Separation of Amino Acids at a Synthetic Ion Exchange Resin

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Ind. Eng. Chem. Res. 1996, 35, 573-585

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Separation of Amino Acids at a Synthetic Ion Exchange Resin: Thermodynamics and Energetics W. Richard Bowen* and Enda Moran Biochemical Engineering Group, Department of Chemical Engineering, University of Wales, Swansea, SA2 8PP UK

The thermodynamics and energetics of the ion exchange of four amino acids at a synthetic ion exchange resin have been studied. Experimental work included determination of ion exchange isotherms and the use of high-sensitivity titration microcalorimetry. A rigorous thermodynamic analysis of the data is developed allowing calculation of the standard free energy, the standard enthalpy, and standard entropy of exchange, and also the differential free energy, incremental enthalpy, and incremental entropy of exchange. The results show that the relative contributions of the enthalpy and entropy to the overall free energy differ markedly for the chosen amino acids. From a knowledge of these fundamental thermodynamic properties, the solution conditions giving enhanced separation are predicted. These predictions are experimentally confirmed. The paper establishes experimental protocols and data analysis methods that allow progression from a single isotherm and a single set of calorimetric data to a selection of process conditions for enhanced selectivity. The experimental techniques and analysis procedures developed are generally applicable to ion exchange separations of biomolecules. Introduction Ion exchange is often the purification method of choice in the downstream processing of the products of biotechnological processes. Amino acids, antibiotics, and proteins are industrial products that include the use of ion exchange in their purification protocols (Pirotta, 1991). The high resolving power and capacity, the low energy requirements, and the relative simplicity of operation of ion exchange are reasons for its widespread use as a unit operation in industry. Ion exchange resins based on cross-linked polystyrene-divinylbenzene copolymers with cation or anion exchange capabilities are the most widely used materials, although ion exchangers manufactured from natural organic polymers such as dextran and cellulose are preferred for the purification of large labile biomolecules such as proteins. Ion exchange resins are used both for the analysis of amino acids (Blackburn, 1983) and in the large scale purification of these compounds, for example, tryptophan (Nakayama, 1985). Previously, researchers have conducted fundamental studies into the ion exchange behavior of amino acids at ion exchange resins. Seno and Yamabe (1960) examined the variation in the uptake of selected amino acids at cation and anion exchange resins with pH of the equilibrium solutions, while Feitelson (1963) measured the distribution coefficients of a number of amino acids at a polystyrenedivinylbenzene cation exchange resin. More recently, Kawakita et al. (1990) showed that the selectivity coefficients of a wide range of primary amino acids for a cation exchange resin in the ammonium form could be correlated reasonably well with a number of physicochemical parameters such as amino acid molecular weight. In their studies of the equilibrium uptake of amino acids at ion exchange resins, Jones and Carta (1993), Dye et al. (1990), and Wang et al. (1989) present models that allow the prediction of the amino acid uptake for multicomponent systems. The kinetics of uptake has also been studied (Saunders et al., 1989). Calorimeters enable the measurement of enthalpy changes in physical and chemical processes. Calori* To whom correspondence should be addressed.

0888-5885/96/2635-0573$12.00/0

metric investigations of ion exchange have been carried out previously (Bonner and Overton, 1961; Boyd et al., 1964), but only the exchange of inorganic ions was considered. Calorimetric studies of the adsorption of proteins at ion exchange materials has been carried out (Bowen and Hughes, 1993). Feitelson (1963) provided enthalpies of ion exchange for the exchange of amino acids at a cation exchange resin, but these enthalpies were calculated by examining the variation in the selectivity coefficient as a function of temperature. Enthalpies of adsorption of caffeine and phenol on polymeric sorbents have been measured (Maity et al., 1992). Otherwise, there is a lack of published information on the thermodynamics of ion exchange of amino acids and other organic molecules at commercially important ion exchange materials. The requirement of specialist equipment for experimentation may be one reason for this. In addition to the charged-based interaction in ion exchange, the selectivity for a particular molecule is often influenced by nonspecific effects within the ion exchanger such as counterion/resin and counterion/ counterion interactions. Such noncharge selectivity has not been rigorously investigated. The objectives of the present study were therefore to investigate in a thorough manner the ion exchange mechanism of a number of amino acids at a polystyrene-divinylbenzene ion exchanger, to quantify and understand the relative contribution of enthalpy and entropy to the overall free energy change on uptake, and to provide an evaluation of the nature and importance of the electrostatic and nonelectrostatic interactions that govern the ion exchange process. This fundamental understanding includes knowledge of equilibrium parameters and the thermodynamics of each process. A method of analysis was developed to calculate the standard enthalpies of ion exchange for each exchange process from experimentally measured enthalpies obtained using a titration microcalorimeter. It will be shown how these thermodynamic data can be used in the optimization of an ion exchange process by indicating the most appropriate solution conditions to cause an increase in the affinity for the desired molecule. Practically, more selective ion © 1996 American Chemical Society

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exchangers lead to increased economy in process time and materials. Furthermore, it will be demonstrated how to better define system conditions based on the consideration of thermodynamic data so as to increase the selectivity for a particular molecule from a multicomponent mixture. This is the basis for an improved separation process. In principle, the analysis procedures and techniques described in this paper can be applied to the development and optimization of ion exchange and adsorption processes for a wide variety of commercially important biomolecules. Materials and Experimental Methods Materials. The ion exchange resin used in this work, Dowex 50W X8 (Dow Chemical Co.), is a gel-type, strongly acidic cation exchanger consisting of sulfonic acid functional groups attached to a polystyrenedivinylbenzene matrix. It has a nominal divinylbenzene content of 8% (8% cross-linking). A nonfunctional adsorbent, BioBeads SM-4 (Bio-Rad Laboratories Ltd., UK), was used for adsorption experiments. This is a polystyrene-divinylbenzene adsorbent with properties similar to those of the Dowex 50W X8 but lacking the charged functional groups characteristic of the ion exchanger. The amino acids L-glycine, L-threonine, L-isoleucine, and L-tryptophan were obtained from Sigma Chemical Co., UK, as crystalline powders with minimum purity of 99%. These amino acids were chosen for their differing physicochemical properties. Experimental Methods. The small-ion capacity (Na+) of the Dowex 50W X8 resin was determined by the method described by Saunders et al. (1989) and the density was determined using the method described by Helferrich (1962). Dry weight measurements were obtained by observing the weight loss upon drying in an oven at 120 °C. Single amino acid solutions were analyzed using the ninhydrin colorimetric assay first developed by Moore and Stein (1948) and subsequently modified by Moore (1968). Tryptophan solutions were quantified by measuring UV absorbance at a wavelength of 218 nm. Analysis of mixtures of amino acids was carried out using the Waters PICO‚TAG amino acid analysis system. This method involved the precolumn derivatization of amino acid samples with phenylisothiocyanate (PITC) followed by separation using reverse phase HPLC and detection using a Waters 990 photodiode array detector measuring UV absorption at 254 nm. To construct ion exchange isotherms, batch equilibrium experiments were carried out in which 10 mL of amino acid solution was added to a series of test tubes containing 0.2 g of ion exchanger. Starting amino acid concentrations in the range 10-100 mM (2.5-40 mM in the case of tryptophan due to its limited solubility) were chosen. After equilibration was reached in a constant temperature room at 25 °C, the pH of each equilibrium solution was recorded and a sample was removed for assay. A mass balance knowing starting and equilibrium concentrations allowed the calculation of the extent of amino acid uptake. A stirred batch vessel was used for isotherm analysis that required temperature control at temperatures other than 25 °C and for amino acid ion exchange in methanol and urea solutions. The glass vessel was constructed with a jacket through which water from a thermostated water bath was circulated. Aliquots of amino acid solution were added to aqueous suspensions of ion exchanger, and periodic sampling provided samples for assay.

Adsorption isotherms for amino acid adsorption on SM-4 were carried out using this technique. Microcalorimetry was carried out using a Thermometric 2277 thermal activity monitor (Thermometric AB, Sweden), an isothermal heat conduction calorimeter. The mode of operation of calorimeters of this type is described by Wadso (1992). Both endothermic and exothermic events can be observed and quantified. Being a high-performance instrument, it has a limit of detectability as low as 0.15 µW with a baseline stability of (0.2 µW. Operating in titration mode, aliquots of amino acid solution were introduced discontinuously by the automatic titration device (Hamilton MicroLab M) into the calorimeter sample vessel containing stirred aqueous suspensions of ion exchanger or adsorbent. Computer control using Digitam 2.0 software allowed much flexibility in experiment design, for example, control of the volume of titrant added and the timing of titrant addition. Results were presented as thermal energy produced per unit time and stored digitally. In general, ion exchange and adsorption calorimetric titrations were performed as follows: 11 80 µL aliquots of amino acid solution were titrated in discrete steps into a 2 mL suspension of ion exchanger or adsorbent stirred at 40 rpm. Time was allowed between successive additions for equilibrium to be reached and the baseline to return to zero. Amino acid dilution experiments involved the titration of amino acid solution into 2 mL of distilled water. All experiments were carried out at 25 °C. Nature of Ion Exchange of Amino Acids Amino acids are amphoteric molecules that can be represented by the empirical formula H2N-CHRCOOH where R is a variable side group. In aqueous solution at neutral pH, amino acids exist almost entirely in zwitterion (dipolar) form, H3+N-CHR-COO-. In acidic solutions amino acids form cations (since -COOH f -COO- + H+ is suppressed) and in basic solutions they form anions, since amino groups are deprotonated and carboxyl groups dissociated. The degree of ionization is dependent on the acid/base strength of the groups, that is, on their pK values. For the amino acids used in the present work, the pK values of the carboxyl groups lie in the range 2.34-2.71 and the pK values of the amino groups lie in the range 9.39-9.68. The nature of the uptake of these molecules at ion exchangers depends on the solution conditions. For the present experiments, the following points are of importance: (i) For each amino acid the pH of the solution varied very little during the uptake, being in the range pH 4.55.0 for all experiments. At such pH values virtually all of the amino acid molecules are in the zwtitterion form in solution. (ii) From a knowledge of the density and hydrogen ion capacity of the ion exchange resin it is possible to calculate the internal pH (Dean, 1969). This is much less than pH 1.0. Hence, the carboxyl groups of the amino acids will be protonated inside the resin. (iii) It has been shown (Yu et al., 1987) that the separation factor for amino acid exchange is similar to the separation factor for amino acid zwitterion exchange at pH values well above the pK of amino acid carboxyl groups (such as those occurring in the present work). Others have also shown that dipolar ions can enter ion exchangers (Seno and Yamabe, 1960).

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Hence, the uptake of amino acid molecules in the present work is best described as the uptake of amino acid zwitterions and the subsequent protonation of the carboxyl group to form an amino acid cation. The amino acid remains bound at the resin functional group by electrostatic attraction with its positively charged amino group:

resin-SO3-H+ + H3+N-CHR-COO- f resin-SO3-H3+N-CHR-COOH (1) This is unlike a conventional ion exchange process in which the resin counterion is released to the bulk solution as part of the uptake mechanism of the preferred ion. Equation 1 is a description of the overall change occurring, such as is required for a thermodynamic analysis. Thermodynamics of Ion Exchange: Theory The uptake of biomolecules by ion exchangers is often described in terms of selectivity coefficients. Since selectivity coefficients are a function of the fractional amount of solute on the resin (Jones and Carta, 1993), their usefulness in the evaluation of the thermodynamic property ∆G°, the standard free energy of ion exchange, is limited. It is necessary to calculate the thermodynamic equilibrium constant, which is a true constant, to enable the evaluation of the standard free energy by the following well-known thermodynamic relationship:

∆G° ) -RT ln K

(2)

where T is the temperature on the kelvin scale and R is the molar gas constant (R ) 8.314 J K-1 mol-1). For the process

resin-H+ + AA( h resin-AA+

(3)

The true thermodynamic equilibrium constant K is (Cruickshank and Meares 1958)

NRAAHfRAAH K) NRHfRHCAAγAA

(4)

NRAAH and NRH are equivalent fractions of the amino acid and hydrogen ion on the resin at equilibrium and fRAAH and fRH are the activity coefficients of these components. CAA is the concentration of the amino acid at equilibrium expressed on the molar scale, and γAA is its associated activity coefficient. In specifying standard states for the case above, the activity coefficients fRAAH and fRH referring to the amino acid and hydrogen ion resinates are defined as being equal to unity for the exchangeable sites on the resin in pure amino acid and hydrogen ion form respectively and in equilibrium with an infinitely dilute solution. The standard state for the solution phase is based on concentration with activity coefficients defined so that as the amino acid concentration tends to zero (CAA f 0), the activity coefficient approaches unity (γAA f 1) (Flett and Meares, 1966; Redinha and Kitchener, 1963). Thus, the overall standard ion exchange process is as follows. A solution of amino acid at a concentration of 1 M is equilibrated with a sample of ion exchange resin containing 1 equiv of exchangeable H+ ions to give a resin containing 1 equiv of amino acid and a solution in which the amino acid concentration is zero. This scenario is hypothetical but thermodynamically correct.

The relative affinity coefficient Ka of the two ions for the resin is defined as (Reichenberg and McCauley, 1955)

Ka )

NRAAH NRHCAAγAA

(5)

While K is a true thermodynamic constant, Ka varies with the composition of the resin; it is a function of NRAAH, the fractional amount of amino acid on the resin. Combining eqs 4 and 5,

K ) Ka(fRAAH/fRH)

(6)

It can then be shown that (Davidson and Argensinger, 1953)

ln K )

∫01ln Ka dNRAAH

(7)

This last relationship allows the calculation of ln K and hence ∆G° from experimentally measurable quantities: ln K is calculated from the integration of ln Ka vs NRAAH plots (Bonner and Overton, 1961). The presence of the solution activity coefficient (γAA) does not necessarily complicate matters since if the equilibrium measurements are performed in dilute solutions (e0.1 M) the activity coefficients are approximately equal to unity (Lilley 1985). Similarly, in the case of amino acid adsorption at nonfunctional adsorbents,

ln KA )

∫01ln Kads dNRAA

(8)

where NRAA is the amount of adsorbed amino acid at each stage in the equilibrium process as a fraction of the total number of adsorption sites and Kads is the relative affinity coefficient of adsorption. The standard free energy of adsorption is

∆G° ) -RT ln KA

(9)

The differential free energy of ion exchange and adsorption is given by (Bonner and Pruett 1959)

∆G*n ) -RT ln Ka or ∆G*n ) -RT ln Kads

(10)

Considering the standard requirements mentioned above of the ion exchange process, the standard enthalpy of exchange is written: 0 0 ∆H° ) (HN - HN ) + (φ0H - φC)1 H ) RAAH)1 RAAH)0

(11)

where φ0H - φC)1 is the relative apparent molar heat H content of a 1 M solution of the amino acid and is equal to its heat of dilution. Heat of dilution curves of the amino acids were constructed from calorimetric data according to the procedure outlined by Harned and Owen (1950). A typical experimentally measured heat of dilution curve is shown in Figure 1. However, it is impossible to measure experimentally the enthalpy change for the exchange of exactly 1 equiv of amino acid ions represented by the first quantity in eq 11 above. In all of the microcalorimetric experiments, titration was carried out so that the resin, initially entirely in protonated form, was fully converted so that all of the exchangeable sites were occupied by an amino acid molecule. Consider the volume (aqueous suspension of ion exchanger) initially in the ampule, VA, and the volume added through titration, VT, separately.

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This expression for ∆H° is now in a form in which all the variables can be experimentally determined. The factor n converts the enthalpy to the number of kilojoules per mole of amino acid exchanged, hence n ) (moles adsorbed)-1. Similarly, it is also possible to calculate the incremental enthalpy changes, that is the successive enthalpy changes as the ion exchanger progresses from an ionic fraction of 0 to 1. For example, if the first addition of titrant gives a resin amino acid fractional loading of N1, then the standard incremental enthalpy change is 0 0 ∆H*1 ) (HN - HN ) + (φ0H - φC)1 H ) (17) RAAH)N1 RAAH)0

Figure 1. Heat of dilution curve for 0.229 M isoleucine.

The amino acid concentration in VA goes from 0 to some concentration x while the concentration in VT goes from T (initial concentration of titrant) to x at the end of the titration. The experimentally measured enthalpy change, ∆Hexpt, is composed of the following three terms:

(HNRAAH)1 - HNRAAH)0) + (i)

(φC)x H

φ0H)

(ii) VA

+

(φC)X H

φC)T H )

(iii) VT

(12) Quantity i is the enthalpy of exchange for a certain amount of amino acid: the amount in moles is determined by correlating previously collected isotherm data with concentrations of amino acid in solution and on the resin during a microcalorimetric titration. φC)x is H the molar heat content of amino acid solution of concentration x. It is apparent that since an infinitely dilute solution is the reference point of zero heat content (φ0H ) 0), then the quantity (φC)x - φ0H) in ii above H which applies to volume VA is equal in magnitude and of opposite sign to the heat of dilution of the amino acid at concentration x. The third term iii in eq 12 which applies to volumeVT can be separated into two measurable components. Then adjusting the terms ii and iii in eq 12 above for volume and concentration to give enthalpies in kilojoules, the final form of the experimental enthalpy change equation becomes

- φ0H) + ∆Hexpt ) (HNRAAH)1 - HNRAAH)0) + VAx(φC)x H VT[x(φC)x - φ0H) - T(φC)T - φC)0 H H H )] (13) If eq 13 is multiplied by a factor n so that the enthalpy change is for the exchange of 1 equiv of amino acid ions, then

This ∆H*1 is the enthalpy change for the exchange of 1 equiv of amino acid ions giving a resin ionic fraction of NRAAH ) N1. Similarly, ∆H* 2 is written as 0 0 - HN ) + (φ0H - φC)1 ∆H*2 ) (HN H ) (18) RAAH)N2 RAAH)N1

The experimentally measured enthalpy change is

∆H1expt ) (HNRAAH)N1 - HNRAAH)0) + VAC1(φC)C1 - φ0H) + V1T[C1[φC)C1 - φC)0 H H H ) T(φC)T - φC)0 H H )] (19) where C1 is the amino acid equilibrium concentration after the first addition of titrant and volume V1T refers to the incremental volume. Hence, in the present experiments V1T ) 80 µL, V2T ) 160 µL, and so on 2 to V11 T ) 880 µL. The expression for ∆Hexpt is

∆H2expt ) (HNRAAH)N2 - HNRAAH)N1) + VA[C2(φC)C2 - φ0H) - C1(φC)C1 - φ0H)] + H H C)T V2T[C2(φC)C2 - φC)0 - φC)0 H H ) - T(φH H )] (20) 2 is the enthalpy change for the In this case ∆Hexpt exchange of a certain number of amino acid ions as the ionic fraction increases from NRAAH ) N1 to NRAAH ) N2 and the equilibrium solution concentration increases from C1 to C2. By multiplying eq 19 by a factor n1 so that the enthalpy change is for the exchange of 1 equiv of amino acid ions,

n1∆H1expt ) n1(HNRAAH)N1 - HNRAAH)0) +

n∆Hexpt ) n(HNRAAH)1 - HNRAAH)0) + nVAx(φC)x - φ0H) + nVT[x(φC)x - φ0H) H H T(φC)T - φC)0 H H )] (14) Since 0 0 - HN ) (15) n(HNRAAH)1 - HNRAAH)0) ) (HN RAAH)1 RAAH)0

eqs 14 and 11 may be combined to give

- φ0H) ∆H° ) n∆Hexpt - nVAx(φC)x H

n1VAC1(φC)C1 - φ0H) + H C)T n1V1T[C1(φC)C1 - φC)0 - φC)0 H H ) - T(φH H )] (21) 0 0 and by eliminating (HN - HN ) from eqs 17 RAAH)N1 RAAH)0 and 21,

∆H*1 ) n1∆H1expt - n1VAC1(φC)C1 - φ0H) H C)T n1V1T[C1(φC)C1 - φC)0 - φC)0 H H ) - T(φH H )] +

(φ0H - φC)1 H ) (22)

nVT[x(φC)x - φ0H) - T(φC)T - φC)0 H H H )] + (φ0H - φC)1 H ) (16)

and similarly for ∆H* 2,

Ind. Eng. Chem. Res., Vol. 35, No. 2, 1996 577

Figure 2. Ion exchange isotherms with best fit polynomial curves for exchange of (a) glycine, (b) isoleucine, (c) threonine, and (d) tryptophan at Dowex 50W X8.

∆H*2 ) n2∆H2expt n2VA[C2(φC)C2 - φ0H) - C1(φC)C1 - φ0H)] H H C)T n2V2T[C2(φC)C2 - φC)0 - φC)0 H H ) - T(φH H )] +

(φ0H - φC)1 H ) (23) and so on for other equilibrium stages. The equations for ∆H* 3, ∆H* 4, etc. are written with minor but obvious adjustments to the equations given. It is possible to calculate the full range of incremental enthalpy values ∆H*n (n ) 1 f 11) aided by heat of dilution curves and calorimetrically measured heats of exchange. The standard entropy of ion exchange, ∆S°, is calculated form the Gibbs energy function describing the standard process:

∆G° ) ∆H° - T∆S°

(24)

and the incremental entropy, T∆S*n, from

∆G*n ) ∆H*n - T∆S*n

(25)

Results: Isotherms and Microcalorimetry Typical equilibrium uptake data for the ion exchange of the four amino acids are shown in Figure 2. Best fit polynomial equations correlate equilibrium solution concentrations with resin amino acid composition. Isotherms for the adsorption of isoleucine and tryptophan at SM-4 are shown in Figure 3. Typical microcalorimetric data are shown in Figure 4 for the titration of each amino acid into suspensions of Dowex 50W X8. All processes shown are exothermic. Each peak represents an 80 µL dose of amino acid titrant. An integration performed by Digitam 2.0 under each of these digitally

Figure 3. Adsorption isotherms and best fit polynomial curves for adsorption of (a) isoleucine and (b) tryptophan at SM-4.

stored peaks quantifies the thermal event in joules of heat energy. The calorimetric data for the adsorption

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Figure 5. Microcalorimetric titration curves for the titration of isoleucine and tryptophan into suspensions of SM-4: (a) 0.05 M Ile/0.05 g of SM-4; (b) 0.025 M Trp/0.05 g of SM-4. Table 1. Equilibrium Uptake Parameters and Standard Thermodynamic Functions for Ion Exchange of Amino Acids at Dowex 50W X8

Figure 4. Microcalorimetric titration curves for titration of amino acids into suspensions of Dowex 50W: (a) 0.666 M Gly/0.15 g of Dowex 50W; (b) 0.191 M Ile/0.04 g of Dowex 50W; (c) 0.420 M Thr/ 0.1 g of Dowex 50W; (d) 0.025 M Trp/0.005 g of Dowex 50W.

of isoleucine and tryptophan at SM-4 are shown in Figure 5. Heats of dilution calorimetric data were of similar graphical form to Figures 4 and 5 with the exception of glycine, whose heat of dilution was endothermic. Analysis and Discussion Thermodynamic Equilibrium Constants and Capacities. The thermodynamic equilibrium constants and saturation capacities for the ion exchange of each amino acid are shown in Table 1. The saturation capacity is taken as the uptake corresponding to the final equilibrium concentration measured for each isotherm. Included in this table are the calculated standard thermodynamic functions for the exchange processes. The equilibrium constant for the exchange of tryptophan (K ) 5580) is approximately 25 times that of the equilibrium constant for threonine (K ) 217). It is apparent that factors additional to the electrostatic interaction are important in affecting the selectivity of

thermodynamic functions

glycine

isoleucine

threonine

tryptophan

K capacity (mmol/g) ∆G° (kJ mol-1) ∆H° (kJ mol-1) T∆S° (kJ mol-1)

772 5.52 -16.47 -9.78 +6.69

820 5.14 -16.63 -3.16 +13.47

217 4.32 -13.33 -5.43 +7.90

5580 4.83 -21.37 -17.75 + 3.6

the ion exchanger for the amino acids. The selectivity for the amino acids increases in the sequence Thr < Gly < Ile < Trp, which is in accordance with the increasing hydrophobicity of the amino acids as defined by Jansen and Ryden (1993). Similar observations were reported for amino acids by Dye et al. (1990) and for other ions by Millar et al. (1964). The hydrophobic interaction, mainly attributed to solvent rearrangements around apolar molecules (Huque 1989), between tryptophan side chains and the resin matrix is augmented by the attractive van der Waals interactions that occur between benzene rings of the side group and the benzene rings of the resin polymeric matrix. This type of interaction (Creighton, 1993) is responsible for the very large K for the tryptophan exchange at Dowex 50W X8. Although isoleucine is more hydrophobic than glycine (+13.23 kJ mol-1 compared to +0.42 kJ mol-1, scale of Jansen and Ryden (1993)), there is relatively little difference between their equilibrium constants of exchange. However, Wolfenden et al. (1981) measured the hydrophilicities of the amino acid chains using model compounds in which the main amino acid backbone was replaced by a hydrogen atom. The hydrophilicity of isoleucine in this scale (-1.01 kJ mol-1) differs little from glycine which is taken as an arbitrary zero. This is consistent with the similar observed ion exchange behavior of these two molecules. Since the side chains

Ind. Eng. Chem. Res., Vol. 35, No. 2, 1996 579 Table 2. Equilibrium Uptake Parameters and Thermodynamic Functions for Adsorption of Isoleucine and Tryptophan at SM-4 thermodynamic functions

isoleucine

tryptophan

K capacity (mmol/g) ∆G° (kJ mol-1) ∆H° (kJ mol-1) T∆S° (kJ mol-1)

478 0.85 -15.28 -6.19 +9.08

961 0.27 -17.02 -44.95 -27.93

of glycine and isoleucine are similar in terms of hydrophilicity, then it is unsurprising that they display similar affinities for the ion exchanger. Threonine, with an hydroxyl group in its side chain, has a hydrophilicity of -30.44 kJ mol-1. This clearly shows that threonine would prefer the aqueous environment to the apolar environment of the Dowex 50W X8 resin and is the reason for its relatively low equilibrium constant. The total ion exchange capacity of the resin is not reached in all cases. Steric hindrance, or blocking of exchange sites by bound molecules, is known to occur in ion exchange processes (Schwarz and Boyd, 1965). This phenomenon will depend mainly on molecular size for a given degree of cross-linking. As expected, the capacity of Dowex 50W X8 is highest for the smallest amino acid, glycine. Hydrophobic and van der Waals interactions of tryptophan side chains at the SM-4 adsorbent surface (also has constituent benzene rings) account for the higher K of adsorption compared to isoleucine (Table 2). Steric factors probably account for the higher capacity of the adsorbent for isoleucine, but in both cases capacity is much lower than the capacities of Dowex 50W X8 for these molecules. It is apparent, therefore, that the charged functional groups of ion exchangers play an important role in determining the uptake capacity of these polymers. Standard Free Energy (∆G°), Enthalpy (∆H°), and Entropy (∆S°). One notable feature of the data in Table 1 is that the ion exchange of tryptophan is clearly an enthalpy-driven process whereas that of isoleucine is essentially an entropy process. The negative values of the free energies and enthalpies for all ion exchange and adsorption processes indicate spontaneous, favorable processes in all cases. The ion exchange process is a complex system with many different contributions to the enthalpy and entropy of exchange. The enthalpies of interaction of the base adenine with itself in solution (-17.58 kJ mol-1 (Eftink and Biltonen (1980))) and phenylalanine at a Dowex 50W X4 ion exchange resin (-18.00 kJ mol-1 (Feitelson (1963))) compare to the standard enthalpy of exchange of tryptophan suggesting that the benzene ring interaction described above is responsible for the large negative enthalpy of ion exchange. In contrast, the (negative) adsorption enthalpy of phenol at polymeric adsorbents is reported to be much smaller in magnitude as π-bonding is not thought to occur in that case (Maity et al., 1992). The standard enthalpy of adsorption of tryptophan at SM-4 is much larger at -44.95 kJ mol-1, possibly indicating that the electrostatic interaction in the ion exchange process hinders to some extent an ideal molecule to surface interaction. The low standard entropy of ion exchange and negative entropy of adsorption are consistent with prominent intermolecular and molecule/surface interaction, since the restriction of translational and rotational freedom of the bound tryptophan molecules causes a substantial decrease in the entropy of the system.

Table 3. Comparison of Calculated and Experimentally Determined Standard Enthalpies for Ion Exchange of Isoleucine at Dowex 50W X8 ∆H° (kJ mol-1) enthalpic effect

calcd

electrostatic protonation pairwise interaction dehydration

-4.57 -1.10 +3.77 -3.52

total

-5.42

exptl

-3.16

The standard enthalpy for the isoleucine exchange is relatively smaller due to its inert branched aliphatic side chain. The hydrophobic interaction between resinbound isoleucine side chains and/or the side chains and the resin surface is based mainly on solvent restructuring around the interacting species and is thus less energetic than the equivalent interactions involving tryptophan. Hydrophobic molecules in aqueous solution order the surrounding water molecules into so-called “icebergs” (Nemethy and Scheraga, 1962). The disruption of these icebergs and release of the water molecules to the bulk solution as the hydrophobic molecule is sorbed by an ion exchanger or adsorbent causes a favorable, positive increase in the system (Woodburn et al., 1992). The positive entropy change due to these hydration changes in both the isoleucine ion exchange and adsorption processes outweighs any negative entropy change resulting from intermolecular and surface interactions. While the tryptophan and isoleucine exchanges can be described as enthalpy- and entropy-driven respectively, there is insufficient difference between the standard enthalpy and standard entropy of both the threonine and glycine exchanges to similarly label these processes. The larger standard enthalpy for the glycine exchange compared to threonine suggests that the additional van der Waals interactions that contribute to the affinity for the resin do not occur in the case of threonine. While attractive interactions may occur between bound glycine molecules, threonine molecules are known to repel each other in solution (Lilley, 1985). In addition, the consideration of the hydrophilicity of the threonine side chain and its anomalous behavior in apolar environments implies that side chain/surface interactions are unlikely to occur. For the ion exchange of an amino acid, the following contributions to the interaction energy should be considered: (i) energy of the electrostatic interaction, (ii) solvation changes as the amino acid is sorbed by the resin, (iii) interactions between amino acids at adjacent sites, (iv) interactions between amino acids and the structural matrix of the ion exchanger, and (v) energy of carboxyl group protonation. In assessing the relative contribution of these factors, the isoleucine exchange is taken as an example. The enthalpy of exchange of the ammonium ion (Bonner and Pruett, 1959) is taken as an approximation of the electrostatic interaction of the isoleucine amino group at an ion exchange site. If it is assumed that the ion exchanger is saturated with isoleucine molecules and that each isoleucine side chain pairs with one other, then this enthalpy (Franks, 1975) will contribute to the overall enthalpy. The enthalpy for the removal of the hydration water as the amino acid enters the hydrophobic environment of the resin and the enthalpy of carboxyl group protonation (Greenstein and Winitz, 1961) must also be included. Table 3 shows that the summation of these enthalpies provides a reasonable estimate of the overall enthalpy of exchange,

Figure 6. Differential free energy, incremental enthalpy and incremental entropy as a function of fractional coverage, NRAAH, for ion exchange of (a) glycine, (b) isoleucine, (c) threonine, and (d) tryptophan at Dowex 50W.

580 Ind. Eng. Chem. Res., Vol. 35, No. 2, 1996

Ind. Eng. Chem. Res., Vol. 35, No. 2, 1996 581

Figure 7. Differential free energy, incremental enthalpy and incremental entropy as a function of fractional coverage, NRAA, for adsorption of (a) isoleucine and (b) tryptophan at SM-4.

suggesting that the more important contributory mechanisms have been considered. However, as the overall standard enthalpy is the sum of several contributions of comparable magnitude but different sign, it would be difficult to calculate accurately the standard enthalpy by such a procedure unless the individual contributions were known precisely. Indeed, of the amino acids used in the present study, it is only for isoleucine that sufficient data are available to enable the present summation. Differential Free Energy (∆G* n), Incremental Enthalpy (∆H*n), and Incremental Entropy (∆S* n). The dependence of the thermodynamic quantities as a function of the fractional amount of amino acids on the ion exchanger and adsorbent is illustrated in Figures 6 and 7. The increase in the absolute value of the differential free energy as an ion exchanger or adsorbent becomes progressively saturated is indicative of the increased tendency for ion exchange or adsorption at sites with nearest sites occupied (Graham, 1959). This phenomenon is observed in all cases. However, analysis of the incremental enthalpy plots is necessary to provide a clearer overview of each process. For example, the approximately constant incremental enthalpy values for the wide range of NRAAH indicates that the enthalpy of

ion exchange of threonine is constant regardless of the presence of threonine molecules already bound on the resin. The absolute value of the enthalpy decreases as tryptophan saturates the ion exchange resin indicating that steric interference from bound molecules hinders to some extent the strong intermolecular interactions that can occur in solution. In contrast, incremental enthalpies for the adsorption of tryptophan on SM-4 increase in magnitude as the material becomes saturated indicating a more favorable and less restricted adsorption at this material. The exchange of isoleucine at Dowex 50W X8 is an endothermic process at high values of fractional loading. When the resin is practically saturated, the more entropy based hydrophobic interactions between isoleucine side chains predominate. The enthalpy data for the glycine exchange shows a trend similar to that of threonine. However, the greater magnitude of the incremental enthalpy (∼-4.0 kJ mol-1) represents the greater propensity for surface and intermolecular interaction compared to threonine. Optimization of Selectivity and Separation Manipulation of Solution Conditions for Single Amino Acids. While some work has been carried out

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Figure 8. Ion exchange isotherms with best fit polynomial curves for exchange of (a) isoleucine and (b) tryptophan at Dowex 50W X8 at different temperatures.

previously to assess the variation in the selectivity coefficient as a function of temperature (Bonner and Pruett, 1959; Kraus and Raridon, 1959), only the ion exchange of inorganic ions was considered. No published information is available describing the effects of various electrolyte conditions on ion exchange selectivity of organic molecules. The fact that the tryptophan and isoleucine exchanges are enthalpy- and entropy-driven processes respectively singles these out for further study. Consideration of the standard thermodynamic properties of these two processes allows prediction of variations in the thermodynamic equilibrium constant. Being strongly exothermic, the negative enthalpy contribution to the standard free energy for the tryptophan exchange should be increased at a lower temperature of 5 °C. In contrast, the large entropic element in the isoleucine exchange implies that increased temperature up to a certain maximum might result in an increased equilibrium constant due to the increased contribution from the T∆S term, even though the process is slightly exothermic. A second possible means of controlling selectivity is the use of a chaotropic agent such as urea that disrupts the normal ordered structure of water (Franks, 1975) and destroys the “iceberg” structures that cause favorable entropy increases in ion exchange processes. Hence, in urea solutions, decreases in the equilibrium constants are expected for both processes but perhaps more so in the case of isoleucine since this exchange is most dependent on the entropic processes of solvent rearrangements. Thirdly, it is possible to decrease the dielectric constant of amino acid solutions by the addition of a low dielectric solvent such as methanol. A lowering of the dielectric constant allows stronger electrostatic and van der Waals interactions

Figure 9. Ion exchange isotherms with best fit polynomial curves for exchange of (a) isoleucine and (b) tryptophan at Dowex 50W X8 in urea solutions of different concentrations. Table 4. Thermodynamic Equilibrium Constants and Standard Free Energies for Ion Exchange of Isoleucine and Tryptophan at Dowex 50W X8 under Different Conditions and in Various Solution Typesa isoleucine solution and physical conditions 5 °C 25 °C 40 °C 1 M urea 3 M urea 5 M urea 7 M urea 5% methanol 40% methanol

tryptophan

K

∆G° (kJ mol-1)

K

∆G° (kJ mol-1)

1160 821 1196 161 64.7 12.9 8.50 367 1990

-16.31 -16.63 -18.44 -12.58 -10.33 -6.35 -5.30 -14.63 -18.81

17660 5560 2807 817 261 109 56.2 5582 4070

-22.60 -21.37 -20.66 -16.61 -13.78 -11.62 -9.98 -21.37 -20.59

a Included for comparison is the equilibrium constant for the exchange in water under normal conditions (25 °C).

to occur (Israelachvili, 1991) thus yielding an increased equilibrium constant at high methanol concentration. The exchange behavior of tryptophan and isoleucine is shown graphically at different temperatures (Figure 8), in urea solutions (Figure 9), and in methanol solutions (Figure 10). The standard free energies of ion exchange and thermodynamic equilibrium constants for these processes are shown in Table 4. Tryptophan follows the predicted decrease in magnitude of ∆G° with increasing temperature. For the isoleucine exchange, the expected increase in magnitude of ∆G° with increasing temperature is also observed. However, the relative changes in the ∆G° for both isoleucine and tryptophan (compared to that at 25 °C) as the concentration of urea is increased are quite similar. For example, in 7 M urea the free energy for the isoleucine exchange is diminished by 11.33 kJ mol-1 and the free energy for the tryptophan

Ind. Eng. Chem. Res., Vol. 35, No. 2, 1996 583

Figure 11. Competitive ion exchange isotherms for exchange of isoleucine and tryptophan in an equimolar mixture at Dowex 50W X8 at 25 °C. Table 5. Standard Free Energies, Thermodynamic Equilibrium Constants, and Separation Factors for Separation of Tryptophan and Isoleucine in an Equimolar Mixture (20 mM) at Dowex 50W under Various Process Conditions ion exchange details 25 °C 5 °C 1 M urea/25 °C

Figure 10. Ion exchange isotherms with best fit polynomial curves for exchange of (a) isoleucine and (b) tryptophan at Dowex 50W X8 in methanol solutions of different concentrations.

exchange is decreased by 11.39 kJ mol-1. Thus, it appears that the changes in the solvation of the two amino acids caused by the addition of urea are comparable. The affinity of tryptophan for the Dowex 50W X8 is quite high in aqueous solution at 25 °C. While there is no benefit in operating this exchange in 40% methanol, the increase in magnitude of ∆G° is substantial for the isoleucine exchange in this concentration of methanol. In addition to the effect mentioned above, the lower dielectric constant promotes the ion exchange of these molecules by decreasing the solubility of the amino acids and repressing the dissociation of the carboxyl group. Changing the solution conditions may also change the structure of the ion exchange resin. Hence, although the trends are clear, great care is needed in the detailed interpretation of the results. Manipulation of Solution Conditions for Mixtures of Amino Acids. The degree of separation achieved between isoleucine and tryptophan in a mixture at Dowex 50W X8 is shown in a competitive isotherm (Figure 11) and quantified using a separation factor (Velayudhan, 1994) in Table 5. The first point of note is that the K for isoleucine in a mixed solution at 25 °C is substantially increased compared to its value in a pure solution (see Table 4), though the value of K for tryptophan remains unchanged. This suggests substantial interaction between isoleucine and tryptophan molecules giving enhanced separation of the former. The data for single amino acid solutions indicate a greater separation factor (enhanced selectivity) for a tryptophan/isoleucine mixture at low temperature. This is confirmed by the data at 5 °C shown in Table 5. Even though isoleucine and tryptophan interact in a mixture, operation at 5 °C gives a 60% increase in the separation factor compared to the value at 25 °C.

amino acids

∆G° (kJ mol-1)

K

RTrp Ile

tryptophan isoleucine tryptophan isoleucine tryptophan isoleucine

-21.37 -18.54 -22.56 -18.83 -17.26 -13.29

5580 1780 17360 3456 1060 214

3.13 5.02 4.96

Finally, Table 5 shows that the addition of 1 M urea to the amino acid mixture at 25 °C gives a 59% increase in the separation factor compared to the value in the absence of urea (a concentration of 1 M urea was chosen as uptake is significantly lowered at higher concentrations). This is probably a result of a decrease in amino acid/amino acid interactions, such as occurs when urea is used to denature proteins. That is, urea interferes with and diminishes the hydrophobic interactions between isoleucine and tryptophan side chains which are dependent on water restructuring around interacting moieties. Such a result could not have been predicted from the thermodynamic data on single amino acid solutions. However, it shows the finely balanced and complex nature of solvation and entropic effects for such ion exchange processes. Conclusions Ion exchange and adsorption isotherms are the classical means of studying the interaction of molecules at charged and uncharged surfaces. The use of isotherms in this work proved useful in the initial assessment of the factors affecting the ion exchange of amino acids at different materials. The uptake data was also a necessary requirement to enable the design of the calorimetric titrations. The microcalorimetry data provided thermodynamic descriptions of each process, and this allowed a more detailed analysis of the principal interactions involved in the ion exchange of the different molecular types. Most notably it was found that although both tryptophan and isoleucine showed high affinity for the resin, the uptake of tryptophan was enthalpy-driven whereas the isoleucine exchange was an entropy-driven process. The aim of the subsequent experimentation, namely, the examination of the ion exchange characteristics of isoleucine and tryptophan in different solution types, was to test the validity of the predictions it was possible

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to make regarding the ion exchange of these molecules if the thermodynamic functions (∆G°, ∆H°, T∆S°) were known. It was found that the effect of variation of temperature was in accord with the thermodynamic analysis, but that the results of manipulation of entropic contributions by addition of urea were complex and difficult to predict. Results for mixtures of amino acids were further complicated by tryptophan/isoleucine interactions. Nevertheless, significant increases in the separation factor for mixtures could be achieved by use of low temperature or addition of urea. The paper shows that the microcalorimetric data can provide new insights into the nature of ion exchange processes. These insights can be useful in the design and optimization of such separation processes. It should be noted that the amino acids studied in the present work may be considered as model compounds. The techniques and analysis procedures described are equally applicable as an aid in the design of ion exchange and adsorption processes for the isolation and purification of other commercially important products such as antibiotics, nucleotides, and vitamins. Acknowledgment We thank Zeneca Bioproducts and the SERC for supporting this work, in particular Dr. John Liddell (Zeneca) for useful suggestions in our discussions. We also thank Mr. Peter Williams of our department for his expert technical assistance with the thermal activity monitor. Nomenclature Ci ) concentration of species i (M) Cn ) successive equilibrium concentrations of amino acid in ampule during an eleven-step calorimetric titration (n ) 1 f 11) (M) fRi ) activity coefficient of ion i in resin 0 HN ) Enthalpy content of resin containing 1 equiv of Ri)1 ions i (kJ mol-1) K ) thermodynamic equilibrium constant of ion exchange KA ) thermodynamic equilibrium constant of adsorption Ka ) relative affinity coefficient of ion exchange Kads ) relative affinity coefficient of adsorption NRi ) amount of ion i on resin as fraction of total capacity of resin for ion i T ) concentration of amino acid titrant (M) V ) volume of solution in microcalorimeter ampule (L) VA ) volume of solution in microcalorimeter ampule before calorimetric titration (L) VT ) added volume of titrant to microcalorimeter ampule after calorimetric titration (L) x ) equilibrium concentration of amino acid in microcalorimeter ampule (M) ∆G° ) standard free energy of ion exchange (kJ mol-1) ∆H° ) standard enthalpy of ion exchange (kJ mol-1) ∆S° ) standard entropy of ion exchange (kJ mol-1) -1 ∆G* n ) differential free energy of ion exchange (kJ mol ) -1 ∆H* n ) incremental enthalpy of ion exchange (kJ mol ) -1 ∆S* n ) incremental entropy of ion exchange (kJ mol ) R ) separation factor ) ratio of K values in competitive exchange γi ) solution activity coefficient of ion i φi ) relative apparent molar heat content of solution of electrolyte i (kJ mol-1)

φcH ) relative apparent molar heat content of amino acid solution of concentration C (kJ mol-1)

Literature Cited Blackburn, S. Amino Acids and Amines. In Handbook of Chromatography; Zweig, G., Sherma, J., Eds.; CRC Press Inc.: Boca Raton, FL, 1983. Bonner, O. D.; Pruett, R. R. Effect of temperature on ion exchange equilibria. II The ammonium-hydrogen and thallous-hydrogen exchanges. J. Phys. Chem. 1959, 65, 1417-1420. Bonner, O. D.; Overton, J. R. Effect of temperature on Ion Exchange Equilibria. IV. Comparison of enthalpy changes calculated from equilibrium measurements and calorimetrically measured values. J. Phys. Chem. 1961, 65, 1599-1602. Bowen, W. R.; Hughes, D. T. Ion Exchange of Proteins: A Microcalorimetric Study of the Adsorption of Bovine Serum Albumin on Anion-Exchange Materials. J. Colloid Interface Sci. 1993, 158, 395-402. Boyd, G. E.; Vaslow, F.; Lindebaum, S. Calorimetric Determinations of the Heats of Ion Exchange Reactions. I. Heats of Exchange of the Alkali Metal Cations in variously crosslinked Polystyrene Sulfonates. J. Phys. Chem. 1964, 68, 590-597. Creighton, T. E. Proteins: Structures and Molecular Properties; W. H. Freeman & Co.: New York, 1993. Cruickshank, E. H.; Meares, P. Thermodynamics of Cation Exchange. I Determination of heats and free energies of exchange by resins. Trans. Faraday Soc. 1957, 53, 1289-1308. Davidson, A. W.; Argensinger, W. J. Equilibrium Constants of Cation Exchange. Ann. N. Y. Acad. Sci. 1953, 57, 105-115. Dean, J. A. Ion Exchange. In Chemical Separation Methods; Van Nostrand Reinhold Co.: New York, 1969; pp 86-125. Dye, S. R.; DeCarli, J. P.; Carta, G. Equilibrium sorption of amino acids by a cation exchange resin. Ind. Eng. Chem. Res. 1990, 29, 849-857. Eftink, M.; Biltonen, R. Thermodynamics of Interacting Biological Systems. In Biological Microcalorimetry; Beezer, A. E., Ed.; Academic Press: London, 1980; pp 343-412. Feitelson, J. Specific effects in the interaction between ion exchange resins and amino acid cations: Influence of resin cross-linkage. J. Phys. Chem. 1963, 67, 2544-2547. Flett, D. S.; Meares, P. Thermodynamics of Cation ExchangesPart 4. Uni-, bi-, and tri-valent ions on Dowex 50W. Trans. Faraday Soc. 1966, 62, 1469-1481. Franks, F. Aqueous Solutions of Amphiphiles and Macromolecules. In Water: A Comprehensive Treatise; Franks, F., Ed.; Plenum Press: London, 1975; Vol. 4. Graham, D. Adsorption Equilibria. Chem. Eng. Prog. Symp. Ser. 1959, 55, 17-23. Greenstein, J. P.; Winitz, M. Chemistry of the Amino Acids; John Wiley & Sons Inc.: New York, 1961; Vol. 1. Harned, H. S.; Owen, B. B. The Physical Chemistry of Electrolytic Solutions; ACS Monograph Series; Reinhold Publishing Co.: New York, 1950. Helfferich, F. Ion Exchange; McGraw Hill Co. Inc.: New York, 1962. Huque, E. M. The Hydrophobic Effect. J. Chem. Educ. 1989, 66, 581-585. Israelachvili, J. Intermolecular and Surface Forces; Academic Press: London, 1991. Jansen, J.-C.; Ryden, L. Protein Separation and Purification. In Biotechnology: Vol. 3 Bioprocessing; Stephanopoulos, G., Ed.; VCH: Weinheim, Germany, 1993; pp 618-642. Jones, I. L.; Carta, G. Ion Exchange of Amino Acids and Dipeptides on Cation Resins with Varying Degree of Crosslinking. 1. Equilibrium. Ind. Eng. Chem. Res. 1993, 32, 107-117. Kawakita, T.; Ogura, T.; Saeki, M.; Hayasaki, H. Selectivity Coefficients of Amino Acids for the Ammonium ion on a Strong Cation Exchange Resin. Agric. Biol. Chem. 1990, 54, 1-8. Kraus, K. A.; Raridon, R. J. Temperature Dependence of some Cation Exchange Equilibria in the range 0-200 °C. J. Phys. Chem. 1959, 63, 1901-1907. Lilley, T. H. Physical Chemistry of Amino Acid Solutions. In Chemistry and Biochemistry of the Amino Acids; Barrett, G. C., Ed.; Chapman & Hall: London, 1985; pp 591-624. Maity, N.; Payne, G. F.; Ernest, M. V.; Albright, R. L. Caffeine adsorption from aqueous solutions onto polymeric sorbents. The effect of surface chemistry on the adsorptive affinity and adsorption enthalpy. React. Polym. 1992, 17, 273-287.

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Received for review December 21, 1994 Revised manuscript received June 21, 1995 Accepted October 23, 1995X IE940755C

X Abstract published in Advance ACS Abstracts, December 15, 1995.