June, 1926
INDUSTRIAL A.VD ENGI,VEERIiVG CHE.VISTRY
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Preparation and Colloidal Properties of Pectin' Preliminary Report By Mary A. Griggs and Ruth Johnstin WELLESLEY COLLEGE, WELLESLEY,M a s s
In the preparation of pure pectin from lemon albedo the following points are emphasized. The albedo is treated with alcohol before grinding, and this extraction must be rapid and at a definite temperature. Dialysis of the pectin sols is the best method of removing electrolytes, especially calcium salts. The pectin is precipitated by the dropwise addition of alcohol to the pectin sol, and not vice versa, the gelatinous precipitate flocculated in an electric field, and filtered through silk cloth. Some colloidal properties of pectin solutions are discussed. These include the optical properties, the charge on the particles and their precipitation, the fact that pectin does not dialyze, the viscosity, and a discussion of
alcohol-pectin gels and sugar-acid-pectin gels. In describing the sugar-acid-pectin gels it is pointed out that heat and evaporation are not necessary for gelation, that there is a time factor in gelation, and that there is probably no compound formation. The writers have indicated in this report the course along which their investigations will proceed. It seems probable that with a standard method of preparing pectin free from impurities, reproducible and valuable results can be obtained. A thorough knowledge of the colloidal behavior of pectin sols must furnish the scientific basis for the commercial utilization of the enormous quantities of pectin now available.
HE ability of pectin sols to form gels is unquestionably the most interesting and important characteristic of these substances. A number of investigators have studied the conditions governing the gelation of sugar-pectinacid mixtures, but it is difficult to bring this work together into any sort of uniformity because in no two cases is the pectin used the same and in only one instance2 has there been any attempt to work with a highly purified pectin. Pectin in water constitutes a colloidal system resembling in some respects the lyophilic sols of gelatin and also of agar-agar. Apparently the pectin-water system differs from these in that the transition from sol to gel state can be effected only by the addition of a third substance or sometimes a fourth. It has been stated repeatedly in the literature that sugar and acid are both necessary for the formation of pectin gels. Certain observations made on sols containing pectin of a much higher degree of purity, and of a much lower ash content than any previously used, have led the authors to believe that the gelation of pectin sols is a simpler process than one is led to believe from a consideration of the complex pectin-sugar-acid-water systems. I n order to study the behavior of colloidally dispersed pectin it seemed desirable to prepare a product of a high degree of purity the physical constants of which could be well defined.
rapidly as possible. If fresh portions of alcohol are added frequently, especially during the first day, readsorption by the albedo of alcohol-soluble material is prevented and the time required for complete extraction is lessened. The completeness of this extraction can be judged from the final form of the pectin, for it cannot be produced as a white powder unless free from these alcohol-soluble and ethersoluble substances. When the extracts become entirely colorless the albedo is dried a t not more than 65' C. According to von Fellenberg,4 this treatment converts much of the insoluble protopectin to soluble pectin. The albedo loses abouf 85 per cent of its weight on extraction and drying. It should be noted in passing that this preliminary extraction with alcohol and ether is of special value in the preparation of apple pectin, in which it has not been generally used. This treatment removes those substances from the apple pulp which oxidize so readily in the air to form sticky dark brown compounds, which when once formed can hardly be separated from the pectin. Pectin prepared from apple pomace or commercial pectin solutions cannot be expected to be free from these organic impurities. EXTRACTION O F PECTIN FROM ALBEDO-The temperature of extraction and the hydrogen-ion concentration of the extracting medium are the conditions which should be carefully defined during this process, since variations in either may profoundly affect the physical state of the resulting product. The effect of heat alone on pectin is not well known, but it has been shown4 that boiling with acid brings about hydrolysis of the pectin to pectic acid and long heating at a lower temperature has the same effect. It therefore seems necessary a t least to define the temperature of preparation. Ninety degrees Centigrade has been chosen in this work as the maximum temperature to which the pectin is ever submitted and the solutions are always brought to this temperature rapidlyin order to avoid long heating. This is probably below the temperature of much hydrolysis, and yet it is great enough to prevent bacterial action and also to allow fairly rapid filtration of the solutions. The preparation of pectin in the cold would be of interest as possibly yielding a product of different properties, but this could only be accomplished with the addition of preservatives. The procedure employed in this work was as follows: 15 grams of the albedo prepared as above were extracted
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Preparation of Pectin
These studies present several new procedures used in the preparation of pectin which have proved to be of real value in removing impurities and in handling this extremely difficult material. Some modifications may still be necessary before an entirely satisfactory method of preparation is established. The material was obtained from the skin of lemons by first removing the outer yellow rind with a stainless steel knife. The albedo was then carefully peeled off so that no pulp adhered and was immediately soaked in alcohol for about 10 minutes. This treatment hardens the skin so that i t can be ground in a meat chopper without any of the darkening due to oxidation and to the action of the metal. EXTRACTION O F ORG.4NIC I~PuRITIEs-The extraction of organic impurities, such as oils, terpenes, resins, etc., with alcohol and then with ether under a reflux condenser, first suggested by Bourquelot and H k r i ~ s e y ,was ~ carried out as 1
Received November 2 7 , 1923. J . Assoc. O?iicial d g r . Chem., 7 , 57 (1923). J . Bharm. c h i m . , [61 7 . 473 (1898).
* Sucharipa, 3
4
Biochem. Z . , 81, 11s (191s).
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successively with three portions (750 ml., 500 ml., 500 ml.) of 0.01 N hydrochloric acid at 90" C. The first portion of acid was added to the albedo cold, stirred for 15 minutes, and then heated with stirring to 90" C. The final yield of purified pectin was about 27 per cent of this albedo, or about 4 per cent of the original albedo. It should be borne in mind that this is an acid-treated pectin. REMOVALOF INORGANIC IMPURITIES-The problem of lowering the ash content of pectin has been attacked by various workers in different ways. Since the ash contains about 95 per cent of calcium oxide, many workers have attempted to remove it with oxalate ions, either extracting the pectin from the albedo with dilute oxalic acid or ammonium oxalate or precipitating with an oxalate solution. I n order to precipitate calcium completely as the oxalate, the solution must be alkaline and pectin hydrolyzes to pectic acid very readily in alkaline solution. Again, calcium oxalate is such a fine precipitate that it will not be retained by filter paper, which also allows the passage of pectin sols. Reprecipitation has been found by some workers, notably Sucharipa,2 to be quite effective for reducing the mineral content. Three reprecipitations, however, from water solutions containing several grams of pectin have left pectin with an ash content of 2.60 per cent, while three reprecipitations from dilute hydrochloric acid solutions have left 0.69 per cent ash. This is not only large but it leaves the pectin with considerable adsorbed acid. These dficulties have led, in this work, to the use of dialysis as the most effective method. DIALYSIS-The pectin sol containing 1 gram in 300 or 400 ml. is dialyzed in a parchment bag with 0.01 N hydrochloric acid. It is heated every 24 hours to about 90" C. to prevent bacterial action. A fresh portion of acid is used daily until it no longer gives a test for calcium. This usually requires a week. The acid can then be dialyzed out with distilled water in 4 or 5 days with daily heating. By this method, however, ash in pectin has been reduced in duplicate dialyses from 2.60 per cent to 0.18 per cent and the pectin has only a slightly acid reaction. About 20 per cent of the pectin is lost in the dialysis. PRECIPITATION-The method of precipitating pectin determines the degree of dispersion of the resulting product, which the writers believe to be the controlling factor in gelation. Not only is complete precipitation of the pectin desirable, but the precipitate should be in a form which is easily filterable and carries down a minimum amount of adsorbed material. The use of dilute solutions-1 gram in 300 or 400 m1.-is important. Two volumes of 95 per cent alcohol should be added to the pectin sol dropwise with thorough stirring a t room temperature. If the pectin sol is added to the alcohol as suggested by T a r 9 and by Halliday and Bailey,G the pectin comes down completely, but in a gummy mass which must adsorb many impurities. Precipitation a t a high temperature results in a gelatinous rather than a flocculent precipitate. Flocculation is aided by the presence of electrolytes, so as these diminish the pectin becomes more gelatinous. This gelatinous mass can be flocculated in a very satisfactory way at the anode on electrolysis with 110-volt d. c. for about 2 hours with platinum electrodes. The effect is identical with that produced by the addition of positive ions without the complication of added electrolytes. At least one reprecipitation is always advisable. The precipitated pectin can be filtered easily by suction through heavy silk cloth stretched over a funnel. It is sucked and pressed as dry as possible with a porcelain spatula, removed from the filter, thoroughly mixed with alcohol for washing, and again pressed dry on the filter. This should 6
Del. Agr. Expt. Sta., Bull. 184 (1923).
*THISJOURNAL, 16, 595 (1924).
Vol. 18, No. 6
be repeated about four times. After the last precipitation the washing with alcohol is followed by similar treatment with ether. The pectin is constantly worked with a porcelain spatula, as suggested by W e n d l e m ~ t h ,on ~ the silk filter with suction, and when it becomes thoroughly dry it is a snow-white hard powder. It contains about 11 per cent moisture and 0.18 per cent ash. Its specific rotation is +BO a t 23" C., which agrees closely with Poore'ss results. It has only a slightly acid reaction unless acid is used iIi the precipitation. Colloidal Properties
There is no doubt that pectin in water solution is colloidally dispersed. The water solutions are opalescent in reflected light and clear in transmitted light. The Tyndall effect has been observed by the authors in a 1 per cent pectin solution with variations in the intensity of the ray with crossed Wicols. I n the ultra-microscope the sols show Brownian movement. It is usually thought that the colloidal particles bear negative charges in water solution. This is borne out by the precipitation of pectin from water solutions by some metallic ions. Lead ions have been found the most effective of many positive ions for this coagulation. A few drops of very dilute (0.01 N ) lead nitrate solution immediately converts a 1 per cent pectin sol to a clear, hard gel. On the other hand, strong alkalies and, on standing, strong acids produce opaque white gels due to hydrolysis of the pectin to pectic acid. The fact that iron a n d aluminum ions having a valence of three are less effective precipitants suggests the possibility that the precipitation is not due solely to a negative charge on the pectin. Moreover, in electrolyzing water-pectin sols practically free from electrolytes, pectin has been found deposited on the cathode, which should indicate a positive charge. However, the addition of alcohol to the nearly pure pectin sols often produces a soft gel in which there is no doubt that the pectin is a negative colloid, for it forms as a solid gel on the anode when electrolyzed. Studies on the dialysis of pectin sols and gels have shown that pectin is but slightly dialyzable and that electrolytes such as acids and calcium salts may be separated from pectin by this method. This leads one to question whether pectin as it occurs in the cell wall is a stable calcium compound, as is often assumed. The viscosity of pectin sols seems to be much greater than the viscosity of gelatin sols of equal concentration. I n a preliminary study of this property the following values for the specific viscosity of lemon pectin sols at 25" C. have been found: Pectin
Per cent
Specific viscosity at 25' C. 1.348 1.834 2.899 4.018 5.190 7.235
The Ostwald viscometer is used to determine the viscosities, and the densities are determined by means of the Sprengel tube. Since viscosity is one of the most important properties of pectin sols, a series of experiments is planned to show the effect on the viscosity of such factors as concentration, pH, age, sugar, alcohol, temperature, method of preparation, etc. At the present time, the conditions governing pectin sol-gel transformation are not weU understood and the structure of the resulting gels has been given very little attention. By means of X-ray and ultra-microscopic studies the authors 7 8
Kolloidchem. Beihefte, 19, 115 (1924). U. S. Dept. Agr., Bull. 1838 (1925).
June, 1926
INDUSTRIAL A N D ENGINEERING CHE.WISTRY
hope to be able to throw some light on this very interesting phase of the subject. At present precipitation seems to be closely related to gelation. By controlling temperature and concentration it has been found possible to produce gels with many of the reagents ordinarily thought to be precipitants of pectin. The alcohol-pectin gels have been prepared quite as successfully as the sugar-acid-pectin gels and they allow the formation of Liesegang rings of both ferric ferrocyanide and silver chromate. The usual pectin gels-containing pectin, water, sugar, and acid-are complicated because of the large number of constituents and the very ease of gelation, which may occur under varying conditions. The four components are essential for this type of gels, and yet Goldthwaiteg has prepared satisfactory gels by substituting glycerol for the sucrose. The authors have substituted 50 per cent dextrose for sucrose and obtained a gel, but only when the solution was heated. That heat is not a necessary factor in the building up of this type of gel struct,ure the writers have shown conclusively by preparing a series of gels containing 50 per cent sugar, 0.5 per cent citric acid, and from 0.125 to 0.5 per cent pectin without any heating. Further, evaporation seems to play no important part, as has often been assumed, because these * THISJOURNAL,1, 333 (1909).
625
gels were left covered and the loss in weight was negligible. It seems probable that the important factors in pectin gel formation are the degree of dispersion of the pectin and the time required for the building up of the gel structure. It has been found that as the percentage of pectin decreased the time required for gelation increased. The high jellying power of this purified pectin should be noted because some authors state that repeated purifications decrease the jellying power. This is doubtless true when the methods of purification bring about the hydrolysis of pectin to the forms containing fewer methoxyl groups. Both Tam5 and Chernofflo have recently suggested the possibility of the formation of definite chemical compounds in sugar-pectin-acid gels in explaining the building up of a structure. SucharipaJ2however, succeeded in washing out most of the sugar and acid from a gel with 75 per cent alcohol, leaving, he believed, the pectin with too little sugar or acid to indicate a chemical compound. The writers have confirmed this repeatedly by dialyzing out from the gels so much sugar that the residue was tasteless and so much acid that a gel could only be made from the residue with the addition of acid as well as sugar. ’OArn. Food J . , 18, 200 (1923).
Nature of Vegetable Tannage’.’ Tanning with Mixtures of Gallotannin and Quinone By Arthur W. Thomas and Margaret W. Kelly COLUXRIAUNIVERSITY, N s w YORK,N. Y.
REVIOUS investigations3 of the change in rate of vegetable tannin fixation by hide substance as a function of the pH values of the solutions have shown that (1) a minimum is always obtained in the region of pH 4 to 5, and (2) the curves rise from this minimum to a maximum a t pH 2 (sometimes pH 1) on the acid side and to a maximum a t pH 7 to 8 on the alkaline side of the point of minimum rate of fixation. Composite curves of results obtained with six commercial vegetable tanning extracts are shown in Figure 1. The explanation of the complgx paths in these graphs in the p H range of 1 to 8 was offered3 on the basis of the ProcterWilson theory of tanning and the postulate of two forms of collagen, the anions of the complex, high-molecular-weight tannin acids combining with collagen cations to form the insoluble substance, leather or “collagen tannate.” It was expected that there would be no tannin fixed from solutions more alkaline than pH 8. However, tannin was fixed even up to the pH value of 12. This could not be accounted for by the Procter-Wilson theory, and since it was more noticeable with tanning extracts rich in nontans, it was suggested3 that the tanning action observed in the alkaline solutions might be attributable to substances of the character of quinone which are capable of oxidizing amino groups in alkaline solution and combining therewith. That the “leathers” tanned in acid and alkaline solutions are different in nature was demonstrated by alcoholic extraction.4
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Presented under the title “Tannage with Mixtures of Gallotannin and Quinone” before the Division of Leather and Gelatin Chemistry at the 67th Meeting of the American Chemical Society, Washington, D. C.. April 21 to 26, 1924. Received October 30, 1925. 2 Contribution NO. 497 from the Department of Chemistry, Columbia University. Thomas and Kelly, THIS JOURNAL, 18, 1148 (1923). I b i d . , 16, 31 (1924).
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If the suggested hypothesis for tannage in alkaline solutions is correct, then tannic acid, which is practically devoid of nontannins, should show no tanning action in solutions more alkaline than p H 8. This was tested and found to be true.5 To be sure, there was a slight degree of tannage in tannic acid e 125
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Figure 1-Rate of T a n n i n g a s a F u n c t i o n of pH Value The 24-hour curve is the average of eleven measurements of six commercial tanning extracts-hemlock bark, quebracho, wattle, gambier, oak bark, aad larch bark, the average concentration being 55 grams total solids per liter. The 2-week curve is the average of six measurements with same extracts, the average concentration being 39 grams total solids.
at pH to 12, but it was in to that obtained with vegetable tanning extracts. It must not be overlooked that there is a certain amount Of decomposition of tannic acid in alkaline solutions due to oxidation. This 8
THIS JOURNAL, 16, 800 (1924).
