T H E HYDRATION AYD PHYSICOCHEMICSL PROPERTIES OF PECTIN AND ITS DERIVATIVES REINHOLD F. STUEWER Department of Chemistry, Stanford Unzversity, California Received July 16, 1937
Although the properties and structure of pectin have been under investigation for many years, no agreement as to the complete structure has yet been reached. It is, however, usually conceded that a polygalacturonic acid is contained in the pectin molecule. According to Ehrlich (2), four galacturonic acid molecules, two of wliich are methylated, constitute the fundamental unit. Other views are those of Nanji, Paton, and Ling (8), who look upon pectin as having a ring structure containing four methylated galacturonic acid molecules and one each of galactose and arabinose. Morell, Baur, and Link (7) have concluded that at least eight or ten acid residues are contained in the pectic acid molecule. If the viem of Nanji, Paton, and Ling be accepted, the normally acidic nature of pectin sols does not follow unless one assumes partial demethoxylation to have occurred in the preparation of the pectin. Spencer (11) explains this acid condition as being due to preferential adsorption of anions. However, since the methods used in preparation of pectins are such that deniethoxylation takes place, it is permissible to assume that all pectins uqed in the making of jellies may be acidic of themselves. While the degree of hydration of pectin is usually considered to be one of the factors involved in its jelling, no previous attempts to measure it directly h a w been made, although qualitative results have been reported (4). In this work, measurements of the hydration in alcohol suspensions were carried out. I t ha. long been k n o m that the presmce of salts is important in the jelling of pthctin. Halliday and Bailey ( G ) showed that the effect of calcium chloride is such that the amount of sugar required to induce jelling is decreased. On the other hand, Spencer (11) finds that in the presencp of acids sodium chloride decreases the jelling ability. It is generally agreed that the cation (4) is of more importance in these salt effects, although definite anion effects may be observed under propu conditions ( 5 ) . Whereas some workers explain the behavior of pectin in the presence of salts as due to sorption effects only (ll),others prefer the hypothesis that exchange of cations between the pectin molecule and the salt is involved 305 THE JOURNAL OF PXYRICALCHEYISTRY, VOL. 42, N O . 3
306
REINHOLD F. STUEWER
(5). Gliickmann (5) has prepared and investigated the properties of pectins containing a number of anions in varying proportions. In the present work an investigation has been made of the acidic character of pectin and of its hydration and sorption characteristics in the acidic and several salt forms.' An apple pectin was used. The acidic form was prepared by thoroughly washing the pectin with a 50 per cent alcohol solution containing hydrochloric acid. This treatment removed practically all of the metallic ions. The pectinic acid described is a product of low-temperature acid hydrblysis of pectin and the pectic acid a product of alkaline hydrolysis of pectin. The salt forms were prepared by the neutralization of the acid with the proper base in the presence of buffer salts containing the same cation. These buffer salts were then washed out by thorough extraction with 50 per cent alcohol. The combining weights of the three acids were found to be 848,425, and 207, respectively, for vacuum-dried pectin, pectinic acid, and pectic acid. EXPERIMENTAL
The conductance of pectic materials and their salts Solutions of the materials were prepared by weighing out the proper amounts and dispersing them in conductivity water. Lower concentrations were prepared by dilution. The sodium salts used in these experiments were prepared by addition of the proper amount of sodium hydroxide to the acidic sol. The conductivities of acidic pectin and pectinic acid are given in table 1. The conductivities of the sodium salts of the two materials are given in table 2. The observed values of pH mere obtained by means of a glass electrode. The calculated values were obtained by computation from the data of tables 1 and 2 and the ionic conductances of sodium and hydrogen. The calculation of the dissociation constant was made on the assumption that the same value obtains for all hydrogens, and, while not constant, it does give an indication of the acid strength of pectin and a pectinic acid. It is interesting to note that the values for the two materials are almost identical. The conductances of the magnesium salts of the two materials and the calcium salt of the pectin were also determined. These values are given in table 3. The data are of interest in that dissociation is far from complete even at extremely low concentrations, and may probably be looked upon as explaining, at least in part, the differences noted in the past in the effects of the various cations on the jelling of pectin. 1 For nomenclature see 9.\T, Thomas: Colloid Chemistry, p. 371, McGraw-Hill Book Co , S e w York (1931).
307
HYDRATION OF PECTIN
TABLE 1 Conductivities o j acidic pectis and pectinic acid QWWALEN1 CONDECTIVITY
NORMALITY
BUBBTANCE
I
Pectin, , , , , . , . . , . , , , , , . . . . . . .
0.00825 0.00330 0.00132 0.000528 0.0001056
-PH
Calou-
lated
Ob-
served
DWOCIATION
CONSTANT x 104
.___
4.79
2.85 3.10 3.38 3.71 4.31
2.9 2.3 1.9 1.2 0.46
2.81 3.10 3.40 3.60 4.30
2.81 3.08 3.38 3.71 4.30
2.9 2.4 2.0 0.94 0.34
64.1 89.3 118.7 138.8 177.8
2.87 3.13 3.43
61.7 83.9 105.0 123.3 154.4
TABLE 2 Conductivities of sodium pectin and sodium pectinate EQUIVALENT CONDUCTIVITY NORMALITY
I
Sodium pectin
0.0100 0.0040 0.0016 0.00064 0.000128 0 ,000256 0.000041 0. oooo256
59.9 65.1 69.2 72.8 82.1
Sodium pectinate
62.2 67.1 70.9 75.3 77.5 78.0
73.7
TABLE 3 Conductances of magnesium pectin, magnesium pectinate, and calcium pectin MAQNESIUM PECTIN NORMALITY
Conductivity
0.0100 0.0020 0.0004 0.00008
38.4 46.1 52.4 60.5
I &:$:& 49 59 67 77
MAQNESIUM P E C l l N A T l
Conductivity
29.1 33.3 39.2 42.2
I
CAICIEM PECTIN
Conductivity
36 42 49 53
36.7 45.7 51.4 56.9
1&
~ ~ ~ & 43 54 60 67
The conclusions from conductivity data were further substantiated by precision measurements2 of the lowering of freezing point made by the
* All these measurements were made by Dr. Kurt H. Andresen, to whom my best thanks are due.
308
REINHOLD F. STUEWER
method of Scatchard (lo), and, for the less dilute solutions, by nicasurements2of the lowering of vapor pressure made by the method of Robinson and Sinclair (9). The results were as given in table 4,where g (the osmotic coefficient) = 1 - j = e/vXm, where v is taken for the purpose of thio calculation as 2 for sodium pectinate and a? 3 for magnesium pectinate. It will be noted that the results from conductivity arid the osmotic data are in close agreement. Hydration of pectic wmterials Experiments with the ultrafiltration method with methyl alcohol and also with sodium chloride as reference substance showed that pcctin is hydrated to the extent of only about one-third of its weight of water. The experiments here described were made as follows, 71 ithout ultrafiltration. Thoroughly dried materials were immersed in alcohol solutions of TABLE 4 Osmotic coeficient of sodium pectinate and magnesium pectinate solutions MAONEBIUM PECTINATE SOLUTIOSS
SODIUM PECTINATE BOLUTIONS
Normality
0.00088 0.00163 0.00241 0.00318 0.00392 0.0108
I ~
1
, ,'
i ~
osmotic coefficient
0.9699 0.9281 0.8901 0.8572
,~
' 0.8273 0.7683*
Osmotic coefficient
Normality
0
0.00417 0.00630
~
0.00981 0.01209
'
I
0
0.4318 0.3902 0.3233* 0.3267*
-__
I____
* By lowering of vapor pressure; all others by freezing point. known concentration and left in contact for about twenty-four hours. Samples of the supernatant liquid were then withdrawn and analyzed for alcohol content. This analysis mas made by carefully distilling and then determining the alcohol content by means of a dipping refractometer. I n cases where the concentration of the alcohol used was legs than 20 per cent, an interferometer was used in analysis. Table 5 gives the data thus obtained. Hydrations are given in per cent of the dry weight of the material. The form of the curves in figure 1 indicates that' extrapolation to zero alcohol concentration is justified. Such extrapolation leads to the following values for the Yarious materials in pure aqueous solution: Pectin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pectic a c i d . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sodium pectate . . . . . . . . . . . . Calcium peotate. . . . . . . . . . . . . . . .
25 per cent 21 per cent 38 per cent 35 per cent
hydration hydration hydration hydration
309
HYDRATION OF PECTIN
The tendency for pectin to disperse in solutions of alcohol of low concentration prevents their use with pectin and its salts; however, the simiTABLE 5 Hydration of pectic materials I
WEIGHT OF SOLUTION
~~~~~~L
BUBRTANCE
CONCENTRAMATERIAL
19.81 34 42 42 07 51 12 58 08 62 42 72 33 82 35 92 52
ilcidic pectin. . . . . . . . . . . . . . . . . . . 18 9 12 6
39 81 03 42 5 55 5 23 1
1
10 0 10 0
Pectic a c i d . . . . . . . . . . . . . . . . . . . . . . . . . .
~
41 49 56 60
13 86 77 15 70 10 80 43
Sodium p e c t a t e . . . . . . . . . . . . . . . . . . . . . . ..i ~
1 Calcium pectate. . . . . . . . . . . . . . . . . . . . . . .
, ~
0 3 0 0 4
1
1
96 79 74 52 43 24 11
41 33 47 47 78 31 33 5 141 1.206
42 14 23 31 10 77 4 939 1 162
1 76.61
10.0 20.0 21.0 10.0
!
10.0 10.0 10.0 10.0 10 0 10.1 10.0 10.0 10.2
95.09 76.61 71.66 51.94 41.79 1 41.79 I 23.31 1 10.77 1 4.939
10.2
1.162
41.79 32.52 23.31
I I I
95 22 95 09 76 61
;: : I1 ; ::
10 20 10 15 14
1
18 3 23 1 18.8 24 8 25 4 23 3 17 1 12 3 2 1
I ~
, ~
6 8 17 1 18 9 17 0 19 0 20.9 20 3 21 0 21.2
80.03 43.29 33.66 25.14
21.4 35.0 35 .O 36.4
96.26 80.03 74.91 54.76 44.38 44.14 24.96 11.50 5.375 1.273
5.9 21.4 22.6 25.7 29.1 26.7 32.6 32.7 34.0 37.0
I
~
1
~
'
1 1
310
REINHOLD F. STUEWER
same hydration value for hydration of acidic pectin as the control in which water was used. Table 6 summarizes the data for a number of materials. In this case all hydration values are for approximately 50 per cent alcohol, and so do not represent the hydrations at zero concentration of the reference substance. I n general the pectates appear to be slightly less hydrated than the salts of pectin or of pectinic acid. Also, the acids are in all cases definitely less hydrated than are the salts, and the calcium salts are less hydrated than either the sodium or the magnesium salts, the latter being hydrated to the 40
-
$ 1
":v;u,' w
11
,
[2:
a
f
100
80
60
ZO
40
ALCOHOL CONCENTRATION IN PER CENT
FIG.1. Hydration of pectin and its derivatives.
0 , pectin; 0 , pectic acid;
X, sodium pectate; 0 , calcium pectate.
TABLE 6 Hydration of the acids and their salts in 60 per cent alcohol PER CENT EPDRATlON OF
Acid
Sodium salt
Magnnm
25
33 34 33
:t
_-pI/-
Pectin. .............................. Pectinic acid. ......................... Pectic a c i d . . ..........................
26 19
33
1
Caloium salt
30 27
311
HYDRATION OF PECTIN
the supernatant liquid were then made to determine the amount of sorption which had taken place. Necessarily, conditions used were at all times such that dispersion of the material did not take place to any appreciable extent. The nature of these materials under such conditions of nondispersion must be similar to that of permutites. They are, therefore, subject to so-called exchange adsorption, which is merely the exchange of cations between the salt present and the colloidal material. Molecular sorption may also take place, and it is this type of sorption with which the following experiments are concerned. Specifically, the effect of various cations in both the sorbent and sorbate was investigated.
TABLE 7 ReEative sorptive powers of pectic materials for potassium iodate I BUBBTANCE
1
BOLUTION UBED
30 per cent alcohol
I
CATION
Potassium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ ........... Magnesium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
...........................
1
’”&~~$” 1
Water
Normality of KIOi
1
0.04 N
IODATE
0.01 N IODATE
2.4 3.9
35.8 9.1
I “,E”,”,,”
!
2.0
8.4
8.6
The data in table 7 were obtained by keeping the materials in contact with the solutions for eleven days with constant shaking. In all cases 14 g. of solution per gram of pectin wsts used. The iodate concentrations given are initial concentrations, and sorptive powers are expressed as the percentage of the iodate sorbed, without allowance for hydration. A similar series of experiments was run in which each pectic material was allowed to remain in contact with the corresponding iodate in 30 per cent alcohol solution for a week with constant shaking. The results were as given in table 8. It is apparent from the data in tables 7 and 8 that the effects of the ions on adsorption of iodate are quite marked. The potassium and sodium salts do not sorb nearly as much of the potassium iodate as do the acid and
312
REINHOLD F. STUEWER
the magnesium salt. The sorption is much greater from sodium chloride than from water solution when pectic acid is the sorbent, but it is not much different when magnesium pectate is used. This a t first sight appears to be in disagreement with the fact that hydrogen ions and magnesium ions are about equally effective in sorption from alcohol solution, but it is explainable on the basis of greater displacement of magnesium than of hydrogen from the pectic molecule by the relatively ineffective sodium ion. Actually such a difference exists even in the sorption from alcohol solution, and the magnesium ion is considerably more effective in promoting sorption than is the hydrogen ion, as shown by table 8, where sorbent and sorTABLE 9 T h e e$ect of total electrolyte concentration o n the progress of adsorption PER CENT SORPTION FROM WATER SOLUTIONS
1
I
PER CENT SORPTION PROM
10N
Final KIOa
Sorption
Initial KIOa
Final KIOa
per cent
per cent
per cent
per cent
per cent
0.0687 0.137 0.279 0.591 1.208 1.991 4.485
0.0685 0.140 0.283 0.588 1.183
0.0591 0.261 1.044 4.427
0.0481 0.234 0.960 4.103
Initial KIOs
,
SODIUM CHLORIDE
-I
Sorption per.cent
18.6 10.4 8.1 7.3
2.1 3.5 4.6
TABLE 10 Rate of sorption of potassium iodate o n magnesium pectate WATER BOLUTIONS
1.0 N SODIUM CHLORIDE SOLUTIONS
bate contain the same cation. That the exchange of magnesiumand sodium does not take place t o a very great extent, however, follows from the fact that magnesium pectate is insoluble in water. The greater acid strength of iodic acid than of pectic acid precludes any very great exchange of potassium and hydrogen in the case of potassium iodate and pectic acid. The effect of total electrolyte concentration on the progress of adsorption is shown in the data in table 9, which were obtained in experiments in which overnight contact between solution and sorbent was allowed. The marked difference at low concentrations is certainly to be attributed to electrical effects, and in the above data is due both t o a difference in the manner in which sorption progresses and to the lack of equilibrium
313
HYDRATION O F P E C T I N
at the low concentrations. It can be shown that in the presence of small electrolyte concentrations an initial negative sorption takes place, which later becomes positive in many cases, whereas in the presence of a greater amount of electrolyte to suppress the Donnan effect, the sorption is at once positive. The data in table 10 serve t o illustrate this point. Sorption is greater at high than at low concentrations in the case of sodium pectate sorbing iodate from alcohol solutions (see table 7 ) . In general, the sorption from alcohol solutions is abnormal in that it does not follow the adsorption isotherm, and the per cent sorption may be greater at high than a t low concentrations. Since pectin itself is readily dispersible, an extensive comparison of its sorptive properties with those of pectic acid could not be carried out; however, a comparison of sorption from alcohol solutions was made. TABLE 11 Sorption of potassium iodate f r o m 40 per cent alcohol solution
1
PER CENT OF IODATE SORBED
Initial K I O ~concentration 0.467 per cent; 3 day8 shaking
SORBENT
1 1
Initial K I O ~con. centration 0.058 per cent; 7 days shaking
Pectin., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pectinic a c i d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pectic a c i d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45.2 29.7 32.7
19 .o 28.7 15.6
Sodium p e c t i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sodium pectinate. ................................ Sodium pectate.. .................................
Dispersed 10.9 9.2
Dispersed 10.5 3.1
Magnesium pectin.. .............................. Magnesium pectinate. ............................ Magnesium pectate ...............................
25.9
27.0 39.6 45.6
37.3 47.2
One may conclude that qualitatively the same effects of cations are to be found with pectin and pectinic acid and their salts as with pectic acid. Insufficient data are obtainable to determine the quantitative relationships precisely, but the data do show that the effects vary in magnitude with the various materials under the conditions of this experiment. GENERAL DISCUSSION
The conductivity data reported substantiate the views held by those who consider pectin to be acidic of itself, and are in agreement with the facts as regards the effect of acidity in pectin jellies. These data show pectin to be a weak acid, but very definitely stronger than acetic acid. The contention of Gliickmann to the effect that the acidic pectin is freed
314
REINHOLD F. STUEWER
from its salts in pectin jellies is justified, although in practice jellies are not always of sufficiently low pH t o cause complete conversion. The fact that the sodium salt is highly dissociated, the salts of divalent metals are considerably less dissociated, and the acid still less is in agreement with the reported facts (11,6,3) that sodium salts hinder jelling while calcium chloride promotes it and that acids are particularly effective in causing jelling in the presence of an organic substance such as sucrose. The data on hydration show that, as Spencer postulates, the addition of acid to pectin decreases the hydration, since the acidic pectin set free is less hydrated than the salt. On the other hand, the postulate that the effect of sugar is one of dehydration of the pectin micelles does not appear to follow, for although a very slight change in hydration occurs with change in alcohol concentration, the great differences which may be obtained in jelling ability with small changes in concentration of the organic precipitant seem not to be accounted for by dehydration. That sorption effects are of only minor importance seems to be indicated by the fact that the effect of acidity is mostly one of pH (12). Differences due to the anion present in the acid have been observed (5, 1). A further indication is the fact that, according to the data here presented, sorption is most marked when hydrogen or a divalent metal is present in the pectin molecule, and almost negligible in the case of monovalent metals. Yet anion adsorption should tend to stabilize the pectin, and so this stabilizing effect is greatest, but still overshadowed, in the presence of cations which promote jelling. In short, pectin is a self-stabilized colloid, its charge resulting from the ionization of the pectic molecule itself. In the form of its highly ionized sodium salt, the charge is great and the stability is accordingly greater than in the case of the less ionized calcium salt, In the acid the ionization is still less and consequently the charge is smaller. An added stabilizing effect is that due to sorption of anions, but this effect appears to be a minor one under ordinary conditions. Its magnitude is dependent upon the anion itself and upon the cations present. The degree of hydration is in the same order as the degree of dissociation in the case of the acid and the calcium and sodium salts; however, the magnesium salt is less dissociated but just as highly hydrated as the sodium salt. The main differences between salts of monovalent and divalent metals are due t o unequal dissociation, and differences between salts of ions of the same valence are probably attributable to differences in hydration which affect stability. SUMMARY
The hydration of pectin and of a number of its derivatives has been measured by the method of a reference substance in water and in aqueous alcohol, in agreement with results by ultrafiltration. It is found that the
HYDRATION OB PECTIN
315
hydrate water amounts to 0.25, 0.21, 0.38, and 0.35 g. of water to 1 g. of pectin, pectic acid, sodium pectate, and calcium pectate, respectively. That of pectinic acid resembles pectin; the three sodium and the three magnesium derivatives are somewhat more hydrated. The sorption of potassium iodate by these materials has been studied. Pectin and pectinic acid are weak acids, definitely stronger than acetic acid. The sodium salts are moderately strong electrolytes, but the magnesium and calcium salts are far from completely dissociated, even in N/10,000 solution. Conclusions are drawn as to the colloid nature of pectin and its derivatives. Our sincere thanks are due to Professor James W. McBain, under whose supervision this work was carried out. REFEREXCES (1) BAKER: Ind. Eng. Chem. 18,89 (1926). Biochem. Z. 212, 162 (1929). (2) EHRLICH: N: 66,64 (1931). (3) G L ~ C K M A NKolloid-Z. (4) GLUCKMANN: Kolloid-Z. 67, 330 (1931). (5) GLUCKMANN: Kolloid-Z. 60,52 (1932). (6) HALLIDAY AND BAILEY:Ind. Eng. Chem. 16, 595 (1924). (7) MORELL,BAUR,AND LINK:J. Biol. Chem. 106, 1 (1934). (8) NANJI, PATON, AND LING:J. SOC.Chem. Ind. 44, 253T (1925). (9) ROBINSON AND SINCLAIR: J. Phys. Chem. 37,495 (1935). (10) SCATCHARD, JONES, AND PRENTISS: J. Am. Chem. SOC.64, 2676 (1932). ‘11) SPENCER: J . Phys. Chem. 33, 1987, 2012 (1929). (12)_,TARR: University of Delaware Agr. Exp. Sta. Bull. 1924, pp. 133, 136.
