328
X. SPEISER, C. H. HILLS, AND C . R. EDDY
(4) FERGUSON, R. H.: Oil &- Soap 21, 6 (1944). ( 5 ) FERGUSOS, R. H., ROSEVEAR, F. B., ASD STILLMAS,R. C . : Ind. Eng. Chem. 35, 1005 (1943). (6) MCBAIN,J. W., BOLDUAN, 0. E. A , AKD Ross, S.: J. Am. Chem. SOC.65, 1873 (1943). ( 7 ) MCBAIN,J. IT., AKD LEE,IT. X.: Ind. Eng. Chem. 35, 78-1 (1943). (8) MCBAIN,J. W.,AND LEE, IT. IT.: Oil & Soap 20, 17 (1943). (9) MCBAIX,J. W., VOLD,R. D., ASD FRICK,>I.: J. Phys. Chem. 44, 1013 ,~0910:. (10) MILLS,IT.: U. S. patent 2,295,594 (September 15, 1942). (11) Ross, S.: Private communication. P. L4., ASD EHRLICH, E.: Z. physik. C'heni. A165, 453 (1933.. (12) THIESSEN, THIESSEN, P. h., ASD SPYCHALSECI, R.: Z.phgsik. Chem. A166,435 (1931.8, THIESSEK, P. A, v . KLESCK,J., GOCKOWIACK, H., A K D STAUF'F, I-.:Z . p h y d c . Chem. A174, 335 (1935). (13) TOLD, AI. J.: J. Am. Chem. SOC. 63, 1427 (1911). (14) VOLD,R. D.: J. Am. Chem. SOC.63,2915 (1941). (15) VOLD,R. D., A N D LYON,LUTHERL. : Ind. Eng. Chem. 37, 497 (1946). (16) TOLD, R. D., AKD YOLD,PII. J.: J. Am. Chem. SOC.61, SOS (1939).
T H E ,ACID BEHBT'IOR OF PECTISIC ACIDS R. SPEISER, C. H. HILLS,
AND
C. R.EDDY
Eastern Regional Research Laboratoryl, Philadelphia, Penn.sylvu!i7n Received February %'i, 1945 ISTRODTJCTIOS
Some time ago theie v a s initiated in this laboratory an investigntiur. of methods for producing pectinic acids of 1011- ester content from apple pomace for use in preparing calcium pectinate jellies of low sugar content (14). These pectinic acids were made by tn-o methods-acid deesterification and enzyme deecterification. Acid deesterification requires treatment of pectinic acid of high ester content for 1 t o 2 days a t 40-50°C. a t a pH less than 1, n-heres- enzyaie deesterification requires only a fen- minutes a t 40°C. and pH 6. The economic advantages of the enzyme method are obvious. Unfortunately 65 per cent sugar jellies and 33 per cent sugar-cdciurn pectinate jellies prepared from enzyme-deesterified pectinic acids were not so strong as those prepared from acid-deesterified pectinic acids. This n-a. t r w even for pectjnic acid samples of the snnie ester content and viscosity (in n-ster dutions) prepared by the above tn-0 methods. Table 1 and figure 1 illustrate this point. It v a s thought that if the factors governing the behavior of pectinic acids were determined, improved strength of enzyme-deesterified pectinate jellies could be obtained and the anomalies in the physicochemical behavior of pectinic acids could also be satisfactorily explained. Therefore, the follon ing studies 1 One of t h e laboratories of the Bureau of hgricultural and Industrial Chemiytry, hgricultural Research hdministration, United States Department of Agriculture
329
ACID BEHAVIOR O F PECTINIC ACIDS
have been made upon pectinic acids prepared by the tTyo methods: acid behavior; kinetics of deesterification; electrophoretic behavior; viscosity, molecular weight, and molecular-weight distribution; and properties of pectin jellies. The strength and other niechanical properties of pectinic acid jellies of high sugar content are yery sensitive to the pH (1, 30) of the jelly and the degree of TABLE 1 Strength of p e c t i n jellies as a j u n c t i o n of method of deesterification
I
JELLY STRENGTH' I
METHOD O F DEESTERIFICATION
54V?PII:
I
I
H59 H7.1
1
Acid Enzyme
jellies
I
I
9er cerht CHIO
I
4.53 4.48
1
35 per cent sugarcalcium pectinate jellies
6 j per cent sugar
ESTER
i
I
,
I
cm.
Cm.
72 24
56 4
~
I I
* The jelly strength, as measured by the Delaware jelly tester (39), is the number of centimeters of water pressure t h a t a jelly can support without rupture. The maximum jelly strength (14) n-as obtained by varying the p H and calcium concentration. I
I
I
I
I
I
dissociation (15) of the pectinic acid. Evjdence has been obtained in this laboratory that the three-dimensional elastic structure of the jelly is built up through extensive hydrogen bonding between the sugar and pectinic acid molecules. Calcium pectinate gel structures are built up through a system of both hydrogen bonds and ionic bonds (1). The stability of these structures is a function of the ionic equjljbria involved, as well as of the dissociation of the pectinic acid.
330
R. SPEISER, C. H . HILLS, AKD C. R. EDDY
T’iscosity, osmotic pressure, and solubility are also greatly influenced by the acid behavior of the pectinic acid. Furthermore, changes in the electrolytic properties of the carboxyl group in passing from the monomer to the polymer molecule are of fundamental interest in the general theory of the behavior of high polymers. For these reasons, a study of the acid behavior of pectinic acid was undertaken.
Constitution of the pectinic acid molecule2 In order to explain the behavior of pectin, it is necessary to have a clear understanding of the constitution and structure of the pectinic acid molecuie. 1-11fortunately, the literature is not in entire agreement even upon this fundamental point (32), a state of affairs not uncommon in the realm of naturally occurring high polymers. The pectinic acid complex extracted from apple pomace consibt- of n long straight-chain skeleton of partially methyl-esterified polygalacturonie acid to which are attached side groups of araban (a branched-chain polyarabino5e) and galactan (a straight-chain polygalactose) (2, 3 , 17, 25, 26, 27, 34, 35, 38). Pre(a r t 0 OCH ),
( o r COOCH,)
-o~o&o,k-&oJ& COOH
COOH (a r CO 0 C H 3 )
H
OH
H
OH
0-
(or C O O C H 3 )
FIG.2. Backbone chain of t h e pectinic acid molecule
viously proposed ring structures (9, 28) have been abandoned in the light of conclusive evidence (from x-ray (40), optical (40), viscosity (35), ultracentrifugal sedimentation (33), and osmotic pressure (35) studies) that the pectinic acid skeleton is essentially linear, resembling that of cellulose to a high degree Data in the literature (29, 32) and data obtained in this laboratory shox that pectinic acid is not honiogeneous and that the methyl ester, araban, and galactan contents of a sample w r y nith the source, method of extraction, and subkeyuent treatment. The molecular n-eight ranges from relatively low value3 to :ippro\imately 300,000 (25, 33, 34). The methyl ester, araban, and galactan cnritentz range from approximately 0 to 11 per cent, 0 to 30 per cent, and 0 to 40 per cent, respectively. It has been suggested (18, 29.34,36) that the araban and gnlactan are attached to the main chain by purely physical forces (that is, mondaiy T alence forces), instead of primary valence bonds. Hox-ever, rate measurements t o be reported in a later paper indicate that a substantial portion of thiq polysaccharide material iequires an activation energy of at least 18,000 c 2 i . for itremoval from the main chain. According to this conception of the pectinic acid molecule, figure 2 reprcqents the main skeleton to which are attached, at undetermined points, the electrolytically inert araban and galactan. 2 The nomenclature used here is that agreed upon by the Anierican Chemical Society Committee for Revision of the Somenclature of Pectic Substances (22).
ACID BEH.1TIOR O F PECTINIC ACIDS
33 1
EXPERIMESTAL
Titration curves were obtained according to the method of Briggs (4). For example, approsinlately 1 g. of pectinic acid was dried t o constant weight in a vacuum oven a t 80°C. The sample m s nioistened with about 1 ml. of alcohol and made up to 100 ml. with water. Measured quantities of 0.01 S sodium hydroxide were added to 5-ml. portions of this solution and then diluted t o 25 ml. The pH of each solution was measured with a Cambridge electron-ray pH meter to ~k0.005pH unit. By this procedure the pH as a function of degree of neutralization was measured a t constant pectinic acid concentration. The same procedure was used with carefully purified d-galacturonic acid.
Preparation of acid-deesterified pectinic acids X commercial 200-grade apple pectin n-as reprecipitated from n-ater d u t i o n n-ith alcohol several times and n-ashed repeatedly with 70 per cent alcohol. A G.i5-kg. portion of the pressed alcohol precipitate was dissolved in 38 liters of distilled water. After the pectin had been stirred for 3 hr. and had completely dissolved, the volume of the solution was made up to 50 liters with distilled u-ater. The teniperature was adjusted to the desired point before the final adjustment to yolume. Enough concentrated hydrochloric acid n-a> added to the pectinic acid solution to make the mixture 0.872 S in hydrochloric acid. The mixture vas then placed in a thermodat a t 40"C., and deesterification wa4 allon-ed to proceed for several days. Portions were removed from time to time, and pectinic acids of various degrees of deesterification were recovered in the following manner: About 2 volumes of 80 per cent alcohol n-as added to the reaction mixture, which was then strongly stirred to break up the lumps of gel. To rernoi-e the ash-forming constituents, the alcohol \vas filtered off, and the precipitate was n-ashed repeatedly with 80 per cent alcohol until the filtrate shoxed a negative test for chloride ion. The pectinic acid was then washed with absolute alcohol to facilitate dehydration. After the alcohol n-as pressed out, the pectinic acid Tvas dried for 1 day a t room temperature and then for 1 or 2 days a t G0"C. in a mechanical convection oven. Finally, the dried pectinic acid was ground to pass a 40-mesh screen and analyzed for ash, carboxyl, and methyl ester content. Preparation of enzyme-deesterified pectinic acid Enzyme deesteiification \vas carried out on another portion of the same pressed alcohol precipitate from which the raw material for the acid deesterification had been taken. d 6.75-kg. portion of this precipitate was dissolved in distilled water, and the volume was made up to 5 i liters. X separate 9-kg. portion of thib solution was used for each of the enzyme deesterifications. The solution was placed in a thermostat maintained a t 40°C. and nas carefully adjusted to pH 6.0, after which the enzyme catalyst (tomato pectase) \vas added. -%s the deesterification proceeded, 2 S sodium hydroxide \\as added slowly and with rapid ,tiiring to maintain the pH at G.O. Xhen the desired degree of deesterification 1 i a 5 attained, the reaction was stopped by immediately lon-ering the pH to about 3.0,
332
I?. S P E I S E R , C . H. HILLS, A S D C. R . EDDY
thereby inactivating the enzyme. The pectase was then destroyed by heating the solution to 65°C. for 20 min., after ivhich the product was purified as above.
Preparation of the enzyme catalyst The pectase enzyme n-as extracted from firm, ripe tomatoeq. The tomatoes were ground to a pulp; sufficient alkali \vas added to adjust the pH to 6.0; and then the juice n-as expressed from the pulp. Suspended material and pigment were removed by decantation and filtration. The clear, yellow solution containing the pectase was then stored at 0°C. under a layer of xylene, added as a preservative. This pectase preparation could be stored under such conditions for several months, although its activity n-as always checked before use. Analysis of the pectinic acid Ash was determined in the usual manner. The methoxyl content was determined by a Zeisel procedure (6) upon samples of pectinic acid which had been treated with water vapor at low pressures to remove all adsorbed alcohol. This precaution is necessary to avoid erroneous results in the analysis for methoxyl content (13). C'alcdations S,the total concentration of carboxyl groups in equi1Talents per liter, was determined from the titration curve of pH versus volume of base added a t constant pectinic acid concentration. -1pH of 7 . 5 vas accepted as the point of equivalence, since complete titration curves have shown this to be the inflection point for concentrations used in this study. Theoretical considerations show that the point of true equivalence occurs at a pH some\yhat higher than that of the inflection point, but the pH of this true equivalence point is a complicated function of S and of the dissociation constants, and the error in using the inflection point i b not large. (COO-), the concentration of dissociated carboxyl groups, was calculated from the relation (COO-)
=
(U-)+ (HT) - (OH-)
in accordance \vith the requirement of electrical neutrality of the solution. (B+) represents the concentration of base in equivalents per liter, corrected for the alkalinity of the ash associated with the pectinic acid. (H+) and (OH-) represent the concentrations of hydrogen and hydroxyl ions, respectively, calculated from the measured pH values on the assumption that activity coefficients are unity. a , the degree of dissociation, was calculated as the ratio of (COO-) to -V. G, the titration constant, \vas calculated from its defining equation
pG is defined as the negative logarithm of G, by analogy with pH.
ACID B E H l T I O R OF PECTIR'IC ACIDS
333
r, the ratio of the number of carboxyl groups in the system to the total number of galacturonide residues, v a s calculated from the relation : r = COOHCOOH + COOCHa
-
N/c
+
hr/ c CH30/3100
nhere c is the concentration of pectinic acid in grams per liter, CH30 is the methoxyl content as customarily expressed in per cent by weight of solid, and 31 is the molecular iveight oi the CH30 residue. The ratio F is the significant quantity in comparing and characterizing pectinic acids of different ester contents and neutralization equivalentq, because r is independent of estraneous materials, such as araban and galactan, which do not contribute directly to the electrolytic behavior but afiect the ester content and neutralization equivalent calculated from the total weight of material. dccii, acy
Estimates of the probable accuracy of the calculated 1-alues reveal that the error in cr is of the order of il per cent and that the error in pG varies, lvith the degree oi neutralization, from f 0 . 0 2 pG unit for lo^ values of a: to ~ 0 . 0 3at a = 0.9, and increases rapidly nithout limit as a: increases from 0.9 to 1.0. Hence, the conc1uAons n-e have dranm are based entirely on the region of a: leb- than 0.9. The importance of errors inherent in the method of calculating pG vems t o have been overlooked by most of the workers dealing n-ith dissociation phenoniena. It qhould be emphasized that the mathematical nature of the culculationb causes small, fixed errors to introduce spuriouq trends in G, \\hich can be of serious magnitude for high values of a and lead to erroneous conclusions about the nature cf the dissociating system. It is therefore esbential to evaluate these errors, RESULTS
Titration data hare been obtained on a nide variety of different pectinic acids of various ester contents, both acid- and enzynie-deesterified. Representative examples are given in tables 2, 3, 4, and 5 and in figure 3, which s h o the ~ variation of pH nith degree of neutralization. Table 3 and figure 4 give a typical example of the effect of concentration. For comparison, table G and figure 3 also shon- the behavior of d-galacturonic acid, Tvhich can be considered the monomer on which the pectinic acid main chain is based. From these figures it is seen that the behavior of pectinic acid le-emble.: qualitatively that of a monobasic acid, in that it has only a single buffel iange. The resemblance is only superficial, however, as can be seen by compari?on TI itli the curve for galacturoiiic acid in figure 3 and with the t n o theoretical cuii-eq in figure 3 calculated fioin the monobasic acid equation
334
R. S P E I S E R , C . H. HILLS, A S D C. R. EDDY
TABLE 2 T i t r a t i o n data f o r acid-dedsterified pectiiiic acid Sample H91B: CHBO content, 6.80 per cent; r = 0.56; ash content, 0.24 per cent; ash alkalinity, 0.034 milliequivalent per gram of pectinic acid. Concentration = 1.94 g. per liter; S = 5.19 X 10-3 equivalents per liter; temperature = 27°C. ~
,
1
0.233 0,258 0.289 0.328 0.377 0.435 0.499
1 1 1
~
P
2.937 3.059 3.196 3.359 3.532 3.695 3.879
I
I
,
H
~
I
3.455 3.522 3.587 3.671 3.751 3.808 3.881
,
1,
1;
'1 '1 I/ 11
i
I
0.566 0.637 0.710 0.860 0.935 0.991 0.995 0.997
I
~
1
I I
P
PH
G ~
4.116 4.280 4.486 4.941 5.545 6.596 6 990 7.220
PG
I
~
4.001 4.036 4.098 4.196 4.390 4.563 4.657 4.108
TABLE 3 T i t r a t i o n d a t a f o r acid-deesterified pectiiiic acid Sample H91E: CH30 content = 1.70 per cent; r = 0.90; ash content = 0.53 per cent; ash alkalinity = 0.064 milliequivalent per gram of pectinic acid
concentration = 1.00 g. per liter; S = 4.15 X 10-3 equivalents per liter; temperature = 27°C. 0 204 0 23i 0.282 0 342 0 419
1
I
3 105 3 280 3 503 3.778 4 038
1
3 695 3 787 3.909 4.063 4 180
'I
I,
0 594 0 780 0 874 0.969 0 988
1
1
4 571 5 145 5.523 6 411 6 980
~
I i
4.405 4.594 4.679 4 908 5.045
Concentration = 2.00 g. per liter; S = 8.36 X 10-3 equivalents per liter; temperature = 27°C. 0.172 0.212 0.259 0.328 0.411
i
I
1
2.882 3.071 3.349 3.642 3.894
~
I
1 I
3.563 3.641 3.806 3.955 4.052
I 1 ,
'I
I,
1
0.683 0.776 0.870 0.964 0.988
I
~
I I
'I ,
I
2.772 2 897 3 021 3 160
1
4.280 4.359 4.486 4.759 5.034
11
3 481 1
I j I
equivalents per liter;
Concentration = 4.00 g. per liter; = 16.82 X temperature = 27°C. 0.164 0.186 0 215 0 247
4.613 4.899 5.310 6.188 6.951
3 538 3 584 3 643
0 499 0 9Oi I,
0 969
1 I
3.932 5 221 6.351
3.934 4.160 4.858 1
-
The heha1 ior of pectinic acid more closely resembles that of citric acid, saccharic acid, and other polybasic acids whose buffer ranges are so close together that they overlap and produce only one composite buffer range, as illustrated in figure 5 .
335
h C I D BEHAVIOR O F P E C T I S I C ACIDS
-4s shoxn in figure 4, an increase in concentration increases the degree of dissociation for a given value of pH. This is typical of the behavior which we hare found for apple pectinic acids of high and low ester content, deesterified by either acid or enzyme treatment. The same effect has been observed by Hinton (15)for orange, currant, and strawberry pectinic acids, and also by Kern (21) for polyncrylic acid and Briggs (4) for arabic acid. In contrast, &galacturonic acid, TABLE 4 T i t r a t i o n data f o r enzyme-deesterijed pectinic acid Sample H91H: CH3O content = 4.48 per cent; r = 0.67; ash content = 0.55 per cent; ash alka!inity = 0.089 milliequivalent per gram of pectinic acid. Concentration = 1.98 g. per liter; N = 5.64 X 10-3 equivalents per liter; temperature = 27°C.
0.248 0.280 0.319 0.365 0.60,C 0.744 0.883
I
I
PH
PG
3.085 3.221 3.376 3.555 4.298 4.690 5.269
3.568 3.632 3.707 3.796 4.107 4.227 4.391
~1 1' I
1,
1
0.953 0.989 0.994 0.996 0.997 0.999
I 1
I
5.973 6.650 7.144 7.479 6.910 7.787
i 1
1 ~
~
4.661 4.710 4.901 5.110 4.364 4.939
I1
TABLE 5 T i t r a t i o n data f o r enzyme-deesterijled pectinic acid Sample FIOlJ: C " 3 0 content = 2.04 per cent; r = 0.86; ash content = 1.01 per cent; ash allialinity = 0.184 milliequivalent per gram of pectinic acid. Concentration = 1.83 g. per liter; S = 6.98 X equivalents per liter; temperature = 27°C. PH
a
PG
I
0.216 0.243 0.275 0.315 0.361 0.410 0.464 0.517
, I I 1
I
3.111 3.250 3.409 3.585 3.741 3.861 4.013 4.159
I
i
I
I
3.671 3.745 3.830 3.922 3.990 4.039 4.079 4.129
I
~1
,
1
~
1 1
'~I I
PH
0 569 0 619 0.682 0.736 0.849 0.906 0 962
I
4 298 4 456 4.604 4 757
5.158 5.449 5.938
~
I I
PG
4.178 4 235 4.274 4 311
I
4.408
I
4.467 4 536
I
like all monobasic acids, s h o w no change in a as concentration is changed at constant pH. The effect of ester content, a- can be seen from figure 3, is to decrease the degree of dissociation Trith increasing r at a given concentration and pH. Unexpectedly. bringing the carboxyls closer together in solution by increasing the concentration causes a to increase, whereas bringing the carboxyls closer together by increasing the number of carboxyls per unit length of chain causes a to decrease. From measurements on a large number of pectinic acids with widely different
336
R. SPEISER,
7
C . H. HILLS, .'SD
I q/ I
I
r
6-
I N
C . R. EDDY
I
DE E ST E RI F I CAT Io N
OH918
056
194
519xlO-'
ACID
w
090
200
835
ACID
X H ~ I H 067
1 9 8
564
ENZYME
OH91J
183
698
ENZYME (Points Only)
213
II 32
H91E
C
086
d-GALACTURONIC ACID d-GALACTURONIC
0
ACID,
CONCENTRATIONS
5-
POINTS
AT
SEVERAL
F R O M I TO 2 0 G/L
I
4-
3-
-
0
02
06
04
IO
08
TABLE 6 Titration data for d-galacturonic acid a-d-Galacturonic acid, recrystallized three times :ash content,0.161 per cent; ash alkalinity, 0.018 milliequivalent per gram of acid; equilibrium rotation, [CY]; = 51.7"". Cancentration = 2.13 g. per liter; S = 11.32 X 10-3 equivalents per liter: temperature = 27°C.
____ a
0.176 0.197 0.212 0.234 0.259 0.287 lverage pli
*
I
I
1 ~
, , =
PH
,
2.749 2.798 2.864 2.019 2.969 3.014 3.419 1 0 . 0 0 9 (I
