE X 0.1 LEAD PRECIPITATION OF TARTARIC ACID Tartaric acid* taken (per Calculated Titration 100 ml. of lead solution) titration diff. diff. found Diff. Grm. ml. ml. ml. 0,298 7 . 5 7 . 7 +0*2 0.447 11.3 11.4 +o- 1 0.596 15.0 14-7 - 0 . 3 * i.e. equivalent to the amount of cream of tartar taken. The slight differences from the theoretical values are very similar to those of citric acid so that in the event of tartaric acid being present the corrections given for citric acid (Table VIII) would apply.There would be no interference with the calculation of the malic acid present in a mixture of the three acids. Succirtic Acid.-Tests carried out on succinic acid in admixture with varying proportions of mono-sodium citrate showed that there was no more lead precipitated than would be due to the citrate alone. Thus any succinic acid present in a mixture of fruit acids would cause the amount of malic acid as given by the calculation earlier to be too high. However, no definite evidence has so far been found that succinic acid occurs in any appreciable quantity in jam fruits. Fumaric Acid.-This acid has been stated to occur as a natural constituent of some fruits. Experiments on the lines of those already carried out with malic and succinic acids showed that fumaric acid when citrate was also present precipitated lead to the extent of about 55 per cent.of the amount required to form the neutral lead salt. Thus any fumaric acid present would appear as malic acid in the calculation of the latter HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 257 However attempts to identify fumaric acid in extracts of plums and greengages in which its presence was at first suspected for other reasons gave negative results. Lactic Acid.-This acid is not supposed to occur naturally in fruits though it may be considered as a natural “food” acid and is in some countries officially recognised as a permissible substance in food preparations. It is used in the manufacture of some commercial pectin preparations which therefore introduce it into jams containing them.Experiments on lactic acid alone and also in admixture with citrate solutions, showed that there was no precipitation of lead whatever by the lactic acid. Thus, any lactic acid present in fruit products would considerably increase the apparent amount of malic acid as given by the calculation from the lead precipitation. Since the calculation depends on the fact that 57 per cent. of the malic acid is unprecipitated whereas the whole of the lactic acid remains so the latter would 100 57 cause the malic to appear too high by - times its equivalent weight as malic acid. In the presence of lactic acid therefore the method of calculating malic acid breaks down unless there is some means of determining the former and allowing for it.AN EXTENSION OF THE LEAD PRECIPITATION PROCESS PRECIPITATION FROM 60 PER CENT. ACETONE SoLuTIoN.-Auerbach and Weber (2. anorg. Chew. 1925, 147 68) in the course of a general study of the solubility of the lead salts of fruit acids found that the lead salts (of citric malic tartaric and succinic acids) were only very slightly soluble in 50 per cent. alcohol. This seemed to hold out the possibility of more definitely characterising the individual acids and the fruit extracts etc. in which they were present and an extension of the lead-precipitation process was accordingly devised. Acetone seemed to offer advantages over alcohol as a medium for the new form of precipitation and a few preliminary experiments showed that the lead salts were just as insoluble in a 50 per cent.(by vol.) acetone medium as in the alcohol mixture of Auerbach and Weber. The amount of lead precipitated by citric acid was however about 10 per cent. more than the theoretical whilst malic acid now gave practically the same precipitation as citric acid also about 10 per cent. more than the theoretical. It is possible that this extra precipitation of lead is connected in some way with a shift of p caused by the acetone (as shown when an indicator was added). It was found in the experiments of Table I that when the $= is not controlled in the aqueous precipitation excess lead up to at least 12 per cent. was precipitated. In the aqueous precipitations it was found that correct precipitation of lead by citric acid was obtained when about 0-5 grm.of citric acid was taken per 100 ml. of 2 per cent. lead acetate (see Tables I1 and IV). The precipitation in the acetone medium was more arbitrary and there was no point at which it could be said that “ correct ” precipitation was obtained. Hence for the purpose of comparisons and practical work it was decided to assume that the precipitation given by 0.5 grm. of citric acid (per 100 ml. of lead solution) was a “correct” one for th 258 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION acetone medium. This was found to average 15.0 ml. of lead-titration difference. On this basis the actual results found for citric acid and a number of mixtures of malic and citric acids in the acetone medium are compared in Table XI1 with the calculated “correct” values.In these tests as in previous ones the citric acid was used in the form of mono-sodium citrate and the malic acid in the partially neutralised condition to correspond with the conditions in fruit products. TABLE XI1 DEVIATION FROM ASSUMED CORRECT LEAD PRECIPITATION IN 50 PER CENT. ACETONE MEDIUM Per 100 ml. of lead solution Mono-sodium citrate Grm. 0.30 0.45 0.60 0.35 0.35 0.35 0.20 0.30 0.40 0.35 0.30 0.25 0.20 0- 15 Mdic acid Grm. -0.104 0 - 208 0.298 0.298 0.074 0.123 0.173 0.222 0.271 0.321 Calc. lead titration diff. ml. 8-85 13.26 1 7 . 7 10.3 13.6 16.8 15.25 18.2 14.1 14.15 14.25 14.3 14.35 14.45 Ti tra tion diff. found ml. 9 - 15 13-35 17.2 10.7 13.65 16.5 15.05 17.4 14.2 14.3 14-3 14.3 14-4 14.45 Difference + 0 * 3 +o.I -0.5 +O-4 +0-05 -0.3 -0.2 -0.8 +o- 1 +0*15 +Om05 0 +0-05 0 The precipitation due to the malic acid in the mixtures has been assumed to be the same as for the equivalent amount (ie. x 3) of citric acid. 67 It can be seen that the lead equivalents of the acids present are subject to the same sort of slight fluctuations as in the aqueous precipitations. The deviations increase rather sharply with titration differences above about 16 ml. probably owing to insufficient excess of lead. It should be noted that the deviations shown by the solutions containing malic acid were similar to those of citric acid alone. This means that the malic acid is precipitated as completely from the 50 per cent.acetone medium as is citric acid. In Table XI11 the deviations are expressed in the form of corrections which can be applied to the titration differences to bring them all to the same basis of assumed correct precipitation. Titration differences falling outside the above range are not very reliable; in such cases it would be advisable t o repeat the test with a different amount of the solution. When the titration differences have been corrected in this way the sum of the citric and malic acids (both expressed as citric) in a mixture of the two can be found by taking a titration difference of 15.0 as representing 0.10 grm. of acid (as citric) precipitated; or what is the same thing 0.50 grm. of acid in the full quantity of solution taken for precipitation by the 100 ml.of 2 per cent. lead acetate solution HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 259 TABLE XI11 CORRECTIONS TO BE APPLIED TO LEAD TITRATION DIFFERENCES (PRECIPITATION IN 50 PER CENT. ACETONE) Lead titration difi. ml. 9-0-11 11-1-13 13.1 - 14 14-1-15 15.1- 15.7 15.8- 16.2 16.3- 16.5 16.6- 16.8 16.9-17.1 Correction ml. -0.3 -0.2 -0.1 -0 + o - 1 +0*2 +0.3 3-0-4 +On6 Thus suppose 25ml. of a solution are treated with 1OOml. of 2 per cent. lead acetate and the mixture is made up to 250ml. with acetone and filtered; 50ml. of the filtrate are then titrated and found to give a difference from the blank of 12.5 ml. The correction on this from Table XIII brings it to 12.3 ml.Hence the amount of combined citric and malic acids in the 25 ml. of solution initially taken is 0.5 x E3 = 0.41 grm. (expressed as citric acid). 15-0 BEHAVIOUR OF OTHER ACIDS IN THE 50 PER CENT. ACETONE MEDIUM.-Tartaric acid which it was seen is precipitated completely like citric acid from aqueous solution is precipitated similarly to citric acid from the 50 per cent. acetone i.e. a slight excess of lead being thrown out (about 15 per cent.). As this acid is not present to any extent in jam products no significant error arises if the lead equivalent of the citric acid is used to cover both acids. Thus the acid precipitated in acetone includes tartaric with the citric and malic acids. Phosphoric acid (used in the form of KH,P04) was found to precipitate from the 50 per cent.acetone medium just the theoretical amount of lead required for the triplumbic phosphate. There was no excess precipitation as with the organic acids. However it must be remembered that the same consideration holds good as in the aqueous precipitation of phosphate; any of the fruit acidity due to phosphoric acid as given by titration to the phenolphthalein end-point has 3/2 times the lead-precipitating power of the organic acids. Consequently if phosphoric acid is present in a mixture the total lead precipitation from acetone will be higher than'it would be if all the acids were organic. Fumaric acid (examined in admixture with citrate) precipitated practically the theoretical amount of lead from the acetone medium but there was a slight solubility effect as a slight further precipitate came out from the filtrate on standing overnight.In any case this acid would be included with sufficient accuracy in the total of the other acids. Lactic acid both alone and in admixture with citrate was found to be unprecipitated from the acetone medium. This was a fact of special importance. It was shown earlier that when present this acid owing to its non-precipitation from aqueous solutions invalidates the calculation of malic acid. In the aceton 260 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION medium however it was alone among the acids considered here in not being fully precipitated. Hence we have a ready means of estimating its amount with some exactness and so allowing for its effect in calculating the malic acid.If T be the grms. of total acid in the solution used (obtained as before mentioned by titration of the free acidity and of the ash) D' the lead titration difference for the 50 per cent. acetone medium (i.e. on one-fifth of T) and K the grms. of lactic acid (reckoned as citric) in the T grms. of total acid then we have 15.0 1 0.1 5 D' = - x - (T - K), since 0.1 grm. of citric acid (or its equivalent in malic tartaric etc.) precipitates 15.0 ml. of 2 per cent. lead acetate solution. From this equation, . . (11) D' K = T - - -30 Reverting to the earlier equation (I) for the calculation of the malic acid from the aqueous lead titration difference the T in that equation must now be replaced by (T-K) since the lactic acid was completely inert in that determination also.So we have M = 1.75 (T - K) - 0.065 D, or substituting for K the value given by equation (11) above, D' 30 M = 1.75 (T - T + -) - 0.065 D = 0.058 D' - 0.065 D Thus the malic acid can now be obtained by a simple calculation from the two lead titrations. It may be noted that for the purpose of determining the malic acid only it is not necessary to know the total acidity of the solution; this is only required if the lactic acid or the total acid (citric + tartaric + phosphoric) are required in addition. It should be pointed out that the preceding calculation includes any phosphoric acid with the citric acid and assumes that it precipitates lead similarly to the latter. Actually as was shown earlier this is not the case.The effect of the assumption is to make the calculated amounts of lactic and malic acids slightly low whilst the remaining acid (i.e. citric + tartaric + phosphoric) is made slightly high. The errors even supposing that all the phosphorus found in the fruit ash is derived from phosphates are not very large-not more than 0-02 per cent. (expressed on the fruit) for the malic and 0.035 per cent. for the (citric + tartaric + phosphoric) acids. The above method for determining the malic acid breaks down of course if there are appreciable amounts of other acids present which are differently precipitated in aqueous and in acetone medium. From other evidence it seems likely that such is not the case with many fruits including most of the soft jam fruits and apples.In any case, for the purpose of the analysis of fruit products there seems no objection in the first instance to classing empirically as malic acid all the acid material which behaves like it in the lead tests. This device while ignoring the slight errors With regard to stone fruits there is more doubt HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 261 that may be caused by traces of phosphoric or other acids has the advantage, from an analytical standpoint of separating the acid constituents of the fruits into three groups: (i) Citric acid and acids which behave similarly (e.g. tartaric acid); (ii) malic acid and acids which behave similarly (if any) ; (iii) lactic acid and acids which behave similarly (if any). Since it will be shown later that the acids of apples and of the stone fruits consist chiefly of malic acid (or of acids behaving like it) it is clear that a means of discriminating between the different groups of acids affords useful information in the analysis of mixed products.the past two or three seasons a considerable number of determinations of lead number has been made on extracts prepared from fresh fruits. At first the aqueous extracts were used for the precipitations without removing the pectin but 'it was found later that the results with stone fruit were more reliable if the pectin was removed. Later determinations were therefore always carried out on the filtrate from the precipitation of pectin with acetone the acetone being removed by evaporation prior to the aqueous lead precipitations.It was found that results on this pectin-free filtrate were practically identical with those on the original extract of soft fruits and apples but were sometimes different (usually higher), and certainly more reproducible with the stone fruits. Later when the lead precipitation from 50 per cent. acetone was worked out the procedure was arranged so as to take advantage of the preliminary precipitation of the pectin with the acetone as will appear in the description of the full method of working eventually adopted. A further modification introduced when the acetone lead precipitation came into use was a fivefold increase in strength of the lead acetate solution (including the contained acetic acid) ; in place of the former 100 ml. only 20 ml.of the stronger solution were of course used. This permitted of the use in the test of sufficiently large amounts of the rather weakly acid extracts obtained from some samples. Also for stone fruits and apples the practice was adopted in the aqueous precipitations of adding a suitable amount of citric acid (in the form of mono-sodium citrate solution or of the free acid) in order to ensure the proper co-precipitation of the malic acid (see BEHAVIOUR OF MALIC ACID p. 251). A still later improvement was the addition to some extracts where the natural malic acid was believed to be deficient (as in strawberries raspberries etc.) of small amounts of malic acid also for the purpose of securing the right degree of co-precipitation. Appropriate allowance was made for these additions of citric or malic acid in calculating the lead numbers of the fruits.In Table XIV is given a summary of the results of determinations of the aqueous lead number on most of the ordinary jam fruits. For stone fruits and apples only the later results obtained on pectin-free extracts and with additions of citrate as above mentioned are included. The extracts of the fruits were prepared by boiling the samples with an equal weight of water for an hour cooling replacing the water lost by evaporation and filtering. In calculating results back to the original fruit correction has been made for the amount of insoluble matter and DETERMINATIONS OF LEAD NUMBER ON FRUIT EXTRACTS.-h the COUTSe O 262 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION also for the fact that the extracts were made up by weight whilst the amounts used for analysis were pipetted.The lead numbers are expressed according to Boseley’s original scheme i.e. the number of ml. of 2 per cent. lead acetate solution precipi-tated by 10 grms. of the sample. In addition to the lead numbers the table shows the average total acid-content of each kind of fruit (from the free acidity and ash titrations). Finally the value obtained by dividing the lead number of each sample by its total acidity is shown. This is really the lead equivalent (2 per cent. lead acetate solution) of each 0.1 grm. of acid (as citric acid). It is also numerically equal to the “lead number per 1 per cent. of acid” in the sample. If all the acid were citric acid this figure would be 13-55.The deviation from this is a measure of the proportion of non-precipitated or partly-precipitated acids (probably chiefly malic acid) present. The figures for “highest” (and “lowest”) values of lead numbers and of lead number per 0.1 grm. of acid do not necessarily belong to the same sample. TABLE XIV LEAD NUMBERS OF JAM FRUITS (AQUEOUS PRECIPITATION) Fruit No. of samples Gooseberries Strawberries . . Raspberries . . Redcurrants . . BIackcurrants . . Apples . . Plums . . Greengages . , Damsons Blackberries . . Apricots 17 15 11 7 10 4 5 2 4 12 2 Highest Lowest Average Highest Lowest Average Highest Lowest Average Highest Lowest Average Highest Lowest Average Highest Lowest Average Highest Lowest Average 1.2. Average Highest Lowest Average Highest Lowest Average 1. 2. Average Lead number ml. 29.9 17.3 25-5 22-7 0 - 8 16.3 34.2 19.0 26.8 43.1 34.8 37.6 64.5 39.5 52.7 10.9 6.9 9 . 4 1s-0 6 . 9 10.2 12.0 8.1 10- 1 19.4 9 . 4 13.0 22.5 7.2 13.9 20.8 14.7 17-8 Total acidity Lead number as citric acid per 0 . 1 grm. acid Per Cent. ml. - 11.7 9.5 2.44 10.4 - 13.3 - 10.5 1.31 12.4 - 14.3 - 12.1 2-02 13.3 - 14.3 - 12.0 2.81 13.4 - 14.4 - 12.2 3.92 13.5 - 7 . 4 6 . 3 1-33 7.1 - 7 - 6 - 2 . 5 2.14 4 . 8 - 5 . 9 - 6 . 5 1.77 5.7 - 7 - 0 - 3 . 0 2-77 4.7 - 10.2 - 8-1 1.51 9 - 2 - 11.0 - 0 .6 1.72 10-3 --From these results it appears that the acid of raspberries redcurrants and blackcurrants is almost entirely citric acid (or belongs to that type). Strawberries contain a slight proportion of other acid gooseberries blackberries and apricot HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 263 rather more whilst apples and the stone fruits probably contain very little citric acid. In a few analyses of raspberries and currants the value for the lead number is even slightly higher than should be obtained if all the acid precipitated its full equivalent of lead. Part of this excess may be due to difficulty in determining the end-point of the titration which is sometimes found in the more strongly coloured fruits; but part may be also due to traces of phosphates which it will be remem-bered precipitate lead more strongly than their titratable acidity warrants.The results of lead-number determinations in the 50 per cent. acetone medium are summarised in Table XV. The data are fewer as this method was only brought into use during the 1932 season. This accounts for the slightly different average figures for acidity compared with those of the preceding Table. TABLE XV LEAD NUMBERS OF JAM FRUITS (50 PER CENT. ACETONE PRECIPITATION) Fruit No. of samples Gooseberries . . Strawberries . . Raspberries Redcurrants . . Blackcurrants . . Apples . . Plums . . Greengages . . Damsons Blackberries . . Apricots . . 2 3 3 2 2 4 3 3 2 2 2 1. 2. Aver age Highest Lowest Average Highest Lowest Average 1.2. Average 1. 2. Average Highest Lowest Average Highest Lowest Average Highest Lowest Average 1. 2. Average 1. 2. Average 1. 2. Average Lead number ml. 40.9 39-8 40.4 27.0 1 5 - 8 21.4 37-4 35-0 36.6 51.8 50-7 51-3 78-2 74.1 76.2 22-2 16.5 19.9 34.4 19.0 26- 1 28.6 15.0 20.3 43.3 34.9 39.1 34.4 82-2 28.3 26.9 23.5 94.7 Total acidity Lead number as citric acid per 0.1 grm. acid Per Cent. ml. - 15.4 - 15.4 2.62 15.4 - 1 5 - 6 - 15-3 1.39 15.4 - 15.6 - 15.0 2-38 15.4 - 17.1 - 16-7 3-04 1 6 - 9 - 17.3 - 16.3 4.53 16.8 15- 1 - 14.9 1.33 15-0 - 14.6 - 1 2 - 3 1-88 13.9 13.0 - 10.3 1.68 12.1 - 15-6 - 14.6 2-59 15- 1 - 15.6 - 15.2 1.84 15-4 - 15.3 - 13.6 1-72 14.4 --In most of the fruits it is evident that the lead number in the acetone medium was very close to that corresponding to complete precipitation of the acids.That is to say the acids fall into the citric or malic acid groups. Most of the results were a little higher in fact as can be seen by comparing the “lead number pe 264 HINTON T H E EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 0.1 grm. of acid” with the theoretical figure of 15.0. With currants the pre-cipitation was appreciably higher than this; so far a satisfactory explanation has not been found but it is possible that phosphates partly account for the excess. The few samples of these examined all gave incomplete precipitations showing the presence of some acid not belonging to either the malic or citric acid groups.The behaviour of this acid in the lead tests according to the scheme elaborated in an earlier section was similar to that of lactic acid. Whether it is lactic acid or not is a matter as yet undecided. Unfortunately its amount is apparently very variable, so that it does not offer very much prospect of assistance in discovering the fruit-content of products containing these fruits. given earlier are applied to the two sets of lead numbers obtained from the fruits, approximate figures for the amount of each of the three acid types are obtained. This has been done for all those samples for which determinations of both lead numbers were available and the apparent amounts of the malic acid and lactic acid types expressed as percentages of the total organic acid are shown in Table XVI.Many of the percentages so found especially of “lactic acid,” are slightly negative. This may possibly arise from the presence of traces of phosphate, The most interesting results were from plums and greengages. DISTRIBUTION O F THE ACID GROUPS I? FRUITS.-If the equations (11) and (111) TABLE XVI APPARENT PROPORTIONS OF Fruit Gooseberry . . Strawberry . . Raspberry . . Redcurrant . . Blackcurrant Apple . . Plum . . Greengage Damson Blackberry Apricot . . ACIDS OF DIFFERENT TYPES IN FRUIT SAMPLES Total acid Per Cent. 2.65 2.58 1.76 1-39 1.01 2.39 2.48 2-27 2 ; 9 i 4-54 4-51 1-47 1.42 1-31 1.10 1-75 1.55 2.35 2-20 1.38 2 .i 8 2.39 2.21 1.46 1-54 1.90 Per cent. of total acid Malic acid Lactic acid group group A I I 41 35 15 8 14 - 5 - 2 12 16 5 14 78 81 77 92 93 70 70 80 70 90 97 67 47 53 15 - 3 - 3 - 2 - 3 - 4 - 4 0 - 3 - 14 - 9 - 15 0 0 0 0 5 18 3 13 17 - 4 2 - 4 - 1 - 2 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 266 which would affect the calculation of both the malic and lactic acid types in a negative direction (see p. 259). Larger negative results are shown by currants which, it was seen had a tendency to give lead numbers above the theoretical in the aqueous precipitation and quite notably above it in the acetone precipitation.Apple and the stone fruits appear as predominantly “ malic acid” fruits, whilst gooseberries blackberries and perhaps apricots appear to have a more even distribution of the acids in the citric and malic groups. Some of these findings so far as relative proportions of citric and malic acids are concerned are similar to the results of Nelson (ANALYST 1924 49 592; 1925, 50 191 295) who made a direct determination of the acids by an ester distillation method. He found in strawberries 10 per cent. raspberries 3 per cent. black-berries 17 per cent. and apricots 70 per cent. of the total acid to be malic acid. Franzen and Helwert (2. Physiol. Chem. see Canning Age 1925 June p.562) reported the acids of apples to be chiefly malic with some citric; whilst currants contained chiefly citric with some malic acid. This again is confirmed by the present results. THE LEAD NUMBER OF JAMs.-we return finally to the motive behind Boseley’s original idea of the lead-precipitation process namely the desire for a means of arriving at the approximate fruit-content of jams. It has been shown that the lead number is due certainly (within experimental limits of accuracy) to the acid constituents of the fruits; further that the acid constituents themselves can by means of the lead precipitation be separated into groups and that the fruits fall broadly into classes containing a preponderance of citric or malic acid. Thus it should be possible within certain rough limits to discover whether the acid constituents of a jam are normal to the class of fruit used provided that any foreign fruit or fruit juice added belongs to a different class.In particular the addition of apple pulp or juice or pomace extract to strawberry raspberry etc. jam should make itself evident by disturbing the normal citric acid preponderance of these fruits. On the other hand such additions to stone fruit jams would not appreciably alter the proportions of the acid groups (unless use can ultimately be made of the “lactic acid” group). For this purpose it is not necessary to calculate from the lead number the actual quantities of citric and malic acids present. The lead numbers themselves can be used and compared with established data for the several kinds of fruits concerned with due allowance for the natural variations.It should here be pointed out what is evident from Tables XIV and XV that it is not the lead number itself that is specially characteristic of a particular fruit. Thus a lead number of 6.5 found for a jam might be given by 40 per cent. of strawberries alone or by 25 per cent. of strawberries and 26 per cent. of apple pulp or by 60 per cent. of apple alone. The characteristic property which makes it possible to gain some idea of the proportions of the constituents in such mixtures is the lead number relative to the acid content or according to the empirical method adopted above for expressing this property the “lead number per 0.1 grm. of acid.” (It should be remembered that total acid is always meant here as obtained from the free acidity and ash titrations.) Hence from a consideration of the average values for this figure For the present the discussion will be confined to the former case 266 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION for the fruits concerned their respective proportions in an unknown mixture or rather the proportions of their acids can be approximately determined.The matter may be made clearer by an example. Suppose the “lead number” of a strawberry and apple jam to be 6.4 and the percentage of total acid in the jam was found to be 0-61 then the acid in the 10 grms. of jam equivalent to the 6.4 ml. of 2 per cent. lead acetate solution is 0.061 grm. and the “lead number per 0.1 grm.of acid” is - = 10.5. Now the averages for strawberries and apples are respectively 12.4 and 7.1 (Table XIV). Hence the proportion 6.4 0.61 of the acids due to strawberries is - 7’1) 100 per cent. of the total = 64 per (12.4 - 7.1) 64 100 cent. Thus the percentage of acid in the jam due to strawberries is 0.61 x - = 0.39 per cent.; and that due to apples will therefore be 0.61 - 0.39 = 0.22 per cent. Taking the average total acid contents of strawberries and apples as 1-31 per cent. and 1.33 per cent. respectively (Table XIV) the amounts of the two fruits in the jam are: Strawberry OS3’ - x 100 = 30 per cent. 1.31 Apple = o’22 - x 100 = 17 ,, 1 -33 The same method can be applied to other mixtures provided the characteristic figures for the fruits concerned “lead number per 0.1 grm.of acid,” are sufficiently far apart. Thus the method breaks down for such mixtures as raspberry and redcurrant plum and apple etc. and is of doubtful value for blackberry and apple, or strawberry and gooseberry (see Table XIV). The calculation can be expressed in the form of a simple formula. Let F and F be the respective percentages of two fruits in a mixed L and L the average values of the “lead number per 0-1 grm. acid” A and A the average values for total acid in the two fruits I the actual “lead number per 0.1 grm. of acid” found in the product, (Table XIV), (Table XIV), sample, lOOa(Z - L,) and a the actual per cent. of total acidity found, (IV) Then F = AdLl - L2) * * (V) 100a(L - I ) A& - b) and F2 = JAMS CONTAINING POMACE EXTRACTS OR PECTINS.-MoSt jams now on the market contain added pectin which is used either in the form of a direct extrac HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 267 from pomace or a specially-prepared proprietary “ fruit pectin ” (usually prepared from apple residues) or as a dry powdered pectin prepared from citrus fruits or apple residues.The latter preparations which are more or less pure pectin should introduce no lead-precipitating acids but themselves into the jam; and as they can be removed along with the natural fruit pectins by a preliminary precipitation with acetone they should cause no complications. The case is different with pomace extracts etc. These when they are aqueous extracts may be considered as apple extracts deprived of a portion of their natural acids.The remaining acids however will still have the same lead-precipitating properties as the acids of the whole fruit (apart from the pectinous constituents which can be removed before the lead test is made). Thus the formulae of the preceding section can still be applied though the apparent percentage of apple juice indicated will be low because of the removal of part of its acid. There will be no interference with the calculation of the amount of the main fruit constituent (FJ. Some commercial pectins however appear to have been prepared by an extraction of pomace with lactic acid. Two of the most popular “fruit pectins” on the market in this country for instance on examination by the lead process, both in the aqueous and the 50 per cent.acetone medium showed a large proportion of lactic acid (or an acid of closely similar type) among their acid constituents. Table XVII shows the essential parts of the analysis of these products and the proportions of citric malic and lactic acid groups calculated from the lead numbers by the formulae already given. TABLE XVII COMPOSITION OF ACID CONSTITUENTS OF COMMERCIAL PECTINS AS CALCULATED FROM THE LEAD NUMBERS Lead number (per 10 grms. of sample) Acids (expressed as citric) A \ Total acid 50 per cent. “citric” “ malic ” “ lactic ” Per Cent. medium medium Per Cent. Per Cent. Per Cent. P I Sample (as citric) Aqueous acetone acid acid acid A. 2.04 3 . 0 6 . 9 0.05 0.41 1-58 B. 1.09 3 . 7 8.7 0.05 0.53 0.51 Thus in one case about half and in the other case three-quarters of the acid was lactic acid the remainder being presumably the natural acids of the apple still remaining in the pomace or apple residues used.Clearly the calculation of the fruit-content from the aqueous lead number by formula (IV) would be erroneous in jams containing such pectin preparations. The effect of the lactic acid would be to depress the “lead number per 0.1 grm. of acid,” making the proportion of apple acids appear too high. The solution of the difficulty is afforded by the second or “acetone” lead number. This gives a value for the total acids excluding the lactic acid so that a corrected value can be obtained for the “ lead number per 0.1 grm. of acid,” which refers only to the malic and citric types of acid.Formula IV can then be applied to these corrected values for a and 1 268 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION This calculation is of use only for those fruits which themselves show no appreciable amount of the lactic type of acid vix. the soft fruits and damsons (see Table XVI). But as plums and greengages have an aqueous lead number so similar to that of apples as to preclude its use in calculating fruit-content in their case the restriction is not a material one. It is desirable to point out here in connection with the calculation of fruit-content in mixtures containing commercial pectins that experience in the analysis of a large number of jam samples of various origin has shown the figure of 7.1 for the “lead number per 0.1 grm.of acid” of apples (Table XIV) to be rather high for general application. A figure giving results more in accordance with other analytical indications is 6.5. This, too is about the figure given by the commercial pectins of Table XVII when due allowance is made for the extraneous lactic acid. Hence for the fruit-content of jams with added l1 pectin” (from apples), formula IV may be simplified to: This was the average from four samples only. (VI) lOOa(Z - 6.5) A,(L - 6.5) F = The a and I of this formula should be suitably corrected for any lactic acid shown to be present by the “acetone” lead number. Values of A and L, appropriate to the various fruits may be obtained from Table XIV. DETAILS OF ANALYTICAL PROCEDURE-The details of the procedure adopted for the various determinations involved in applying the lead-precipitation method t o the analysis of jams are as follows: Preparation of Extract.-Weigh 250 grms.of the sample into a beaker and add 250 ml. of water. Mix well to break up the jam then heat to boiling with continual stirring and boil gently for an hour with occasional stirring keeping the beaker covered and maintaining the volume by adding water if necessary. Cool transfer to a 500-ml. measuring flask make up to volume shake well and filter through a coarse filter. Preparation of Pectin-free Filtrate.-Transfer 250 ml. of the aqueous extract to a 500-ml. flask and add acetone (while swirling round without entangling too much air) to the mark. Mix well and filter through a large dry filter with precautions to avoid loss by evaporation.Titration of Free Acid-Pipette 40 ml. of the pectin-free filtrate into a large beaker add about 500 ml. of boiled and cooled distilled water and titrate with N/10 sodium hydroxide solution using phenolphthalein as indicator. Carry out a blank titration of 500 ml. of the water similarly and deduct this from the jam titration. The difference multiplied by 0.07 gives the percentage of free acid (expressed as hydrated citric acid) in the jam. Ash ofJam.-Evaporate 50 or 100 ml. of the pectin-free filtrate to dryness in a platinum dish on a water-bath char over an Argand burner (protecting the contents of the dish from the gas fumes) and ash at a dull red heat preferably in an electric muffle.Titration of Combined Fruit Acid.-Dissolve the ash in a measured 15 ml. of N/10 hydrochloric acid filter into a 175-ml. conical flask and wash through Cool and (if required) weigh HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION 269 thoroughly. Cool add a drop of methyl orange solution, titrate to yellow with N/10 sodium hydroxide solution and then to the neutral tint with N/10 hydrochloric acid. Calculate the amount of acid consumed by the ash to the number of ml. of N/10 acid per 100 grms. of jam; this is the “methyl orange alkalinity” of the ash. (Pfyl 2. Nahr. Genussm. 1922 43 313.) Next acidify the titrated solution with about 2 ml. of N/10 hydrochloric acid, and evaporate to about 15 ml. (It is sometimes advisable to boil on a sand-bath, on account of bumping.) Cool and neutralise carefully to methyl orange with N/10 sodium hydroxide solution.Add a few drops of phenolphthalein solution and 10 ml. of a strong neutral calcium chloride solution. Boil again for a few minutes and titrate to the phenolphthalein end-point with N/10 sodium hydroxide solution. Calculate the number of ml. of N/10 sodium hydroxide solution per 100 grms. of jam and multiply by 3/2. This gives the phosphoric acid in the ash as its equivalent of N/10 sodium hydroxide solution. Then find the “total alkalinity” of the ash equivalent to all the alkali and alkaline earth metals present, by adding to the “methyl orange alkalinity” one-third of the phosphoric acid equivalent. Finally multiply this total alkalinity by 0.007 to obtain its value as percentage of combined fruit acid as citric acid.Total Fruit Acid.-The total fruit acid (including any phosphates present in the extract) is the sum of the free acid and combined acid found as above and is expressed as the percentage of total citric acid (hydrated). Aqueous Lead Number.-Take an amount of pectin-free filtrate (to the nearest 5 or 10 ml.) containing approximate amounts of total fruit acids according to the following scheme : Boil for a few minutes. GITll. (i) Gooseberry apricot or blackberry jams . . . . 0.50 (ii) Strawberry raspberry redcurrant or blackcurrant jams . . 0.35 (iii) Apple cherry plum greengage or damson jams . . . . 0.65 Remove the acetone by distillation transfer the residue to a 250-ml. measuring flask and cool.Then add in the case of groups (i) and (ii) 3.0 ml. of 10 per cent. malic acid solution or in the case of group (iii) 3.0 ml. of 5 per cent. citric acid solution (pipetted accurately). (The strength of the acid used should be correct to within 1 per cent. of the total.) Dilute to about 200 ml. Ensure that the temperature of the solution is at about 16” to 20” C. then add from a pipette while rotating the flask 20 ml. of lead acetate solution (containing 100 grms. of normal lead acetate pure crystals and 12.5 grms. of glacial acetic acid per litre) and make up to volume with water. Shake well and filter without delaying more than a few minutes. Titrate 50 ml. of the filtrate (diluted with 50 ml. of water) at or near the boiling-point with ammonium molybdate solution (9-3 grms.per litre) using a 0.5 per cent. solution of tannic acid as an outside indicator (by spotting on a tile). The first appearance of a distinct yellow colour in the test drop (or a definite increase in a slight existing yellowish colour) marks the end-point. For a blank titration dilute 20 ml. of the lead acetate solution to 250 ml., and titrate 50 ml. of this + 50 ml. of water in the same way. Correct the difference between the two titrations for any lack of correct strength in the lead or molybdat 270 HINTON THE EXAMINATION OF FRUITS AND JAMS BY LEAD PRECIPITATION solutions. If the difference so corrected is not in the range 11-14 ml make a further correction according to Table VIII. (Should the difference lie outside the range there allowed for repeat the determination on a larger or smaller quantity, as the case may require of pectin-free filtrate.) From the corrected titration difference deduct 3.6 ml.(when malic acid was initially added) or 4.1 ml. (when citric was added). Calculate the remainder back to the number of ml. of 2 per cent. lead acetate solution which would be completely precipitated by 10 grms. of the original sample (or 40 ml. of the pectin-free filtrate). This is the “lead number” (aqueous) of the jam according to Boseley’s original definition; that is if the amount of pectin-free filtrate taken for the test be P ml. and the corrected titration difference D then 200D Lead number (aqueous) L = -P The “lead number per 0.1 grm. of acid” is then found by simply dividing L by the percentage of total fruit acid (a) in the sample (VII) L I = -a Acetone (50 Per Cent.) Lead Number.-Take an amount of pectin-free filtrate (to the nearest 5 or 10 ml.) containing approximate amounts of total fruit acid as follows: (i) Gooseberry strawberry rasp berry redcurran t blac kcurrant Grm* apricot or blackberry jams .. . . 0.50 (ii) Apple cherry plum greengage or damson jams . . . . . . . . 0.40 (If more than 200 ml. would be required the amount must be restricted to this figure. ) Place the required amount in a 250-ml. measuring flask and in the case of jams of group (ii) add 3.0 ml. of 5 per cent. citric acid solution (measured accurately). (Should it have been necessary to limit the amount of pectin-free filtrate taken to 200 ml. the deficiency of fruit acid may be made up by a suitable addition of 5 per cent.citric acid solution its effect being allowed for later.) Add acetone while rotating the flask to make up the total amount of acetone present to 125 ml. Then add from a 20 ml. of 10 per cent. lead acetate solution (as in the aqueous test) and make up to volume with water. Mix and filter taking precautions t o avoid loss by evaporation. Titrate 50ml. of the filtrate diluted with 50 ml. of water as in the aqueous test. Correct the difference between the titration and the blank (that obtained earlier will suffice) for any factors of the lead or molybdate solutions. If the difference so corrected is not approximately 14-15 ml. make a further correction according to Table XIII. From the corrected titration difference deduct 1-5 ml.for each 1 ml. of added 5 per cent. citric acid solution if any. Calculate the remainder as before back to the number of ml. of 2 per cent. lead acetate solution completely precipitated by 10 g-rms. of the sample. This i DISCUSSION ON THE ANALYSIS OF FRUITS AND JAMS the “lead number” (acetone) L’ of the jam, acid is then obtained as before, The lead number per 0.1 (VIII) L’ 1’ =-a 27 1 grm. of Interpretatiofi of Reszc1ts.-If I’ is appreciably less than 15 and plums or green-gages are not present lactic acid from a commercial pectin is probably present, and its amount can be approximately found by a formula adapted from the equation (11) given earlier: T I where k is the percentage of lactic acid in the sample.(see VII) is then obtained by deducting the lactic from the total acid: A corrected value for I - - (XI L I (corrected) = -a - k The value for I corrected or not as required and the total acidity a with any lactic acid deducted are then used in formula VI to find the fruit-content of the sample. For mixed jams coming within the scope of the method formulae IV and V may be used though it should be noted that if pomace extract or liquid pectin is present it will be included with the fruit of lower lead number. DISCUSSION The PRESIDENT remarked that all would agree with him that they had had very interesting papers read to them and that the methods described were extremely ingenious and the results of unquestionable utility. They had now agreed standards for the fruit-content of jam and also the National Mark Standards were in existence and it was therefore very essential that any proposed methods of analysis by means of which the true fruit-content of jam could be determined should be closely studied.He himself had had practically no experience of the examination of fresh fruits and this must be the case with many other analysts, and therefore the results which Mr. Hughes and Miss Maunsell had given would be of great value. He admired the ingenuity those authors had displayed in the method of calculation. They had concluded their paper by observing that it was really necessary to examine a number of jams before coming to any definite conclusion. The Public Analyst usually had to draw his results from one sample, and this was perhaps unfortunate.He was particularly pleased to hear Mr. Hinton’s paper because as Mr. Hinton had mentioned Boseley had experi-mented with a lead process and almost on the last occasion on which he was present at a meeting of the Society he had referred to his process which he enthusiastically believed would enable the fruit-content of jam to be accurately determined. Mr. T. RENDLE congratulated the Society on having such an excellent evening and the authors on papers containing such useful information. All who handled fruit and fruit products had felt the lack of satisfactory methods of analysis and every fresh figure was a considerable help. He would like to know whether the figures for apricots referred to the fresh dried or canned fruit.He believed that Macara’s published figures for insoluble solids showed very considerable differences for different varieties of the same fruit and for the same variety grow 272 DISCUSSION ON THE ANALYSIS OF FRUITS AND JAMS in different districts. Mr. Hughes and Miss Maunsell had put forward figures for one variety only of raspberries-“ Lloyd George.” He felt that it would add to the value of the paper if further information on other varieties could be given and if the maximum and minimum figures as well as the average were included. He would also like to know whether there was less variation between the maximum and minimum values for non-sugar solids than with the figures for insoluble solids on the same samples. With regard to Mr. Hinton’s paper it was customary in the manufacture of pectin from pomace to adjust the final acidity by the addition of citric or tartaric acid.Would the use in a jam of pectin containing these acids influence the lead number ? Mr. G. N. GRINLING referred to a pamphlet issued by the Research in Canning Section of the University of Bristol whereby the ingredients of canned fruit were calculated., Mr. J. R. NICHOLLS queried how accurate the figure for non-sugar solids was likely to be. Presumably when one got differing total solids and sugars all the errors would be thrown on the non-sugar solids. Mr. YOUNG asked what was the possibility of the action of sulphites on the lead number. Mr. WILSON (from California) said what a great pleasure it had been to him to be present at that meeting.He had done some work on these lines himself, and certainly in California they found that many factors such as degree of ripeness variety and district affected these natural products. It was always a pleasure to him to attend such a meeting and he extended a hearty welcome to any member who could attend one of their meetings in America. Dr. L. H. LAMPITT said that when a mass of figures such as those they had had that evening was shown upon the screen it was practically impossible at the time seriously to consider them and put forward any useful criticism. He did not know whether Mr. Hinton had considered the large number of other acids which occurred in small quantities and how far they were likely to affect his results of mixed acids when found in jams.Knowing how interested Mr. Boseley had been Dr. Lampitt thought that it would be a very good thing if chemists in the fruit trade could try this method on a large number of samples. It was only by gradual accumulation of a large number of figures that they were likely to get satisfactory information of a biological nature. He would like to thank Mr. Hinton for putting forward another possible tool which might prove of great use. Mr. T. MACARA congratulated Mr. Hughes and his colleague on the very ingenious way in which they had worked out the value of the non-sugar solids in fruits and applied the results to the analysis of jams. He had given figures in his own paper on the “Composition of Fruits” (ANALYST 1931 56 39) from which the non-sugars could be calculated and although these figures were not obtained by the same methods they could be used in the way indicated by Mr.Hughes and Miss Maunsell. It was a pity that the Society had not found it possible to publish all the tables of analyses of fruits which he had submitted, as the results for individual fruits were frequently helpful in dealing with difficult cases. He was pleased to hear the authors stress the necessity for the examination of more than one sample before coming to a decision. This was necessary to meet cases of bad distribution of the fibre and seeds in filling the jam into jars. There were also occasions when a manufacturer was obliged to skim off seeds or pips when these floated too freely to the surface of the jam. There were of course other cases where pips might be deliberately added and it was then necessary to consider other constituents of the fruit e.g.the acid. In this connection he referred to the He hoped that the new results would prove helpful to others DISCUSSION ON THE ANALYSIS OF FRUITS AND JAMS 273 case of a sample of gooseberry and raspberry jam submitted to him for analysis. This contained sufficient pips to represent 100 per cent. of raspberries but he had reported it as containing only 10 per cent. At first the manufacturer professed indignation at this result but afterwards admitted that he had purposely “faked” the jam to see whether it could be detected. He stated that he had added 12 per cent. of raspberries. The result reported was arrived at after a consideration of the figures for constituents other than that for insoluble matter particularly the acid figure in conjunction with a microscopical examination.It must be recognised however that it was possible to make a report of this sort for commercial purposes when a Public Analyst might naturally hesitate to do so. He was not quite sure that the authors fully realised the Analyst’s position in this respect. Like all other methods proposed it could not by itself give the true fruit-content of a jam. It was however another tool which did help especially with jams made from a mixture of fruits containing different acids. Mr. A. L. BACHARACH said that he would like to add his tribute to the ingenuity of the calculations. During the last few years he had become increasingly im-pressed by the practical value of statistical methods.Mr. Hughes and Miss Maunsell that evening had done one extremely useful thing (among others) by showing that in fact these methods could be employed to give certain figures and results that were perfectly intelligible and gave one the only really sound basis for the control of many manufacturing processes. Miss MAUNSELL replying to Mr. Rendle said that the apricots referred to in the tables were fresh fruit except in Table VII where canned apricots provided the data for an example of calculation. For all fruits the analyses of many varieties were included and the non-sugar solids figures for each kind of fruit fell into the statistical distribution calculated. The fruit was obtained from England Scotland Holland Germany Belgium France Italy and (in the case of apricots) Spain. South African apricots also contained non-sugar solids in agreement with the figure for English and Spanish fruit. The same applied to South African and American apples and pears. With regard to the question about varieties of raspberries and whether they varied according to district “ Lloyd George ” raspberries had been selected to illustrate the difference between those grown in England and those grown in Scotland. Different varieties of this fruit did not vary very much; one obtained possibly high results for one season but all the varieties would give high results for that district. All varieties of raspberries fell within the limits given and so varieties could not be differentiated by chemical analysis. The pamphlet mentioned by Mr. Grinling was of interest but the results were based on physical data; it might be helpful to use both sets of figures. As to limits of accuracy in Table IX the calculated composition of the jam was given as well as the actual analysis and it would be seen there that the greatest difference in non-sugar solids was 0.1 per cent. vix. non-sugar solids, 3.9 per cent. calculated and 3.8 per cent by analysis. Mr. HINTON replying said that the citric or tartaric acid used in extracting pectin had no appreciable disturbing effect. He did not think that bearing in mind the small amounts used the amounts of acid would fall outside the range of natural variations in the fruits. Mr. Young mentioned the possibility of disturbance by sulphites. Sulphite was quite easily dealt with by boiling the sulphur dioxide off. If oxidised it might cause a small amount of lead precipi-tation; but if much sulphate had been formed the effect on the p value would be so enormous that the sample would be detected as abnormal at once. Dr. Lampitt had mentioned traces of other acids. They had never found them to occur in sufficiently large quantities to disturb the results. In any case any traces of unusual acids fell into one of the three groups shown. Mr. Hinton’s paper described another very useful method