Langmuir 2005, 21, 8743-8750
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Amino Acid Adsorption on Zeolite β John E. Krohn and Michael Tsapatsis* Department of Chemical Engineering and Material Science, University of Minnesota, Minneapolis, Minnesota 55455-0132 Received May 2, 2005. In Final Form: June 29, 2005 A thermodynamic equilibrium model has been developed to describe amino acid adsorption on microporous materials. The model addresses electrostatic, hydrophobic and steric interactions. A procedure for fitting the model’s parameters is presented and should be applicable to the majority of the common 20 amino acids. The approach is demonstrated using experimental measurements of L-phenylalanine and L-arginine on zeolite β. Between the adsorption mechanisms of ion exchange and physisorption, the first can contribute as much as two-thirds of the phenylalanine adsorbed at saturation. For the materials tested, ion exchange is maximized when the zeolite’s silicon-to-aluminum ratio is 12. When this atom ratio is raised to 100, ion exchange no longer plays a significant role, but the amount physisorbed increases by 30%.
Introduction Amino acids are industrially important as supplements to stock feed and in the improvement of protein quality in food technology.1 Commercial production of amino acids typically involves extraction from natural products, chemical or enzymatic synthesis, or microbiological fermentation.2 In all of these processes, a product separation mechanism is required. Amino acids can then be used in the production of oligopeptides and larger biopolymers, but a challenging separation step is again required to remove unreacted amino acids from dipeptides.3 Among the applicable chromatographic techniques, processes using organic ion-exchange resins are most commonly employed. These processes often require a series of adsorption/desorption steps that involve significant losses of the desired product. As part of an effort to improve efficiency, zeolites have been investigated as an alternative adsorption medium. In 1994, a patent2 described a process in which pH is used to control the adsorption of amino acids, from aqueous solution, using various zeolites in powder or pellet form. Size selectivity, resistance to swelling, and thermal stability are among the advantages that were cited in favor of using zeolites in this application. These advantages are routinely claimed for zeolite powders and pellets, and many of these advantages can be claimed for zeolite membranes as well. Recent advances in large-pore zeolitic membrane synthesis4,5 make it increasingly attractive to pursue a steady-state separation of amino acids. In 2001, Munsch et al.1 expounded upon the effect of pH in the adsorption of amino acids on zeolites and presented a significant amount of data specifically dealing with zeolite β in this application. To build upon these findings, we have conducted adsorption experiments in an attempt to improve our quantitative understanding of the driving forces involved. While focusing on separations, it is our intent to add to the general understanding of interactions between amino (1) Munsch, S.; Hartmann, M.; Ernst, S. Chem. Commun. 2001, 19781979. (2) Yonsel, S.; Schaefer-Treffenfeldt, W.; Kiss, A.; Sextl, E.; Naujok, H. DE Patent 4217203, 1993. (3) Stockhammer, S.; Schaefer-Treffenfeldt, W.; Knaup, G.; Drauz, K.; Sextl, E. DE Patent 19535751, 1997. (4) Jeong, H.; Krohn, J.; Sujaoti, K.; Tsapatsis, M. J. Am. Chem. Soc. 2002, 124, 12966-12968. (5) Tsapatsis, M. AIChE J. 2002, 48, 654-660.
acids and microporous materials in order to advance related fields such as the templating of new porous materials6-8 and the study of reactions in confined spaces.9 Background Amino Acids. Amino acids are comprised of basic amino and acidic carboxyl functional groups as well as a characteristic side chain. Aside from participating in condensation reactions that lead to peptide formation, the amino and carboxyl groups give the amino acid unusual electrolytic properties. At high pH, the carboxyl group tends to be dissociated, giving the compound a negative charge. At low pH, the amino group, as well as the overall molecule, becomes positively charged. At an interim pH known as the isoelectric point, amino acid in solution has, on average, a net charge of zero. At this point, a solution contains positively and negatively charged amino acid ions in equal quantities. A solution at the isoelectric point, pI, also has a large concentration of ionic species that are comprised of both charged amino and carboxyl groups. This dually charged species, referred to as zwitterions, can be considered to be net-neutral with respect to charge. The interconversion of these charged species is detailed in Figure 1. The side chain, designated in Figure 1 with an R, can be a dissociating functional group as well. In the case of arginine and lysine (see Figure 2) the side chain has an amino group allowing the molecule to carry a +2 charge at low pH. Glutamic acid has two carboxyl groups allowing for a -2 charged ion. In contrast, phenylalanine’s side chain contains a phenyl group. This nondissociating, lowpolarity side chain makes phenylalanine more hydrophobic than most of the 20 common amino acids. In light of the equilibria given in Figure 1 and assuming R is nondissociating, the effect of pH on the charged species concentrations can be determined by use of dissociation equilibrium relations,
K1 )
mA ( mH+ mA+
K2 )
mA-mH+ mA(
(1)
Here mA+, mA-, mA(, and mH+ are the cationic, anionic (6) Che, S.; Garcia-Bennett, A. E.; Yokoi, T.; Sakamoto, K.; Kunieda, H.; Terasaki, O.; Tatsumi, T. Nat. Mater. 2003, 2, 801-805. (7) Belton, D.; Paine, G.; Patwardhan, S. V.; Perry, C. C. J. Mater. Chem. 2004, 14, 2231-2241. (8) Davis, M. E.; Lobo, R. F. Chem. Mater. 1992, 4, 756-768. (9) Zamaraev, K. I.; Salganik, R. I.; Rommannikov, V. N.; Vlasov, V. A.; Khramtsov, V. V. Dokl. Chem. 1995, 340, 56-57-58.
10.1021/la0511788 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/18/2005
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Figure 1. Amino acid dissociation equilibrium.
Figure 2. Pertinent amino acids.
and zwitterionic amino acid concentrations respectively, followed by the free proton concentration. Given the definition for the total amino acid concentration
mAT ≡ mA+ + mA- + mA(
(2)
each individual charged species concentration can be determined in terms of the free proton and total amino acid concentrations, e.g.
mA- )
1 mAT 1 + mH+/K2 + mH+2/K1K2
(3)
Figure 3 shows the relative concentrations, e.g.,
xA- ) mA-/mAT, obtained using published dissociation constants.10 While the phenylalanine plot is generated as given above, production of the other plots requires consideration of a fourth species of +2 or -2 charge. Comparison of the plots in Figure 3 highlights some important differences resulting from variation of the amino acid side chain. Phenylalanine has a pI of 5.48 and carries a very low net charge over much of the moderate pH range. Over this same pH range, lysine and arginine are largely cationic in nature. Lysine’s pI is 9.74 while arginine’s is 10.76. Because zeolite frameworks carry a negative charge, it is reasonable to suspect this difference in amino acid charge will induce variation in interactions between adsorbate and adsorbent. While adsorption results are deferred to a later section, differences in adsorption characteristics have been reported and are the basis for our focus on phenylalanine and the bi-amino-group species, arginine and lysine. Comparisons of properties other than charge characteristics may also be made. On the basis of bond length and angle, phenylalanine, lysine and arginine have been approximated as cylinders having dimensions 9.7 × 4.7, 11.3 × 3.5, and 11.0 × 4.7 Å respectively.11 Hydrophobicity presents a more stark dissimilarity between these amino acids. Free energies of transfer between organic and water phases12 reveal phenylalanine to be one of the most hydrophobic of the common amino acids while lysine and arginine are among the most hydrophilic. Electrostatic, hydrophobic and steric interactions are all likely to be important effects in amino acid adsorption on microporous materials.
Figure 3. Relative concentrations of ionic species. Data presented for phenylalanine (Phe), arginine (Arg), glutamic acid (Glu) and lysine (Lys). xA+, xA-, and xA( represent fractions of amino acid in solution existing as cationic, anionic and zwitterionic species, respectively. Species with charges of +2 and -2 are also shown.
Amino Acid Adsorption on Zeolite β
Figure 4. Amino acid adsorption on zeolite β (Si/Al ) 25). Adapted from ref 1.
Zeolites. Zeolites are microporous crystalline aluminosilicates having narrow pore size distributions with average dimensions ranging 0.3-3 nm.13,14 The zeolite framework consists exclusively of corner-sharing AlO4 and SiO4 tetrahedra. The presence of aluminum produces a negative charge balanced by nonframework cations that are readily exchanged. Ion exchange can be carried out with various cationic species, and protonation is possible allowing the production of highly acidic surfaces. Furthermore, the number of charged sites in the zeolite, and therefore the capacity for cation exchange, can often be raised by increasing the aluminum content of the framework. However, this increase in aluminum concentration also decreases the zeolite’s hydrophobicity. Because of this additional effect, a change in aluminum content can have varying effects on amino acids of different hydrophobicity. Zeolite β was used in all adsorption experiments reported in the current paper. Zeolite β has three mutually orthogonal and interconnected 12-member-ring pore systems with linear channels along two axes and nonlinear channels along the third.15 Its largest pore openings are 6.6 × 6.7 Å.16 Adsorption. The results most pertinent to our work come from the publication by Munsch et al.1 Some of their findings on protonated zeolite β with Si/Al ) 25 have been reproduced in Figure 4. Important features include the following: (1) Phenylalanine is adsorbed to a greater extent at low pH than it is at high. While not shown in Figure 4, this is also true for glutamic acid. (2) Phenylalanine adsorption also displays a region of pH insensitivity with a relatively sudden transition occurring at moderate pH conditions. (3) Lysine’s loading increases with increasing pH and exceeds the loading of phenylalanine under alkaline conditions. Furthermore, the lysine results demonstrate no plateau within the pH range investigated. In the same report, the authors speculated as to the role steric and hydrophobic effects played on adsorption. With phenylalanine on aluminum-free zeolite β, a pronounced step change occurred in loading vs solution (10) Lide, D. R., Ed. CRC Handbook of Chemistry and Physics, 85th ed.; Section Title: Biochemistry; CRC Press: Boca Raton, FL, 2004/ 2005. (11) Ching, C. B.; Hidajat, K.; Uddin, M. S. Sep. Sci. Technol. 1989, 24, 581-597. (12) Radzicka, A.; Wolfenden, R. Biochemistry (N.Y.) 1988, 27, 16641670. (13) Barrer, R. M. In Zeolites and Clay Minerals as Sorbents and Molecular Sieves; Academic Press: London, 1978; p 496. (14) Sherman, J. D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 34713478. (15) Newsam, J. M.; Treacy, M. M. J.; Koetsier, W. T.; De Gruyter, C. B. Proc. R. Soc. London, Series A: Math., Phys. Eng. Sci. 1988, 420, 375-405. (16) Baerlocher, C., Meier, W. M., Olson, D. H., Eds. Atlas of Zeolite Framework Types; Elsevier: Amsterdam, 2002; p 308.
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concentration. This was rationalized as a result of interactions between the adsorbed-species with the step occurring when adsorbate clustering becomes significant. With H-ZSM-5, they argued that restricted pore necks diminish interaction between adsorbed phenylalanine molecules, resulting in unexpectedly lower adsorption relative to zeolite β. Interestingly, a more recent report17 suggests that amino acid net charge has little effect on adsorption of phenylalanine on Na-ZSM-5. Matsui et al.18 carried the zeolite separation technique to larger biomaterials and speculated in great detail as to the forces involved: Below the isoelectric point, the driving force for adsorption could primarily be Coulombic in nature. At and above the pI, hydrophobic interactions and pore structure assume prominent roles. While adsorption of monomeric amino acids is influenced by the same forces governing peptide adsorption, electrostatics is likely to play a more prominent role. New information presented in this paper is organized as follows: (1) experiment description, (2) theory used in analysis of experimental findings, (3) experimental findings, and (4) modeling results. In each of these sections, phenylalanine information is presented prior to addressing arginine. Experimental Section Zeolite β powder was obtained from Zeolyst International with the following specifications: Si/Al mole ratio ) 12.5, 48, or 150; Na2O wt % ) 0.05 and surface area ) 680, 650, or 620m2/g, respectively. Elemental analysis of the three materials provided Si/Al values of 12, 48, and 100, respectively. Of these three materials, the first was received in an ammonium cation form, and was calcined prior to use in adsorption experiments. The calcination was carried out using a temperature ramping rate of 1.8 °C/min. The samples were first raised to 178 °C and held for 2 h. They were then raised to 450 °C and held for 5 h. Air circulation was maintained throughout the heating, and XRD (Siemens D5005) was used after calcining to confirm that no drastic structural change occurred. The other two materials, with Si/Al ) 48 and 100, were received in their protonated form, checked by XRD and ramped to 180 °C in air over a period of 4 h prior to use. Amino acid adsorption was carried out using L-arginine (BioChemika Ultra g99.5%) and l-phenylalanine (Aldrich 99%). Hydrochloric acid (Mallinckrodt 37%) and sodium hydroxide (Mallinckrodt 99%) were used for pH adjustment. A typical experiment would consist of first mixing the zeolite powder, deionized water and HCl or NaOH. After an hour of mixing, the amino acid(s) would be added and allowed to stir for 8-12 h at a temperature of 21-23 °C. Note that the equilibration time used was found to provide results within approximately 3% of the adsorption measured after 3 days of mixing. A sample, no larger than 0.5% of the total experiment volume, would then be withdrawn. This sample would be filtered twice with the second filter’s mesh size being 0.2 µm. Note that tests at pH values of 2-12 have confirmed that filtering does not result in amino acid loss. The amino acids were converted, using o-phthalaldehyde, to materials detectable or more reproducibly detected at ultraviolet wavelengths. High performance liquid chromatography, using an Agilent 1100 series instrument with a Zorbax Eclipse XDB column and UV detector, would then provide the equilibrated solution-phase amino acid concentration. Adsorption was determined by the change in solution-phase amino acid concentration as a result of zeolite contact. By drying and weighing the filters before and after use, an estimate would be made for the zeolite loss occurring upon removal of the HPLC sample. By accounting for losses, error was reduced when the original mixture was then adjusted to new conditions, mixed for another 8-12 h (17) Titus, E.; Kalkar, A. K.; Gaikar, V. G. Colloids Surf. As Physicochem. Eng. Asp. 2003, 223, 55-61. (18) Matsui, M.; Kiyozumi, Y.; Yamamoto, T.; Mizushina, Y.; Mizukami, F.; Sakaguchi, K. Chem.sEur. J. 2001, 7, 1555-1560.
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Phe+. One way to reflect this limitation is to introduce ms, the concentration of ion exchange sites with sufficient space to be a viable host for cationic phenylalanine adsorption. Ion exchange must then satisfy two site balances, eq 4 and
mPhe+ + ms ) mso Figure 5. Phenylalanine solubility limit: (0) solubility in water, as reported in the literature; (2) measured solubility with varying pH; (-) theoretical solubility with varying pH; (O) measured solubility in 1 M NaCl. and sampled again. The adjustment would be the dosing of additional amino acid or the addition of sodium chloride. All experiments were carried out with the same total solution volume and zeolite mass. Solubility Limit. Because adsorption was determined by solution-phase concentration change, the solubility limit was checked to ensure precipitation was not occurring. Solubility data were measured for phenylalanine and arginine. To determine the solubility limit, an amino acid was added to stirring water in sufficient quantity to produce an opaque slurry at a temperature of 21-23 °C. Adjustment of pH was carried out at this point. After stirring for approximately 20 min, the sample was then checked to ensure that a dense slurry was still present. The sample was then filtered twice as described above and analyzed by HPLC. Figure 5 displays the experimentally determined and theoretical pH dependencies of phenylalanine solubility. Also shown is a published19 value for phenylalanine in pure water. The theoretical solubility is determined first by assuming equilibrium has been achieved between the solid phase amino acid and the solution phase zwitterionic species. The equilibrium solution phase concentration of the zwitterionic species is established by the published, pure water solubility. As the pH is varied, the zwitterion concentration is held constant by solid/liquid phase equilibrium, but the other charged amino acid species can increase in relative concentration. This results in an overall increase in solution phase concentration with increasingly extreme pH conditions. An additional point is included in Figure 5 to demonstrate the solubility effect of NaCl addition. The pH dependence of arginine’s solubility limit (not reported) was similarly measured.
Results and Discussion Phenylalanine and arginine were chosen as the subject amino acids because of the dramatic difference in their response to pH variation. As illustrated in Figure 4, rising pH ultimately leads to diminished adsorption for phenylalanine. Arginine, as will be shown below, behaves similar to lysine, i.e., its adsorption rises as pH rises from 2 to 10. Adsorption Model. Ion Exchange Modeling. To begin building a thermodynamic equilibrium model for ion exchange of phenylalanine, we first assume that no anionic adsorbate species enters the zeolite. Electroneutrality for the zeolite would then require that all aluminum be paired with protons, sodium cations or cationic phenylalanine as follows:
mH+ + mNa+ + mPhe+ ) mAl-
(4)
Note that an overbar is used to designate a species within the zeolite while corresponding symbols lacking the overbar refer to bulk solution phase species. Steric hindrance can prevent phenylalanine from balancing the entire framework charge, i.e., with sufficiently low Si/Al, not all cations are exchangeable by (19) Nozaki, Y.; Tanford, C. J. Biol. Chem. 1971, 246, 2211-2217.
(5)
where mso is the initial concentration of this new type of site. h + Phe+ T H+ + Phe+, Given the ion exchange, H+ + S a selectivity coefficient is defined as
KPhe+ ≡
mH+mPhe+
(6)
mH+msmPhe+
Also, for H+ + Na+ T H+ + Na+,
KNa+ ≡
mH+mNa+
(7)
mH+mNa+
Note that equilibrated ion exchange is described without consideration of the electric double layer. For the purpose of the current work, this simplification introduces no significant error. Solution-phase electroneutrality,
mNa+ + mH+ + mPhe+ ) mCl- + mOH- + mPhe- + Q (8) and water’s equilibrium dissociation
Kw ≡ mH+mOH-
(9)
are also required in the model. Here, Q is the silanolgroup charge accumulation at the interface between the zeolite and bulk solution. Details regarding the treatment of this term are provided by Nikolakis et al.20 for the case of silicalite-1. We have adopted the dissociation equilibrium constant and site density values of this study. This estimation seems adequate given the relatively minor impact Q has on the model. The approach to modeling arginine adsorption is essentially the same as with phenylalanine with the exception that arginine is capable of assuming a +2 charge state. This new species is assumed to adsorb by the ion h + Arg2+ T 2H+ + Arg2+, and this exchange, 2H+ + S introduces the need for an additional selectivity coefficient
KArg2+ ≡
mH+2mArg2+ mH+2msmArg2+
(10)
The site balances now are
mH+ + mNa+ + mArg+ + 2mArg2+ ) mAl-
(11)
mArg+ + mArg2+ + ms ) mso
(12)
and
(20) Nikolakis, V.; Tsapatsis, M.; Vlachos, D. G. Langmuir 2003, 19, 4619-4626.
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Figure 6. pH Dependence of phenylalanine adsorption on H-β: (-[-) Si/Al ) 12; (-9-) Si/Al ) 48. Error bars are set at (2σ. Relevant solution conditions are summarized below.
Zwitterion Adsorption Modeling. We describe the adsorption of zwitterions as A+/- + B T A+/-, where B is a site available exclusively for physisorption of a zwitterion, A+/-. Langmuir assumptions are made; i.e., complexities such as adsorbate-adsorbate interaction are not addressed. With adsorption proceeding as written above, an adsorption equilibrium coefficient, K+/-, is defined as
K+/- )
mA+/-
(13)
mA+/-mB
and the adsorption site balance is noted as
mA+/- + mB ) mBo
(14)
Here mBo and mB respectively designate the initial and final unoccupied site concentrations. Finally, the ion exchange and zwitterion adsorption models are coupled by the amino acid material balance,
mAT + mAT ) mATo
(15)
Phenylalanine Experimental Results. Phenylalanine-adsorption pH profiles, on zeolite β with silicon-toaluminum ratios of 12 and 48, are shown in Figure 6. Each pH profile presented represents a collection of samples of common initial amino acid concentration. The amount of adsorption is given in millimoles of amino acid per gram of zeolite measured at room temperature and at the indicated pH. Error bars, included in Figure 6 and throughout this report, represent an estimate of (2σ based on HPLC analysis of 150 standards as well as four adsorption reproductions. The Si/Al ) 12 profile in Figure 6 is similar to the previously published data shown in Figure 4. Both profiles show phenylalanine displaying relative insensitivity of adsorption to pH across a broad regime on the acidic side of the pH scale and encompassing the amino acid’s isoelectric point. Both profiles also support that adsorption begins to decrease as the pH rises above a value of approximately 6. Our phenylalanine adsorption values are, in general, slightly lower than those previously reported,1 despite our zeolite having half the Si/Al. A comparison of absolute values is problematic because solution concentrations were not provided in ref 1. For the experiment used to create the Si/Al ) 12 profile presented in Figure 6, the final (equilibrated) solution phenylalanine concentra-
Figure 7. Phenylalanine adsorption isotherms on H-β: (-[-) Si/Al ) 12; pH ) 3.4-4.5; (-9-) Si/Al ) 48; pH ) 3.4-4.5; (2) Si/Al ) 12; pH ) 9.1-9.4. Error bars are set at (2σ.
tions were in the range 3.2 × 10-2 to 6.8 × 10-2 mol/L. For the experiment used to create the Si/Al ) 48 profile, the equilibrated concentration range was 3.5 × 10-2 to 4.5 × 10-2 mol/L. Aside from a difference in solution concentration, differences between zeolite protonation steps are among the possible contributing reasons for the adsorption discrepancy between ref 1 and the current study. We also found a small relative difference in behavior below a pH of 3. Our work indicates a small decrease in adsorption as HCl is used to drop the pH from 3 to 2, while such a decrease was not found in ref 1. We have found no evidence, from XRD and elemental analysis, to suggest that the zeolite at pH < 3 has lost crystallinity or experienced dealumination. The increase in adsorption, seen in Figure 6 across much of the pH scale, with the change from Si/Al ) 48 to 12 is readily explained with an ion exchange argument. Increased aluminum provides additional sites for the adsorption of cationic phenylalanine. The intersection of the two profiles under alkaline conditions suggests that the concentration of cationic phenylalanine has diminished to a level such that ion exchange is no longer a significant adsorption mechanism. This condition does not occur until the relative concentration for cationic phenylalanine is more than 2 orders of magnitude smaller than its value at the isoelectric point. Comparing the equilibrated solution phenylalanine concentrations, given above, with the adsorption isotherms shown in Figure 7, it is apparent that the above pH profiles were generated at or near adsorption site saturation. Still, while saturation is evident in the adsorption isotherm at pH > 9, reducing the pH of samples in this saturated regime increases the adsorption. This suggests the existence of at least two types of adsorption sites. Furthermore, adsorption at the high pH is driven by interactions weaker than the electrostatics of ion exchange, e.g., hydrophobic interactions, as evidenced by the lower initial slope of the high-pH isotherm. For the two low-pH isotherms given in Figure 7, pH was not controlled. Initially, the pH was roughly 3.4, but the addition of phenylalanine gradually drove both samples to a pH of 4.5. Still, this should not complicate the interpretation of the isotherm. Phenylalanine’s adsorption is not sensitive to pH in the range of the aforementioned pH drift. On the other hand, NaOH was used to maintain the pH of the adsorption isotherm obtained at pH > 9. This also should not complicate the results since phenylalanine adsorption, at this high pH, appears not to involve ion exchange. As long as the pH is held constant, the added sodium ions should have no effect on phenylalanine’s adsorption. Additional data will be presented below to support this argument.
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Figure 8. NaCl Response of phenylalanine adsorption on H-β: (-]-) No NaCl; (9) 0.1 M NaCl; (2) 1 M NaCl. Arrows designate the transition of particular samples from pre-salt to saltadjusted conditions.
Also shown in Figure 7, is the effect of changing Si/Al. An increase in Si/Al from 12 to 48 appears to reduce, but not eliminate, cationic phenylalanine adsorption. The initial slope remains steep, relative to the other isotherms, while the saturation loading is reduced. This reduction is possibly dampened by Si/Al’s effect on hydrophobicity of the zeolite, as discussed in the background section. Because variation of Si/Al induces competing effects on the total phenylalanine adsorbed, NaCl addition has been used to help clarify the effect of ion interactions. Figure 8 shows the response observed when NaCl was added. The salt was added to obtain 0.1 or 1 M NaCl concentrations prior to adsorption. For samples with a pH between 5.5 and 8.5, salt addition decreased the pH without significantly changing phenylalanine adsorption. When the initial pH was less than 4, 0.1 M NaCl produced little effect, while the higher dose of salt induced desorption of phenylalanine. Most interesting was the response to NaCl when the samples were initially at a pH above 9. These samples respond with increased phenylalanine adsorption. Furthermore, they appear to respond by moving along the no-NaCl pH profile. Together, these observations suggest that phenylalanine adsorption, at this high alkalinity, responds only to pH and not to the presence of added sodium ions. This finding, coupled with the fact that Si/Al has no significant effect at this pH, supports the idea that cationic phenylalanine is not available for ion exchange and that amino acid adsorption involves only zwitterions for pH > 9. Figure 9 depicts an indirect interaction, between added sodium ions and phenylalanine, which would lead to the observed increase in amino acid adsorption. Any salt-induced departure from the no-NaCl pH profile, as seen with pH < 9, is an indication of direct competition between added sodium ions and cationic phenylalanine. Figure 10 compares adsorption isotherms for zeolite β at conditions that minimize ion exchange. The similarity in results between Si/Al ) 12 and 48, at pH > 9, is anticipated given the overlap of results in Figure 6. With ion exchange of phenylalanine not playing a role, the influence of hydrophobic interaction will be increased. Therefore, the decrease in zeolite hydrophilicity, as a result of increasing the Si/Al from 48 to 100, is a likely reason for the increase in phenylalanine adsorption. Figure 10 also demonstrates that, for Si/Al ) 100, ion exchange of phenylalanine does not contribute substantially to adsorption even when the concentration of cationic phenylalanine is increased by lowering the pH from >9 to ∼6. Arginine Experimental Results. Arginine is chemically similar to lysine in that it has two amino functional
Figure 9. Effect of NaCl addition on phenylalanine adsorption at pH > 9. The addition of NaCl increases mNa+, which promotes desorption of protons by means of ion exchange. Desorbing protons reduce the solution pH and, at pH > 9, convert phenylalanine from its anionic state to its zwitterionic as depicted by Figure 3. Increasing mPhe+/- promotes zwitterionic adsorption, i.e., increasing mPhe+/-≈ mPheT.
Figure 10. Phenylalanine adsorption isotherms on H-β: (2) Si/Al ) 12; pH ) 9.1-9.4; (9) Si/Al ) 48; pH ) 9.2-9.9; (O) Si/Al ) 100; pH ) 6-6.5; (b) Si/Al ) 100; pH ) 9.4-9.8. Note that total phenylalanine adsorption is plotted vs the concentration of zwitterions in solution. Error bars are set at (2σ.
Figure 11. pH Dependence of arginine adsorption on H-β: (2) Si/Al ) 12; (9) Si/Al ) 48. Error bars are set at (2σ.
groups that can each gain a positive charge. This quality gives the two amino acids an extremely hydrophilic nature and the highest isoelectric points of all common amino acids. Similar to previously published lysine data,1 arginine adsorption increases as the solution pH rises toward the isoelectric point (see Figure 11). Phenylalanine Adsorption Modeling. Model parameter fitting is greatly simplified for the case of phenylalanine if we consider that for Si/Al ) 100, at any pH, and for Si/Al ) 12 and 48, at pH > 9, adsorption by ion exchange is negligible. Therefore, the data of Figure 10 can be used to estimate K+/- and mBo. Figure 12
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Figure 12. Models for phenylalanine loading isotherms on H-β: (2) Si/Al ) 12, pH ) 9.1-9.4; (9) Si/Al ) 48, pH ) 9.29.9; (O) Si/Al ) 100, pH ) 6-6.5; (b) Si/Al ) 100, pH ) 9.4-9.8. Modeling results are presented as lines. Note that total phenylalanine adsorption is plotted vs the concentration of zwitterions in solution. Table 1. Parameter Estimates for Zwitterionic Phenylalanine Adsorption on Zeolite β mBo
K+/Si/Al
est (mol/L)-1
CI (mol/L)-1
est (mmol/g)
CI (mmol/g)
12 48 100
100 150 150
40 a 40
0.45 0.45 0.59
0.07 a 0.05
a The small size of the Si/Al ) 48 data set confers little statistical merit on the fitted parameter estimates, and we report no confidence interval for these estimates.
Figure 14. Modeling phenylalanine loading isotherms given variations in Si/Al and pH. Modeling results are presented as lines. (a) Si/Al ) 12: ([) pH ) 3.4-4.5; (2) pH ) 9.1-9.4. (b) Si/Al ) 48: ([) pH ) 3.4-4.5; (0) pH ) 9.3; (×) pH ) 9.8. (c) Si/Al ) 100: ([) pH ) 6-6.5; (O) pH ) 9.8
Table 2. Parameter Estimates for Cationic Phenylalanine Adsorption mso
KPhe+
CI est CI est Si/Al (mmol/g)-1 (mmol/g)-1 (mmol/g) (mmol/g) 12 48 100
200 200
100
0.47 0.06 0
0.03 0.08
KNa+ est CI 0.003 0.001 0.003
presents Figure 10 overlaid with model results. Note that, in these figures, total phenylalanine adsorption is plotted vs the concentration of zwitterions in solution. Parameter estimates were obtained by nonlinear regression and are summarized in Table 1. Also included in Table 1 is the half-width of the 95% confidence interval associated with each estimate. The table shows that as Si/Al increases, o
both K+/- and mB increase. The remaining parameters can then be determined from experiments at conditions where ion exchange is significant. Table 2 presents parameter values used to fit data included in Figures 1315. While nonlinear regression was used to obtain all parameter estimates for Si/Al ) 12, values were assumed for the Si/Al ) 48 parameters, KPhe+ and KNa+.
Figure 13. Modeling a change in Si/Al and its effect on the pH profile. Modeling results are presented as lines: ([) Si/Al ) 12; (9) Si/Al ) 48; (-) mPheT, (--) mPhe+/-for Si/Al ) 12.
Figure 15. Modeling NaCl’s effect on phenylalanine’s pH profile. Points in this figure are from Si/Al ) 12 H-β: (]) no NaCl; (9) 0.1 M NaCl; (-) model of pre-salt conditions; (--) model for 0.1 M NaCl
Figure 13 reproduces Figure 6 and overlays modeling results. The predicted total phenylalanine adsorbed, mPheT ) mPhe+ + mPhe+/-, is represented by solid lines. While the model can reflect the change in adsorption caused by a change in Si/Al, a significant error is evident in the model’s ability to reflect the effect of pH as conditions become highly acidic. Complications such as adsorbateadsorbate interactions, not addressed by the current model, may be responsible for this deviation. Figure 14 shows that the model captures adsorption isotherm behavior over a wide range of pH, amino acid loading and Si/Al. In Figure 15, the model appears to predict reasonably well the final state of the NaCl-modified adsorption results from Figure 8. Arginine Adsorption Modeling. The above parameter fitting procedure cannot be applied in its entirety to arginine data. With phenylalanine, estimation of mBo and K+/- is facilitated by the existence of a pH range where cationic amino acid does not affect adsorption. Obtaining a similar condition for arginine would require a pH well above the point where dealumination of the zeolite becomes a problem. The uncertainty of mBo and K+/- then cascades to the remaining parameters. Despite this difficulty, we
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arginine’s 10.76. Therefore, our model and procedure for parameter estimation should be applicable to most of the common amino acids. Conclusion
Figure 16. Modeling arginine adsorption vs pH. Points in this figure are from Si/Al ) 12 H-β. Modeling results are presented as lines: (-) mArgT, (- -) mArg2+, (- - -) mArg+, and (- -) mArg+/-. Table 3. Parameter Values for Arginine Adsorption of Si/Al ) 12 Zeolite β KArg2+ KArg+ mso mBo K+/(mmol/g)-2 (mmol/g)-1 (mmol/g) (mol/L)-1 (mmol/g) 0.002
0.003
1.1
15
0.4
KNa+ 0.003
can demonstrate (see Figure 16) that the above model is capable of fitting arginine’s pH profile using parameter values listed in Table 3. Because arginine has the highest isoelectric point of the common 20 amino acids, it serves as the worst case scenario with respect to the problem of exposing the zeolite to highly alkaline conditions. Of the common 20 amino acids, 17 have pI values of 6.3 or less, compared with
A thermodynamic equilibrium model has been developed to describe amino acid adsorption on microporous materials. The model involves both cation exchange and zwitterion adsorption, with each mechanism being limited by spatial constraints. The procedure presented for fitting the model’s parameters should be applicable to the majority of the common 20 amino acids. Experiments were conducted, with phenylalanine, to locate the transition between the regime dominated by zwitterionic adsorption and the regime that includes ion exchange as a significant phenomenon. The effects, on the pH profile, of varying Si/Al and salt concentration suggest that ion exchange continues to be a significant adsorption mechanism, for phenylalanine, up to a pH of approximately 8.5-9. This is notable given the fact that phenylalanine’s isoelectric point is 5.48, and at a pH of 8.5, cationic phenylalanine exists in solution with a relative concentration on the order of 10-7. For Zeolite β with Si/Al ) 12, ion exchange accounts for two-thirds of mPheT under conditions of saturation loading and pH ) 4-7. When Si/Al is raised to 100, ion exchange no longer plays a significant role but the amount physisorbed increases by 30%. LA0511788
