IAVDUSTRIAL A K D ENGINEERING CHEMISTRY
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Vol. 18, No. 4
The Determination of Pectin' Titration Method By C. F. Ahmann and H. D. Hooker D~PARTHZNT os AGRICWLTVULCHEMISTRY, UNIVERSITY OF MISSOURI, COLUMBIA, Mo.
:KCE pectin is an ester, readily saponifiable in the cold, as established by Fellenberg,2 acidity must develop on saponification and the amount of sodium hydroxide neutralized during saponification must be a measure of the amount of pectin present.
S'
Materials Used
The pectin used in this investigation was apple pectin supplied by the Leo Greenwald Vinegar Co., Topeka, Kan. The commercial product was dissolved in water, precipitated by alcohol, redissolved in dilute hydrochloric acid, reprecipitated, and the process repeated until a white product was obtained. It was possible by this process to reduce the ash to 0.2 per cent. By standard combustion methods this material showed the following composition on an ash-free basis: carbon 42.73, hydrogen 5.57, and oxygen 51.70 per cent. A water solution containing 0.2 gram in 100 cc. of water with a pH value of 6.2 to 6.4 has a pH value of 5.3. Precautions were taken to remove all traces of hydrochloric acid by washing until an alcohol extract gave no test for chlorides with silver nitrate. I n the earlier part of this investigation thrice distilled water with a pH range of 6 to 6.4 was used. Later, boiled distilled water was used for comparison. A carbon dioxidefree sodium hydroxide solution, prepared from sodium and carbon dioxide-free water according to the method of Carn ~ gwas , ~ used. I n the earlier part of the work the titrations were made electrometrically with a Bovie hydrogen-ion potentiometer. During saponification all samples were protected from carbon dioxide by paraffined stoppers or by soda-lime tubes.
reaction, as there is a large variation in the apparent amount of pectin present when determined at 23' and a t 55' C. Pectin Gram 0.0 0.10
Received October 30, 1925. Biochem. Z., 86, 118 (1918). a J . A m . Chem. SOC..43, 2575 (1921).
c.
50 23 50 55 65 23 .55 23 55 23 55
0.075 0.025
NaOH cc. 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
HCI Cc. Difference 0.0 20.6 22.1 4.5 5.6 21.0 6.0 20.6 7.7 18.9 23.2 3.4 22.2 4.4 2.3 24.3 23.5 3.1 1.0 25.0 25.1 1.5
equiv. Cc. 0.0 3.6 4.5 4.8 0.2 2.7 3.5 1.8 2.5 0.8 1.2
However, comparable results are obtained for a given series if the temperature is held constant. At 23" C. as the amount of pectin is increased the amount of alkali used increases proportionally but gives an incorrect value for the amount of pectin present if the factor for any other temperature is used as is shown by Table 11. If, however, the factor for any given temperature is determined and the saponification is carried on a t that temperature, comparable results are obtained, as shown by Figures 1 and 2. Table 11-Comparison Temzerature C. 23
55
35 34
1
Temperature
0.050
Effect of Temperature
Aqueous solutions containing 1.00, 0.075, 0.05, and 0.025 gram of purified pectin were treated with 50 'cc. of 0.424 N sodium hydroxide for 12 hours a t 23") 50', 55") and 60" C., respectively. They were then cooled to 23' C. and made up to 250 cc. Aliquots of 25 cc. were taken and titrated electrometrically with 0.08 N hydrochloric acid. The end point used was the pH of sodium pectate prepared by saponification of pectin with sodium hydroxide at 55' C. for 12 hours, the sodium salt being precipitated and washed with alcohol until no test for alkalinity was obtained. Titrations using phenolphthalein as indicator checked the results obtained by the electrometric titration, since the pH of the sodium pectate used was 8.6. The amount of acidity developed during saponification is represented by the difference between the amount of hydrochloric acid needed if there were no pectin present and the amount actually used. This difference, expressed as cubic centimeters of normal sodium hydroxide, represents the amount of alkali combined with the pectin to form sodium pectate. The effect of temperature is shown by the different amounts of alkali used for a given weight a t various temperatures. From Table I it is evident that temperature influences the
of Temperature on Saponi5cation of Pectin 0.1 N NaOH 0.424 N 0.08 N
Table I-Effect
of Factors for Varying Amounts of Pectin a t 2 3 O a n d 5 5 O C. Pectin 0.1 N NaOH Gram cc. Factor 0.100 3.6 8.93 0.075 2.7 6.97 0.050 1.8 6.94 0.025 0.8 7.81 0.100 4.8 5.20 0.075 3.5 5.35 0.050 2.5 5.00 0.025 1.2 5.25
c
33
32
28 27
Curve I - No pectin Curve IT- 0.05 qram p e c f h Curve D7- 0.10 qram pectln Curve g- 0.15 qram pecfin Curve T- 0.20 gram pertin
fn
VALUE
Figure I-Titration Curves of Acidity Developed after Saponification of Pectin for 12 Hours a t 23' C.
The effect of temperature is also shown in Table TI1 by the change in the saponification equivalent of pectin saponified at different temperatures.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
April, 1926 T a b l e 111-Change
of SaponiBcation Equivalent w i t h Change in Temperature Saponification Temgerature equivalent C. of pectin 23 285.6 35 250.2 55 208.9 65 175.0
Effect of Time
Pectin solutions prepared similarly to those described above were allowed to saponify for different lengths of time. After the saponification had proceeded for 3 hours the solutions were made up to 250 cc. Aliquots of 25 cc. were taken and titrated. At the end of 12, 24, and 36 hours the titrations were repeated. The amount of alkali combined with the pectin is represented, as before, by the difference in the amount of hydrochloric acid needed when no pectin was present and the amount used in the respective samples. of T i m e o n Saponification of Pectin
Table IV-Effect Pectin Gram 0.0 0.10
0,072
0.0:o 0.CiT5 0 . 0.X
Time Hours 24 3 12 24 3 12 24
Temp.
'C. 50 23 23 23 23 23 23 23 23 23
3 12 36 3 12 24 3 12 24
55 55 55
55
0.424 N NaOH Cc. 5.0 5.0 .5.0 5.0 5.0 5.0 5.0 5.0 5.0
5.0 5.0 5.0
5.0 5.0
D.3
5.0
55
5.0
0.08 N HCI Cc. 26.6 22.3 22.2 22.1 23.2 23.2 23.1 24.3 23.3 24.1 22.7 22.2 21.0 23.9 23.5 23.0
0.1
Difference 0.0 4.3 4.4 4.5 3.4 3.4 3.5 2.3 2.3 2.5 3.9 4.4 6.6 2.7 3.1 3.6
h-
NaOH equiv. Cc. 0.0 3.4 3.5 3.6 2.7 2.7 2.8 1.8 1.8 2.0 3.1 3.5 5.2 2.1 2.5 2.8
Table IV shows little variation in the saponification at 23" C. with changes in time. At 55" C., however. there is a marked change with time. Saponification a t 65" C., or even higher temperatures, for 12 hours or more w-cluld insure complete saponification, but carbohydrates at 60" or 70" C. on prolonged standing give rise to acids of small molecular weight. The method proposed for the determination of pectin depends on the increased acidity due to the formation of pectic acid from pectin; hence the results will be unreliable if other acids are formed in addition t o pectic acid. Those solutions kept a t 60" or 70" C. for 12 hours or more develop a n odor of caramel, which indicates a decomposition of the pectin. It was therefore necessary to determine the length of time required to complete saponification without decomposing the pectic acid. At the end of 12 hours' saponification at 55" C. there was no odor of caramel, indicating that the decomposition of pectin for this length of time is negligible. Table V shows the equivalent weight of pectin saponified at 55" C. for different lengths of time. Table V-Effect.
of T i m e on Saponification of Pectin at 5 5 O C. Saponification Time equivalent Hours of pectin 3 234.0 12
24
208.9 170.0
Pectic Acid Produced at 23" and 55" C. I n order t o determine the conditions which give results that represent the quantity of pectin present most accurately, pectic acid was prepared by treating pectin a t 23" C. and also at 55" C. Sodium pectate was produced by saponifying pectin with sodium hydroxide for 12 hours. The solution of sodium pectate was then acidified with hydrochloric acid, precipitating pectic acid. The pectic acid thus formed was dissolved and reprecipitated, the process being repeated
413
until the ash content was less than 0.1 per cent and an alcoholic extract gave no test for chlorides with silver nitrate. If there is a difference in degree of saponification there will be a difference in the amount of alkali necessary to neutralize a given weight of acid. As pointed out before, there may be a n increased acidity produced with time due to further breaking down of pectin to acids of low molecular weight, but these would be removed in the process of purifying the pectic acid. Two-tenths of a gram of pectic acid prepared by saponification at 23" C. required 7.9 cc. of 0.1 N sodium hydroxide. Two-tenths of a gram of pectic acid prepared a t 55" C. required 10.3 cc. 0.1 N sodium hydroxide. The neutral equivalent for pectic acid a t 23" C. is 261.0; a t 55' C., 194.9. The conditions of saponification that produce the pectic acid of the lower neutral equivalent would be the best. From Table V it is seen that saponification at 55" C. for 12 hours gives a saponification equivalent for pectin of 208.9, which corresponds to a pectic acid with ''ra neitral equivalent of 194.9. L o n g e r hydrolysis at 55" C. therefore gives inaccurate results. Titration curves of pectin solutions s a p o n i f i e d a t 23" \ a n d 55' C. a l s o show t h a t when uLi conditions are held constant comparable results are obCurve I - No pecfin Curve - 0.05 9rom pecfin tained. However, Curve iF-4.10 gram pecfln reliable results canCurve LV-0.15 qram pecfin not be o b t a i n e d Curve T-4.20 qram pec6n u n l e s s the neutral equivalent has been determined for the c o n d i t i o n s under pff VALUE which the saponifiFigure 2-Titration Curves of Acidity Deafter Saponification of Pectin for 12 cations are c a r r i e d veloped Hour@a t 5 5 ' C . out. Method of Determining Pectin
s
On the basis of the preceding investigations the following method of estimating pectin is proposed: To pectin solutions containing from 0.26 to 1.0 gram in 200 cc. of solution a known amount of alkali (50 cc.) is added from a pipet so that the concentration of the alkali will be about 0.1 normal. The solutions are then made up to volume (250 cc.) and allowed to stand a t 55" C. for 12 hours, the flasks being sealed during hydrolysis in order to prevent the entrance of carbon dioxide. Aliquots are then pipetted off and titrated with hydrochloric acid. The sodium hydroxide should be about four times as strong as the hydrochloric acid used for the back titration, in order that the difference will be large and so reduce the percentage error. From the number of cubic centimeters of alkali combined with the pectin thus found the amount of pectin is calculated. Taking the neutral equivalent of pectic acid a t 55' C. as 194.9, which is equivalent to 208.9 grams of pectin, the amount of pectin can be calculated from the amount of alkali used, thus: NaOH: Pectin = Weight of alkali combined: X 40
208.9
PREcAuTIoNs-In case very small quantities of pectin are found (0.010 gram in 50 cc. of solution) about a 200-cc. sample should be taken. As the alkali is four times as strong
INDUSTRIAL A N D ENGINEERING CHEMISTRY
414
as the acid, care must be taken in pipetting the alkali for saponification, since a drop more in one than in another makes a n error of about 0.2 cc. Comparison of Precipitation Method with Titration Method
This method of determining pectin was checked against the method developed by Carre and Haynes 4 The samples for comparison were taken from a stock solution of pectin containing 2.00 grams per liter. The pectin was estimated on 25cc. aliquots. According to the method of Carre and Haynes, the pectin is saponified a t room temperature; however, the precaution was taken to keep the temperature a t 29” C. during the saponification. The pectin in the samples to be determined by the titration method was I
4
Biochcm. J . , 16, 60 (1922).
Vol. 18, No. 4
saponified a t 55” C. Table VI compares the results obtained by the two methods. Table VI-comparison Precipitation method Gram 0,0450 0.0484 0.0486 0.0473
of Precipitation Method with Titration Method Titration method Gram 0.0520 0.0525 0.0515 0.0512
Actual weight of pectin Gram 0.050 0.050 0.050 0.050
Both methods give reliable estimates of pectin when working with pure pectin. However, when working with plant material, which contains salts that are occluded by the calcium pectate gel, as pointed out by Carre and Haynes, the precipitation method becomes less reliable, while by the titration method the interference of occluded material is avoided. The titration method as developed in this study is also more rapid.
The Chemical Unsaturation of Rubber under the Action of Heat, Trichloroacetic Acid, UltraViolet Light, and Mastication’.’ By H. L. Fisher and A. E. Gray THE B. F. GOODRICHCo., AKRON,OHIO
l’he changing of the chemical reactivity of rubber to hydrogenate rubber was by various physical agents in recent work on hydrogenon account of its physical conries a n d Nagel3 o n ation is discussed. dition-that is, “its degree shellac, and of PumThe effect of various agents on the chemical unsatof dispersion or aggregation.” merer and Burkard,4 Stauduration was studied, and it is shown (1) that heat, in the H e t h e r e f o r e h a d rubber inger and F r i t ~ c h iand , ~ Harabsence of air, at vulcanization temperatures, around strongly plasticized on a mill, ries6 on the hydrogenation of 141’ C., even for 8 hours, causes no appreciable drop in and was then able to hydrorubber, it would appear that the chemical unsaturation, whereas at high temperagenate it in the presence of there is some connection betures, 245’ to 360’ C., there is a lowering of the unplatinum black. (It should tween the physical state of saturation, the amount depending on the time and be noted, however, that he these substances and their temperature (verification of the work of Staudinger) ; has never published any anac h e m i c a l reactivity. Har(2) that trichloroacetic acid causes a definite lowering lytical data.) Pummerer and ries and Nage13 found that, of the unsaturation; (3) that ultra-violet light causes Burkard4 tried “to bring into whereas the ordinary alcoholno lowering of the unsaturation; and (4) that mastireaction the most highly possoluble shellac yielded the cation in air causes a lowering in the unsaturation sible d e p o l y m e r i z e d a n d maximum of 30 per cent of possibly due to absorption of oxygen, but in an attherefore reactive rubber” by aleuritic acid by cold hydrolmosphere of carbon dioxide, it causes no change in unusing very dilute solutions ysis with potassium hydroxsaturation, although there is, of course, a decided of h i g h l y purified rubber. ide, t r e a t m e n t with an change in the physical properties. They were very successful. ethereal solution of hydrogen So were S t a u d i n g e r and chloride caused the shellac ~ used high temperatures (270’ C.) a i d high to be insoluble in alcohol and, although it was still solu- F r i t ~ c h i ,who ble in potassium hydroxide, cold hydrolysis with this re- pressures (93 atmospheres). It is of special interest that all agent then gave no more than 3 per cent of aleuritic acid. these experimenters did their work independently yet with By boiling with glacial acetic or formic acid the insoluble the same fundamental idea of changing the rubber into a variety could be reconverted into the soluble variety and it more reactive state. The hydrorubber in all cases is apparwould once again yield the maximum amount of aleuritic ently the same. It is like a paraffin hydrocarbon in chemacid upon hydrolysis. Harries,6 with this work as a back- ical properties, has a very high indeterminate molecular ground, thought that the reason why he had never been able weight, and gives typical colloidal solutions. The nuccess of these investigations was dependent, ac1 Presented before the joint meeting of the Division of Xubber Chemistry and the Akron Section of the American Chemical Society, Akron, Ohio, cording to the different authors, upon “disaggregating” or February 22 and 23, 1926. “depolymerizing” the large rubber molecule into smaller 1 Part of this paper was given by the senior author under the title dispersed particles or particles of lower molecular size which, “The Physical State of Colloidal Organic Substances and Their Chemical in each case, are considered to be more reactive chemically. Reactivity,” at the First National Symposium on Organic Chemistry, Rochester, N,Y.,December 29 to 31, 1925. Heat and pressure are familiar agents for promoting chemical d - 247 ~ . ,(192.3). a ~ n .~ ,S B 3833 , (1922);~ ~ i i ~ i ss, reactions, but dilution is seldom, if ever, used for the same 4 B n . , 66, 3458 (1922). purpose; in fact, dilution generally retards a reaction. If we Heloelica Chirn. Acta, 6 , 785 (1922). consider that we are dealing with colloidal systems, the meth0 Ber., 66, 1048 (1923).
ROM the work of Har-
F
