The Solubility of Amino Acids in Various Solvent Systems The Solubility of Amino Acids in Various Solvent Systems

OF ( ABSTRACT The effect of varying so_lvent systems on the solubilities of glycine, L-alanine, L-valine, L-phenylalanine, and DL-amino octanoic acid at z50 C. was studied. The entire concentration spec trum from pure water to pure semipolar solvent was used for each of. the solve~t systems of methanol-water, ethanol-water, n-propanol water, isopropanol-water,, and tertiary butanol-water. Further, the effect of pH variation on the solubilities of the amino acids in each of the solvent systems was studied. Aliquots were withdrawn from each solvent system studied and analyzed gravimetrically to determine the resultant solubility. It was found that the solubility. behavior of those amino acids studied was a function of the constant effect of the -amino carboxylic acid portion of the molecule and the independent interactions of the remaining neutral portion of the molecule. Maxi mum solubility was found in pure water with a reduction to low solubility in the semipolar solvents in the order of a second degree polynomial equation. To each percent strength of the hydroalcoholic solvent sys tems, the ratio of water to alcoholic molecules per amino acid molecule remains constant. This would indicate that the lengthening of the non polar portion of the chain from the hydrogen of glycine to the methyl

In an aquea.is system, the total solubility is equal t_ o the sum of the original zwitterion solubility plus the solubility of the salt that was found. For those amino acids studied, only single salts were formed.
In the hydroalcoholic solvent systems, variation of pH' produced minimum solubility at the isoelectric point with no distinct isoelectric band seen. As the percent alcohol increased in those solvent systems studied, similar increments of acid or base added to the system produced a proportionally greater increase in the magnitude of total solubility of the amino acid. This might be attributed to an increase in the importance of the charge species as the polarity of the solvent system decreased as well as the increased affinity of the amino acid for the Na+ or Clion present.    14.     13.
T ~e Solubility of the Arn.ino Acids in Pure Ethanol as a Function of pH' The Solubility of the Amino Acids in Pure n -Propanol as a Function of pH' The Solubility of the Amino Acids in Pure Isopropanol as a Function of pH' The Solubility of the Amino Acids in Pure Tertiary Butanol as a Function of pH' 16. The importance of amino-acids in viable biological systems is due to the unusual properties o f this class of chemical compounds.
These substances, possessing both acidic and basic properties within the same molecule, afford the basic linkages leading to complex peptides and proteins, the basic materials of life. Further, ten such amino-acids have been shown to be essential for life processes.
Again, due to the basic and acidic c hara cter in a given molecule, these substances are dipolar compounds, with either zwitterion species formation or anionic or cationic species formation depending upon the acidity of the fluid environment. The relative concentration of these species, which coexist with each other, depend completely upon the value of pH.
Since there are dissociable groups in these molecules, many studies have been performed to determine the acid and base dissociation constants for the individual amino acids. Cohn and Edsall (1) have tabulated the constants for a large series of these compounds.
In 1933, Edsall and Blanchard (2) postulated that dissociation 1 r of the alpha amino acids in aqueous solution was ·as follows: They believed ~hat the charged and uncharged forms were in equilibrium. Further, they expanded a method developed by Ebert (3) to calculate the Kz of these systems. This ratio of charged to uncharged ions in solution· was f;und to be in the range of 104 to 10 6 for most aliphatic amino acids. This theory of the zw:ltterion or dipolar type configuration has been further substantiated by Fox and Foster (4).
An important property of the amino acids is that they exhibit an isoelecfric point. This is the point in an electric field at which the zwitterion form will not migrate to either the anode or ca. tho de (5 ).
Also, at the isoelectric point, the sum of the net charge is zero. If we represent the isoelectric point as pl and the dissociation constants as pK 1 and pK 2 , then (6) .  For the purposes of this study, label purity of the manufacturer was accepted and no pretreatment of the solvents was felt to be necessary .

6
The water used was deionized using a mixed bed column to a concentration of less than 10 ppm of NaCl to minimize the possibility of complex formation with any metals usually present (9). The other 6 7 solvents used were absolute anhydrous methanol, absolute ethanol, n-propanol? iso-propanol 9 and tertiary-butano1. Methods -Two types of systems were used in this study. The first type of system was the pure and mixed solvents including water, methanol, ethanol, n-propanol, isopropanol and tertiary butanol and mixtures of each with water . The solubility of each of the amino acids used in the study was found in the pure solvent systems and in the mixed solvents systems of Oo/o, 10%, 30%, 5;0%, 70%, 90% and 100% (v /v) of methanol -water, ethanol-water, n-propanol-water, isopropanolwater, and tertiary butanol-water . In the second type system, the solubility of each of the series of amino acids was varied by addition of either acid or base .
Procedure -· For the first part, the same basic procedure was used for all solvent systems . For each l?ercent strength which was evaluated, three samples of equal volume were prepared from a stock solution.
Into ea c h sample bottle, an excess amount of amino acid was added, and the bottle was covered with a cap lined with an inert teflon liner.
Since initial studies indicated that the amino acids reached a maximum solubility in one -half to 1 hour , a preliminary rotation period of one hour at room temperature was performed to insure that excess amino acid remained. The samples were then placed into a water bath, which was maintained at a temperature of 25° + O. 2°, and allowed to reach equilibrium. In 24 hours, either one ml. or five ml. samples were withdrawn from the sample bottles using a pipette with a pledget of ( 8 glass wool on the end as a filtering medium. The size of the sample withdrawn from the bottles was determined by preliminary experimentation in order to minimize the possible error during gravimetric analysis. The withdrawn sample was immediately transferred to a tared vial which was then weighed to determine density. The series of tared vials was then placed in an oven to be evaporated and dried to constant weight. The evaporations were made at a temperature of 95° C. or less to avoid decomposition of the amino acids. (10) Immediately after sample withdrawal, the pH or pH' of each solvent system was taken.
In the second part, the interest was twofold and a procedure was designed to maximize the experimental precision in each case.
Firstly, in the pure aqueous solvent systems, the interest was in salt formation as well as the variation of total solubility as a function of pH. A procedure was developed to quantitate the amount of acid or base present at any given pH. One hundred milliliters of water, a . specific amount of an HCl or NaOH standardized solution, and an excess of amino acid was mixe{l in an erlenmeyer flask which was then capped and placed on a magnetic stirrer and allowed to reach equilibrium. In twenty-four hours, a series of at least fifteen 5-milliliter samples were withdrawn from the flask and placed in tared vials. To those samples previously mixed with HCl, 5 accurately measured different amounts of ( ( 9 NaOH solution were added in sets of 3 each, so that the pH increased to below that of the isoelectric point. After precipitation, these samples were allowed to equilibrate for 24 hours . The arn.ount of the precipitate as well as the amount of the combined an1ino acids ren-iaining in the solutions and the pH of the solutions were determined using the procedure de scribed in section one for gravimetric analysis. A sirnilar process was followed for the samples mixed with standard NaOH except that HCl was added in incremented amounts to a pH above that of the isoelectric point.
In the non -aqueous solvents, the relationship between total amino acid solubility and _pH' was the main concern. Initially, four samples of equal volume were prepared for each strength to be evaluated.
Excess amino acids were added to eac_:h sample, the preliminai·y rotation performed, and the samples placed into the water bath at 25° C. and allowed to equilibrate. In 24 hours, one ml. samples were withdrawn, the pH or pH 1 taken, and the samples were analyzed gravirnetr ically. Into two o.f the samples, incrernental amounts of L acid were added in succeeding 24 hour periods and the pH or pH' as well as a gravirnetric analysis performed on the aliquots taken during each period. Into the remaining samples, base was added and a similar analysis prepared. The sample size and number of sarnples was adjudged sufficient since good agreement was f ound among succeeding sarn.ples £or the increase in total solubility of the an1ino acids.

!IL RESULTS AND DISCUSSION
The solubilities of the amino acids studied were determined in water, ,methanol, ethanol, n-propanol, isopropanol, and tertiary butanol at 25° C.
To illustrate the physical and chemical relationships of the amino acids studied, their structural formulas are shown in Table 1.
It can be seen that all the amino acids studied were alpha amino acids.
The D or L prefixed to the name of the amino acid indicates the configurational relationship of the structural g roups about the asymetric carbon atom. A DL prefix indicates a racemic compound or optically ( inactive mixture o f the stereoisomers. A(+) or(-:.) after the initial letter prefix may be used to show the direction of rotation of polarized ~ight, but is not usually necessary since no differences in physical or chemical properties are induced by this property. (11) It can be seen that variation in solubility charac teristics of these I. amino acids can be attributed to differences in their chemical structure.  .
By use of Raman Spectroscopy (12,13), melting point data (14), changes in molecular volume (15), and dielectric constant measurements (16), the amino acid has been shown to exist as dipolar ions in solution. The zwitterionic character of the amino acid gives it a high aqueous solubility due to its attraction to the dipolar water molecules. Table 2 shows the solubility of the amino acids studied in water at 25° C. These values correspond to a good degree with the range of literature values previously reported. (17,18) Since glycine is being considered the basic common structural unit in each of the amino acids, its use as the standard in com.paring solubility differences of the other amino acids would logically follow. Table 3 illustrates the ratio of solubility of an amino acid in gm . /ml. ring is approximately equivalent to three CH2 groups (19). Then the correlation of this aromatic amino acid into the smooth non-linear line of decreasing polarity further substantiates the use of glycine as a basic · solubility unit. Now that it has been shown that an increase in the number of carbon atoms decreases solubility (20,21) and that glycine is the basic soJ..ubility unit, then it would seem that the total amino acid solubility is dependent on both the polar and nonpolar portions of the molecule.
The addition of a semi-polar liquid to the polar aqueous solvent to form a binary solvent system would be expected to cause a change in the solubility behavior of the amino acids by providing an environment of continuously decreasing polarity. As the concentration of semi-polar liquid is increased, the binary solvent medium would continually decrease the polarity to the point of the pure semi-polar liquid.
In this way, the polarity of the system is changed in discrete steps by increasing the alc,oholic content.
In a solvent system of decreasing polarity, the differences in solubility of these amino acids studied would be dependent on the basic              Using diele c tric constant as a measure of polarity, the pure solvents can be arranged in this order of decreasing polarity : methanol, ethanol, n -propanol, and te r tiary butanol. These semi -polar solvents in combination with water will produce a series of solvent systems with the same order of decreasing polarity. For example, at 30% (v /v) of semi-polar solvent -water, the order of decreasing polarity remains methanol -water , ethanol -water, n-propanol -water, and tertiary butanol -water . However, it can be seen in Figure 7 that glycine throughout the concentration scale shows. a decreasing solubility in the order of n -propanol, tertiary butanol, ethanol and methanol. As illustrated in Figure 8, the addition of a methyl group to form L-alanine       Table 14, the ( proxi1nity of the Y intercept values which range from 2. 65204 to 2. 79148 with a true solubility of 2. 9 M. in pure water can be seen.
2 The X and X values also show a similarity. In Table 15    In Table 18 ™™"""""-·--=----            The Solubility of the Amino Acids in Pure Ethanol as a Function of pH! C olunm A -GJycine, C olurn.n D -L -Ala nine , Colunrn C -L -Valine, Colum.n D L-Phcnylalaninc, Colnm.n E -DL -Arninoocta noic Acid.    Table 10) However, between 70% and 90% the ethanol molecules begin to outnumber the water molecules and this in turn corresponds to the increased efficiency of salt formation in the solvent systems. In a solvent system as the dielectric constant of the medium is reduced, the interionic forces become more important since they compose an increasingly greater portion o~ the attractive charge between oppositely char ged ions. These ions of Na or Cl which are added separately to different solvent systems, as HCl or NaOH, can be attracted to the dipolar ions in the form of cations or anions to form the amino acid salts or can form associations with the hydrogen or hydroxyl ions with which they were added. However, in considering the activity coefficients for pure HCl (38) and pure NaOH (39) solutions at the molar concentrations of acid or base which were added to the amino acids in the scn1i -polar solvents; it was found that at the highest conc entration of ( ( 74 acid or base added the a ct ivity coefficients were reported to be approximately O. 86 and O. 85 respectively. Therefore it would seem that at the low concentrations of acid or base added in solutions of low dielectric constant, the c1and Na+ ions would provide the major attractive force for the predominately charged amino acids. This increased attraction as the polarity of the solvent system is reduced would seem to explain the greater efficiency of salt formation. Fur~her, the comparison of the ratio of S to S 0 with the quantity of acid or base added seems to provide an indication of a reduction in the isoelectric band. S i nce by definition the species present within the isoelectric band are predominately dipolar and produce an invariant solubility, it would seem' that an increase in total solubility could be attributed to salt formation. The increase in the S/S 0 ratio as the percent alcohol was increased would indicat~ a f:'eduction in the isoelectric band as a function of decreasing polarity. This reduction was due to an increased a ffinity of the amino acids for the Na+ and c1ions. 4 . For those amino acids studied in aqueous solvent systems, a band of minimum solubility is found from a pH above and below _ the isoelectric point. A distinct increase in total solubility is seen as the pH exceeds the limits of this isoelectric band. 5. In aqueous systems, the increase in total solubl.lity of those amino acids studied is dfrectly proportional to the moles of acid or base added . . . . Total solubility = (slope)(moles of acid or base added) + initial solubility of the amino acid.
The slope which represents the increase in total solubility as a function of acid or base added shows an increase as the non-polar to polar<-ratio of the amino acids increases. This would indicate that as the non-polar portion of the amino acid molecule increases, the anion or cation species is more readily available for salt formation .
6. The total solubility of the amino acids studied at various pH's in aqueous solutions is the sum of the ori ginal zwitterion