Acidic Isolation
of Low-Ester Pectinic Acids R . M . McCready, H . S . Owens, A . D . Shepherd, and W . D . Maclay WESTERN REGIONAL HESEiRCH LABORQTORY, U. S. DEPARTMENT O F AGRICULTURE, ALBANY, CALIF.
I
NCREASED production An acidic method for isolating pectinic acids of low methyl equipment resistant to acid a t in the past few years of ester content which requires neither concentration of 40" C. or higher. The encitrus and apple juices and the pectinic acid solution nor the use of organic precipizymic method, while rapid concentrates and the concomitating agents is shown to be effective. The various factors and easily controlled, has a tant increase in waste maaffecting the precipitability of this type of material are disadvantagein that theprodterials have stimulated invesevaluated and a recommended proredure of isolation is uct requires a critical amount tigations that would utilize presented. of calcium in the preparation these wastes. Production of of lowsolids gels. This may one of the important bybe overcome by use of larger products, pectin, increased from 2 million t o more than 5 million amounts of polyphosphates (D, and probably other calciumpounds per year in the past decade, an appreciable portion of this sequestering agents may be wed to reduce the concentration of increase being due to abnormal requirements resulting from the calcium and thus overcome the disadvantage. war. In order to maintain and expand the production of pectic In this study no products made by use of acidic catalysts have materials, it is necessary to broaden their market by developing been employed, because they are similar to the alkali-prepared products of greater versatility than ordinary pectin or by reducproducts in physical behavior ( 1 7 ) . Samples identified with a B ing the cost of the latter. One such class of products is the lowwere prepared by the action of ammonium hydroxide a t pH 10.8 methoxyl pectins which form gel structures through reaction a t 5" C. (8) on a low-ash citrus pectin which contained 84% uronic with calcium and other polyvalent cations. Methods of preparaanhydride on the moisture- and ash-free basis and 10% methoxyl tion and the versatility of lox-methoxyl pectins for use in the groups. The samples identified with an E were prepared by in low-solids gel field have been widely discussed in recent years situ de-esterification with lemon pectin esterase (11). 911 of the (1, 3, 7 , 8, 11, 15). products were precipitated in aqueous alcohol at pH 1 and washed Isolation of pectins has always been an expensive process. free of chloride ion in aqueous alcohol, dehydrated in alcohol, One of the most widely used methods is vacuum.concentration of then dried in vacuo a t 60" C,. overnight, and ground to pass a 60the clarified extract, followed by precipitation with aqueous alcomesh screen. Degraded samples were prepared by heating a soluhol, dehydration in alcohol, and drying of the isolated pectin in tion of sample 3.3B at pH 3.3 and 97" C., removing samples a t vacuo. An alternative method has been to precipitate with caldefinite time intervals, and cooling rapidly to room temperature. cium or basic aluminum ions and to de-ash the pectin with acidiMETHODS fied aqueous alcohol before drying it. More recent methods have involved the use of copper or nickel ions, removal of these ions The important variables in the acid precipitation process are with ion exchange resins, and spray or drum drying of the conester content, pH, temperature, the intrinsic viscosity used as a measure of chain length, and the concentration, which have not centrate. In some cases drying of a vacuum concentrate without been investigated before. purification of the pectin solutions has been used. ,411 of these Methoxyl content was determined by the saponification prqcesses necessitate the recovery of large quantities of alcohol method of Hinton (4). This method is adequate when short times or removal, by heat in vacuo, of large quantities of water and are of saponification are used (0). Relative viscosities were measured in calibrated Ostwald-Cannon-Fenske pipets at 25' C. rather expensive, either in equipment, energy, or both. ProducThe intrinsic viscosities m r e obtained from the intercept of tion of low-methoxyl pectins or pectic acid by these methods the curves of log vsli/c against c (9,IO). Measurements of pH were would indicate a selling price of at least $1.50 per pound, the curmade with a Bcckman met'er. rent price for 200-grade, high-methoxyl pectin. The properties of The procedure for the acid precipitation was to add ten volumes of pectin solution of known concentration a t pH 4 to one low-methoxyl pectins are sufficiently different from those of highvolume of hydrochloric acid sufficiently concentrated to yield methoxyl pectins so that modification of the older methods or dethe desired pH; this is different from the method used by Paul velopment of new ones is warranted. The method presented in and Grandseigne ( I S ) . The solutions were gently stirred for 5 this paper involves direct precipitation by acid. I t has been studminutes, then allowed t o stand for an additional 15 minutes. The gel was broken by gentle stirring, strained on cheesecloth, ied extensively on a laboratory scale, and the results show that and pressed. The strained liquor was clarified by filtration. appreciable concentration of pectinic acid can be accomplished by Analysis of the filtrate required a rapid and fairly accurate purely mechanical means, such as screening and pressing. The method. The specific rotation of the pectin used is reproducible, advantages are sufficient that engineering studies have been unnot appreciably influenced by pH, and independent of concentration in the range of concentrations used in this investigation. dertaken, and plans for use of the method on pilot-plant scale are The rotations of the original solution and the filtrate were obunder xay. served, and the latter corrected for dilution. The ratio of the difDE-ESTERIFICATION ference between the rotations of the original solution and the filtrate to that of the original multiplied by 100 gives the percentage The starting material for the preparation of low-methoxyl pecof the pectinic acid precipitated. tins may be either the raw material, such as apple pomace or citrus waste, or the pectin extract from it. Any of the three general PRECIPITABILITY catalysts, acid, base, or enzyme (pectin esterase), may be used. INFLUENCE OF DE-ESTERIFICATION. The effect of removal of These catalysts for de-esterification have been compared (8). ester groups on the precipitability of pectin by acid is shown in The results may be briefly summarized as follows: The advanTable I. Data on samples de-esterified by alkali and by citrus tages of the alkaline method are rapid rate, low cost of equipment, pectin esterase are included for comparison. and ease of control. The acidic method is slower and requires 1254
December, 1946
'
INDUSTRIAL AND ENGINEERING CHEMISTRY
As the methoxyl content decreases, the percentage of pectinic acid that can be precipitated increases. With alkali-treated.pectinic acids, the increme is fairly gradual, not reaching 90% precipitation until the methoxyl content is decreased t o about 4%. The esterase-treated samples at a methoxyl content of 7 to 8% show a rather abrupt increase in the amount precipitated. There was some precipitation with sample 7.9E, but the gel particles were so small they passed through the cheesecloth. The precipitation of a maximum of 95% of enzymatically de-esterified material is due to its heterogeneity with respect to methoxyl content. The molecules having high methoxyl contents do not precipitate. These behaviors are not unpredictable. It has been established that hydrogen bonds involving carboxyl groups are stronger than those involving alcohol or water groups (14); therefore, as the number of carboxyl groups are increased, the possibility of intermolecular bonding increases. The production of large 8ggregates with a small charge reduces their stability, and precipitation ensues. Previous data indicate that adjacent carboxyl groups are liberated by the action of the esterase t o give these portions of the chain pectic acidlike qualities (5, 17). These portions of the chains probably function as centers of precipitation in the pres ence of acid. The transition between precipitability and nonprecipitability by acid occurs a t 5 to 6% methoxyl for pectinic acids de-esterified with alkali, and at 7 to 8% for unfractionated, enzymatically de-esterified pectinic acids, which is the same as that found for the transition in viscosity behavior ( 1 7 ) . EFFECTOF pH. Table I1 shows the influence of p H on the precipitation of samples of different methoxyl contents. It appears that, as the methoxy content is decreased from 4.4 to 0, the p H a t which precipitation just occurs is changed very little. It was noted before that, as the p B of solutions of low-methoxy1 pectin is decreased below 4, the viscosity-pH curve goes through a minimum, and a further decrease in pH causes the viscosity to increase (17). This has been interpreted t o indicate submicroscopic aggregation. Further reduction in pH t o about 2.5 apparently permits a sufficient number of cross links to be formed, so that the aggregates become too large t o remain dispersed. At this p H less than 10% of the carboxyl groups are ionized; the repulsive forces between the molecules are thus decreased and the chance for hydrogen bonding between them is enhanced. Changes in hydration of the pectinic acid with pH a p pear to be too small (16) to account for the change in stability. I n this connection it is interesting that the p H a t which highsolids gels can just be formed with low-methoxyl pectins is not very different from that required for acid precipitation (12). Table I1 also shows that change in concentration has little influence on the pH required for precipitation. INFLUENCE OF TEMPERATURE. Although decreased ionization and increased number of carboxyl groups increase the chance for hydrogen bonding and cross linking between pectinic acid molecules, increased thermal agitation tends to break such linkages (Table 111). The differential heat of solution is of the order of 10 kg.-cal. Although i t is an attractive hypothesis t o relate this value to the energy required to break a carboxyl to carboxyl hydrogen bond, 8 kg.-cal. (14), the polar nature of both the solute and solvent invalidate such an assumption. Undoubtedly many types of hydrogen bonds are involved in the formation of these aggregates, and certainly more than one such bond per molecule is required. INFLUENCE OF CHAINLENGTH. There is some suggestion in Table I that a decrease in intrinsic viscosity (a measure of an average molecular weight, 10) decreases the precipitability of pectinic acid by acid. Table IV confirms this finding by showing that, as the intrinsic viscosity decreases from 3.4 to 0.2,the percentage of pectinic acid 3.3B precipitated a t pH 1.3 decreases from 95 to 24%. Although the molecular heterogeneity of pectinic acid precludes more than semiquantitative treatment of the data, it is apparent that a certain chain length is necessary to permit sufficient amount of cross linking to aause precipitation. With a logarithmic dis-
1255
TABLE I. EFFECTOF METHOXYLCONTENTON PRECIPITATION OF PARTLY DE-ESTERIFIED PECTINIC ACIDSFROM 1% SOLUTIONE AT
pH 1.4
Pectinio PrecipitaAcid No.. [VI tion, % Remarks 6.2B 3.5 0 Viscous solution 5.8B 3.5 0 Viscous solution 5.2B 3.4 32 Not easily workable 4.8B 3.3 72 Not easily workable 4.4B 3.3 83 Not easily workable 4.2B 3.3 90 Workable gel 3.7B 3.2 93 Workable gel 3.2B 3.2 95 Workable gel 2.8B 3.2 97 Workable gel 7.93 4.8 Not easily workable 7.OE 85 Workable gel 5.8E s:i 85 Workable gel 4.5E 4.1 85 Workable gel 4.4E 6.0 90 Workable gel 3.5E 4.7 90 Workable gel 2.0E 2.2 90 Workable gel 0.2E 3.9 95 Workable gel 0 Numbers refer to methoxyl content, letters to method of de-esterificstion (B = base; E enzyme).
..
-
TABLE11. EFFECTOF pH AND CONCENTRATION OF PARTLY DE-ESTERIFIED PECTINIC ACIDBON PRECIPITATION FROM ACID SOLUTION Pectinic Acid No. 3.7B
... ... ... ...
[VI 3.2
... ... ... ... ... ... ... ... .... .> ... . . I
... ... ... ... ... ...
4.4B
3:i
2.8B
3.2
0. OB
. . .. . ..
1.9
4'.4E
6.0
... ...
... ... ...
...
... .*. .
I
.
... ...
... ... ... ...
... ... ... ... ... ...
Concn.,
%
PH
2.0
2.8 2.7 2.5 1.9 1.4 2.5 2.2 1.4 0.9 2.6 2.5 2.3 1.3 0.8 2.4 1.3 0.8 2.8 2.6 2.1 1.4 0.9 2.5 2.26 2.0 1.5 1.6 1.5 1.5 3.8 3.2 2.4 1.5
...
... ... ... 1.0 ... ... ... 0.5 .. .. .. ... 1.0 ... i:o
..*
1.0
... ..*
2:o 1.0 0.5 1.0
... .. ..
Pptn.,
% 0
..
77 91 95 0 83 92 93 0
..
79 93 95
..
94 95
..
80 94 96 97 0 82 84 93 94 90 90 0
.. ..
90
Remarks Viscous soln.
Viscous s o h . Very soft gel Firm gel Workable gel Workable gel Very soft gel Workable gel Workable gel Very soft gel Firm gel Workable gel Workable gel Workable sel Viscous s o h . Workable gel Workable gel Workable gel Workable gel Workable gel Workable gel Viscous soln. Soft gel Softgel Workablegel
tribution of molecular weights (19), the value for B is 1.1 (16) in the equation M, = M ~ ~ O . W where M , = number-average molecular weight Mo = molecular weight at maximum of distribution curve Assuming that there is an abrupt transition between the chain length of molecules that precipitate and those that do not, calculations can be made yielding a value fgr the molecular weight which permits precipitation. For the sample having the lowest intrinsic viscosity and a molecular weight of about 7 X los (IO), the molecular weight which just permits precipitation is about 10 X 108. Thgse values are only rough approximations, especially since the precipitation is not an abrupt phenomenon. As a check on these calculations a diffusionexperiment was i u n on the filtrate from sample 3.3B refluxed 4 hours. Shverborn (16) showed that the diffusion constant of pectin does not vary a p preciably with concentration, so a single concentration was used. h) obtained was 12.8 X lO-'sq. cm. The diffusion constant (Do, per second, Using a frictional ratio of 1.8 calculated from the intrinsic viscosity, the molecular weight is 3 X 108. Application of Simhe's equation (18) for the viscous behavior of elongated rods yields values for the molecular weight of pectin which lie between the number- and the weight-average molecular weights (10).
'
INDUSTRIAL AND ENGINEERING CHEMISTRY
1256
Vol. 38, No. 12:
ACKNOWLEDGMENT
TABLE111. EFFECTOF TEMPERATURE ON PRECIPITATION OF PARTLY
Pectinic Acid No.
DE-ESTERIFIED P E C T I K I C ACID FROM 1 y o SOLUTION Temp., Ill1
O
c.
PH
Pptn., %
The authors are indebted to the following members of thie laboratory: W. H. Ward for determination of the diffusion constant and H. A. Swenson for some of the analyses. LITERATURE CITED
(1) Baker, G. L., and Goodwin, M.W., Del. 4gr. Expt. Sta., Bull.. 234, Tech. KO.28 (1941); Bull. 246, Tech. No. 31 (1944). (2) Eastern Regional Research Lab., private communication, 1948. (3) Hills, C. H., White, J. W.,Jr., and Baker, G. L., Proc. Inst. Food Tech., 1942, 47. (4) Hinton, C. L., “Fruit Pectins”, 1st Am. ed., p. 33, New York,. Chemical Pub. Co., 1940. TABLE IV. EFFECTOF INTRISSIC VISCOSITY ON PRECIPITATION(5) Jansen, E. F., and hracDonnel1, L. R., -4ich. Biochem., 8 , {j? (1946). O F PARTLY DE-ESTERIFIED PECTINIC ACID3.3B FROM 1% SOLU(6) Jansen, E. F., TT:aisbrot, S. Fa, and Rietz, E., IND.ESG. CHEM., TION AT pH 1.3 ANAL.ED.,16, 523 (1944). Minutes Heated Minutes Heated (7) Kaufman, C. JT‘., Fehlberg, E. R., and Olsen, A. G., Food I n d m a t 97‘ C. and Pptn., at 97O C. and Pptn.. ’ tries, 14 (12), 57-8, 109 (1942); 15 ( l ) , 58-60 (1943). pH 3.3 Iril % pH 3.3 [VI % (8) McCready, R. M., Owens, H. S., and llaclay, W. D., Ibid., 16, 794, 906 (1944). (9) Owens, H. S., Lotakar, H., Merrill, R. M.,and Peterson, .I., J. Am. Chem. SOC.,66, 1178 (1944). (10) Owens, H. S., Lotakar, H., Schultz, T. H., and Maclay, W. D , , Ibid., 68, 1628 (1946). (11) Owens, H. S., McCready, R. M., and Maclay, W.D., IND.ENO CHEM.,36, 936 (1944). (12) Owens, H. S., and Maclay, W. D., J . Colloid Sci., 1, No. 4, 313, Calculations for the refluxed sample gave a molecular weight of (1946). 7 X lo3. All of these results have the same order of magnitude (13) Paul, R., and Grandseigne, R. H., French Patent 695,204 (Aug. 24, 1929). and suggest that samples with molecular weights much below (14) Pauling, L. C., “Nature of Chemical Bond”, p. 307, Ithaca,. 7 X l o 3 cannot be handled by this technique. Cornell Univ. Press, 1940. The data indicate that precipitation of low-methoxyl pectins (15) Pollari, V. E., Murray, 1%’. G., and Baker, G. L., F m i t Products by acid is favored by pH values below 2, temperatures below J., 25 ( l ) , 6-8 (1945). (16) Siiverborn, S., dissertation, Uppsala, 1945. 25’ C., and a methoxyl content below 4. Considering these (17) Schultz, T. H., Lotakar, H., Owens, H. S., and Maclay, W. I).. data, many samples both of pectin in extracts and in solutions J . Phys. Chem., 49, 564 (1945). prepared from isolated pectin have been de-esterified and precipi(18) Simha, R., Ibid.,44, 25 (1940). tated by the following procedure: (19) Svedberg, T., and Pederson, K. O., “The bltraoentrifuge”, pp349-53, New York, Oxford Univ. Press, 1940.
Fifteen hundred grams of commercial citrus pectin (moisture 7.07,, methoxy19.0yo, uronic anhydride 83y0 corrected for moisture and ash) were dissolved in 30 liters of water a t 25’ C. The solution was cooled to 13” C., followed by the addition of 1200 ml. of ammonia hydroxide (28y0“8) solution. The temperature rose to 15” C., where it was maintained for 3 hours and 20 minutes. The ammoniacal solution was poured into 20 liters of solution containing 800 ml. of concentrated sulfuric acid. The mixture was stirred slowly to break up lumps and ensure complete precipitation of the pectinic acid. The resulting pH was about 1.3. The free liquid was drained from the acid gel by screening in a cloth-lined reel. The drained gel was freed of more solution by pressing in cloth bags in a hydraulic press to a solids content of 307,. Excess acid and ammonium salts were washed from the gel by suspending it in 40 liters of water. After thorough stirring, the wash water was again drained from the gel followed by hydraulic pressing. The procedure was repeated. The press cake was then disintegrated, spread on trays, and dried at 66” C. in vacuo. The dried flakes of pectinic acid were ground in a hammer mill to pass a 100mesh screen, Drying in vacuo is not essential to the process, since tray drying in air up to 80” C. has been used successfully. A yield of dried poffdered pectinic acid of 1275 grams or 95% (corrected for moistureand loss of methoxyl) was obtained. To increase its dispersibility, the material was stirred in an atmosphere of ammonia until a 1% solution gave a pH of about 4.5, This required only about 10 minutes.
Free Evaporation into Air of Water from a Free Horizontal Quiet SurfaceCorrection Attention is called to an error in Equation 11 of the paper by Boelter, Gordon, and Griffin, appearing in INDUSTRIAL AND ENGINEERIXG CHEMISTRY, 38, 596 (1946). In the original paper by Hinchley and Himus [Trans. Inst. Chem. Engrs. (London), 2, 57 (1924)l their equation is given as:
where W = rate of evaporation, kg./sq. m./hr p = pressure, mm. Hg In the notation and units employed by Boelter, Gordon, and Griffin this becomes
e
(Puw- Pvm)1.2
and is in better agreement with the results of Boelter, Gordon, and Griffin’s investigations given in Equation 13 as:
SUMMARY
Low-methoxyl pectins de-esterifird by alkali 01 acid can be precipitated in yields greater than 90% by use of acid below pH 2 a t temperatures below 2.5” C., when the methoxyl content of the pectin is below 4 and the intrinsic viscosity is greater than 2. Enzymatically de-esterified materials can be obtained in 80% yields or above a t methoxyl contents less than 7%. The isolated material can be pressed to a solids content of 30% or more, saving alcohol or heat to that extent This press cake can be iedissolved a t pH values between 4 and 5 to yield a usable concentrate, or it can be dried, ground, and partly neutralized.
= 0.091
e
=
0.067
- P,,)l.z
than Equation 11 as shown. The mistake may have arisen from a printer’s error in a later aper by Himus [Trans. Inst. Chem. Engrs. (London), 7, 166 1929)],where the correction is incorrectly stated as
P
w = 0.02 (P‘
- PJ1.2
which becomes e
=
0.20 (Psw- Py,)1,2
in Boelter, Gordon, and Griffin’s units,
