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An opportunity for simultaneous purification and deesterification of pectic substances
Food Hydrocolloids Vol.? no.2 pp.103-112, 1993
An opportunity for simultaneous purification and deesterification of pectic substances Mariana I.Popova, Christo G.Kratchanov and Ivan N.Panchev Department of Organic Chemistry, Higher Institute of Food and Flavor Industry, 26 Maritsa Blvd, Plovdiv 4000, Bulgaria Abstract. The possibility of simultaneous purification and deesterification of apple pectin under conditions of acid chromatographic elution in a column filled with the pectic substances subjected to purification was investigated. Elution was done with diluted hydrochloric solutions of ethanol and isopropanol and the effect of temperature, acid concentration and process duration was studied. Two types of commercial pectins with different degrees of esterification were used. The dependence between purity, degree of esterification (DE) and the gel strength (GS) of the purified pectic preparations was investigated. It was shown that the gel strength increases with increasing purity, but only to a finite value of DE. The GS maximum as a function of DE depends on the type of initial pectin. The process of deesterification was studied kinetically and the rate constants were calculated on the basis of an equation describing reactions of the first order. The results obtained indicate that deesterification takes place faster in an isopropanol medium than in an ethanol medium for both types of pectin. The rate constants of deesterification also depend on the initial degree of esterification, deesterification taking place more quickly with highly esterified pectins than with moderately esterified ones.
Introduction It is well known that pectic substances find a vast application as gel-forming thickening and stabilizing agents in the food industry (1,2). They can also be used as detoxicants in cases of heavy metal poisoning (3,4) and as adsorbents and ion exchangers (5-9). Degree of esterification (DE) and purity are determining factors for the application of pectic substances in these fields. In previous investigations (10) we have shown how commercial pectins can be purified by washing them in a column with hydrochloric organic solvents at room temperature (25°C). It has been observed that along with increasing the purity of the pectin, its DE decreases, i.e. deesterification in a heterogeneous medium takes place. The acid-catalyzed deesterification of pectic substances is carried out under industrial conditions both in a homogeneous and heterogeneous medium in order to obtain medium and low esterified pectins (1,11-13). Pectin deesterification taking place in a heterogeneous medium has been studied kinetically (13,14) and the rate constants within the 30-40°C temperature range have been determined. Changes in the gelling properties of pectin have also been observed with acid deesterification (15,16). It has been established that by decreasing the DE from 75 to 50-55% the gel strength (GS) increases and then rapidly decreases. Obviously the changes in the pectin GS depend on the DE. It can be concluded that the ratio of free and esterified carboxyl groups has a very significant effect. It is possible that their equivalent ratio provides for a better arrangement of the macromolecules in the gel favoring a better pectin GS. The decrease of the GS is due above all to depolymerization of the pectin macromolecule as a result of acid
103
M.I.Popova, C.G.Kratchanov and I.N.Panchev
hydrolysis of the glycoside bonds. It is accepted that by decreasing the amount of ballast substances the gel strength is also increased. The aim of the present work is to study the process of purification and acid deesterification of apple pectin under dynamic conditions, and to establish the effect of purification conditions (temperature, HCl concentration, process duration and solvent polarity) on the quality of pectin preparations. The process took place in a column. Pectin played the role of an immobile phase, while purification and deesterification were achieved by passing diluted hydrochloric ethanol and isopropanol through the column. Materials and methods
Materials and reagents
Two types of apple pectin were used with DE 74.3 and 61.7% (produced by the Pectin state company). The organic solvents (ethanol and isopropanol) and the hydrochloric acid were of a p.a. grade. Experimental methods
The pectin preparations were prepared for washing as previously described (10). We used 40% ethanol and isopropanol containing HCl (2.2 and 3.1%) and thermostated columns with diameter 14 x 10- 3 m and height = 0.5 m at temperatures of 25, 40 and 50°C. The pectin played the role of an immobile phase and diluted hydrochloric ethanol and isopropanol were used as eluents. The effect of the HCl concentration (2.2 and 3.1%) on the processes of purification and deesterification taking place under these conditions was studied kinetically for 48 h. The loss of substance as a result of elution was controlled by weight in all the experiments. It was found to be commensurable with the decrease in the ballast substances of pectin determined during the analysis of the washed pectin and expressed by an increase in its purity. The absolute loss of pectic substance in the experiments was usually within the 1-3% range, but in rare cases reached 6%. The f-h swelling of the pectin preparations was carried out at the same temperature at which the elution was done. Method of analysis
The DE and the purity (anhydrouronic acid content) of the pectin preparations were determined by the neutralization method with phenol red as the indicator (17). The gel strength was determined by the Tan-Baker method using standard 65% sugar jellies (18), at the optimal pH for the respective DE (the optimal pH for pectin gelling decreased with decreasing DE). The ash content was determined by weight after incinerating the pectin sample. The IR spectra of the pectin preparations were done on UR-20 apparatus, (Karl Zeiss, lena, FRG) KBr in tablets. The rate constants of deesterification were determined using the equation for a first order reaction: 104
Table I. Purification of highly esterified apple pectin with hydrochloric ethanol
Temperature ("C) Time indices (h)
2.2% HCI in 40% ethanol 40°C 0 4 8
12
24
48
DE(%) Purity (%) Ash content (%) GS"TB
74.3 58.1 3.16 178
59.8 78.2 0.23 220
58.4 82.7 0.12 215
46.8 85.6 0.10 182
DE(%) Purity (%) Ash content (%) GsoTB
3.1% HCI in 40% ethanol 74.3 65.2 58.7 75.4 58.1 68.0 0.40 0.38 3.16 210 178 208
70.3 62.1 0.45 190
63.4 76.0 0.38 210
50°C 4
8
12
24
48
61.4 64.0 0.37 180
58.9 70.6 0.28 225
49.5 75.5 0.13 200
46.1 82.7 0.13 200
41.4 85.0 0.06 163
--
'"3' 0:
S' ::s
'"0:0
'" 'C 53.4 76.5 0.22 194
50.3 83.2 0.20 180
44.3 84.8 0.12 160
59.0 67.0 0.30 206
56.6 73.0 0.27 199
48.5 76.5 0.21 178
42.8 83.2 0.12 165
37.9 86.9 0.10 161
0:
:l.
,.,
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a
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'"
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ee
::s
...,
0
'C I-'
0
U1
a::s'" '"
M.I.Popova, C.G.Kratchanov and I.N.Panchev
(1)
where (DE)o is the degree of esterification at the initial moment of reaction t = and k is the rate constant. The rate constants were calculated using the least squares method (19) in accordance with the experimental measurements of DE as a function of time. The modified LIN programme described by Johnson (20) was used for this purpose, and the adequacy check for equation (1) was done in accordance with Fisher's statistical criterion (19).
o and t represents time
Results and discussion Initially experiments with highly esterified apple pectin were carried out. The results obtained are presented in Table 1. A parallel increase in the purity and GS of the pectin to the specified DE value «58-59%) was observed, after which the GS decreased and the purity continued to increase at a slower rate. It is to be noted that with these experiments the rate of change in the GS depended significantly neither on the temperature nor on the acid concentration. These factors primarily influenced the rate of deesterification. The change in the GS of the highly esterified pectin as a function of DE as presented in Figure 1 is interesting to note. It can be seen that after the maximum (59%) the GS slowly decreased to -49-52% DE after which it dropped more sharply. The observed
230
200 190 180 170 f6 150 80
60
50
1,0
JO
J)£%
Fig. 1. Purification of highly esterified apple pectin with hydrochloric ethanol: • 40°C, 2.2% HCI; x SO°C, 2.2% HCI; 6. 40°C, 3.1% HCI; • SO°C, 3.1% HC!.
106
Simultaneous purification and deesterification of pectins
phenomenon could be explained taking into account the fact that with acid treatment not only deesterification but also pectin depolymerization is catalyzed (16). The second reaction undoubtedly resulted in decreasing the GS. Therefore it should be accepted that deesterification resulted in increasing the GS. It would be useful to establish for what DE values the pectin molecule would have the highest GS if deesterification took place without its parallel process of depolymerization. This will be the subject of a future investigation. The results obtained from our investigations with highly esterified pectin (HEP) led us to carry out analogous investigations with another batch of commercial pectin different in its DE, purity and GS parameters. Table II presents the results from the laboratory experiments carried out with medium esterified pectin (MEP). Here as well pectin deesterification was fastest at the beginning of the treatment after which the changes were more gradual. The GS of the purified pectin preparations had maximum DE values of 55-53% after which it dropped and the GS value of the initial pectin was repeated at DE 50%. The observation that deesterification was speeded up with an increase in temperature was confirmed. Figure 2 presents GS as a function of DE. It can be seen that the maximum for MEP was shifted in the direction of a lower DE compared to that for HEP. This indicates that the effect of the deesterification process on the GS depended on the initial DE value. With a view to clarifying the effect of the type of alcohol on the rate of deesterification of pectin and on its gelling properties, a number of experiments were carried out using diluted hydrochloric isopropanol as the eluent under Table II. Purification of medium esterified apple pectin with hydrochloric 40% ethanol Duration indices (h)
O'
6
12
24
48
50T; 2.2% HCI DE (%) Purity (%) Ash content (%) GsoTB
61.7 67.5 2.0 174.0
53.3 82.3 0.37 200.0
51.5 83.5 0.33 175.0
47.5 87.5 0.30 167.0
42.7 88.0 0.28 154.0
25°C; 3.1% HC] DE (%) Purity (%) Ash content (%) GsoTB
61.7 67.5 2.0 174.0
56.9 77.8 0.33 190.0
54.7 78.0 0.20 186.0
53.8 77.7 0.20 181.0
51.6 77.8 0.20 172.0
40°C; 3.1% HCI DE(%) Purity (%) Ash content (%) GSo-rB
61.7 67.5 2.0 174.0
55.9 70.5 0.38 179.0
50.3 77.8 0.24 173.0
48.1 78.1 0.20 168.0
44.1 79.8 0.07 149.0
50°C; 3.1% HCl DE (%) Purity (%) Ash content (%) GsoTB
61.7 67.5 2.0 174.0
52.8 76.0 0.38 183.0
47.9 80.6 0.28 169.0
46.2 82.1 0.20 159.0
42.3 88.3 0.20 151.0
• Control values.
107
M.I.Popova, C.G.Kratchanov and I.N.Panchev
GS Or, 210 200 190 18 110 160
15°80
io])£%
"0
70
Fig. 2. Purification of medium esterified apple pectin with hydrochloric ethanol: • 25°C, 3.1% HCl; x 50°C, 2.2% HCI; 6. 40°C, 3.1% HCl;. 50°C, 3.1% HC!.
Table III. Purification of medium esterified apple pectin with hydrochloric 40% isopropanol Duration indices (h)
O'
6
12
24
48
50°C; 2.2% HCl DE(%) Purity (%) Ash content (%) GS"TB
61.7 67.5 2.0 174.0
50.0 85.2 0.40 213.0
43.4 87.0 0.40 170.0
38.1 90.8 0.37 145.0
33.1 97.0 0.30 134.0
25°C; 3.1% HCl DE(%) Purity (%) Ash content (%) GsoTB
61.7 67.5 2.0 174.0
55.2 82.6 0.20 178.0
53.7 83.0 0.17 186.0
52.0 84.3 0.17 188.0
49.0 84.6 0.13 188.0
40°C; 3.1% HCl DE(%) Purity (%) Ash content (%) GS"TB
61.7 67.5 2.0 174.0
55.2 77.0 0.90 182.0
47.9 82.6 0.25 178.0
42.1 88.1 0.20 148.0
32.4 88.9 0.13 132.0
50°C; 3.1% HCl DE (%) Purity (%) Ash content (%) GS"TB
61.7 67.5 2.0 174.0
49.5 82.5 0.30 191.0
43.2 83.4 0.27 160.0
37.9 88.2 0.20 139.0
31.7 95.0 0.20 129.0
* Control values.
108
Simultaneous purification and deesterification of pectins
GS TB 230 220 2~0
200
NO f80
/TO 160 150
Ho 130 120
80
70
60
50
"0
.10
])E%
Fig. 3. Purification of medium esterified apple pectin with hydrochloric isopropanol: x 50°C, 2.2% HCl;. 25°C, 3.1% HCI; 6. 40°C, 3.1% HCI;. 50°C, 3.1% HCl.
analogous conditions. The results obtained (presented in Table III) indicate that in this case deesterification took place more quickly and the GS changed more slowly. Obviously depolymerization in a medium of isopropanol was slower than with ethanol, other conditions being comparable. This also had an effect on the dependence of the GS on the DE (graphically represented in Figure 3). In this case the maximum value of GS was observed for 50% DE which should be attributed to the slowed-down depolymerization under these conditions. Comparing the graphically presented dependence of the GS on the DE (Figures 1-3), it can be seen that the pectin GS depended strongly on DE and the maximum of this function was influenced by the depolymerization taking place simultaneously. Under the conditions of suppressing the latter, this maximum would obviously be for a DE below 50%. This conclusion brought about two suggestions: (i) the availability of free carboxyl groups over 50% was necessary for non-ion gels as well; (ii) when suitable deesterification conditions were found (i.e. strong suppression of depolymerization) low esterified pectins (i.e. low methoxyl pectin) with a very good GS could be obtained. In this sense the favorable effect of isopropanol should be taken into account. 109
M.I.Popova, C.G.Kratchanov and I.N.Panchev
It was interesting to check whether pectin purification and deesterification under the adopted conditions were accompanied by essential structural changes. That is why IR spectra of the initial pectin and the purified pectin preparations were done in KBr tablets. Some of the obtained spectra are presented in Figure 4. The data indicate that there were differences only in the ratio of the intensity of free and esterified carboxyl group signals (1700-1750 crn"}. No other
2
1
Fig. 4. IR spectra of chromatographically purified pectin with 2.2% HCl in 40% ethanol as the eluent at a temperature of 50"C with various durations of elution: 1,4 h; 2,8 h ; 3, 24 h; 4, 48 h.
110
Simultaneous purification and deesterification of pectins
essential differences were observed either in the samples from one and the same series or in the samples treated with different organic solvents. The kinetic study of the deesterification process according to equation (1) enabled us to calculate the rate constants of the reaction. The results are presented in Table IV. The data indicate that the rate constants depended on the initial degree of esterification. The reaction took place much faster with both solvents for initial HEP compared to the deesterification of MEP. The type of solvent also had a strong effect on the rate of the process. The rate constants obtained for deesterification in an isopropanol medium were considerably higher than those obtained for deesterification in an ethanol medium. This was probably due to differences in the solvation of the two solvents in relation to the pectin macromolecules. Conclusions
1. It has been shown that a simultaneous purification and deesterification of pectic substances through chromatographic elution with diluted hydrochloric solutions is possible. 2. The interdependence of GS and DE in acid deesterification of commercial samples of apple pectin has been studied. It has been observed that at the beginning deesterification results in an increase in GS, the position of the maximum being dependent on the type (DE) of initial pectin and the de esterification conditions. 3. The rate constants of pectin deesterification increase with increasing acid concentration and temperature. It has been established that the rates of deesterification also depend on the initial degree of esterification of the Table IV. Rate constants of acid deesterification of apple pectin No. of expo
Temperature eC)
Eluent
Apple pectin, DE = 74.3% 1 25 2 40 3 50 4 40 5 50 6 40 7 50
3.1 3.1 3.1 2.2 2.2 3.1 3.1
HCI HCI HCI HCI HCI HCI HCI
in in in in in in in
40% 40% 40% 40% 40% 40% 40%
ethanol ethanol ethanol ethanol ethanol isopropanol isopropanol
1.11 5.45 8.75 4.65 8.63 7.70 11.81
Apple pectin, DE = 61.7% 8 25 9 40 10 50 11 25 12 50 13 25 14 40 15 50 16 25 17 50
3.1 3.1 3.1 2.2 2.2 3.1 3.1 3.1 2.2 2.2
HCI HCI HCI HCl HCI HCI HCI HCI HCI HCI
in in in in in in in in in in
40% 40% 40% 40% 40% 40% 40% 40% 40% 40%
ethanol ethanol ethanol ethanol ethanol isopropanol isopropanol isopropanol isopropanol isopropanol
0.83 1.77 1.87 0.50 1.66 1.11 3.61 3.82 0.94 3.28
± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ±
0.03 0.15 0.35 0.13 0.34 0.29 0.49
0.17 0.25 0.33 0.13 0.33 0.19 0.19 0.44 0.15 0.23
111
M.I.Popova, C.G.Kratchanov and I.N.Panchev
pectin and the type of solvent. The reaction takes place at a faster rate with HEP compared to MEP. The accelerating action of isopropanol in comparison with ethanol concerning deesterification is connected with the better solvation capacity of isopropanol in relation to the pectin macromolecules. References 1. Pilnik.W., Voragen,A.G. (1970) In Hulme,A.C. (ed.), Biochemistry of Fruits, Their Products I. Academic Press, London, p. 53. 2. Be Miller,J.N. (1986) Chemistry and Function of Pectins, ACS Symposium Series No. 310, pp. 2-12. 3. Kohn,R., Tibensky,V. (1971) Coli. Czech. Chem. Comm., 36, 92. 4. Stantchev .St., Kratchanov ,Chr., Popova.M., Kirtschev.N. and Martschev.M. (1979) Z. ges Hyg. H., 8, 285. 5. Kohn,R. and Furda,I. (1968) Acta Chem. Scand., 22, 3098. 6. Kratchanov.Chr. and Popova.M. (1968) J. Chromatogr., 37, 297. 7. Kohn,R. and Furda,I. (1967) Call. Chem. Comm., 32, 4470. 8. Kratchanov.Chr. and Popova.M, (1982) J. Chromatogr., 24, 197. 9. Ivanov,K., Popova,M., Kratchanov.Chr. and Maneva.D. (1990) Lebensm. Unters. Forscli., 191, 210. 10. Kratchanov.Chr. and Popova.M. (1990) 1. Chromatogr., 509, 239. 11. Doesburg,J.J. (1965) Pectic Substances in Fresh and Preserved Fruits and Vegetables, JBVT Comm 25, Wageningen, The Netherlands. 12. Spciser.R., Eddy,C.R. and Hills,H. (1945) J. Phys. Chem., 49, 563. 13. Kratchanov.Chr. and Balakireva.B. (1972) Tr. Sci. Inst. Technol. Super., Plovdiv, 19 (Ill), 81. 14. Kratchanov,Chr. (1982) Lebensm. Ind., 29, 174. 15. Smith,C.J.B. and Bryant,E.F. (1968) 1. Food Sci., 33, 262. 16. Kirtchev,N., Pantchev.I. and Kratchanov.Chr. (1989) Intern. 1. Food Sci. Technol., 24, 479. 17. Owens,H.S., Comb,R.M., Shepherd,A.D., Shultz,T.H., Pippen,E.L., Swenson,H.A., Miers, LC.. Erlandsen,R.T. and Maclay,W.D. (1952) Methods Used at Western Regional Research Laboratory, Agricultural Industrial Chemistry Report, No. 340. Western Regional Research Laboratory, Albany, CA, pp. 1-24. 18. Bender,W.A. (1949) Analyt. Chem., 21, 408-411. 19. Bzandt.S. (1970) Statistical and Computational Methods in Data Analysis. New York, London, pp. 146, 228. 20. Johnson,K.J. (1983) Numerical Methods in Chemistry. Marcel Dekker, Inc, New York and Basel, p. 252.
Received on 7 August 1992; accepted on 11 January 1993
112
An opportunity for simultaneous purification and deesterification of pectic substances Mariana I.Popova, Christo G.Kratchanov and Ivan N.Panchev Department of Organic Chemistry, Higher Institute of Food and Flavor Industry, 26 Maritsa Blvd, Plovdiv 4000, Bulgaria Abstract. The possibility of simultaneous purification and deesterification of apple pectin under conditions of acid chromatographic elution in a column filled with the pectic substances subjected to purification was investigated. Elution was done with diluted hydrochloric solutions of ethanol and isopropanol and the effect of temperature, acid concentration and process duration was studied. Two types of commercial pectins with different degrees of esterification were used. The dependence between purity, degree of esterification (DE) and the gel strength (GS) of the purified pectic preparations was investigated. It was shown that the gel strength increases with increasing purity, but only to a finite value of DE. The GS maximum as a function of DE depends on the type of initial pectin. The process of deesterification was studied kinetically and the rate constants were calculated on the basis of an equation describing reactions of the first order. The results obtained indicate that deesterification takes place faster in an isopropanol medium than in an ethanol medium for both types of pectin. The rate constants of deesterification also depend on the initial degree of esterification, deesterification taking place more quickly with highly esterified pectins than with moderately esterified ones.
Introduction It is well known that pectic substances find a vast application as gel-forming thickening and stabilizing agents in the food industry (1,2). They can also be used as detoxicants in cases of heavy metal poisoning (3,4) and as adsorbents and ion exchangers (5-9). Degree of esterification (DE) and purity are determining factors for the application of pectic substances in these fields. In previous investigations (10) we have shown how commercial pectins can be purified by washing them in a column with hydrochloric organic solvents at room temperature (25°C). It has been observed that along with increasing the purity of the pectin, its DE decreases, i.e. deesterification in a heterogeneous medium takes place. The acid-catalyzed deesterification of pectic substances is carried out under industrial conditions both in a homogeneous and heterogeneous medium in order to obtain medium and low esterified pectins (1,11-13). Pectin deesterification taking place in a heterogeneous medium has been studied kinetically (13,14) and the rate constants within the 30-40°C temperature range have been determined. Changes in the gelling properties of pectin have also been observed with acid deesterification (15,16). It has been established that by decreasing the DE from 75 to 50-55% the gel strength (GS) increases and then rapidly decreases. Obviously the changes in the pectin GS depend on the DE. It can be concluded that the ratio of free and esterified carboxyl groups has a very significant effect. It is possible that their equivalent ratio provides for a better arrangement of the macromolecules in the gel favoring a better pectin GS. The decrease of the GS is due above all to depolymerization of the pectin macromolecule as a result of acid
103
M.I.Popova, C.G.Kratchanov and I.N.Panchev
hydrolysis of the glycoside bonds. It is accepted that by decreasing the amount of ballast substances the gel strength is also increased. The aim of the present work is to study the process of purification and acid deesterification of apple pectin under dynamic conditions, and to establish the effect of purification conditions (temperature, HCl concentration, process duration and solvent polarity) on the quality of pectin preparations. The process took place in a column. Pectin played the role of an immobile phase, while purification and deesterification were achieved by passing diluted hydrochloric ethanol and isopropanol through the column. Materials and methods
Materials and reagents
Two types of apple pectin were used with DE 74.3 and 61.7% (produced by the Pectin state company). The organic solvents (ethanol and isopropanol) and the hydrochloric acid were of a p.a. grade. Experimental methods
The pectin preparations were prepared for washing as previously described (10). We used 40% ethanol and isopropanol containing HCl (2.2 and 3.1%) and thermostated columns with diameter 14 x 10- 3 m and height = 0.5 m at temperatures of 25, 40 and 50°C. The pectin played the role of an immobile phase and diluted hydrochloric ethanol and isopropanol were used as eluents. The effect of the HCl concentration (2.2 and 3.1%) on the processes of purification and deesterification taking place under these conditions was studied kinetically for 48 h. The loss of substance as a result of elution was controlled by weight in all the experiments. It was found to be commensurable with the decrease in the ballast substances of pectin determined during the analysis of the washed pectin and expressed by an increase in its purity. The absolute loss of pectic substance in the experiments was usually within the 1-3% range, but in rare cases reached 6%. The f-h swelling of the pectin preparations was carried out at the same temperature at which the elution was done. Method of analysis
The DE and the purity (anhydrouronic acid content) of the pectin preparations were determined by the neutralization method with phenol red as the indicator (17). The gel strength was determined by the Tan-Baker method using standard 65% sugar jellies (18), at the optimal pH for the respective DE (the optimal pH for pectin gelling decreased with decreasing DE). The ash content was determined by weight after incinerating the pectin sample. The IR spectra of the pectin preparations were done on UR-20 apparatus, (Karl Zeiss, lena, FRG) KBr in tablets. The rate constants of deesterification were determined using the equation for a first order reaction: 104
Table I. Purification of highly esterified apple pectin with hydrochloric ethanol
Temperature ("C) Time indices (h)
2.2% HCI in 40% ethanol 40°C 0 4 8
12
24
48
DE(%) Purity (%) Ash content (%) GS"TB
74.3 58.1 3.16 178
59.8 78.2 0.23 220
58.4 82.7 0.12 215
46.8 85.6 0.10 182
DE(%) Purity (%) Ash content (%) GsoTB
3.1% HCI in 40% ethanol 74.3 65.2 58.7 75.4 58.1 68.0 0.40 0.38 3.16 210 178 208
70.3 62.1 0.45 190
63.4 76.0 0.38 210
50°C 4
8
12
24
48
61.4 64.0 0.37 180
58.9 70.6 0.28 225
49.5 75.5 0.13 200
46.1 82.7 0.13 200
41.4 85.0 0.06 163
--
'"3' 0:
S' ::s
'"0:0
'" 'C 53.4 76.5 0.22 194
50.3 83.2 0.20 180
44.3 84.8 0.12 160
59.0 67.0 0.30 206
56.6 73.0 0.27 199
48.5 76.5 0.21 178
42.8 83.2 0.12 165
37.9 86.9 0.10 161
0:
:l.
,.,
::l
a
o'
::s co ::s
Q. Q.
'"
'" '" ;;... s,., co
ee
::s
...,
0
'C I-'
0
U1
a::s'" '"
M.I.Popova, C.G.Kratchanov and I.N.Panchev
(1)
where (DE)o is the degree of esterification at the initial moment of reaction t = and k is the rate constant. The rate constants were calculated using the least squares method (19) in accordance with the experimental measurements of DE as a function of time. The modified LIN programme described by Johnson (20) was used for this purpose, and the adequacy check for equation (1) was done in accordance with Fisher's statistical criterion (19).
o and t represents time
Results and discussion Initially experiments with highly esterified apple pectin were carried out. The results obtained are presented in Table 1. A parallel increase in the purity and GS of the pectin to the specified DE value «58-59%) was observed, after which the GS decreased and the purity continued to increase at a slower rate. It is to be noted that with these experiments the rate of change in the GS depended significantly neither on the temperature nor on the acid concentration. These factors primarily influenced the rate of deesterification. The change in the GS of the highly esterified pectin as a function of DE as presented in Figure 1 is interesting to note. It can be seen that after the maximum (59%) the GS slowly decreased to -49-52% DE after which it dropped more sharply. The observed
230
200 190 180 170 f6 150 80
60
50
1,0
JO
J)£%
Fig. 1. Purification of highly esterified apple pectin with hydrochloric ethanol: • 40°C, 2.2% HCI; x SO°C, 2.2% HCI; 6. 40°C, 3.1% HCI; • SO°C, 3.1% HC!.
106
Simultaneous purification and deesterification of pectins
phenomenon could be explained taking into account the fact that with acid treatment not only deesterification but also pectin depolymerization is catalyzed (16). The second reaction undoubtedly resulted in decreasing the GS. Therefore it should be accepted that deesterification resulted in increasing the GS. It would be useful to establish for what DE values the pectin molecule would have the highest GS if deesterification took place without its parallel process of depolymerization. This will be the subject of a future investigation. The results obtained from our investigations with highly esterified pectin (HEP) led us to carry out analogous investigations with another batch of commercial pectin different in its DE, purity and GS parameters. Table II presents the results from the laboratory experiments carried out with medium esterified pectin (MEP). Here as well pectin deesterification was fastest at the beginning of the treatment after which the changes were more gradual. The GS of the purified pectin preparations had maximum DE values of 55-53% after which it dropped and the GS value of the initial pectin was repeated at DE 50%. The observation that deesterification was speeded up with an increase in temperature was confirmed. Figure 2 presents GS as a function of DE. It can be seen that the maximum for MEP was shifted in the direction of a lower DE compared to that for HEP. This indicates that the effect of the deesterification process on the GS depended on the initial DE value. With a view to clarifying the effect of the type of alcohol on the rate of deesterification of pectin and on its gelling properties, a number of experiments were carried out using diluted hydrochloric isopropanol as the eluent under Table II. Purification of medium esterified apple pectin with hydrochloric 40% ethanol Duration indices (h)
O'
6
12
24
48
50T; 2.2% HCI DE (%) Purity (%) Ash content (%) GsoTB
61.7 67.5 2.0 174.0
53.3 82.3 0.37 200.0
51.5 83.5 0.33 175.0
47.5 87.5 0.30 167.0
42.7 88.0 0.28 154.0
25°C; 3.1% HC] DE (%) Purity (%) Ash content (%) GsoTB
61.7 67.5 2.0 174.0
56.9 77.8 0.33 190.0
54.7 78.0 0.20 186.0
53.8 77.7 0.20 181.0
51.6 77.8 0.20 172.0
40°C; 3.1% HCI DE(%) Purity (%) Ash content (%) GSo-rB
61.7 67.5 2.0 174.0
55.9 70.5 0.38 179.0
50.3 77.8 0.24 173.0
48.1 78.1 0.20 168.0
44.1 79.8 0.07 149.0
50°C; 3.1% HCl DE (%) Purity (%) Ash content (%) GsoTB
61.7 67.5 2.0 174.0
52.8 76.0 0.38 183.0
47.9 80.6 0.28 169.0
46.2 82.1 0.20 159.0
42.3 88.3 0.20 151.0
• Control values.
107
M.I.Popova, C.G.Kratchanov and I.N.Panchev
GS Or, 210 200 190 18 110 160
15°80
io])£%
"0
70
Fig. 2. Purification of medium esterified apple pectin with hydrochloric ethanol: • 25°C, 3.1% HCl; x 50°C, 2.2% HCI; 6. 40°C, 3.1% HCl;. 50°C, 3.1% HC!.
Table III. Purification of medium esterified apple pectin with hydrochloric 40% isopropanol Duration indices (h)
O'
6
12
24
48
50°C; 2.2% HCl DE(%) Purity (%) Ash content (%) GS"TB
61.7 67.5 2.0 174.0
50.0 85.2 0.40 213.0
43.4 87.0 0.40 170.0
38.1 90.8 0.37 145.0
33.1 97.0 0.30 134.0
25°C; 3.1% HCl DE(%) Purity (%) Ash content (%) GsoTB
61.7 67.5 2.0 174.0
55.2 82.6 0.20 178.0
53.7 83.0 0.17 186.0
52.0 84.3 0.17 188.0
49.0 84.6 0.13 188.0
40°C; 3.1% HCl DE(%) Purity (%) Ash content (%) GS"TB
61.7 67.5 2.0 174.0
55.2 77.0 0.90 182.0
47.9 82.6 0.25 178.0
42.1 88.1 0.20 148.0
32.4 88.9 0.13 132.0
50°C; 3.1% HCl DE (%) Purity (%) Ash content (%) GS"TB
61.7 67.5 2.0 174.0
49.5 82.5 0.30 191.0
43.2 83.4 0.27 160.0
37.9 88.2 0.20 139.0
31.7 95.0 0.20 129.0
* Control values.
108
Simultaneous purification and deesterification of pectins
GS TB 230 220 2~0
200
NO f80
/TO 160 150
Ho 130 120
80
70
60
50
"0
.10
])E%
Fig. 3. Purification of medium esterified apple pectin with hydrochloric isopropanol: x 50°C, 2.2% HCl;. 25°C, 3.1% HCI; 6. 40°C, 3.1% HCI;. 50°C, 3.1% HCl.
analogous conditions. The results obtained (presented in Table III) indicate that in this case deesterification took place more quickly and the GS changed more slowly. Obviously depolymerization in a medium of isopropanol was slower than with ethanol, other conditions being comparable. This also had an effect on the dependence of the GS on the DE (graphically represented in Figure 3). In this case the maximum value of GS was observed for 50% DE which should be attributed to the slowed-down depolymerization under these conditions. Comparing the graphically presented dependence of the GS on the DE (Figures 1-3), it can be seen that the pectin GS depended strongly on DE and the maximum of this function was influenced by the depolymerization taking place simultaneously. Under the conditions of suppressing the latter, this maximum would obviously be for a DE below 50%. This conclusion brought about two suggestions: (i) the availability of free carboxyl groups over 50% was necessary for non-ion gels as well; (ii) when suitable deesterification conditions were found (i.e. strong suppression of depolymerization) low esterified pectins (i.e. low methoxyl pectin) with a very good GS could be obtained. In this sense the favorable effect of isopropanol should be taken into account. 109
M.I.Popova, C.G.Kratchanov and I.N.Panchev
It was interesting to check whether pectin purification and deesterification under the adopted conditions were accompanied by essential structural changes. That is why IR spectra of the initial pectin and the purified pectin preparations were done in KBr tablets. Some of the obtained spectra are presented in Figure 4. The data indicate that there were differences only in the ratio of the intensity of free and esterified carboxyl group signals (1700-1750 crn"}. No other
2
1
Fig. 4. IR spectra of chromatographically purified pectin with 2.2% HCl in 40% ethanol as the eluent at a temperature of 50"C with various durations of elution: 1,4 h; 2,8 h ; 3, 24 h; 4, 48 h.
110
Simultaneous purification and deesterification of pectins
essential differences were observed either in the samples from one and the same series or in the samples treated with different organic solvents. The kinetic study of the deesterification process according to equation (1) enabled us to calculate the rate constants of the reaction. The results are presented in Table IV. The data indicate that the rate constants depended on the initial degree of esterification. The reaction took place much faster with both solvents for initial HEP compared to the deesterification of MEP. The type of solvent also had a strong effect on the rate of the process. The rate constants obtained for deesterification in an isopropanol medium were considerably higher than those obtained for deesterification in an ethanol medium. This was probably due to differences in the solvation of the two solvents in relation to the pectin macromolecules. Conclusions
1. It has been shown that a simultaneous purification and deesterification of pectic substances through chromatographic elution with diluted hydrochloric solutions is possible. 2. The interdependence of GS and DE in acid deesterification of commercial samples of apple pectin has been studied. It has been observed that at the beginning deesterification results in an increase in GS, the position of the maximum being dependent on the type (DE) of initial pectin and the de esterification conditions. 3. The rate constants of pectin deesterification increase with increasing acid concentration and temperature. It has been established that the rates of deesterification also depend on the initial degree of esterification of the Table IV. Rate constants of acid deesterification of apple pectin No. of expo
Temperature eC)
Eluent
Apple pectin, DE = 74.3% 1 25 2 40 3 50 4 40 5 50 6 40 7 50
3.1 3.1 3.1 2.2 2.2 3.1 3.1
HCI HCI HCI HCI HCI HCI HCI
in in in in in in in
40% 40% 40% 40% 40% 40% 40%
ethanol ethanol ethanol ethanol ethanol isopropanol isopropanol
1.11 5.45 8.75 4.65 8.63 7.70 11.81
Apple pectin, DE = 61.7% 8 25 9 40 10 50 11 25 12 50 13 25 14 40 15 50 16 25 17 50
3.1 3.1 3.1 2.2 2.2 3.1 3.1 3.1 2.2 2.2
HCI HCI HCI HCl HCI HCI HCI HCI HCI HCI
in in in in in in in in in in
40% 40% 40% 40% 40% 40% 40% 40% 40% 40%
ethanol ethanol ethanol ethanol ethanol isopropanol isopropanol isopropanol isopropanol isopropanol
0.83 1.77 1.87 0.50 1.66 1.11 3.61 3.82 0.94 3.28
± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ±
0.03 0.15 0.35 0.13 0.34 0.29 0.49
0.17 0.25 0.33 0.13 0.33 0.19 0.19 0.44 0.15 0.23
111
M.I.Popova, C.G.Kratchanov and I.N.Panchev
pectin and the type of solvent. The reaction takes place at a faster rate with HEP compared to MEP. The accelerating action of isopropanol in comparison with ethanol concerning deesterification is connected with the better solvation capacity of isopropanol in relation to the pectin macromolecules. References 1. Pilnik.W., Voragen,A.G. (1970) In Hulme,A.C. (ed.), Biochemistry of Fruits, Their Products I. Academic Press, London, p. 53. 2. Be Miller,J.N. (1986) Chemistry and Function of Pectins, ACS Symposium Series No. 310, pp. 2-12. 3. Kohn,R., Tibensky,V. (1971) Coli. Czech. Chem. Comm., 36, 92. 4. Stantchev .St., Kratchanov ,Chr., Popova.M., Kirtschev.N. and Martschev.M. (1979) Z. ges Hyg. H., 8, 285. 5. Kohn,R. and Furda,I. (1968) Acta Chem. Scand., 22, 3098. 6. Kratchanov.Chr. and Popova.M. (1968) J. Chromatogr., 37, 297. 7. Kohn,R. and Furda,I. (1967) Call. Chem. Comm., 32, 4470. 8. Kratchanov.Chr. and Popova.M, (1982) J. Chromatogr., 24, 197. 9. Ivanov,K., Popova,M., Kratchanov.Chr. and Maneva.D. (1990) Lebensm. Unters. Forscli., 191, 210. 10. Kratchanov.Chr. and Popova.M. (1990) 1. Chromatogr., 509, 239. 11. Doesburg,J.J. (1965) Pectic Substances in Fresh and Preserved Fruits and Vegetables, JBVT Comm 25, Wageningen, The Netherlands. 12. Spciser.R., Eddy,C.R. and Hills,H. (1945) J. Phys. Chem., 49, 563. 13. Kratchanov.Chr. and Balakireva.B. (1972) Tr. Sci. Inst. Technol. Super., Plovdiv, 19 (Ill), 81. 14. Kratchanov,Chr. (1982) Lebensm. Ind., 29, 174. 15. Smith,C.J.B. and Bryant,E.F. (1968) 1. Food Sci., 33, 262. 16. Kirtchev,N., Pantchev.I. and Kratchanov.Chr. (1989) Intern. 1. Food Sci. Technol., 24, 479. 17. Owens,H.S., Comb,R.M., Shepherd,A.D., Shultz,T.H., Pippen,E.L., Swenson,H.A., Miers, LC.. Erlandsen,R.T. and Maclay,W.D. (1952) Methods Used at Western Regional Research Laboratory, Agricultural Industrial Chemistry Report, No. 340. Western Regional Research Laboratory, Albany, CA, pp. 1-24. 18. Bender,W.A. (1949) Analyt. Chem., 21, 408-411. 19. Bzandt.S. (1970) Statistical and Computational Methods in Data Analysis. New York, London, pp. 146, 228. 20. Johnson,K.J. (1983) Numerical Methods in Chemistry. Marcel Dekker, Inc, New York and Basel, p. 252.
Received on 7 August 1992; accepted on 11 January 1993
112