T H E PURIFICATIOX AXD ESTIMATIOS OF PECTIN BY GENE SFENCER
Ash Composition of Apple Pectin The spectrogram of apple pectin ash with which we worked gave the lines of the following elements: Na, K, Ca, Mg, Fe, Al, Mn, Si, and P. Chemical analysis shows the presence of chlorides, sulphates, carbonates and organic acid anions. According to Wiley, 56y0of the ash of apples, expressed as oxides, is potassium, 9% is phosphorus; Mg, Ca, S, are each present in the neighborhood of 3 7 0 ; the other elements are present in quantities less than one per cent. There are three recognized methods for removing electrolyte impurities from colloid sols: electrodialysis ; reprecipitation; washing by decantation with a non-peptizing solvent. Methods of Purification Reprecipitatzon by Alcohol: Taking ash content as criterion of efficiency, reprecipitation by alcohol is, in our opinion, the least satisfactory of the three methods. It involves the handling of large volumes of filtrate to obtain a relatively small yield of purified pectin, and there is an unavoidable loss of pectin in the filtrate. The recovery of this pectin may be accomplished by filtrate concentration followed by alcohol treatment. If heat is used in evaporation of the filtrate, there is an appreciable loss of pectin by hydrolysis as may be shown by the jelly-forming power of the product. This recovery does not seem to be worth while on a laboratory scale, at least. The best results by this reprecipitation method are obtained if the following facts are recognized: I . Experimentally the more jelly-like a precipitate, the larger is the amount of ash retained. This, theoretically, is due to the greater surface exposed for salt adsorption and to a greater occlusion and adsorption of mother liquor. A more granular, compact, and easily filterable precipitate was obtained by adding the alcohol to the pectin rather than in the reverse order. 2. The removal of mother liquor by pressure filtration is experimentally equivalent to several reprecipitations, as is shown by the ash reduction. Removal of mother liquor by centrifuge is not sufficient since the pectin retains many times its weight of liquor. Even after pressing the pectin between adsorbent materials until it crumbles, the loss in weight by air drying may be more than the weight of the pectin. Obviously the inorganic substances held by the retained liquor will increase the ash content of the purified material. 3 . Precipitation must be immediately followed by filtration. It was found that if the precipitate were allowed to stand in the mother liquor,
43 0
GENE SPENCER
or the mother liquor permitted to evaporate from the filtered pectin, instead of carrying the process through as quickly as possible, the ash content was appreciably greater. The smaller the bulk of the pectin precipitate in a given operation the greater the efficiency of purification, The manipulation and proportions giving the simplest technique and most acceptable results are: 500 cc. of a 2 % pectin s01,in a 2-liter separatoryfunnel, are treated with 600 cc. of 95% alcohol, acidified by 2 . 5 cc. concentrated hydrochloric acid. The alcohol is added in I O O cc. increments and the funnel shaken vigorously after each addition. The final mix then contains approximately 1% pectin, 50% alcohol and . 2 5 % hydrochloric acid, the remainder being water. The precipitate is emptied directly into a carefully washed and dried canton flannel filter cloth, supported on a hollow square rack. The pectin is pressed with a porcelain spatula until the filtrate comes slowly. The filter cloth is then removed from the rack and folded over the pectin to form a complete envelope container. This pack is subjected to pressure filtration until the liquor comes away dropwise. The filter cake is transferred to a clean dry cloth, broken up and again wrapped and pressed in the filter press between blotting paper or cotton until no further liquor can be pressed out. Before this filter cake material is repeptized preparatory to the next precipitation, it should be crumbled and air-dried; otherwise peptization is never satisfactory, due to the adsorbed alcohol. If this precaution is not taken, the next addition of alcohol gives the jelly-like precipitate which is to be avoided. The proportions suggested in this method were arrived at by carrying through a series of purifications in which the concentration of the constituents, pectin, acid, alcohol, was varied regularly. Percent ash in the purified pectin, after one precipitation, was plotted against the percent of the variable, in the final mixture. Purification, of course, increased with decreasing pectin; a lowering of pectin below 1% in the final mix was considered impracticable, because of the work involved in obtaining a small amount of pectin and also because of high percent loss in the filtrate. The lower the percent of pectin used the higher our percentage loss of pectin. I n the second series, the curve for per cent ash against per cent acidity began to flatten a t approximately 0.370 hydrochloric acid. I n the third series, the ash against per cent alcohol curve began to flatten between 50% and 60%. As to the number of reprecipitations: A series of experiments was carried out using the manipulation and proportions indicated. The per cent ash plotted against the number of precipitations gave a curve which began to flatten between the third and fourth reprecipitation. The first precipitation removed about soy0 of the ash. With each subsequent precipitation the efficiency of purification was definitely lessened, as was to be expected. Electro-dialysis: Electro-dialysis is a satisfactory method for purification but should, in our opinion, be used as a supplement to purification by decan-
PCRIFICATION AND ESTIMATION O F PECTIN
43 I
tation. When one attempts to electro-dialyze a high-ash pectin, the apparatus needs careful supervision to keep the temperature down. An apparatus, which ran continuously with practically no attention save for the refilling of the water reservoirs is shown in Fig. I . The heat produced in the purification of a 2y0 ash pectin, is removed by the cooled air which stirs the sol, and by a constant dropwise flow of wash-water through the electrode chambers. I n the apparatus used this amounts to approximately I liter of water per hour over each electrode.
1EC-I-4I
.\. Pectin solution.
B. Electrode Chamber, made of fat extraction thimbles impre nated with collodion, C. SiDhon and constant level control t r a m for electrode cham%er 0. D. Likbig condenser for cooling oxygen free air. E . Reservoir supplyin wash water to the electrode chamber. F. Ammeter. G. Jfoltmeter. H. Platinum Electrodes.
Since pectin is negativelycharged, there will be atendencyforit to pass into the anode chamber and for water to pass into the cathode chamber. For this reason nTater builds up faster in the cathode chamber and there is concomitantly somelossof pectin by cataphoresis, though in general pectin accumulates on the outside of the anode chamber and does not get through the membrane. This pectin layer must be removed occasionally from the outside of the anode chamber, since the air stirring does not take care of it entirely. That there is some loss of pectin by cataphoresis is shown by evaporation and alcohol precipitation of the anode liquor. The cathode liquor gave no evidence of pectin. The electrode chambers may be made either of animal membrane or collodion. If collodion is used, greater permanence is obtained if fat extraction thimbles are carefully washed and dried, and then impregnated with 3y0
43 2
G E X E SPEXCER
mitro-cellulose sol in 2 5 parts of alcohol and 7 5 parts of ether. Satisfactory membranes were obtained by immersing the dry thimbles in this collodion sol twice without intermediate drying. When the thimbles, so covered, had become dry to the touch, that is, in about 20 minutes, they were washed in water until they had no odor of ether. The alkali, concentrated in the cathode chamber, will in time harden the membrane so that it breaks. It is, therefore, desirable with high-ash pectin to substitute a 0.1% nitric acid solution for the water running into the chamber. The nitrate ions will pass through the sol toward the anode. In doing SO they should theoretically displace the more readily adsorbed fruit acid anions, by reason of their higher concentration, and should thus augment purification. If nitric acid is used, at the beginning of the process, water is returned to the cathode reservoir toward the end of the process to wash out excess nitric acid. One of the advantages of electro-dialysis, as compared with other methods, is the more efficient removal of fruit acid anions. The resistance which the cell, used in Fig. I , offers to the passage of a I I O volt drop, varies of course, with the concentration of the pectin and its electrolyte content. A 1% apple pectin containing .37G ash passed a current of about 0.1ampere. The fall in ammeter reading is taken as a criterion of the progress of purification. Theoretically when the ammeter reading is the same as that given by the cell filled with water, purification is complete. Experimentally this point was not reached. The process was stopped when there was no further fall in ammeter reading. Pectin, purified by electro-dialysis, is stored in dry form rather than as a sol on account of the danger of spoilage. Precipitation is accomplished by shaking the sol with twice its volume of 9570 alcohol. The difficulty of spoilage during the electro-dialysis was overcome by adding a few cc. of mineral oil to the sol, This film over the top prevented the entrance of air molds and also cut down frothing caused by the air stirring. The action of mold in the destruction of jelly-forming power of pectin is quick and sure. To reduce a 0.37~ ash pectin to 0.17~ ash in this apparatus required 7 2 hrs. Washing by Decantation: By far the simplest method of purification and the one which yields the lowest ash pectin, is washing powdered pectin by decantation with 6 0 7 ~alcohol, acidified to rYo with nitric acid, until I O O cc. of the wash liquor evaporated to I cc. gives no test for calcium with a pocket spectroscope. The flame test cannot be relied upon since potassium will still be present because of its high concentration in apples. After the calcium is removed, the washing is continued with 8 0 7 ~neutral alcohol until the supernatant liquid gives no reaction with blue litmus. The ash of apple pectin was reduced from 6.8Yo to 0.1087~ in three days with from 3 to 4 decantations per day. If the first step in this purification were continued until K, instead of Ca, were eliminated, the soluble ash left would be nil. Ash Composition of Purified Pectin The study of the ash composition of pectin purified by these three methods indicates that the elements which are preventing the preparation of an
PURIFICATIOS .4ND ESTIMATION O F PECTIN
433
ash-free pectin are silicon and aluminum. These two elements are the chief constituents of the ash left in the pectin purified by each process. For this reason there may be a tendency for a given method to yield a “constant ash pectin” with a pectin sample from the same source. This is liable to happen in the electro-dialysis method due to the retention of certain inorganic substances in the colloid form. Thus the persistence of aluminum and silicon may be explained. “constant, ash” tendency should be recognized as a possible limitation of the method and not necessarily as of chemical significance. By electro-dialyses we were able to purify pectin to a 0 . 1 4 7 ~ash. Johnston and Denton reduced the ash to 0.1876 by this method. X spectrogram of the ash of our purified sample showed that approximately of it was X1, Si and Ti, the remaining 2Yc being Fe, M g , Ca and V. Ti and V were not evident in the more complex spectrogram of the ash of unpurified pectin. If the A1 and Si are present as the colloidal silicate, it is doubtful if electrodialysis in our apparatus could ever reduce the ash much below 0.14. h membrane which would permit the passage of the silicate by cataphoresis would probably give rise to loss of pectin. Pectin purified by three reprecipitations by alcohol usually retained 0.3 5; ash. The spectrogram of this ash, compared with that of the unpurified pect’in, showed that the elements which resisted removal were Fe, Si, Al. When the pectin sol purified in this way vias subjected to electrolysis using a revolving cathode about I cm. from the anode, a potential drop of I O volts gave a current of 0.15 amperes. In four hours the ash of this sol was reduced about 20‘3 and in eleven hours, about 40%. The chief deposit on t’he electrode was iron. The greaest degree of purification was obtained by decantation washing with acidified alcohol. The ash was hereby reduced to 0.1087~ for which the spectrogram showed the same elements which were present in the unpurified pectin. The Ca, Mg, S a were removed more completely t>hanAl, Fe, Si and K. This high K, in the purified pectin is in keeping with the fact that j67C of the ash of apples is potassium oxide. Electro-dialysis of this pectin should give an ash, containing chiefly A1 and Si. A $
Jelly-forming Power of Purified Pectin Pectin purified by all three methods had a satisfactory jelly-forming power as compared to that of the original pectin, but this was not taken too seriously because the amount of adsorbed acid was changed as well as the amount of adsorbed ash during purification. I n both the reprecipitation method of purification and the decantation method, acid was used in the purification. Consequently the pectin resulting from these two methods required either less sugar or less acid to give a jelly of the same strength as the control jelly. Pectin purified by the electro-dialysis method required either more sugar or more acid to bring the jelly strength up to that of the control jelly. At first glance this might seem t,o indicate that the pectin had been improved
43 4
G E S E SPEKCER
in the two methods using acid and deteriorated in the electro-dialysis method. This does not necessarily follow. The whole difference may be in the initial acidity of the pectin sols and in the nature of the electrolytes retained by each method. Quantitative Determination of Pectin As yet we have no satisfactory method for the quantitative estimation of pectin. The methods’ which have been variously suggested in the literature depend largely upon precipitation, with its inherent error in incomplete precipitation, precipitation of non-pectin substances, and inability to wash out excess precipitating agent without repeptizing the pectin. Volumetric measurements of alcohol precipitates of pectin are subject to the error involved in the type of precipitate, which varies with manipulation. The salt adsorption error characteristic of the gravimetric measurements of alcoholic precipitates may in a measure be corrected by ignit’ion determinations, but the fundamental error of loss in precipitation and the precipitation of non-pectin substances makes this correction insignificant. Efforts to apply titration methods to products of pectin hydrolysis fail because of fading end point. Whatever the chemical reaction may be, the colloidal properties of the sol obscure it and make it valueless so far as an analytical method is concerned. Jelly-making tests as a criterion of pect,in measurements are of the greatest practical value but useless for scientific work because so many factors other than pectin affect jelly formation. Possibilities f o r a Colorimetric Quantitative Method for Pectin: Botanists? have recognized certain dyes as specific for pectin in plant tissues, such as phenosafranin, methyl blue, bismarck brown, para violet. On this basis a valuable colorimetric method might be developed. Many difficulties would arise in the development of such a method, for instance, the dye might not distinguish pectin from the intermediate “partially methylated” derivatives which are peptized by water; or the inorganic impurities might complicate the subject by forming lakes. However, it seems worth considering by someone who knows this phase of optical chemistry. Bibliography Determination of Pectin. RlcXair: J. Phys. Chem., 20, 633 (1916); Carre and Haynes: Biochem. J., 16, 60 (1922); Johnston and Denton: Ind. Eng. Chem., 15, 778 (1923); Campbell: J. Ind. Eng. Chem., 12, j j 8 (1920); Paine: Am. Food J., 17, No. 3 I I (t922). Wickmann: J. Am. Official Agr. Chem., 6, 34 (1922); 7, 107 (1923); Farneil: Int. Sug& J., 25, 248, 630 (1923); 26, 4 (1924); Emmett and Carre: Biochem. J., 20, 6 (1926); King: h a l y s t , 50, 371 (1925). Stains. IVood: Ann. Bot., 38, 273 (192 ); Mangin Compt. rend., 109, 579 (1890); Tobler: Chem. Centralbl., 1906, 1020; Howe:$ot. Gas., 7 2 , 313 (1921); LaMarllero: Rev. g6n. Bot., 18, 209 906); H u n t : Wash. Agr. Expt. Sta., 48, Z O I (1918); Sucharipa: J. Ass. Off. Agr. Chem., (1923); Paine: Am. Food J., 17, No. 3, I I (1922); von Fellenberg: J. Chem. Soc., 11 351 (19 5); Koydl: Chem. Abs., 8, 3725 (1914); Xanj, Paton and Ling: J. SOC. Chen,. 11 .., 44, 35zT (1925); Correns: Faserforschung: 1, 2 2 (1921);Ahmann and Hooker: Ind. Eng. Chem., 18, 412 (1926); Kanji and Norman: J. SOC. Chem. Ind., 45. 337T (1929). 3 Purification. Poore: U.S. Dept. Agr. De t Bull. 1323, I (192j); Emmett: Biochem. J., 20, 564 (1926); Griggs and Johnson: Ind. %ng. Chem., 18, 623 (1926); Johnston and Denton: Ind. Eng. Chem., 15, 778 (1923). Cornell L‘nicersity
’ Quantitative
