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Synthesis of chitosan-amino acid conjugates and their use in heavy metal uptake
Synthesis of chitosan-amino acid conjugates and their use in heavy metal uptake Hiroshi Ishii*, M a i k o M i n e g i s h i , B o o n m a L a v i t p i c h a y a w o n g and Tomoyo iitani
Department of Chemical Science and Engineering, Tokyo National College of Technology 1220 Kunugida. Hachiouji, Tokyo 193, Japan Received 15 June 1994; revised 20 August 1994
Chitosan-amino acid conjugates were prepared by coupling amino acid esters to the carboxyl group of glyoxylic acid-substituted chitosan. The removal of heavy metals (Cu, Ni, Co and Mn) was increased by introduction of amino acids to chitosan, especially for Mn. Heavy metals were almost completely removed by chitosan-amino acid conjugates from solutions at an initial concentration of 100 parts per million.
Keywords: chitosan; amino acid; heavy metal uptake
It is now well known that chitosan (deacetylated chitin) has a promising ability to adsorb many kinds of heavy metal ions 1-6. To increase the adsorption capacity and/or selectivity for metal ions, various chemically modified chitosans have been prepared 7-16. Chemical modification was achieved for both amino groups and primary hydroxyl groups. From the point of view of biodegradability, which is related to the structure of the compound, amino acids are suitable for use as substituting groups for modification. In this paper, we report on the preparation of chitosan-amino acid conjugates and their use in heavy metal uptake.
metal(s). The mixture was shaken for 2 days at 30°C and the concentration of the remaining metal ion(s) was measured by SEIKO SAS-722 and SAS-7500 spectrometers. As ions co-existing in the solution affect
OH
OH
NH2
\
Methods Equilibrium adsorption experiments were performed as follows. Highly swollen chitosan beads (1 ml, net dry chitosan weight 0.11 g) were added to 19 ml of an aqueous solution containing 100 parts per million (ppm) of heavy * To whom correspondence should be addressed
0141-8130/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
I~1 /n CH I (CH2)3
\
I~ /m II OH I COOH
reQldue
Experimental Materials Chitosan was purchased from Katakura Chikkarin Co. Ltd and used without any purification. Chitosan beads were prepared as described previously 17. The amino acids used were of the L (laevo) configuration. Esters of amino acids, i.e. Gly-OBzl.TosOH, AlaOBzl-TosOH, Ser-OMe-HC1, Leu-OBzl.TosOH, ProOMe.HCI, Phe-OBzl.TosOH, Asp(OBzl)-OBzl.TosOH and Aib-OMe-HC1, were synthesized by standard methods. Heavy metals were used in the sulfate form.
OH
OH
OH
H2N-CHR-COOR' EEDOJDMF x
N II
/n
x
N
/m
II
CH
CH
I
I
(CH2)3
CONHCHR-COO.'
resldue
OH
OH
-_ N /n II CH I (CH2)3
\
/m Ii CH I CONHCHRCOOH
k
resldue
Figure 1 Synthetic scheme for production of chitosan-amino acid conjugates: R=side chain of amino acid, R'=alcohol component of ester
Int. J. Biol. M a c r o m o l . V o l u m e 1 7 N u m b e r 1 1 9 9 5
21
Heavy metal uptake by modified ch,tosan: t4. Ishfi et al. heavy metal a d s o r p t i o n b e h a v i o u r 17, no buffer was used for the uptake experiment. C o m p e t i t i v e uptake experiments were carried out using solutions c o n t a i n i n g two or three kinds of metal ions (each at a c o n c e n t r a t i o n of 100 ppm).
and glyoxylic acid-substituted chitosan beads. The mixture was stirred for 2 days, and beads were collected and washed successively with m e t h a n o l and distilled water.
Synthesis of glyoxylic acid-substituted chitosan
A m i n o acid ester-substituted chitosan beads were added to a q u e o u s alkaline solution and the mixture was stirred overnight. Beads were collected and washed. Th e co n t en t of the a m i n o acid i n t r o d u c e d was determined by b a c k - t i t r a t i o n of the c a r b o x y l g r o u p using N a O H and HC1. F o r all a m i n o acids used, the degree of substitution was ~ 0.4.
Saponification of chitosan- amino acid esters
A q u e o u s g l u t a r a l d e h y d e solution (3.8%; 600 ml) and a q u e o u s glyoxylic acid solution (16%; 600ml) were simultaneously added dropwise at r o o m t e m p e r a t u r e to 300 ml of swollen chitosan beads. The mixture was stirred overnight, and red c o l o u r e d beads were collected and washed with distilled water. T h e synthetic scheme is shown in Figure 1.
Results and discussion
Coupling amino acid esters to glyoxylic acid-substituted chitosan
Uptake./i'om solutions containing a single metal ion Table 1 lists the heavy metal u p t a k e results at metal ion
T h e benzyl or methyl ester of a m i n o acids, ' ethoxycarbonyl-2-ethoxy- 1,2-dihidoroquinoline (EEDQ), and N - m e t h y l m o r p h o l i n e (two equivalents each per carboxyl g r o u p of the chemically modified chitosan beads) were added to a m i x t u r e of d i m e t h y l f o r m a m i d e
c o n c e n t r a t i o n s of 100 ppm. Th e o r d er of extent of r e m o v a l by n o n - m o d i f i e d chitosan was C u > Ni > C o > Mn. This result could be explained by the relative o r de r of metal o r g an i c chelate b o n d strength, i.e. the Irving Williams series. The extent of r e m o v a l by the glyoxylic acid-substituted chitosan was lower than that by nonmodified chitosan except for Mn. This is due to a decrease in the n u m b e r of free a m i n o groups. It is p r o b a b l e that the carboxyl function could not en h an ce the chelating ability of the modified chitosan. All metal ions (Cu, Ni, C o and Mn) were r e m o v e d almost completely by the chitosan a m i n o acid c o n j u g a t e under the conditions investigated. The extent of r e m o v a l was not influenced by the type of a m i n o acid introduced, indicating that metal ions interact with the b a c k b o n e of the a m i n o acid residue and chitosan molecule.
Extent of removal (%) of heavy metals by chitosan and modified chitosan beads for single-ion solutions Table I
Adsorbent Chitosan Glyoxylic acid-chitosan Gly-chitosan Ala-chitosan Ser-chitosan Leu-chitosan Pro-chitosan Phe--chitosan AslY-chitosan Aib~zhitosan
Cu
Ni
Co
Mn
98.3 58.0 99.7 99.7 99.8 99.8 99.6 99.9 99.6 99.2
78.5 31.5 99.9 100 100 100 100 100 99.9 100
21.0 17.0 100 100 100 99.9 99.9 99.9 99.9 99.9
7.0 28.0 98.2 100 100 100 99.9 100 99.7 99.9
Uptake/i'om a solution containing two or three types O~ ions (competitit~e uptake) Tables 2 and 3 list the heavy metal uptake results from
Initial concentration - 100 ppm Aib=a-amino isobutyric acid Table 2
Extent of removal (%) of heavy metals by chitosan and modified chitosan beads for two-ion solutions Cu and Co
Adsorbent Chitosan Glyoxylic acid-chitosan Gly-chitosan Ala~zhitosan Asp~chitosan
Cu and Ni
Cu and Mn
Ni and Co
Ni and Mn
Co and Mn
Cu
Co
Cu
Ni
Cu
Mn
Ni
Co
Ni
Mn
Co
Mn
100 74.3 99.9 100 99.8
26.4 8.7 100 100 100
100 75.t 100 100 100
76.5 13.7 100 100 100
99.8 81.0 99.0 99.4 99.3
7.0 2.5 94.5 92.6 84.0
87.4 28.9 100 100 99.9
33.4 17.1 100 100 100
91.2 34.8 99.9 1(X) 99.8
15.1 18.6 100 100 100
48.6 22.8 100 100 100
14.8 17.0 1(X) 100 100
Initial concentration = 100 ppm for each metal ion Table 3 Extent of removal (%) of heavy metals by chitosan and modified chitosan beads for three-ion solutions Cu, Ni and Co Adsorbent Chitosan Glyoxylic acid~zhitosan Gly chitosan Ala~,:hitosan Asl'~chitosan
Cu, Ni and Mn
Ni, Co and Mn
Cu
Ni
Co
Cu
Ni
Mn
Cu
Co
Mn
Ni
Co
Mn
99.7 75.3 100 99.9 99.8
87.2 12.8 99.8 99.5 100
23.2 3.7 99.8 99.8 99.8
100 75.6 99.8 99.9 99.6
90.2 t 3.2 99.5 99.6 99.5
0.0 0.0 97.7 98.6 98.8
100 75.8 99.8 100 99.0
37.9 6.9 99.7 99.5 99.5
10.7 8.4 99.5 99.3 99.3
93.0 30.0 99.7 99.6 99.6
43.7 16.9 99.6 99.4 99.4
f4.t) 16.4 99.6 99.3 99.3
Initial concentration- 100 ppm for each metal ion
22
Cu, Co and Mn
Int. J. Biol. Macromol. Volume 17 Number 1 1995
Heavy metal uptake by modified chitosan: H. Ishfi et al. solutions containing two or three types of metal ions. Under competitive uptake by non-modified chitosan, the extent of removal did not decrease, while a decrease in the extent of removal was observed for the glyoxylic acid-substituted chitosan. In contrast with the results using glycoxylic acid-substituted chitosan, metal ions can be removed almost completely under competitive conditions using amino acid ester-substituted chitosans. It is worth noting that Mn, which was not removed efficiently by chitosan, could be removed by chitosan-amino acid conjugates. As suggested by their structure, the chitosanamino acid conjugates show biodegradability and effective uptake ability for heavy metals.
References 1 2 3 4 5 6 7 8 9
Muzarelli, R.A.A. 'Chitin', Pergamon Press, Oxford, 1976 Covas, C.P., Alvarez, L.W. and Monal, W.A.J. Appl. Polym. Sci. 1992, 46, 1147 Findon, A., McKay, G. and Blain, H.S.J. Environ. Sci. Health 1993, A28, 173 Deans, J.R. and Dixon, B.G. Water Res. 1992, 26, 469 Udaybhaskar, P., Iyengar, L. and Rao, A.V.S.P.J. Appl. Polym. Sei. 1990, 39, 739 Onsoyen, E. and Skaugrud, O. J. Chem. Tech. Bioteeh. 1990, 49, 395 Mitani, T., Moriyama, A. and Ishii, H. Biosci. Biotech. Biochem. 1992, 56, 985 Lasko, C.L., Pesic, B.M. and Oliver, D.J.J. Appl. Polym. Sei. 1993, 48, 1565 Monal, W.A. and Covas, C.P. Angew. Makromol. Chem. 1993, 207, 1
10 11 12
Conclusions
13
Chitosan-amino acid conjugates were synthesized using glyoxylic acid-substituted chitosan as an intermediate. For the heavy metals used, especially Mn, the chitosanamino acid conjugates were more effective adsorbents than the non-modified chitosan.
14 15 16 17
Muzarelli, R.A.A. Carbohydr. Polym. 1992, 19, 231 Saneedo, I., Guibal, E.G., Roulph, C. and Cloirec, P.L. Environ. Technol. 1992, 13, 101 Rorrer, G.L., Hsien, T.Y. and Way, J.D. Ind. Eng. Chem. Res. 1993, 32, 2170 Kawamura, Y., Mitsuhashi, M., Tanibe, H. and Yoshida, H. Ind. Eng. Chem. Res. 1992, 32, 386 Holme, K.R. and Hall, L.D. Can. J. Chem. 1991, 69, 585 Kurita, K.,Koyama, Y.andChikaoka, S. Polym.J. 1988,20,1083 Muzarelli, R.A.A., Tanfani, F., Emanuelli, M. and Bolognini, L. Biotech. Bioeng. 1985, 27, 1115 Mitani, T., Fukumuro, N., Yoshimoto, C. and Ishii, H. Agric. Biol. Chem. 1991, 55, 2419
Int. J. Biol. Macromol. Volume 17 Number 1 1995
23
Department of Chemical Science and Engineering, Tokyo National College of Technology 1220 Kunugida. Hachiouji, Tokyo 193, Japan Received 15 June 1994; revised 20 August 1994
Chitosan-amino acid conjugates were prepared by coupling amino acid esters to the carboxyl group of glyoxylic acid-substituted chitosan. The removal of heavy metals (Cu, Ni, Co and Mn) was increased by introduction of amino acids to chitosan, especially for Mn. Heavy metals were almost completely removed by chitosan-amino acid conjugates from solutions at an initial concentration of 100 parts per million.
Keywords: chitosan; amino acid; heavy metal uptake
It is now well known that chitosan (deacetylated chitin) has a promising ability to adsorb many kinds of heavy metal ions 1-6. To increase the adsorption capacity and/or selectivity for metal ions, various chemically modified chitosans have been prepared 7-16. Chemical modification was achieved for both amino groups and primary hydroxyl groups. From the point of view of biodegradability, which is related to the structure of the compound, amino acids are suitable for use as substituting groups for modification. In this paper, we report on the preparation of chitosan-amino acid conjugates and their use in heavy metal uptake.
metal(s). The mixture was shaken for 2 days at 30°C and the concentration of the remaining metal ion(s) was measured by SEIKO SAS-722 and SAS-7500 spectrometers. As ions co-existing in the solution affect
OH
OH
NH2
\
Methods Equilibrium adsorption experiments were performed as follows. Highly swollen chitosan beads (1 ml, net dry chitosan weight 0.11 g) were added to 19 ml of an aqueous solution containing 100 parts per million (ppm) of heavy * To whom correspondence should be addressed
0141-8130/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
I~1 /n CH I (CH2)3
\
I~ /m II OH I COOH
reQldue
Experimental Materials Chitosan was purchased from Katakura Chikkarin Co. Ltd and used without any purification. Chitosan beads were prepared as described previously 17. The amino acids used were of the L (laevo) configuration. Esters of amino acids, i.e. Gly-OBzl.TosOH, AlaOBzl-TosOH, Ser-OMe-HC1, Leu-OBzl.TosOH, ProOMe.HCI, Phe-OBzl.TosOH, Asp(OBzl)-OBzl.TosOH and Aib-OMe-HC1, were synthesized by standard methods. Heavy metals were used in the sulfate form.
OH
OH
OH
H2N-CHR-COOR' EEDOJDMF x
N II
/n
x
N
/m
II
CH
CH
I
I
(CH2)3
CONHCHR-COO.'
resldue
OH
OH
-_ N /n II CH I (CH2)3
\
/m Ii CH I CONHCHRCOOH
k
resldue
Figure 1 Synthetic scheme for production of chitosan-amino acid conjugates: R=side chain of amino acid, R'=alcohol component of ester
Int. J. Biol. M a c r o m o l . V o l u m e 1 7 N u m b e r 1 1 9 9 5
21
Heavy metal uptake by modified ch,tosan: t4. Ishfi et al. heavy metal a d s o r p t i o n b e h a v i o u r 17, no buffer was used for the uptake experiment. C o m p e t i t i v e uptake experiments were carried out using solutions c o n t a i n i n g two or three kinds of metal ions (each at a c o n c e n t r a t i o n of 100 ppm).
and glyoxylic acid-substituted chitosan beads. The mixture was stirred for 2 days, and beads were collected and washed successively with m e t h a n o l and distilled water.
Synthesis of glyoxylic acid-substituted chitosan
A m i n o acid ester-substituted chitosan beads were added to a q u e o u s alkaline solution and the mixture was stirred overnight. Beads were collected and washed. Th e co n t en t of the a m i n o acid i n t r o d u c e d was determined by b a c k - t i t r a t i o n of the c a r b o x y l g r o u p using N a O H and HC1. F o r all a m i n o acids used, the degree of substitution was ~ 0.4.
Saponification of chitosan- amino acid esters
A q u e o u s g l u t a r a l d e h y d e solution (3.8%; 600 ml) and a q u e o u s glyoxylic acid solution (16%; 600ml) were simultaneously added dropwise at r o o m t e m p e r a t u r e to 300 ml of swollen chitosan beads. The mixture was stirred overnight, and red c o l o u r e d beads were collected and washed with distilled water. T h e synthetic scheme is shown in Figure 1.
Results and discussion
Coupling amino acid esters to glyoxylic acid-substituted chitosan
Uptake./i'om solutions containing a single metal ion Table 1 lists the heavy metal u p t a k e results at metal ion
T h e benzyl or methyl ester of a m i n o acids, ' ethoxycarbonyl-2-ethoxy- 1,2-dihidoroquinoline (EEDQ), and N - m e t h y l m o r p h o l i n e (two equivalents each per carboxyl g r o u p of the chemically modified chitosan beads) were added to a m i x t u r e of d i m e t h y l f o r m a m i d e
c o n c e n t r a t i o n s of 100 ppm. Th e o r d er of extent of r e m o v a l by n o n - m o d i f i e d chitosan was C u > Ni > C o > Mn. This result could be explained by the relative o r de r of metal o r g an i c chelate b o n d strength, i.e. the Irving Williams series. The extent of r e m o v a l by the glyoxylic acid-substituted chitosan was lower than that by nonmodified chitosan except for Mn. This is due to a decrease in the n u m b e r of free a m i n o groups. It is p r o b a b l e that the carboxyl function could not en h an ce the chelating ability of the modified chitosan. All metal ions (Cu, Ni, C o and Mn) were r e m o v e d almost completely by the chitosan a m i n o acid c o n j u g a t e under the conditions investigated. The extent of r e m o v a l was not influenced by the type of a m i n o acid introduced, indicating that metal ions interact with the b a c k b o n e of the a m i n o acid residue and chitosan molecule.
Extent of removal (%) of heavy metals by chitosan and modified chitosan beads for single-ion solutions Table I
Adsorbent Chitosan Glyoxylic acid-chitosan Gly-chitosan Ala-chitosan Ser-chitosan Leu-chitosan Pro-chitosan Phe--chitosan AslY-chitosan Aib~zhitosan
Cu
Ni
Co
Mn
98.3 58.0 99.7 99.7 99.8 99.8 99.6 99.9 99.6 99.2
78.5 31.5 99.9 100 100 100 100 100 99.9 100
21.0 17.0 100 100 100 99.9 99.9 99.9 99.9 99.9
7.0 28.0 98.2 100 100 100 99.9 100 99.7 99.9
Uptake/i'om a solution containing two or three types O~ ions (competitit~e uptake) Tables 2 and 3 list the heavy metal uptake results from
Initial concentration - 100 ppm Aib=a-amino isobutyric acid Table 2
Extent of removal (%) of heavy metals by chitosan and modified chitosan beads for two-ion solutions Cu and Co
Adsorbent Chitosan Glyoxylic acid-chitosan Gly-chitosan Ala~zhitosan Asp~chitosan
Cu and Ni
Cu and Mn
Ni and Co
Ni and Mn
Co and Mn
Cu
Co
Cu
Ni
Cu
Mn
Ni
Co
Ni
Mn
Co
Mn
100 74.3 99.9 100 99.8
26.4 8.7 100 100 100
100 75.t 100 100 100
76.5 13.7 100 100 100
99.8 81.0 99.0 99.4 99.3
7.0 2.5 94.5 92.6 84.0
87.4 28.9 100 100 99.9
33.4 17.1 100 100 100
91.2 34.8 99.9 1(X) 99.8
15.1 18.6 100 100 100
48.6 22.8 100 100 100
14.8 17.0 1(X) 100 100
Initial concentration = 100 ppm for each metal ion Table 3 Extent of removal (%) of heavy metals by chitosan and modified chitosan beads for three-ion solutions Cu, Ni and Co Adsorbent Chitosan Glyoxylic acid~zhitosan Gly chitosan Ala~,:hitosan Asl'~chitosan
Cu, Ni and Mn
Ni, Co and Mn
Cu
Ni
Co
Cu
Ni
Mn
Cu
Co
Mn
Ni
Co
Mn
99.7 75.3 100 99.9 99.8
87.2 12.8 99.8 99.5 100
23.2 3.7 99.8 99.8 99.8
100 75.6 99.8 99.9 99.6
90.2 t 3.2 99.5 99.6 99.5
0.0 0.0 97.7 98.6 98.8
100 75.8 99.8 100 99.0
37.9 6.9 99.7 99.5 99.5
10.7 8.4 99.5 99.3 99.3
93.0 30.0 99.7 99.6 99.6
43.7 16.9 99.6 99.4 99.4
f4.t) 16.4 99.6 99.3 99.3
Initial concentration- 100 ppm for each metal ion
22
Cu, Co and Mn
Int. J. Biol. Macromol. Volume 17 Number 1 1995
Heavy metal uptake by modified chitosan: H. Ishfi et al. solutions containing two or three types of metal ions. Under competitive uptake by non-modified chitosan, the extent of removal did not decrease, while a decrease in the extent of removal was observed for the glyoxylic acid-substituted chitosan. In contrast with the results using glycoxylic acid-substituted chitosan, metal ions can be removed almost completely under competitive conditions using amino acid ester-substituted chitosans. It is worth noting that Mn, which was not removed efficiently by chitosan, could be removed by chitosan-amino acid conjugates. As suggested by their structure, the chitosanamino acid conjugates show biodegradability and effective uptake ability for heavy metals.
References 1 2 3 4 5 6 7 8 9
Muzarelli, R.A.A. 'Chitin', Pergamon Press, Oxford, 1976 Covas, C.P., Alvarez, L.W. and Monal, W.A.J. Appl. Polym. Sci. 1992, 46, 1147 Findon, A., McKay, G. and Blain, H.S.J. Environ. Sci. Health 1993, A28, 173 Deans, J.R. and Dixon, B.G. Water Res. 1992, 26, 469 Udaybhaskar, P., Iyengar, L. and Rao, A.V.S.P.J. Appl. Polym. Sei. 1990, 39, 739 Onsoyen, E. and Skaugrud, O. J. Chem. Tech. Bioteeh. 1990, 49, 395 Mitani, T., Moriyama, A. and Ishii, H. Biosci. Biotech. Biochem. 1992, 56, 985 Lasko, C.L., Pesic, B.M. and Oliver, D.J.J. Appl. Polym. Sei. 1993, 48, 1565 Monal, W.A. and Covas, C.P. Angew. Makromol. Chem. 1993, 207, 1
10 11 12
Conclusions
13
Chitosan-amino acid conjugates were synthesized using glyoxylic acid-substituted chitosan as an intermediate. For the heavy metals used, especially Mn, the chitosanamino acid conjugates were more effective adsorbents than the non-modified chitosan.
14 15 16 17
Muzarelli, R.A.A. Carbohydr. Polym. 1992, 19, 231 Saneedo, I., Guibal, E.G., Roulph, C. and Cloirec, P.L. Environ. Technol. 1992, 13, 101 Rorrer, G.L., Hsien, T.Y. and Way, J.D. Ind. Eng. Chem. Res. 1993, 32, 2170 Kawamura, Y., Mitsuhashi, M., Tanibe, H. and Yoshida, H. Ind. Eng. Chem. Res. 1992, 32, 386 Holme, K.R. and Hall, L.D. Can. J. Chem. 1991, 69, 585 Kurita, K.,Koyama, Y.andChikaoka, S. Polym.J. 1988,20,1083 Muzarelli, R.A.A., Tanfani, F., Emanuelli, M. and Bolognini, L. Biotech. Bioeng. 1985, 27, 1115 Mitani, T., Fukumuro, N., Yoshimoto, C. and Ishii, H. Agric. Biol. Chem. 1991, 55, 2419
Int. J. Biol. Macromol. Volume 17 Number 1 1995
23