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Solubilities ofL -cystine,L -tyrosine,L -leucine, and glycine in sodium chloride solutions at various pH values
J. Chem. Thermodynamics 1998, 30, 379]387
Solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in sodium chloride solutions at various pH values Renzo Carta a Dipartimento di Ingegneria Chimica e Materiali Uni¨ ersita’ di Cagliari, 09123 Cagliari, Italy
The solubilities of four amino acids ŽL-cystine, L-tyrosine, L-leucine, and glycine. at T s 298 K and at the pH values of 0.00, 1.00, 2.00, 11.00, 12.00, and 13.00 in water solutions of sodium chloride, have been measured. The concentration of NaCl was varied from Ž0.0 to 3.0. mol . dmy3 . The solubility of L-cystine increased as the concentration of salt was raised from Ž0.0 to 3.0. mol . dmy3 . For the same variation of sodium chloride concentration the solubilities of L-tyrosine and L-leucine decreased. The solubility of glycine was not influenced significantly by the presence of NaCl. A simple relation between the ratio of the solubilities of the amino acids with, and without, salt and NaCl concentration is presented. The relation predicts well the solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in aqueous solutions of NaCl starting from known values of the solubilities in pure water. Q 1998 Academic Press Limited
KEYWORDS: solubility; amino acids; sodium chloride; equilibrium; thermodynamic model
1. Introduction Amino acids play an important role both in animal metabolism and in industrial processes. Since they are rarely found in nature in a free form, they must be obtained from hydrolysis of protein-containing materials, or by fermentation. These production methods often result in aqueous mixtures containing various solutes including several types of amino acids and inorganic salts at different concentrations. From a theoretical point of view the interactions between ions and small molecules with biological macromolecules are of considerable importance in determining the behaviour of macromolecules.Ž1,2. In particular, information on the thermodynamic properties of polar non-electrolytes in aqueous salt solutions helps to understand the conformational changes of molecules in solution produced by the addition of denaturants, or by the transport of charged solutes across membranes. The design and optimization of purification processes, especially those based on crystallization which are used extensively in the purification of mixtures of amino a
ŽE-mail: [email protected]..
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Q 1998 Academic Press Limited
380
R. Carta
acids, require the knowledge of thermodynamic data, and suitable thermodynamic models, which in their turn depend on the availability of adequate data. The solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in aqueous solutions can also give an indication on the influence of the impurities and, therefore, on the tendency of the possible incorporation of these impurities during crystallization processes. Solubility data of many amino acids in pure aqueous solutions Ž3 ] 5. and in aqueous solutions containing acids or bases Ž6 ] 8. are available in the literature. The solubility behaviour of amino acids in water and aliphatic alcohols solutions was investigated by Orella and KirwanŽ9. but increase in the availability of experimental solubility data helps in the comprehension of the chemistry of these molecules. We report here experimental results on the solubility of L-cystine, L-tyrosine, L-leucine, and glycine in solutions containing sodium chloride in concentrations c from Ž0.0 to 3.0. mol . dmy3 and at the pH values 0.00, 1.00, 2.00, 11.00, 12.00, and 13.00. These data provide a useful base for the development of a thermodynamic model that predicts Žif used for example with the data in reference 6. the solubilities of the amino acids studied here in NaCl aqueous solutions.
2. Experimental Acid solutions were prepared by adding known quantities of hydrochloric acid ŽCarlo Erba RPE 1.0 mol . dmy3 . to h.p.l.c. water, to reach the target pH values; basic solutions were prepared by diluting sodium hydroxide ŽCarlo Erba RPE 0.1 mol . dmy3 . with h.p.l.c. water. The final pH values of these solutions were measured using a pH meter ŽMetrohm 691, accuracy "0.01 units. calibrated using the two points method. For acid solutions the first point was a buffer solution at pH s 1.00, and the second point a buffer solution at pH s 4.00. When calibrating for basic solutions, two buffer solutions at the pH values of 10.00 and 13.00 were used, respectively Žbuffer solutions Carlo Erba RPE.. Sodium chloride was added to these solutions up to the required molar concentrations of salt. These solutions were then mixed with solid L-cystine ŽAldrich Chemie, mass fraction s 0.99, C 6 H 12 O4 S 2 , FW 240.30, CAS-num. 56-893., L-tyrosine ŽAldrich Chemie, mass fraction s 0.99, C 9 H 11 NO 3 , FW 181.19, CAS-num. 60-18-4., L-leucine ŽJansen, mass fraction ) 0.99, C 6 H 13 NO 2 , FW 131.18, CAS-num. 61.90.5., and glycine ŽJansen, mass fraction ) 0.99, C 2 H 5 NO 2 , FW 75.07, CAS-num. 56-40-6.. The label purity of the manufacturer was accepted and no pretreatment of the chemicals was considered necessary. The flasks containing the acid or basic solutions and the added amino acid were transferred to a thermostatic bath kept at T s Ž298 " 0.1. K. The solutions were continuously stirred by a magnetically-driven agitator. The target of T s 298 K was approached with two different, but similar, methods. In the first case the solution was kept at T s 303 K for 48 h, and in the second one the solution was kept at T s 293 K for the same period of time. The two solutions were then brought to T s 298 K and kept for 48 h at this temperature. Differences in the measured solubilities were within the accuracy of the analytical methods.
Solubilities of four amino acids in NaCl solutions
381
The hydration of the solid phase was checked using the procedure described by Carta and Tola.Ž6. The differences between the mass of the amino acids before, and after 48 h contact with h.p.l.c. water Žtaking into account the mass of the solid q the dissolved amount. were less than 1 per cent of the mass used for the test. The stirring time of 48 h was established via a trial and error procedure in order to achieve equilibrium conditions. In fact, L-cystine and L-tyrosine exhibited a very long dissolution time Ž)24 h., especially when the amount of solid amino acid was only a little greater than that required for the saturation of the solutions. Although the equilibrium for L-leucine and glycine was achieved in shorter times, these solutions were also equilibrated for 48 h. After 48 h stirring was discontinued and the remaining solid was allowed to precipitate. A sample of about 10 cm3 was taken using a syringe when the separation was almost complete. This sample was filtered in a constant temperature filter Žpaper Whatman a52. and the clear liquid was taken for analysis. Solutions containing L-cystine and L-tyrosine were analysed stectrophotometrically Žspectrophotometer Shimatzu UV 160A., according to the procedures of Carta and Tola.Ž6. Solutions containing L-leucine and glycine were analysed by drying known amounts of samples to constant weight. This procedure, known as the dry weight method, and extensively used,Ž4,10. was tested as previously reported.Ž6. The analysis was carried out by completely evaporating 3 cm3 of saturated liquid at T s Ž323 to 333. K, at atmospheric pressure, and weighing the mass of solid obtained. This maximum temperature was chosen to avoid an excessively rapid evaporation with possible loss of amino acids and thermal decomposition. The mass of sodium chloride in these solutions was taken into account in calculating the solubilities of the amino acids. Each solubility was obtained by averaging the concentration of three series of samples taken from three difference flasks. The maximum deviations of the nine samples were "1 . 10y5 mol . dmy3 for L-cystine and L-tyrosine, analysed by the spectrophotometer; "2.5 . 10y4 mol . dmy3 for L-leucine; and "5 . 10y4 mol . dmy3 for glycine, analysed by the dry weight method.
3. Results and discussion The measured solubilities of the four amino acids studied as a function of the sodium chloride concentration at the pH values 0.00, 1.00, 2.00, 11.00, 12.00, and 13.00 are shown in tables 1 to 4; these values relate to the stock solutions, and not the equilibrium ones Žthat are reported in the same tables in parentheses .. Actually the stock solutions pH values differ from those of the equilibrated solutions Žparticularly for the solutions containing L-leucine and glycine because of their high solubilities ., however, they give information on the amount of acid and base dissolved. The addition of salt has two effects on the solubility of non-electrolyte substances: the first one is associated with a decrease of the relative permittivity of the solvent,Ž11. and the second one with the solvation.
382
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TABLE 1. Experimental solubilities s for L-cystine as a function of sodium chloride conconcentration c at T s 298 K. ŽThe pH values in parentheses belong to the equilibrated solutions. pH: crŽmol . dmy3 .
0.00
1.00
0
0.09412 Ž0.00. 0.10315 Ž0.01. 0.10911 Ž0.01. 0.11864 Ž0.02. 0.12719 Ž0.02. 0.14087 Ž0.03. 0.14399 Ž0.02.
0.01701a Ž1.03. 0.02039 Ž1.05. 0.02365 a Ž1.05. 0.02398 Ž1.06. 0.02401 Ž1.06. 0.02447 Ž1.07. 0.02437 Ž1.08.
0.5 1.0 1.5 2.0 2.5 3.0 a
2.00 11.00 srŽmol . dmy3 . 0.00145 Ž2.11. 0.00156 Ž2.12. 0.00172 Ž2.14. 0.00189 Ž2.14. 0.00201 Ž2.15. 0.00234 Ž2.17. 0.00247 Ž2.17.
0.00161 Ž7.81. 0.00169 Ž7.81. 0.00181 Ž7.79. 0.00211 Ž7.79. 0.00233 Ž7.76. 0.00228 Ž7.79. 0.00242 Ž7.78.
12.00
13.00
0.00450 Ž10.93. 0.00465 Ž10.93. 0.00517 Ž10.92. 0.00552 Ž10.90. 0.00567 Ž10.90. 0.00593 Ž10.88. 0.00596 Ž10.89.
0.02825 a Ž12.28. 0.03125 Ž12.32. 0.03392 a Ž12.35. 0.03716 Ž12.31. 0.03722 Ž12.30. 0.04087 Ž12.29. 0.04212 a Ž12.32.
From reference Ž6..
Many substances reduce the relative permittivity of water when they are dissolved in it. For example, in table 5, some mixtures which produce this effect are shown. The same table shows the variation of the solubility of glycine Žcolumn 4. and L-leucine Žcolumn 5. as the mass fraction of the second solvent in the solutions increases from 0 to the maximum value. Although sodium chloride reduces the relative permittivity of water by an
TABLE 2. Experimental solubilities s for L-tyrosine as a function of sodium chloride conconcentration c at T s 298 K. ŽThe pH values in parentheses belong to the equilibrated solutions. pH: crŽmol . dmy3 .
0.00
1.00
0
0.10341 Ž0.02. 0.09943 Ž0.02. 0.09048 Ž0.01. 0.08671 Ž0.02. 0.08051 Ž0.00. 0.07804 Ž0.03. 0.06801 Ž0.01.
0.03575 a Ž1.17. 0.03489 Ž1.09. 0.03073 Ž1.21. 0.03016 Ž1.19. 0.02752 Ž1.17. 0.02625 Ž1.11. 0.02407 Ž1.17.
0.5 1.0 1.5 2.0 2.5 3.0 a
From reference Ž6..
2.00 11.00 srŽmol . dmy3 . 0.00695 Ž6.97. 0.00653 Ž6.96. 0.00657 Ž6.96. 0.00577 Ž6.95. 0.00541 Ž6.97. 0.00504 Ž6.94. 0.00455 Ž6.95.
0.00361 Ž7.79. 0.00359 Ž7.77. 0.00351 Ž7.77. 0.00211 Ž7.73. 0.00233 Ž7.76. 0.00228 Ž7.75. 0.00242 Ž7.74.
12.00
13.00
0.00780 Ž10.34. 0.00465 Ž10.38. 0.00717 Ž10.33. 0.00662 Ž10.35. 0.00667 Ž10.34. 0.00612 Ž10.36. 0.00582 Ž10.37.
0.06002 a Ž12.13. 0.05925 Ž12.08. 0.05912 a Ž12.03. 0.05216 Ž12.07. 0.04822 Ž12.06. 0.04687 Ž12.03. 0.04674 a Ž12.04.
383
Solubilities of four amino acids in NaCl solutions
TABLE 3. Experimental solubilities s for L-leucine as a function of sodium chloride conconcentration c at T s 298 K. ŽThe pH values in parentheses belong to the equilibrated solutions. pH: crŽmol . dmy3 .
0.00
1.00
0
0.9237 a Ž0.63. 0.8507 Ž0.59. 0.6053 a Ž0.69. 0.5972 Ž0.55. 0.5421 Ž0.55. 0.4922 Ž0.54. 0.4514 a Ž0.54.
0.3582 Ž2.25. 0.3208 Ž2.22. 0.3002 Ž2.21. 0.2382 Ž2.20. 0.2251 Ž2.21. 0.1981 Ž2.18. 0.1492 Ž2.12.
0.5 1.0 1.5 2.0 2.5 3.0 a
2.00 11.00 srŽmol . dmy3 . 0.2154 a Ž7.15. 0.1852 Ž7.05. 0.1598 Ž7.11. 0.1502 Ž7.04. 0.1252 Ž7.06. 0.1194 Ž7.05. 0.0994 Ž7.08.
0.1881 Ž7.17. 0.1707 Ž7.22. 0.1439 Ž7.21. 0.1177 Ž7.23. 0.0994 Ž7.23. 0.0962 Ž7.22. 0.0890 Ž7.24.
12.00
13.00
0.1893 Ž7.99. 0.1684 Ž8.03. 0.1556 Ž7.97. 0.1205 Ž7.98. 0.1160 Ž8.01. 0.1031 Ž7.99. 0.0888 Ž8.03.
0.3357 a Ž10.13. 0.2852 Ž10.17. 0.2412 a Ž10.17. 0.2302 Ž10.21. 0.2152 Ž10.19. 0.1794 Ž10.21. 0.1832 a Ž10.22.
From reference Ž6..
amount similar to that shown in table 5, it does not decrease the solubility of glycine, which remains approximately constant. So, it can be concluded that in aqueous solutions of NaCl the solubilities of L-cystine, L-tyrosine, L-leucine, and glycine, with respect to those in pure water change mainly as a consequence of the solvation. If in a liquid phase only water and sodium chloride are present, water molecules
TABLE 4. Experimental solubilities s for glycine as a function of sodium chloride conconcentration c at T s 298 K. ŽThe pH values in parentheses belong to the equilibrated solutions. pH: crŽmol . dmy3 .
0.00
1.00
0
3.504 a Ž2.31. 3.499 Ž2.34.
2.769 Ž2.42. 2.723 Ž2.43.
2.721a Ž4.92. 2.701 Ž4.98.
3.580 a Ž2.34. 3.489 Ž2.32. 3.502 Ž2.33. 3.428 Ž2.34. 3.313 a Ž2.32.
2.841 Ž2.42. 2.687 Ž2.50. 2.782 Ž2.49. 2.759 Ž2.41. 2.731 Ž2.43.
2.756 Ž5.01. 2.738 Ž5.03. 2.729 Ž4.99. 2.702 Ž5.02. 2.638 Ž5.00.
0.5 1.0 1.5 2.0 2.5 3.0 a
From reference Ž6.
2.00 11.00 srŽmol . dmy3 .
12.00
13.00
2.706 Ž7.07. 2.718 Ž7.11.
2.765 Ž7.28. 2.729 Ž7.26.
3.052 a Ž8.38. 3.061 Ž8.29.
2.702 Ž7.09. 2.657 Ž7.10. 2.689 Ž7.09. 2.703 Ž7.11. 2.691 Ž7.09.
2.741 Ž7.25. 2.755 Ž7.28. 2.763 Ž7.27. 2.751 Ž7.25. 2.731 Ž7.26.
3.045 a Ž8.32. 3.048 Ž8.37. 3.055 Ž8.35. 3.005 Ž8.33. 3.083 a Ž8.35.
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TABLE 5. Per cent variation of the solubilities s of glycine and L-leucine in some mixed solvents with respect to that in pure water
System Water]methanol Water]ethanol Water]Eth. Gl
Max. mass fraction of second solvent 1.0 1.0 0.90
Variation of the relative permittivity «r
Variation of glycine
Variation of L -leucine
78.54]32.70 78.54]23.54 78.54]47
y95 y99.75 y94.36
b
a
y100 y70.83
References 9 12 13
Eth. Gl s Ethylene glycol. a Approximated. b Not available.
are the only agents of solvation. But if other species are present in the solution, these too contribute to the solvation in proportion to their dielectric properties and concentrations. When non-electrolyte amino acid molecules possessing strong dipoles are added to an aqueous sodium chloride solution, two main factors influence their solubility with respect to that in pure water. First, in the presence of salt molecules less free water will be available to dissolve the amino acid: some water molecules will be captured by the ions into their solvation sheath, with consequent reduction of the solubility, and second, amino acid molecules give a solvation contribution which competes with that of the water molecule so that their solubility will increase. This effect will increase as the charges on the amino acid molecules grow. In our case the solubility of L-cystine increases as the concentration of NaCl rises from Ž0.0 to 3.0. mol . dmy3 because L-cystine, with its high charge, contributes to the solvation more than the other amino acids examined in this study. Actually, L-tyrosine and mainly L-leucine, which have a lower electric charge than L-cystine, are incorporated to a lesser extent into the solvation sheath of the salt, so that the salting out effect prevails in their solution. For glycine the decrease of solubility due to the decrease of the water available is easily compensated for by an increase in the contribution to the solvation of the ions of the salt. In fact, the hydrophobic part of the glycine molecule is less important than that of the molecule of L-leucine that has the same molecular charge. Therefore, the decrease of the number of molecules of free water gives a lower decrease of the solubility of glycine with respect to, for example, that of L-leucine. As shown by Carta and Tola,Ž6. the experimental solubilities can be correlated with the linear relation lg Ž srs w . s K so c,
Ž 1.
where s is the solubility of the amino acid in aqueous solutions of sodium chloride with concentration c, and s w is the solubility in pure water. Values of the salting inrout constant K so for L-cystine, L-tyrosine, L-leucine, and glycine are: L-cystine, K so s 0.06840; L-tyrosine, K so s y0.04823; L-leucine, K so s y0.1139; glycine, K so s y0.0016.
Solubilities of four amino acids in NaCl solutions
385
FIGURE 1. Logarithm of the ratio of solubilities s and s W with and without NaCl at T s 298 K for against sodium chloride concentration c at various pH values: `, pH s 0; I, pH s 1; e, , calculated using equation Ž1.. pH s 2; =, pH s 11; ^, pH s 12; \, pH s 13; L-cystine
FIGURE 2. Logarithm of the ratio of solubilities s and s W with and without NaCl at T s 298 K for against sodium chloride concentration at c various pH values: `, pH s 0; I, pH s 1; e, pH s 2; =, pH s 11; ^, pH s 12; \, pH s 13; , calculated using equation Ž1.. L-tyrosine
386
R. Carta
FIGURE 3. Logarithm of the ratio of solubilities s and s W with and without NaCl at T s 298 K for against sodium chloride concentration c at various pH values: `, pH s 0; I, pH s 1; e, , calculated using equation Ž1.. pH s 2; =, pH s 11; ^, pH s 12; \, pH s 13; L-leucine
FIGURE 4. Logarithm of the ratio of solubilities s and s W with and without NaCl at T s 298 K for glycine against sodium chloride concentration c at various pH values: `, pH s 0; I, pH s 1; e, pH s 2; =, pH s 11; ^, pH s 12; \, pH s 13; , calculated using equation Ž1..
Solubilities of four amino acids in NaCl solutions
387
The measured values for the solubilities s of the four amino acids studied are shown in tables 1 to 4. Calculated using equation Ž1.4 and experimental values of lgŽ srs w . are plotted against the concentration of sodium chloride in figures 1 to 4. In these figures the dimensions of the axes are equal to allow comparison between the different results for the studied amino acids. The calculated solubility of L-cystine, L-tyrosine, L-leucine, and glycine in sodium chloride solutions containing hydrochloric acid, or sodium hydroxide, differs from the experimental values. These differences have their absolute maximum at 18.88 per cent for L-cystine at pH s 1.00 and at a concentration of sodium chloride of 1.0 mol . dmy3 . Since many of these differences are greater than the experimental errors, equation Ž1. does not appear to be a correct representation of the influence of sodium chloride concentration on the solubilities of the amino acids studied in the regions outside the isoelectric zone Žzone with 3r5 pH units around the isoelectric point.. However, it should be observed that outside the isoelectric zones, the solubilities of amino acids increase significantly. For example, in the solutions of L-leucine as the pH decreases from 7 to 0 the solubility increases by more than five times Žfrom about 0.18 mol . dmy3 to about 1 mol . dmy3 ..Ž6. This increase occurs also in aqueous solutions containing sodium chloride. Therefore, the separation processes by crystallization from broths produced, for example, by acid or basic hydrolysis of protein-containing materials, must be operated near a pH of 7 because at this value the solubilities of these amino acids are at a minimum. For these purposes the primary interest lies in a precise knowledge of the solubilities in the isoelectric regions where the equation presented gives the best results. Moreover, equation Ž1., which predicts solubility values with a reasonable approximation, can be used to calculate the solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in solutions with concentrations of sodium chloride from Ž1.0 to 3.0. mol . dmy3 and could be employed in the design and optimization of purification process. REFERENCES 1. Schrier, E. E.; Robinson, R. A. J. Solution Chem. 1974, 3, 493]501. 2. Brigg, C. C.; Lilley, T. H.; Rutherford, J.; Woodhead, S. J. Solution Chem. 1974, 3, 649]658. 3. Fasman, G. D. Handbook of Biochemistry and Molecular Biology: 3rd edition. CRC Press: Boca Raton. 1976, Vol. I, p. 115. 4. Needman, T. E.; Paruta, A. N.; Gerraughty, R. J. J. Pharm. Sci. 1971, 60, 565]567. 5. Zumstein, R. C.; Rousseau, R. W. Ind. Eng. Chem. Res. 1989, 28, 1226]1231. 6. Carta, R.; Tola, G. J. Chem. Eng. Data 1996, 41, 414]417. 7. Danehy, J. P.; Moorehead, T. J. J. Sulphur Chem. 1972, 2, 121]123. 8. Gatewood Brown, M.; Rousseau, R. W. Biotechnol. Prog. 1994, 10, 253]257. 9. Orella, C. J.; Kirwan, D. J. Biotechnol. Prog. 1989, 5, 89]91. 10. Jin, X. Z.; Chao, K. C. J. Chem. Eng. Data 1992, 37, 199]203. 11. Bockris, J. O’M.; Reddy, A. K. N. Modern Electrochemistry. Plenum Publishing Corporation 227: New York. 1970, Vol. 1, Chap. 2. 12. Dey, B. P.; Lehiri, S. C. J. Indian Chem. Soc. 1992, 69, 552]557. 13. Mozaki, K.; Tanford, C. J. Biol. Chem. 1965, 240, 3568]3573.
(Recei¨ ed 12 February 1997; in final form 9 October 1997)
WA97r014
Solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in sodium chloride solutions at various pH values Renzo Carta a Dipartimento di Ingegneria Chimica e Materiali Uni¨ ersita’ di Cagliari, 09123 Cagliari, Italy
The solubilities of four amino acids ŽL-cystine, L-tyrosine, L-leucine, and glycine. at T s 298 K and at the pH values of 0.00, 1.00, 2.00, 11.00, 12.00, and 13.00 in water solutions of sodium chloride, have been measured. The concentration of NaCl was varied from Ž0.0 to 3.0. mol . dmy3 . The solubility of L-cystine increased as the concentration of salt was raised from Ž0.0 to 3.0. mol . dmy3 . For the same variation of sodium chloride concentration the solubilities of L-tyrosine and L-leucine decreased. The solubility of glycine was not influenced significantly by the presence of NaCl. A simple relation between the ratio of the solubilities of the amino acids with, and without, salt and NaCl concentration is presented. The relation predicts well the solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in aqueous solutions of NaCl starting from known values of the solubilities in pure water. Q 1998 Academic Press Limited
KEYWORDS: solubility; amino acids; sodium chloride; equilibrium; thermodynamic model
1. Introduction Amino acids play an important role both in animal metabolism and in industrial processes. Since they are rarely found in nature in a free form, they must be obtained from hydrolysis of protein-containing materials, or by fermentation. These production methods often result in aqueous mixtures containing various solutes including several types of amino acids and inorganic salts at different concentrations. From a theoretical point of view the interactions between ions and small molecules with biological macromolecules are of considerable importance in determining the behaviour of macromolecules.Ž1,2. In particular, information on the thermodynamic properties of polar non-electrolytes in aqueous salt solutions helps to understand the conformational changes of molecules in solution produced by the addition of denaturants, or by the transport of charged solutes across membranes. The design and optimization of purification processes, especially those based on crystallization which are used extensively in the purification of mixtures of amino a
ŽE-mail: [email protected]..
0021]9614r98r030379 q 09 $25.00r0rct970313
Q 1998 Academic Press Limited
380
R. Carta
acids, require the knowledge of thermodynamic data, and suitable thermodynamic models, which in their turn depend on the availability of adequate data. The solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in aqueous solutions can also give an indication on the influence of the impurities and, therefore, on the tendency of the possible incorporation of these impurities during crystallization processes. Solubility data of many amino acids in pure aqueous solutions Ž3 ] 5. and in aqueous solutions containing acids or bases Ž6 ] 8. are available in the literature. The solubility behaviour of amino acids in water and aliphatic alcohols solutions was investigated by Orella and KirwanŽ9. but increase in the availability of experimental solubility data helps in the comprehension of the chemistry of these molecules. We report here experimental results on the solubility of L-cystine, L-tyrosine, L-leucine, and glycine in solutions containing sodium chloride in concentrations c from Ž0.0 to 3.0. mol . dmy3 and at the pH values 0.00, 1.00, 2.00, 11.00, 12.00, and 13.00. These data provide a useful base for the development of a thermodynamic model that predicts Žif used for example with the data in reference 6. the solubilities of the amino acids studied here in NaCl aqueous solutions.
2. Experimental Acid solutions were prepared by adding known quantities of hydrochloric acid ŽCarlo Erba RPE 1.0 mol . dmy3 . to h.p.l.c. water, to reach the target pH values; basic solutions were prepared by diluting sodium hydroxide ŽCarlo Erba RPE 0.1 mol . dmy3 . with h.p.l.c. water. The final pH values of these solutions were measured using a pH meter ŽMetrohm 691, accuracy "0.01 units. calibrated using the two points method. For acid solutions the first point was a buffer solution at pH s 1.00, and the second point a buffer solution at pH s 4.00. When calibrating for basic solutions, two buffer solutions at the pH values of 10.00 and 13.00 were used, respectively Žbuffer solutions Carlo Erba RPE.. Sodium chloride was added to these solutions up to the required molar concentrations of salt. These solutions were then mixed with solid L-cystine ŽAldrich Chemie, mass fraction s 0.99, C 6 H 12 O4 S 2 , FW 240.30, CAS-num. 56-893., L-tyrosine ŽAldrich Chemie, mass fraction s 0.99, C 9 H 11 NO 3 , FW 181.19, CAS-num. 60-18-4., L-leucine ŽJansen, mass fraction ) 0.99, C 6 H 13 NO 2 , FW 131.18, CAS-num. 61.90.5., and glycine ŽJansen, mass fraction ) 0.99, C 2 H 5 NO 2 , FW 75.07, CAS-num. 56-40-6.. The label purity of the manufacturer was accepted and no pretreatment of the chemicals was considered necessary. The flasks containing the acid or basic solutions and the added amino acid were transferred to a thermostatic bath kept at T s Ž298 " 0.1. K. The solutions were continuously stirred by a magnetically-driven agitator. The target of T s 298 K was approached with two different, but similar, methods. In the first case the solution was kept at T s 303 K for 48 h, and in the second one the solution was kept at T s 293 K for the same period of time. The two solutions were then brought to T s 298 K and kept for 48 h at this temperature. Differences in the measured solubilities were within the accuracy of the analytical methods.
Solubilities of four amino acids in NaCl solutions
381
The hydration of the solid phase was checked using the procedure described by Carta and Tola.Ž6. The differences between the mass of the amino acids before, and after 48 h contact with h.p.l.c. water Žtaking into account the mass of the solid q the dissolved amount. were less than 1 per cent of the mass used for the test. The stirring time of 48 h was established via a trial and error procedure in order to achieve equilibrium conditions. In fact, L-cystine and L-tyrosine exhibited a very long dissolution time Ž)24 h., especially when the amount of solid amino acid was only a little greater than that required for the saturation of the solutions. Although the equilibrium for L-leucine and glycine was achieved in shorter times, these solutions were also equilibrated for 48 h. After 48 h stirring was discontinued and the remaining solid was allowed to precipitate. A sample of about 10 cm3 was taken using a syringe when the separation was almost complete. This sample was filtered in a constant temperature filter Žpaper Whatman a52. and the clear liquid was taken for analysis. Solutions containing L-cystine and L-tyrosine were analysed stectrophotometrically Žspectrophotometer Shimatzu UV 160A., according to the procedures of Carta and Tola.Ž6. Solutions containing L-leucine and glycine were analysed by drying known amounts of samples to constant weight. This procedure, known as the dry weight method, and extensively used,Ž4,10. was tested as previously reported.Ž6. The analysis was carried out by completely evaporating 3 cm3 of saturated liquid at T s Ž323 to 333. K, at atmospheric pressure, and weighing the mass of solid obtained. This maximum temperature was chosen to avoid an excessively rapid evaporation with possible loss of amino acids and thermal decomposition. The mass of sodium chloride in these solutions was taken into account in calculating the solubilities of the amino acids. Each solubility was obtained by averaging the concentration of three series of samples taken from three difference flasks. The maximum deviations of the nine samples were "1 . 10y5 mol . dmy3 for L-cystine and L-tyrosine, analysed by the spectrophotometer; "2.5 . 10y4 mol . dmy3 for L-leucine; and "5 . 10y4 mol . dmy3 for glycine, analysed by the dry weight method.
3. Results and discussion The measured solubilities of the four amino acids studied as a function of the sodium chloride concentration at the pH values 0.00, 1.00, 2.00, 11.00, 12.00, and 13.00 are shown in tables 1 to 4; these values relate to the stock solutions, and not the equilibrium ones Žthat are reported in the same tables in parentheses .. Actually the stock solutions pH values differ from those of the equilibrated solutions Žparticularly for the solutions containing L-leucine and glycine because of their high solubilities ., however, they give information on the amount of acid and base dissolved. The addition of salt has two effects on the solubility of non-electrolyte substances: the first one is associated with a decrease of the relative permittivity of the solvent,Ž11. and the second one with the solvation.
382
R. Carta
TABLE 1. Experimental solubilities s for L-cystine as a function of sodium chloride conconcentration c at T s 298 K. ŽThe pH values in parentheses belong to the equilibrated solutions. pH: crŽmol . dmy3 .
0.00
1.00
0
0.09412 Ž0.00. 0.10315 Ž0.01. 0.10911 Ž0.01. 0.11864 Ž0.02. 0.12719 Ž0.02. 0.14087 Ž0.03. 0.14399 Ž0.02.
0.01701a Ž1.03. 0.02039 Ž1.05. 0.02365 a Ž1.05. 0.02398 Ž1.06. 0.02401 Ž1.06. 0.02447 Ž1.07. 0.02437 Ž1.08.
0.5 1.0 1.5 2.0 2.5 3.0 a
2.00 11.00 srŽmol . dmy3 . 0.00145 Ž2.11. 0.00156 Ž2.12. 0.00172 Ž2.14. 0.00189 Ž2.14. 0.00201 Ž2.15. 0.00234 Ž2.17. 0.00247 Ž2.17.
0.00161 Ž7.81. 0.00169 Ž7.81. 0.00181 Ž7.79. 0.00211 Ž7.79. 0.00233 Ž7.76. 0.00228 Ž7.79. 0.00242 Ž7.78.
12.00
13.00
0.00450 Ž10.93. 0.00465 Ž10.93. 0.00517 Ž10.92. 0.00552 Ž10.90. 0.00567 Ž10.90. 0.00593 Ž10.88. 0.00596 Ž10.89.
0.02825 a Ž12.28. 0.03125 Ž12.32. 0.03392 a Ž12.35. 0.03716 Ž12.31. 0.03722 Ž12.30. 0.04087 Ž12.29. 0.04212 a Ž12.32.
From reference Ž6..
Many substances reduce the relative permittivity of water when they are dissolved in it. For example, in table 5, some mixtures which produce this effect are shown. The same table shows the variation of the solubility of glycine Žcolumn 4. and L-leucine Žcolumn 5. as the mass fraction of the second solvent in the solutions increases from 0 to the maximum value. Although sodium chloride reduces the relative permittivity of water by an
TABLE 2. Experimental solubilities s for L-tyrosine as a function of sodium chloride conconcentration c at T s 298 K. ŽThe pH values in parentheses belong to the equilibrated solutions. pH: crŽmol . dmy3 .
0.00
1.00
0
0.10341 Ž0.02. 0.09943 Ž0.02. 0.09048 Ž0.01. 0.08671 Ž0.02. 0.08051 Ž0.00. 0.07804 Ž0.03. 0.06801 Ž0.01.
0.03575 a Ž1.17. 0.03489 Ž1.09. 0.03073 Ž1.21. 0.03016 Ž1.19. 0.02752 Ž1.17. 0.02625 Ž1.11. 0.02407 Ž1.17.
0.5 1.0 1.5 2.0 2.5 3.0 a
From reference Ž6..
2.00 11.00 srŽmol . dmy3 . 0.00695 Ž6.97. 0.00653 Ž6.96. 0.00657 Ž6.96. 0.00577 Ž6.95. 0.00541 Ž6.97. 0.00504 Ž6.94. 0.00455 Ž6.95.
0.00361 Ž7.79. 0.00359 Ž7.77. 0.00351 Ž7.77. 0.00211 Ž7.73. 0.00233 Ž7.76. 0.00228 Ž7.75. 0.00242 Ž7.74.
12.00
13.00
0.00780 Ž10.34. 0.00465 Ž10.38. 0.00717 Ž10.33. 0.00662 Ž10.35. 0.00667 Ž10.34. 0.00612 Ž10.36. 0.00582 Ž10.37.
0.06002 a Ž12.13. 0.05925 Ž12.08. 0.05912 a Ž12.03. 0.05216 Ž12.07. 0.04822 Ž12.06. 0.04687 Ž12.03. 0.04674 a Ž12.04.
383
Solubilities of four amino acids in NaCl solutions
TABLE 3. Experimental solubilities s for L-leucine as a function of sodium chloride conconcentration c at T s 298 K. ŽThe pH values in parentheses belong to the equilibrated solutions. pH: crŽmol . dmy3 .
0.00
1.00
0
0.9237 a Ž0.63. 0.8507 Ž0.59. 0.6053 a Ž0.69. 0.5972 Ž0.55. 0.5421 Ž0.55. 0.4922 Ž0.54. 0.4514 a Ž0.54.
0.3582 Ž2.25. 0.3208 Ž2.22. 0.3002 Ž2.21. 0.2382 Ž2.20. 0.2251 Ž2.21. 0.1981 Ž2.18. 0.1492 Ž2.12.
0.5 1.0 1.5 2.0 2.5 3.0 a
2.00 11.00 srŽmol . dmy3 . 0.2154 a Ž7.15. 0.1852 Ž7.05. 0.1598 Ž7.11. 0.1502 Ž7.04. 0.1252 Ž7.06. 0.1194 Ž7.05. 0.0994 Ž7.08.
0.1881 Ž7.17. 0.1707 Ž7.22. 0.1439 Ž7.21. 0.1177 Ž7.23. 0.0994 Ž7.23. 0.0962 Ž7.22. 0.0890 Ž7.24.
12.00
13.00
0.1893 Ž7.99. 0.1684 Ž8.03. 0.1556 Ž7.97. 0.1205 Ž7.98. 0.1160 Ž8.01. 0.1031 Ž7.99. 0.0888 Ž8.03.
0.3357 a Ž10.13. 0.2852 Ž10.17. 0.2412 a Ž10.17. 0.2302 Ž10.21. 0.2152 Ž10.19. 0.1794 Ž10.21. 0.1832 a Ž10.22.
From reference Ž6..
amount similar to that shown in table 5, it does not decrease the solubility of glycine, which remains approximately constant. So, it can be concluded that in aqueous solutions of NaCl the solubilities of L-cystine, L-tyrosine, L-leucine, and glycine, with respect to those in pure water change mainly as a consequence of the solvation. If in a liquid phase only water and sodium chloride are present, water molecules
TABLE 4. Experimental solubilities s for glycine as a function of sodium chloride conconcentration c at T s 298 K. ŽThe pH values in parentheses belong to the equilibrated solutions. pH: crŽmol . dmy3 .
0.00
1.00
0
3.504 a Ž2.31. 3.499 Ž2.34.
2.769 Ž2.42. 2.723 Ž2.43.
2.721a Ž4.92. 2.701 Ž4.98.
3.580 a Ž2.34. 3.489 Ž2.32. 3.502 Ž2.33. 3.428 Ž2.34. 3.313 a Ž2.32.
2.841 Ž2.42. 2.687 Ž2.50. 2.782 Ž2.49. 2.759 Ž2.41. 2.731 Ž2.43.
2.756 Ž5.01. 2.738 Ž5.03. 2.729 Ž4.99. 2.702 Ž5.02. 2.638 Ž5.00.
0.5 1.0 1.5 2.0 2.5 3.0 a
From reference Ž6.
2.00 11.00 srŽmol . dmy3 .
12.00
13.00
2.706 Ž7.07. 2.718 Ž7.11.
2.765 Ž7.28. 2.729 Ž7.26.
3.052 a Ž8.38. 3.061 Ž8.29.
2.702 Ž7.09. 2.657 Ž7.10. 2.689 Ž7.09. 2.703 Ž7.11. 2.691 Ž7.09.
2.741 Ž7.25. 2.755 Ž7.28. 2.763 Ž7.27. 2.751 Ž7.25. 2.731 Ž7.26.
3.045 a Ž8.32. 3.048 Ž8.37. 3.055 Ž8.35. 3.005 Ž8.33. 3.083 a Ž8.35.
384
R. Carta
TABLE 5. Per cent variation of the solubilities s of glycine and L-leucine in some mixed solvents with respect to that in pure water
System Water]methanol Water]ethanol Water]Eth. Gl
Max. mass fraction of second solvent 1.0 1.0 0.90
Variation of the relative permittivity «r
Variation of glycine
Variation of L -leucine
78.54]32.70 78.54]23.54 78.54]47
y95 y99.75 y94.36
b
a
y100 y70.83
References 9 12 13
Eth. Gl s Ethylene glycol. a Approximated. b Not available.
are the only agents of solvation. But if other species are present in the solution, these too contribute to the solvation in proportion to their dielectric properties and concentrations. When non-electrolyte amino acid molecules possessing strong dipoles are added to an aqueous sodium chloride solution, two main factors influence their solubility with respect to that in pure water. First, in the presence of salt molecules less free water will be available to dissolve the amino acid: some water molecules will be captured by the ions into their solvation sheath, with consequent reduction of the solubility, and second, amino acid molecules give a solvation contribution which competes with that of the water molecule so that their solubility will increase. This effect will increase as the charges on the amino acid molecules grow. In our case the solubility of L-cystine increases as the concentration of NaCl rises from Ž0.0 to 3.0. mol . dmy3 because L-cystine, with its high charge, contributes to the solvation more than the other amino acids examined in this study. Actually, L-tyrosine and mainly L-leucine, which have a lower electric charge than L-cystine, are incorporated to a lesser extent into the solvation sheath of the salt, so that the salting out effect prevails in their solution. For glycine the decrease of solubility due to the decrease of the water available is easily compensated for by an increase in the contribution to the solvation of the ions of the salt. In fact, the hydrophobic part of the glycine molecule is less important than that of the molecule of L-leucine that has the same molecular charge. Therefore, the decrease of the number of molecules of free water gives a lower decrease of the solubility of glycine with respect to, for example, that of L-leucine. As shown by Carta and Tola,Ž6. the experimental solubilities can be correlated with the linear relation lg Ž srs w . s K so c,
Ž 1.
where s is the solubility of the amino acid in aqueous solutions of sodium chloride with concentration c, and s w is the solubility in pure water. Values of the salting inrout constant K so for L-cystine, L-tyrosine, L-leucine, and glycine are: L-cystine, K so s 0.06840; L-tyrosine, K so s y0.04823; L-leucine, K so s y0.1139; glycine, K so s y0.0016.
Solubilities of four amino acids in NaCl solutions
385
FIGURE 1. Logarithm of the ratio of solubilities s and s W with and without NaCl at T s 298 K for against sodium chloride concentration c at various pH values: `, pH s 0; I, pH s 1; e, , calculated using equation Ž1.. pH s 2; =, pH s 11; ^, pH s 12; \, pH s 13; L-cystine
FIGURE 2. Logarithm of the ratio of solubilities s and s W with and without NaCl at T s 298 K for against sodium chloride concentration at c various pH values: `, pH s 0; I, pH s 1; e, pH s 2; =, pH s 11; ^, pH s 12; \, pH s 13; , calculated using equation Ž1.. L-tyrosine
386
R. Carta
FIGURE 3. Logarithm of the ratio of solubilities s and s W with and without NaCl at T s 298 K for against sodium chloride concentration c at various pH values: `, pH s 0; I, pH s 1; e, , calculated using equation Ž1.. pH s 2; =, pH s 11; ^, pH s 12; \, pH s 13; L-leucine
FIGURE 4. Logarithm of the ratio of solubilities s and s W with and without NaCl at T s 298 K for glycine against sodium chloride concentration c at various pH values: `, pH s 0; I, pH s 1; e, pH s 2; =, pH s 11; ^, pH s 12; \, pH s 13; , calculated using equation Ž1..
Solubilities of four amino acids in NaCl solutions
387
The measured values for the solubilities s of the four amino acids studied are shown in tables 1 to 4. Calculated using equation Ž1.4 and experimental values of lgŽ srs w . are plotted against the concentration of sodium chloride in figures 1 to 4. In these figures the dimensions of the axes are equal to allow comparison between the different results for the studied amino acids. The calculated solubility of L-cystine, L-tyrosine, L-leucine, and glycine in sodium chloride solutions containing hydrochloric acid, or sodium hydroxide, differs from the experimental values. These differences have their absolute maximum at 18.88 per cent for L-cystine at pH s 1.00 and at a concentration of sodium chloride of 1.0 mol . dmy3 . Since many of these differences are greater than the experimental errors, equation Ž1. does not appear to be a correct representation of the influence of sodium chloride concentration on the solubilities of the amino acids studied in the regions outside the isoelectric zone Žzone with 3r5 pH units around the isoelectric point.. However, it should be observed that outside the isoelectric zones, the solubilities of amino acids increase significantly. For example, in the solutions of L-leucine as the pH decreases from 7 to 0 the solubility increases by more than five times Žfrom about 0.18 mol . dmy3 to about 1 mol . dmy3 ..Ž6. This increase occurs also in aqueous solutions containing sodium chloride. Therefore, the separation processes by crystallization from broths produced, for example, by acid or basic hydrolysis of protein-containing materials, must be operated near a pH of 7 because at this value the solubilities of these amino acids are at a minimum. For these purposes the primary interest lies in a precise knowledge of the solubilities in the isoelectric regions where the equation presented gives the best results. Moreover, equation Ž1., which predicts solubility values with a reasonable approximation, can be used to calculate the solubilities of L-cystine, L-tyrosine, L-leucine, and glycine in solutions with concentrations of sodium chloride from Ž1.0 to 3.0. mol . dmy3 and could be employed in the design and optimization of purification process. REFERENCES 1. Schrier, E. E.; Robinson, R. A. J. Solution Chem. 1974, 3, 493]501. 2. Brigg, C. C.; Lilley, T. H.; Rutherford, J.; Woodhead, S. J. Solution Chem. 1974, 3, 649]658. 3. Fasman, G. D. Handbook of Biochemistry and Molecular Biology: 3rd edition. CRC Press: Boca Raton. 1976, Vol. I, p. 115. 4. Needman, T. E.; Paruta, A. N.; Gerraughty, R. J. J. Pharm. Sci. 1971, 60, 565]567. 5. Zumstein, R. C.; Rousseau, R. W. Ind. Eng. Chem. Res. 1989, 28, 1226]1231. 6. Carta, R.; Tola, G. J. Chem. Eng. Data 1996, 41, 414]417. 7. Danehy, J. P.; Moorehead, T. J. J. Sulphur Chem. 1972, 2, 121]123. 8. Gatewood Brown, M.; Rousseau, R. W. Biotechnol. Prog. 1994, 10, 253]257. 9. Orella, C. J.; Kirwan, D. J. Biotechnol. Prog. 1989, 5, 89]91. 10. Jin, X. Z.; Chao, K. C. J. Chem. Eng. Data 1992, 37, 199]203. 11. Bockris, J. O’M.; Reddy, A. K. N. Modern Electrochemistry. Plenum Publishing Corporation 227: New York. 1970, Vol. 1, Chap. 2. 12. Dey, B. P.; Lehiri, S. C. J. Indian Chem. Soc. 1992, 69, 552]557. 13. Mozaki, K.; Tanford, C. J. Biol. Chem. 1965, 240, 3568]3573.
(Recei¨ ed 12 February 1997; in final form 9 October 1997)
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