Distribution of Isomorphic Amino Acids between a Crystal Phase and

We present distribution coefficients and crystal purity data for systems containing two of the three near-isomorphic amino acids L-leucine (LEU), L-is...
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Ind. Eng. Chem. Res. 2002, 41, 1873-1876

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Distribution of Isomorphic Amino Acids between a Crystal Phase and an Aqueous Solution Jeffrey Givand,† Bong-Kyu Chang,‡ Amyn S. Teja,* and Ronald W. Rousseau School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0100

We present distribution coefficients and crystal purity data for systems containing two of the three near-isomorphic amino acids L-leucine (LEU), L-isoleucine (ILE), and L-valine (VAL) in aqueous solutions. The data validate our earlier observation that the extent of impurity incorporation in amino acid crystals is related to the ratio of the pure-component solubility of the primary solute (or product) to that of the impurity in the same solvent. A simple quantitative relationship between the impurity distribution coefficients in aqueous systems was also obtained. This relationship makes it possible to predict purity data in isomorphic systems in a common solvent. Introduction work,1

we have shown that the incorIn our earlier poration of impurities by lattice substitution in amino acid crystals nucleated and grown from impure solutions is controlled, to a large extent, by the thermodynamics of the solid-liquid system. More specifically, we have shown that the extent of impurity incorporation is related to the ratio of the pure-component solubility of the primary solute (or product) to that of the impurity in the same solvent. We demonstrated the validity of this hypothesis by crystallizing a model solute (Lisoleucine, ILE) from solutions containing a model impurity (L-leucine, LEU). The solvents used were mixtures of water with various cosolvents and/or additives selected to affect crystal purity. The ILE/LEU system was chosen in our earlier study because biologically active ILE is often synthesized from fermentation broths, and small quantities of the isomorphic impurities LEU, L-valine (VAL), and R-aminobutyric acid are also produced in that process. Evidence from prior experiments has shown that lattice substitution is the dominant mechanism by which the given impurities are incorporated into ILE crystals.2,3 There is, therefore, an incentive to limit lattice substitution in this and similar amino acid systems. It is well-known that many crystallization characteristics are related to solute solubility. As a result, relationships for the solubility of several amino acids have been developed as functions of temperature, pH, and electrolyte or cosolvent concentration (for example, see refs 4-8). Bezard et al.9 attributed variations in the distribution coefficient of an impurity between a crystal phase and an aqueous solution to the solubility of that species in the solution. On the other hand, our previous work1 has shown that the relative distribution of two amino acids depends on their relative pure-component solubilities in the same solvent. o of two The pure-component relative solubility R23 solutes, where component 2 is the desired product and * To whom correspondence should be addressed. E-mail: [email protected]. Tel: 404-894-3098. Fax: 404-8942866. † Current address: Merck and Company. ‡ Current address: Department of Chemical Engineering, Auburn University.

component 3 is an impurity (component 1 is the solvent), is defined as

Ro23 ) xo2/xo3

(1)

where xoi is the pure-component solubility of species i in a given solvent. The distribution coefficient Ki is defined as

Ki ) zi/xi

(2)

where zi is the mole fraction of amino acid i in the crystal and xi is the mole fraction of amino acid i on a solvent-free basis in the mother liquor. The relative distribution coefficient of the impurity is then defined by

β32 ) K3/K2

(3)

We have shown previously that for a pair of amino acids in a variety of solvents

β32 ) f [Ro23]

(4)

This functional relationship allowed us to identify cosolvents and/or electrolytes that affect the relative solubility and, therefore, the crystal purity. In the present work, we explore the relationship o for pairs of amino acids. We also between β32 and R23 present experimental data on the purity of crystals obtained from aqueous solutions containing ILE/VAL, VAL/ILE, VAL/LEU, LEU/VAL, ILE/LEU, and LEU/ ILE (where the first-named component is the product and the second-named amino acid is the impurity in the crystal). Note that, in three of these systems (VAL/ILE, VAL/LEU, and ILE/LEU), the product amino acid is the more soluble species in water; in the remaining three systems, the product amino acid is the less soluble species in water. Experimental Section Crystallization experiments were conducted in a sterilized 150-mL jacketed glass vessel kept at a constant temperature by water circulating through the

10.1021/ie010759z CCC: $22.00 © 2002 American Chemical Society Published on Web 03/08/2002

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jacket of the vessel from a programmable water bath. The bath provided temperature control of (0.01 K. Amino acid solutions were prepared in a sterilized glass vessel by adding preweighed quantities of the amino acids, pure deionized water, and cosolvent and/ or electrolyte (as needed) in order to achieve the desired mole fraction or ionic strength of the components in the solution. These solutions contained 5, 10, or 15 mol % impurity amino acid (on a solvent-free basis). The container was then sealed, and the contents were heated and stirred until the solution was homogeneous. The final conditions were held constant for about 30 min to ensure that no solid amino acid was present in the solution. One hundred milliliters of the hot unsaturated amino acid solution was then transferred to the sterilized equilibrium vessel using a heated syringe. The solution was filtered through a 0.45-µm filter to remove any contaminants. The equilibrium vessel was also maintained at the elevated temperature of the solution being transferred, and its contents was continuously monitored for any signs of crystallization. The experiment was terminated if crystals were found to be present at the elevated temperature. The set-point temperature of the circulating water bath was then lowered until crystals of amino acid could be visually observed. The final temperature was held constant for at least 8 h to ensure equilibrium, after which a sample was withdrawn from the liquid phase and analyzed. Additional details of the sampling and analysis procedures are available elsewhere.10 Because the composition of the solid crystals was also desired, a method of extraction, filtration, washing, drying, and analysis of the crystals was developed for these studies. In addition, these tests were run in duplicate to obtain estimates of the variability of the crystallization process and experimental errors. After the liquid was sampled, the bath circulator was turned off and the contents of the equilibrium vessel poured through a filter under vacuum. Once all the mother liquor had been drawn through the filter, the wet crystals on the filter paper were rinsed three times with a total of about 100 mL of water in order to wash away any adhering mother liquor. A vacuum was drawn on the funnel and its contents for at least 15 min in order to dry the crystals sufficiently. The filter paper with adhered amino acid crystals was then removed from the funnel, and two 0.1-g samples of amino acid crystals were scraped from the filter paper. The samples were collected on weighing paper and added to separate empty 100-mL volumetric flasks. The amino acid samples were then dissolved in approximately 100 mL of purified water. After dissolution, 0.8 mL of each sample was pipetted into a vial and the relative amounts of amino acids were determined by high-performance liquid chromatography (HPLC) analysis. This sampling procedure was then repeated for a second sample. Several analytical techniques were employed to quantify and characterize the various liquid and crystalline samples taken during the experiments. The concentrations of amino acid in the saturated liquid samples were measured via HPLC analysis. Similarly, initial and final amino acid concentrations in the liquid from the crystalpurity experiments were measured using HPLC. The degree of incorporation of amino acid impurity in the

Table 1. Distribution of ILE between LEU Crystals and Water at 23.7 °C ILE solvent-free mol % in solution

ILE mol % in crystals

KILE

KLEU

βILE/LEU

5.82 5.90 11.72 11.69 16.92 17.35 22.91 22.90

1.88 1.63 3.14 3.22 5.52 4.28 6.89 7.19 average )

0.32 0.28 0.27 0.28 0.33 0.25 0.30 0.31 0.29

1.04 1.05 1.10 1.10 1.14 1.16 1.21 1.20 1.12

0.31 0.26 0.24 0.25 0.29 0.21 0.25 0.26 0.26

Table 2. Distribution of LEU between ILE Crystals and Water at 23.7 °C LEU solvent-free mol % in solution 5.41 5.55 9.89 9.90 14.11 14.21 18.86 18.75

LEU mol % in crystals

KLEU

KILE

βLEU/ILE

7.58 7.37 13.01

1.40 1.33 1.32

0.98 0.98 0.97

1.43 1.35 1.36

18.35 17.74 23.26 23.53 average )

1.30 1.25 1.23 1.25 1.30

0.95 0.96 0.95 0.94 0.96

1.37 1.30 1.30 1.33 1.35

Table 3. Distribution of VAL between ILE Crystals and Water at 25 °C ILE solvent-free mol % in solution

ILE mol % in crystals

KVAL

KILE

βVAL/ILE

1.35 1.38 6.42 6.47 12.67 12.40

0.25 0.28 1.39 1.33 2.49 2.72 average )

0.19 0.20 0.22 0.21 0.20 0.22 0.20

1.01 1.01 1.05 1.05 1.12 1.11 1.06

0.18 0.20 0.21 0.19 0.18 0.20 0.19

Table 4. Distribution of ILE between VAL Crystals and Water at 25 °C VAL solvent-free mol % in solution

VAL mol % in crystals

KILE

KVAL

βILE/VAL

4.74 4.74 9.44 9.28 12.95 13.12

5.19 5.47 12.24 12.23 16.73 16.91 average )

1.09 1.15 1.30 1.32 1.29 1.29 1.24

1.00 0.99 0.97 0.97 0.96 0.96 0.97

1.10 1.16 1.34 1.36 1.35 1.35 1.28

product crystals was also quantified using this chromatographic technique. Gas chromatography (GC) analysis was used to check for evidence of cosolvent incorporation in the amino acid crystals. Details of the HPLC and GC procedures are available elsewhere.10 Results As mentioned previously, crystallizations from aqueous solutions containing varying concentrations of pairs of these amino acids were conducted in the present work. The results of these experiments are presented in Tables 1-9. Pure-component solubilities of the three amino acids in aqueous solutions were measured in our earlier work1 and are shown in Figure 1. Relative o ) at approximately 25 °C are given in solubilities (R23 Tables 10 and 11. Figure 2 shows a plot of the ILE/LEU data in several aqueous solutions. The impurity (i.e., leucine) content

Ind. Eng. Chem. Res., Vol. 41, No. 7, 2002 1875 Table 5. Distribution of LEU between VAL Crystals and Water at 25 °C VAL solvent-free mol % in solution

VAL mol % in crystals

KLEU

KVAL

βLEU/VAL

6.67 6.67 10.43 10.74 14.14 14.61

10.05 10.31 16.93 16.56 23.44 23.26 average )

1.51 1.55 1.62 1.54 1.66 1.59 1.58

0.96 0.96 0.93 0.93 0.89 0.90 0.93

1.56 1.61 1.75 1.65 1.86 1.77 1.70

Table 6. Distribution of VAL between LEU Crystals and Water at 25 °C LEU solvent-free mol % in solution

LEU mol % in crystals

KVAL

KLEU

βVAL/LEU

7.36 7.55 12.94 13.07 18.99 19.12

0.34 0.33 0.76 0.72 1.11 1.02 average )

0.05 0.04 0.06 0.06 0.06 0.05 0.05

1.08 1.08 1.14 1.14 1.22 1.22 1.15

0.04 0.04 0.05 0.05 0.05 0.04 0.05

Figure 1. Solubilties of VAL (b), ILE ([), and LEU (2) in water.

Table 7. Distribution of LEU between ILE Crystals and Aqueous (NH4)2SO4 (I ) 1.6) at 23.7 °C LEU solvent-free mol % in solution

LEU mol % in crystals

KLEU

KILE

βLEU/ILE

5.66 5.67 9.75 9.76 14.51 14.48

6.62 6.51 11.98 11.90 17.32 17.32 average )

1.17 1.15 1.23 1.22 1.19 1.20 1.19

0.99 0.99 0.98 0.98 0.97 0.97 0.98

1.18 1.16 1.26 1.25 1.23 1.24 1.22

Table 8. Distribution of LEU between ILE Crystals and Aqueous Ethylene Glycol (20 mol %) at 23.7 °C LEU solvent-free mol % in solution

LEU mol % in crystals

KLEU

KILE

βLEU/ILE

4.55 4.55 9.18 9.11 13.80 13.72

6.03 5.94 11.35 11.26 17.36 17.25 average )

1.33 1.31 1.24 1.24 1.26 1.26 1.27

0.98 0.99 0.98 0.98 0.96 0.96 0.97

1.35 1.32 1.27 1.27 1.31 1.31 1.30

Table 9. Distribution of LEU between ILE Crystals and Aqueous DMSO (20 mol %) + CaCl2 (I ) 3.1) at 23.7 °C LEU solvent-free mol % in solution

LEU mol % in crystals

KLEU

KILE

βLEU/ILE

6.12 6.24 15.33 15.52 10.59 10.58

5.78 5.70 16.37 16.36 11.18 11.09 average )

0.94 0.91 1.07 1.05 1.06 1.05 1.01

1.00 1.01 0.99 0.99 0.99 0.99 1.00

0.94 0.91 1.08 1.06 1.06 1.05 1.02

of the product crystals is plotted on the y axis versus the impurity content of the mother liquor (on a solventfree basis) on the x axis. Note that the leucine-toisoleucine ratio in isoleucine crystals is about 30% greater than that in the equilibrium mother liquor when the solvent is water. This is also true when the solvent consists either of a 20 mol % solution of ethylene glycol in water or an ammonium sulfate solution of ionic strength I ) 1.6 mol kg-1. The data follow an approximately linear relationship between the impurity content of product crystals and the equilibrium mother liquors, and the lines through the data in Figure 2 are

Figure 2. Impurity (LEU) content of ILE crystals as a function of the LEU content of the mother liquor when the solvent is water (b), water + 20 mol % ethylene glycol (0), water + ammonium sulfate (9), and water + 20 mol % DMSO + CaCl2 ([). Table 10. Relative Solubilities and Distribution Coefficients of ILE/LEU in Aqueous Solutions at 23.7 °C solution

Ro23

K3

K2

β32

H2O (NH4)2SO4 (I ) 1.6) 20% ethylene glycol 20% DMSO + CaCl2 (I ) 3.1)

1.56 1.56 1.49 1.35

1.30 1.19 1.27 1.01

0.96 0.98 0.97 1.00

1.35 1.22 1.30 1.01

Table 11. Relative Solubilities and Distribution Coefficients of Amino Acid Pairs in Water at 25 °C system: product 2/ impurity 3

Ro23

K3

K2

β32

VAL/LEU VAL/ILE ILE/LEU LEU/ILE ILE/VAL LEU/VAL

2.70 1.72 1.56 0.64 0.58 0.37

1.58 1.24 1.30 0.29 0.20 0.05

0.93 0.97 0.96 1.12 1.06 1.15

1.70 1.28 1.35 0.26 0.19 0.05

linear fits through the origin. The slopes of these lines are the distribution coefficients K defined in eq 2. Distribution coefficients may also be calculated from the data given in Tables 1-9 and are summarized in Tables 10 and 11. Note that the distribution coefficient of leucine in the aqueous solutions mentioned above is approximately 1.3, confirming that there is a 30% enhancement in impurity content when isoleucine is crystallized from

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these aqueous solutions in the presence of leucine. By contrast, no enhancement in impurity content is obtained in the ILE/LEU system when the solvent is an aqueous solution of 20 mol % dimethyl sulfoxide (DMSO) and calcium chloride at an ionic strength I ) 3.1 mol kg-1. This particular solution is of interest because the relative solubility ratio of isoleucine to leucine in this o ) 1.35, which is significantly different solution is R23 o ) ∼1.5) in the previously mentioned from its value (R23 aqueous solvents (Table 10). The enhanced crystal purity obtained with this solvent conforms to the hypothesis stated in eq 4. As shown in our earlier work, the tendency of LEU to contaminate ILE crystals is greater than the tendency of ILE to contaminate LEU crystals because the solubility of ILE in pure water is higher than that of LEU in water. This behavior was also confirmed in the present work for other pairs of amino acids crystallized from pure water. Furthermore, the data in Table 11 also show that (i) in systems in which the product amino acid is less soluble in water than the impurity

βLEU/VAL ) βILE/VALβLEU/ILE

(5)

because βLEU/VAL ) 0.05, βILE/VAL ) 0.19, and βLEU/ILE ) 0.26 and (ii) in systems in which the product amino acid is more soluble in water than the impurity

βVAL/LEU ) βVAL/ILEβILE/LEU

(6)

because βVAL/LEU ) 1.70, βVAL/ILE ) 1.28, and βILE/LEU ) 1.35. Therefore, purity data need only be obtained for two out of the three pairs of amino acids in a common solvent. The third pair may be predicted using eqs 5 and 6. Similar relationships also hold for the distribution coefficients. Note that, in the case of the purecomponent solubility ratio, we may write

Roij ) Roik Rokj

(7)

which suggests that (eq 4)

solubilities are in the order xoi < xoj < xok (eq 5) or xoi > xoj > xok (eq 6). Conclusions We have shown that the prediction of the purity of crystals recovered from mother liquors containing small concentrations of nearly isomorphic impurities is possible from a knowledge of the relative solubilities of the impurity and product. Our work confirms that the solvent plays a significant role in determining crystal purity and provides a basis for adjusting the solvent composition to enhance crystal purity. Simple relationships have also been derived for pairs of amino acids in a common solvent (eqs 5 and 6). Acknowledgment Partial funding of this research by Ajinomoto, Inc., and the Georgia Research Alliance is gratefully acknowledged. Literature Cited (1) Givand, J.; Teja, A. S.; Rousseau, R. W. Effect of relative solubility on amino acid crystal purity. AIChE J. 2001, 47, 2705. (2) Koolman, H. C.; Rousseau, R. W. Effects of isomorphic compounds on the purity and morphology of L-isoleucine crystals. AIChE J. 1996, 42, 147. (3) Zumstein, R. C.; Rousseau, R. W. Solubility of L-isoleucine in and recovery from neutral and acidic aqueous solutions. Ind. Eng. Chem. Res. 1989, 28, 1226. (4) Nass, K. Representation of the solubility behavior or amino acids in water. AIChE J. 1988, 34, 1257. (5) Chen, C.; Zhu, Y.; Evans, L. B. Phase partitioning of biomolecules: solubilities of amino acids. Biotechnol. Prog. 1989, 5, 111. (6) Orella, C. J.; Kirwan, D. J. The solubility of amino acids in mixtures of wter and aliphatic alcohols. Biotechnol. Prog. 1989, 5, 89. (7) Orella, C. J.; Kirwan, D. J. Correlation of amino acid solubilities in aqueous aliphatic solutions. Ind. Eng. Chem. Res. 1991, 30, 1040. (8) Gupta, R. B.; Heidemann, R. A. Solubility models for amino acids and antibiotics. AIChE J. 1990, 36, 333. (9) Bezard, L.; Otto, W.; Beckmann, W. In Crystal Growth of Organic Materials 4; Ulrich, J., Ed.; Shaker Verlag: Aachen, Germany, 1997; p 131. (10) Givand, J. The Effect of Relative Solubility on Crystal Purity. Ph.D. Dissertation, Georgia Institute of Technology, Atlanta, GA, 1999.

(8)

Received for review September 10, 2001 Revised manuscript received January 23, 2002 Accepted February 1, 2002

However, eq 8 is valid only if the pure-component

IE010759Z

βij ) βikβkj