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Study of the Adsorption of Some Amino Acids by Silica Chemically Modified with Aminobenzenesulfonic and Phosphate Groups
JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.
183, 453–457 (1996)
0568
Study of the Adsorption of Some Amino Acids by Silica Chemically Modified with Aminobenzenesulfonic and Phosphate Groups LAURO T. KUBOTA, 1, * ALESSANDRA GAMBERO,* ANTONIO SANTANA SANTOS,*
AND
JOSE´ M. GRANJEIRO†
*Instituto de Quimica, Unicamp, PO Box 6154, 13083-970, Campinas, SP, and †Depto de Bioquimica, FOB, USP, Bauru, SP, Brazil Received April 12, 1996; accepted June 11, 1996
Two silica gels, one modified with aminobenzenesulfonic (SABS) groups and the other with phosphate (SZP) groups, were prepared to adsorb some amino acids. Chemical analysis of the modified silica gave 0.65 mmol g 01 aminobenzenesulfonic groups and 0.56 mmol g 01 phosphate groups. The maximum adsorption capacities for amino acids determined by batch experiments for SABS were 1.37, 0.67, 0.76, and 0.59 mmol g 01 for glycine, lysine, histidine, and leucine, respectively, and those for SZP were 0.75, 0.58, 0.44, and 0.75 mmol g 01 for glycine, lysine, histidine, and leucine, respectively. The adsorption capacity of SABS was significantly affected by the solution pH, showing a higher selectivity than SZP. The materials were very stable, allowing their use several times without changes in adsorption capacity. q 1996 Academic
and elution processes because they affect the net charge and complementary interaction between mobile and stationary phases (17, 18). Amino acids may be very useful in investigating these properties because of their ionic equilibria. Some proteins have affinity with phosphate and sulfonic groups (19); thus, studies of amino acid interaction with these groups are very important to aid the understanding of this characteristic process and the use of this material as a stationary phase for amino acid and protein separation (20). The preparation of a silica gel modified with sulfonic groups and another with phosphate groups is described. Amino acid adsorption by this material was studied. A comparison between them is also reported in this work.
Press, Inc.
Key Words: modified silica; amino acid adsorption; aminobenzenesulfonic groups.
EXPERIMENTAL
Preparation of Silica Gel Modified with 4-Aminobenzenesulfonic Groups INTRODUCTION
In recent years, modification of the silica gel surface has been extensively investigated (1–3) because of its characteristics such as high surface area, porosity, rigidity, and mechanical resistance (4). These studies have been made to prepare materials able to adsorb some species such as catalysts (5, 6), organic compounds (7), and metal ions (8, 9). These materials have been used as a stationary phase for chromatography (10), principally in HPLC (11–13). There are many studies of the adsorption properties of some metal ion by modified silica gel (14, 15); however, amino acid adsorption has not been studied in this context, although many investigations of amino acid separations using modified silica have been made (16). In ion-exchange chromatography, the charged stationary phase can adsorb species with opposite charge. These species can be eluted by changing the pH or ionic strength or by using a counterion that presents stronger interaction with stationary phase. The pH and ionic strength are very important to the adsorption 1
To whom correspondence should be addressed.
Silica gel (Fluka) with a surface area of 500 m2 g 01 , ˚ , and particle size 0.2–0.7 average pore diameter of 60 A mm was previously degassed at 1507C under high vacuum. This silica presents a narrow range of distribution of pore diameter, representing an amount of silanol groups of about 3.5 mmol g 01 . About 50 g of this activated material was immersed in a solution consisting of 10 ml of 3-aminopropyltriethoxysilane in 400 ml of toluene. This mixture was refluxed with constant agitation for 10 h at 907C. Afterward, the material was filtered and washed with toluene, ethanol, and acetone. The resulting material was denominated as aminopropyl silica gel (APSG). In a second step, 6 g of aminobenzenesulfonic acid (sodium salt) and 12 g of sodium nitrite were dissolved in 200 ml of a 4 mol liter 01 HCl solution, in an ice bath. This solution was shaken for 10 min to obtain the diazonium compound. Then, 20 g of APSG was added into this solution and shaken for 10 min. The material obtained was filtered, washed with demineralized water, and dried in an oven for 2 h at 707C. The quantity of organic groups immobilized on the silica surface was 0.65 mmol g 01 , determined by elemental analysis. This modified silica is hereafter denominated as SABS.
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The infrared spectrum of this material was obtained by the autosupported disk method as described by Gushikem and Moreira (21), using a Perkin–Elmer Model 1600 IR spectrometer with Fourier transform. Preparation of Silica Gel Modified with Zirconium Phosphate The procedure for coating the silica gel surface with zirconium phosphate was carried out in two steps. In the first step, about 50 g of degassed silica gel (as described above) was immersed in a solution consisting of 11.6 g of pure ZrCl4 dissolved in 300 ml of dry ethanol. The mixture was refluxed for 8 h at 807C and the resulting material was washed by decantation and then heated at 1507C for 4 h. This material was hydrolyzed by immersing the solid in demineralized water. The solid was washed with water to remove all chloride ions and dried in an oven at 1207C for 5 h. The amount of attached zirconium on the surface, analyzed by X-ray fluorescence, was 3.8% (w/w) ZrO2 . About 25 g of this material was added to 200 ml of a 0.1 mol liter 01 H3PO4 solution and shaken for 10 h at 907C. Then, the solid was washed with demineralized water and dried in an oven at 1007C for 4 h. The amount of phosphorus was 0.56 1 10 03 mol g 01 , determined by X-ray fluorescence. This material is hereafter denominated as SZP.
to determine the adsorption capacity of the material used for several times. RESULTS AND DISCUSSION
Characteristics of the Materials The silica chemically modified with aminopropyl groups presented a quantity of 0.67 1 10 03 mol g 01 of amino groups. When this material was reacted with aminobenzenesulfonic groups, the material showed an orange color, characteristic of a compound with a {N|N{ bond. The reaction can be represented by the equations ©O © O ©Si©(CH¤)‹©NH¤ 1 H¤N©
©SO‹Na
HCl/NaNO¤
©O
©O © O ©Si©(CH¤)‹©N
N©
©SO‹H
©O (1)
The amount of aminobenzenesulfonic groups was 0.65 1 10 03 mol g 01 , showing a high efficiency in the synthesis reactions. The infrared spectrum (Fig. 1) shows bands at
Batch Adsorption of Amino Acids The isotherms for adsorption of amino acids by the chemically modified silicas were obtained at room temperature, using a batch technique. Four-tenths gram of the modified silica was added to 50 ml of a solution containing amino acids at different concentrations and shaken for 30 min. Then the supernatant was decanted and analyzed, by a spectrophotometric method (22), using a DU2000 (Pharmacia) spectrophotometer ( l Å 490 nm) and benzoquinone as chromogenic reagent, to quantify the adsorbed amino acid. The amount of amino acid adsorbed by solid phase, N f , was calculated by applying the equation (23) N f Å (Na 0 Ns )/m, where Na is the initial number of moles of amino acid, Ns is the number of moles of amino acid in equilibrium with the solid phase, and m is the mass of the solid phase. The adsorption isotherms were also determined at different solution pH and equilibrium times. Reutilization and Stability of the Material The material was used four times to verify its stability and the possibility of reuse of the material. These experiments were carried out by batch technique as describe above,
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FIG. 1. Infrared sprectrum of SABS, obtained by autosupported disk.
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ADSORPTION OF AMINO ACIDS BY SILICA
455
groups. The maximum adsorption capacities were 0.75, 0.58, 0.44, and 0.75 mmol g 01 for glycine, lysine, histidine, and leucine, respectively. In this case, small differences between adsorption isotherms were observed. This behavior may be attributed to the differences in the affinity between SZP and amino acid. A comparison between two silicas shows that the adsorption capacities are different for each case. This behavior can be assigned to the differences in the affinities between the amino acid and modified silica. In general, the amino acid adsorption by SABS presented an adsorption capacity slightly higher than that of SZP. This behavior may be assigned to the quantity of grafted functional groups, although in both cases the quantity of adsorbed amino acid was higher than the number of functional groups, suggesting that some amino acids can be adsorbed by the remaining silanol groups and/or trapped in some smaller pores. The FIG. 2. Curve of glycine adsorption by SABS as a function of time. Glycine concentration: 5.0 10 03 mol liter 01 , temperature: 25.07C, solution pH: 3.0.
1435, 1467, and 1528 cm01 , characteristic of the aromatic compound, and the band at 1370 cm01 was assigned to the propyl group. This material presented good stability in neutral solution, but at pH values lower than 2 a small amount of leaching was observed. The adsorption capacity decreased about 10% after the material was used four times. For the silica gel modified with zirconium(IV) phosphate, no visual change was observed after chemical modification. Analysis of the material gave an amount of phosphate groups of 0.56 1 10 03 mol g 01 , indicating good performance of the preparation reaction. The stability of this material was checked and, even in acid medium, no significant change was observed in its adsorption capacity. Adsorption Properties In Fig. 2, the quantity of adsorbed glycine is plotted as a function of time, showing that equilibrium is reached after 30 min. This time is very short, when compared with other materials (24), indicating that the adsorption sites are well exposed on the silica surface and the pore size distribution is very narrow. Figures 3A and B show the isotherms for adsorption of glycine, leucine, histidine, and lysine by silica chemically modified with aminobenzenesulfonic groups. From these isotherms the maximum adsorption capacities for each amino acid were determined as 1.37, 0.67, 0.76, and 0.59 mmol g 01 for glycine, lysine, histidine, and leucine, respectively. The isotherms showed similar aspects for the amino acids, except for the highest adsorption capacity of glycine. These characteristics may be attributed to molecular size, suggesting that SABS has a good affinity for amino acids. Figures 4A and B show the isotherms for adsorption of the same amino acids by the silica modified with phosphate
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FIG. 3. Adsorption isotherms of the amino acids by SABS. (A) Glycine(a) and leucine(b); (B) histidine(c) and lysine(d). Temperature: 257C, pH: 4.
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order of the adsorption capacity between SABS and SZP, however, is different. This behavior is very important because it shows a difference in the affinities between the materials. The higher capacity for adsorption of leucine of the phosphate groups shows that leucine has a better affinity with phosphate groups than with sulfonic groups. The effect of the solution pH on amino acid adsorption by silica modified with sulfonic groups is presented in Fig. 5A and B. The differences observed between the best adsorption pH for each amino acid may be attributed to the acid–base equilibrium of the carboxylic, amino, and R groups. A high adsorption capacity is observed at pH 1, except for leucine. This behavior can be assigned to the charged form of the amino acids. At a pH of about 1 the amino acids are in a more positively charged form (25); thus, the adsorption should be
FIG. 5. Curve of amino acid adsorption as a function of solution pH for SABS. (A) Glycine(a) and leucine(b); (B) histidine(c) and lysine(d). Temperature: 257C.
FIG. 4. Adsorption isotherms of the amino acids by SZP. (A) Glycine(a) and leucine(b); (B) histidine(c) and lysine(d). Temperature: 257C, pH: 4.
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high. Figure 5A shows a better affinity for glycine than leucine by SABS, suggesting that the affinity is not based only by electrostatic interaction, since the pK values for both amino acids are very similar. The difference in behavior verified between glycine and leucine in Fig. 5A and for histidine and lysine in Fig. 5B may be attributed to charge and another interaction type. For lower pH values, the charge interaction should predominate, considering the first p K values (1.82 for histine and 2.18 for lysine). At higher pH the charge does not explain the behavior observed, indicating that the affinity depends on other factors. This aspect is very important to preconcentrate and then separate the amino acids in a chromatographic column. However, for silica modified with phosphate groups the differences between the best adsorption pH is lower, suggesting that the affinity is as important as the charge. Figures 6A and B show amino acid adsorption as a
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ADSORPTION OF AMINO ACIDS BY SILICA
CONCLUSIONS
The modified silica may be very useful in improving selectivity, employing it in the separation of several biological compounds. Amino acid adsorption on solid surfaces provides some important information about the interaction of the charged species on modified surfaces. The interaction depends on the charge and the affinity of the species for the surface. The stability of the silica and exchanger groups can be very important for use in amino acid and protein separation. ACKNOWLEDGMENTS The authors thank Fapesp for the financial support and Professor Carol Collins for the English revision of the manuscript. A.S.S. is indebted to SAE for a fellowship.
REFERENCES
FIG. 6. Curve of amino acid adsorption as a function of solution pH for SZP. (A) Glycine(a) and leucine(b); (B) histidine(c) and lysine(d). Temperature: 257C.
function of pH for the silica modified with phosphate groups. In these cases, the amino acids presented a high adsorption at a pH of about 1, indicating that at this pH the material adsorbs all amino acids without selectivity. At pH between 3 and 5 a significant adsorption was observed showing a small selectivity. In this case, the values of first pK for the amino acid can be correlated in the same sequence for the pH values when the maximum adsorption occurs. The higher selectivity of SABS, in contrast to SZP, suggests its greater potential as an affinity chromatography support. Similarly, p-aminobenzylphosphonic acid–Sepharose was successfully used for low-molecular-weight acid phosphatase purification (26). In this way, SABS could be an alternative material for use as a stationary phase in chromatography, since this material has better physical properties than Sepharose. This study is under investigation in our laboratory.
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1. Kubota, L. T., and Gushikem, Y., J. Electroanal. Chem. 362, 219 (1993). 2. Frenzel, W., and Krekler, S., Anal. Chim. Acta 310, 437 (1995). 3. Kubota, L. T., Gushikem, Y., Mansanares, A., and Vargas, H., J. Colloid Interface Sci. 173, 372 (1995). 4. Iler, R. K., ‘‘The Chemistry of Silica.’’ Wiley, New York, 1979. 5. Kubota, L. T., Gushikem, Y., Perez, J., and Tanaka, A. A., Langmuir 11, 1009 (1995). 6. Kennedy, J. F., and Cabral, J. M. S., Trans. Met. Chem. 15, 197 (1990). 7. Bronner, W. E., and Becher, G. R., J. Chromatogr. A 705, 247 (1995). 8. Kubota, L. T., Gushikem, Y., and Moreira, J. C., Analyst 116, 281 (1991). 9. Sutthivaiyakit, P., and Kettrup, A., Anal. Chim. Acta 169, 331 (1985). 10. Moriguchi, S., Naito, K., and Takei, S., J. Chromatogr. 686, 101 (1994). 11. Rirkle, W. H., and Selim, A. I., J. High Resolut. Chromatogr. 18, 353 (1995). 12. Scholl, J. P., J. Liq. Chromatogr. 17, 3369 (1994). 13. Kirkland, J. J., van Straten, M. A., and Claessens, H. A., J. Chromatogr. A 691, 3 (1995). 14. Wasiak, W., Chromatographia 41, 107 (1995). 15. Krukler, S., Frenzel, W., and Schulze, G., Anal. Chim. Acta 296, 115 (1994). 16. Oi, N., Kitahara, H., and Aoki, F., J. Chromatogr. A 694, 129 (1995). 17. Ovalle, R., Anal. Biochem. 229, 1 (1995). 18. Nesterenko, P. N., Elefterov, A. I., Tarasenko, D. A., and Shpigun, O. A., J. Chromatogr. A 706, 59 (1995). 19. Croe, K., McCarthy, P. T., and Flanagan, R. J., J. Chromatogr. A 693, 289 (1995). 20. Hansen, P., and Lindeberg, G., J. Chromatogr. A 690, 155 (1995). 21. Gushikem, Y., and Moreira, J. C., J. Colloid Interface Sci. 107, 70 (1985). 22. Zaia, D. A. M., Barreto, W. J., Santos, N. J., and Endo, A. S., Anal. Chim. Acta 277, 89 (1993). 23. Peixoto, C. R. M., Gushikem, Y., and Baccan, N., Analyst 117, 1029 (1992). 24. Bond, G. C., Flamerz, S., and Shukri, R., Faraday Discuss. Chem. Soc. 87, 65 (1989). 25. Campbell, M. K., ‘‘Biochemistry.’’ Saunders, Philadelphia, 1991. 26. Dissing, J., and Srensmark, O., Biochim. Biophys. Acta 1041, 232 (1990).
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0568
Study of the Adsorption of Some Amino Acids by Silica Chemically Modified with Aminobenzenesulfonic and Phosphate Groups LAURO T. KUBOTA, 1, * ALESSANDRA GAMBERO,* ANTONIO SANTANA SANTOS,*
AND
JOSE´ M. GRANJEIRO†
*Instituto de Quimica, Unicamp, PO Box 6154, 13083-970, Campinas, SP, and †Depto de Bioquimica, FOB, USP, Bauru, SP, Brazil Received April 12, 1996; accepted June 11, 1996
Two silica gels, one modified with aminobenzenesulfonic (SABS) groups and the other with phosphate (SZP) groups, were prepared to adsorb some amino acids. Chemical analysis of the modified silica gave 0.65 mmol g 01 aminobenzenesulfonic groups and 0.56 mmol g 01 phosphate groups. The maximum adsorption capacities for amino acids determined by batch experiments for SABS were 1.37, 0.67, 0.76, and 0.59 mmol g 01 for glycine, lysine, histidine, and leucine, respectively, and those for SZP were 0.75, 0.58, 0.44, and 0.75 mmol g 01 for glycine, lysine, histidine, and leucine, respectively. The adsorption capacity of SABS was significantly affected by the solution pH, showing a higher selectivity than SZP. The materials were very stable, allowing their use several times without changes in adsorption capacity. q 1996 Academic
and elution processes because they affect the net charge and complementary interaction between mobile and stationary phases (17, 18). Amino acids may be very useful in investigating these properties because of their ionic equilibria. Some proteins have affinity with phosphate and sulfonic groups (19); thus, studies of amino acid interaction with these groups are very important to aid the understanding of this characteristic process and the use of this material as a stationary phase for amino acid and protein separation (20). The preparation of a silica gel modified with sulfonic groups and another with phosphate groups is described. Amino acid adsorption by this material was studied. A comparison between them is also reported in this work.
Press, Inc.
Key Words: modified silica; amino acid adsorption; aminobenzenesulfonic groups.
EXPERIMENTAL
Preparation of Silica Gel Modified with 4-Aminobenzenesulfonic Groups INTRODUCTION
In recent years, modification of the silica gel surface has been extensively investigated (1–3) because of its characteristics such as high surface area, porosity, rigidity, and mechanical resistance (4). These studies have been made to prepare materials able to adsorb some species such as catalysts (5, 6), organic compounds (7), and metal ions (8, 9). These materials have been used as a stationary phase for chromatography (10), principally in HPLC (11–13). There are many studies of the adsorption properties of some metal ion by modified silica gel (14, 15); however, amino acid adsorption has not been studied in this context, although many investigations of amino acid separations using modified silica have been made (16). In ion-exchange chromatography, the charged stationary phase can adsorb species with opposite charge. These species can be eluted by changing the pH or ionic strength or by using a counterion that presents stronger interaction with stationary phase. The pH and ionic strength are very important to the adsorption 1
To whom correspondence should be addressed.
Silica gel (Fluka) with a surface area of 500 m2 g 01 , ˚ , and particle size 0.2–0.7 average pore diameter of 60 A mm was previously degassed at 1507C under high vacuum. This silica presents a narrow range of distribution of pore diameter, representing an amount of silanol groups of about 3.5 mmol g 01 . About 50 g of this activated material was immersed in a solution consisting of 10 ml of 3-aminopropyltriethoxysilane in 400 ml of toluene. This mixture was refluxed with constant agitation for 10 h at 907C. Afterward, the material was filtered and washed with toluene, ethanol, and acetone. The resulting material was denominated as aminopropyl silica gel (APSG). In a second step, 6 g of aminobenzenesulfonic acid (sodium salt) and 12 g of sodium nitrite were dissolved in 200 ml of a 4 mol liter 01 HCl solution, in an ice bath. This solution was shaken for 10 min to obtain the diazonium compound. Then, 20 g of APSG was added into this solution and shaken for 10 min. The material obtained was filtered, washed with demineralized water, and dried in an oven for 2 h at 707C. The quantity of organic groups immobilized on the silica surface was 0.65 mmol g 01 , determined by elemental analysis. This modified silica is hereafter denominated as SABS.
453
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0021-9797/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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The infrared spectrum of this material was obtained by the autosupported disk method as described by Gushikem and Moreira (21), using a Perkin–Elmer Model 1600 IR spectrometer with Fourier transform. Preparation of Silica Gel Modified with Zirconium Phosphate The procedure for coating the silica gel surface with zirconium phosphate was carried out in two steps. In the first step, about 50 g of degassed silica gel (as described above) was immersed in a solution consisting of 11.6 g of pure ZrCl4 dissolved in 300 ml of dry ethanol. The mixture was refluxed for 8 h at 807C and the resulting material was washed by decantation and then heated at 1507C for 4 h. This material was hydrolyzed by immersing the solid in demineralized water. The solid was washed with water to remove all chloride ions and dried in an oven at 1207C for 5 h. The amount of attached zirconium on the surface, analyzed by X-ray fluorescence, was 3.8% (w/w) ZrO2 . About 25 g of this material was added to 200 ml of a 0.1 mol liter 01 H3PO4 solution and shaken for 10 h at 907C. Then, the solid was washed with demineralized water and dried in an oven at 1007C for 4 h. The amount of phosphorus was 0.56 1 10 03 mol g 01 , determined by X-ray fluorescence. This material is hereafter denominated as SZP.
to determine the adsorption capacity of the material used for several times. RESULTS AND DISCUSSION
Characteristics of the Materials The silica chemically modified with aminopropyl groups presented a quantity of 0.67 1 10 03 mol g 01 of amino groups. When this material was reacted with aminobenzenesulfonic groups, the material showed an orange color, characteristic of a compound with a {N|N{ bond. The reaction can be represented by the equations ©O © O ©Si©(CH¤)‹©NH¤ 1 H¤N©
©SO‹Na
HCl/NaNO¤
©O
©O © O ©Si©(CH¤)‹©N
N©
©SO‹H
©O (1)
The amount of aminobenzenesulfonic groups was 0.65 1 10 03 mol g 01 , showing a high efficiency in the synthesis reactions. The infrared spectrum (Fig. 1) shows bands at
Batch Adsorption of Amino Acids The isotherms for adsorption of amino acids by the chemically modified silicas were obtained at room temperature, using a batch technique. Four-tenths gram of the modified silica was added to 50 ml of a solution containing amino acids at different concentrations and shaken for 30 min. Then the supernatant was decanted and analyzed, by a spectrophotometric method (22), using a DU2000 (Pharmacia) spectrophotometer ( l Å 490 nm) and benzoquinone as chromogenic reagent, to quantify the adsorbed amino acid. The amount of amino acid adsorbed by solid phase, N f , was calculated by applying the equation (23) N f Å (Na 0 Ns )/m, where Na is the initial number of moles of amino acid, Ns is the number of moles of amino acid in equilibrium with the solid phase, and m is the mass of the solid phase. The adsorption isotherms were also determined at different solution pH and equilibrium times. Reutilization and Stability of the Material The material was used four times to verify its stability and the possibility of reuse of the material. These experiments were carried out by batch technique as describe above,
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FIG. 1. Infrared sprectrum of SABS, obtained by autosupported disk.
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ADSORPTION OF AMINO ACIDS BY SILICA
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groups. The maximum adsorption capacities were 0.75, 0.58, 0.44, and 0.75 mmol g 01 for glycine, lysine, histidine, and leucine, respectively. In this case, small differences between adsorption isotherms were observed. This behavior may be attributed to the differences in the affinity between SZP and amino acid. A comparison between two silicas shows that the adsorption capacities are different for each case. This behavior can be assigned to the differences in the affinities between the amino acid and modified silica. In general, the amino acid adsorption by SABS presented an adsorption capacity slightly higher than that of SZP. This behavior may be assigned to the quantity of grafted functional groups, although in both cases the quantity of adsorbed amino acid was higher than the number of functional groups, suggesting that some amino acids can be adsorbed by the remaining silanol groups and/or trapped in some smaller pores. The FIG. 2. Curve of glycine adsorption by SABS as a function of time. Glycine concentration: 5.0 10 03 mol liter 01 , temperature: 25.07C, solution pH: 3.0.
1435, 1467, and 1528 cm01 , characteristic of the aromatic compound, and the band at 1370 cm01 was assigned to the propyl group. This material presented good stability in neutral solution, but at pH values lower than 2 a small amount of leaching was observed. The adsorption capacity decreased about 10% after the material was used four times. For the silica gel modified with zirconium(IV) phosphate, no visual change was observed after chemical modification. Analysis of the material gave an amount of phosphate groups of 0.56 1 10 03 mol g 01 , indicating good performance of the preparation reaction. The stability of this material was checked and, even in acid medium, no significant change was observed in its adsorption capacity. Adsorption Properties In Fig. 2, the quantity of adsorbed glycine is plotted as a function of time, showing that equilibrium is reached after 30 min. This time is very short, when compared with other materials (24), indicating that the adsorption sites are well exposed on the silica surface and the pore size distribution is very narrow. Figures 3A and B show the isotherms for adsorption of glycine, leucine, histidine, and lysine by silica chemically modified with aminobenzenesulfonic groups. From these isotherms the maximum adsorption capacities for each amino acid were determined as 1.37, 0.67, 0.76, and 0.59 mmol g 01 for glycine, lysine, histidine, and leucine, respectively. The isotherms showed similar aspects for the amino acids, except for the highest adsorption capacity of glycine. These characteristics may be attributed to molecular size, suggesting that SABS has a good affinity for amino acids. Figures 4A and B show the isotherms for adsorption of the same amino acids by the silica modified with phosphate
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FIG. 3. Adsorption isotherms of the amino acids by SABS. (A) Glycine(a) and leucine(b); (B) histidine(c) and lysine(d). Temperature: 257C, pH: 4.
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order of the adsorption capacity between SABS and SZP, however, is different. This behavior is very important because it shows a difference in the affinities between the materials. The higher capacity for adsorption of leucine of the phosphate groups shows that leucine has a better affinity with phosphate groups than with sulfonic groups. The effect of the solution pH on amino acid adsorption by silica modified with sulfonic groups is presented in Fig. 5A and B. The differences observed between the best adsorption pH for each amino acid may be attributed to the acid–base equilibrium of the carboxylic, amino, and R groups. A high adsorption capacity is observed at pH 1, except for leucine. This behavior can be assigned to the charged form of the amino acids. At a pH of about 1 the amino acids are in a more positively charged form (25); thus, the adsorption should be
FIG. 5. Curve of amino acid adsorption as a function of solution pH for SABS. (A) Glycine(a) and leucine(b); (B) histidine(c) and lysine(d). Temperature: 257C.
FIG. 4. Adsorption isotherms of the amino acids by SZP. (A) Glycine(a) and leucine(b); (B) histidine(c) and lysine(d). Temperature: 257C, pH: 4.
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high. Figure 5A shows a better affinity for glycine than leucine by SABS, suggesting that the affinity is not based only by electrostatic interaction, since the pK values for both amino acids are very similar. The difference in behavior verified between glycine and leucine in Fig. 5A and for histidine and lysine in Fig. 5B may be attributed to charge and another interaction type. For lower pH values, the charge interaction should predominate, considering the first p K values (1.82 for histine and 2.18 for lysine). At higher pH the charge does not explain the behavior observed, indicating that the affinity depends on other factors. This aspect is very important to preconcentrate and then separate the amino acids in a chromatographic column. However, for silica modified with phosphate groups the differences between the best adsorption pH is lower, suggesting that the affinity is as important as the charge. Figures 6A and B show amino acid adsorption as a
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ADSORPTION OF AMINO ACIDS BY SILICA
CONCLUSIONS
The modified silica may be very useful in improving selectivity, employing it in the separation of several biological compounds. Amino acid adsorption on solid surfaces provides some important information about the interaction of the charged species on modified surfaces. The interaction depends on the charge and the affinity of the species for the surface. The stability of the silica and exchanger groups can be very important for use in amino acid and protein separation. ACKNOWLEDGMENTS The authors thank Fapesp for the financial support and Professor Carol Collins for the English revision of the manuscript. A.S.S. is indebted to SAE for a fellowship.
REFERENCES
FIG. 6. Curve of amino acid adsorption as a function of solution pH for SZP. (A) Glycine(a) and leucine(b); (B) histidine(c) and lysine(d). Temperature: 257C.
function of pH for the silica modified with phosphate groups. In these cases, the amino acids presented a high adsorption at a pH of about 1, indicating that at this pH the material adsorbs all amino acids without selectivity. At pH between 3 and 5 a significant adsorption was observed showing a small selectivity. In this case, the values of first pK for the amino acid can be correlated in the same sequence for the pH values when the maximum adsorption occurs. The higher selectivity of SABS, in contrast to SZP, suggests its greater potential as an affinity chromatography support. Similarly, p-aminobenzylphosphonic acid–Sepharose was successfully used for low-molecular-weight acid phosphatase purification (26). In this way, SABS could be an alternative material for use as a stationary phase in chromatography, since this material has better physical properties than Sepharose. This study is under investigation in our laboratory.
AID
JCIS 4435
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6g16$$$364
09-27-96 09:02:32
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coida
AP: Colloid