1. Introduction

Knowledge of the existence of amino acids dates back over a century in many cases, as doe’s knowledge of their extended in proteins. When amino acids were discovered, their identity was established by isolating and purring the individual compounds and obtaining elemental analysis. After the advent of paper chromatography, this technique was used with a variety of different solvents to identify elution characteristic and demonstrate the purity of isolated compounds. Amino acids were located by the use of a reagent that produced colour with the compound. The most common reagent used for locating amino acids is ninhydrin. Which produces a purple colour with imines, a pink or yellow color with amino acids, and various intermediate colors, with compounds containing an amino group and a sulfonid group and so on. It also reacts with small peptides such as glutathione. The techniques of paper chromatography were applied to the separation of mixtures of amino acids, such as the components of the protein after hydrolysis, and then to the separation of free amino acids in physiologic fluids and tissues.

It was extended by the use of two dimensional chromatography, in which a different solvent was used in each direction[1]. Later electrophoresis was employed as one of the separating techniques. After locating the amino acids by reaction with ninhydrin, the spots could be cut out, eluted with alcohol or acetone and measured spectrophotometrically[2]. In order to measure the free amino acids in biological tissues and fluids, all proteins must be removed. Various methods have been employed to accomplish this, such as ultra filtration, equilibrium dialysis, and proton precipitation. A Variety of protein precipitation has been employed for this purpose, the most common being picric acid. For extracting tissues the most common methods employ 10% trichloroacetic acid, usually by blending or homogenizing with 510 vol of acid followed by a high speed centrifugation[2]. Plasma, cerebrospinal fluid and urine are most commonly deproteinized using 5 vol 3% sulphosalicylic acid as protein precipitated, again followed by centrifugation. The precipitated proteins can be purified by washing with hot trichloroacetic acid to remove nucleic acids and with a Varity of organic solvents to remove lipids. The proteins can then be hydrolysed, usually with 6N HCl under vacuum at 120℃ for 24 h or more, and their amino acid composition determined[3].

Many publications have done on the behaviour of weak acids in anhydrous solvents. Interesting work has been done by Kolthoff et al.[4,5]. Aleksandrov et al.[6,7] studied the dissociation of salicylic acid in butane-2-one. Kreshkov et al.[8] studied the dissociation of amino acids (as weak) acids) in mixtures of formic and ethylmethylketone and in mixtures of acetic acid-ethylmethylketone.

Gomaa et al.[9] studied association, dissociation and hydrogen bonding of salicylic acid in water-N, N- dimethylformamide mixtures from solubility measurements.

The aim of this work is to evaluate the solubility of phenylalanine in different solvents and discuss in details the solvation parameters for the solubility process. Study the effect of different solvents of the solubility and the physical behaviour accompanied is very important for the biochemical analysers. Also apply a theoretical model for evaluating different microscopic interactions of phenylalanine is necessary.

The aim of this work is to evaluate the solubilities of phenylalanine in different solvents and discuss in details the solvation parameters for the solubility processes for phenylalanine which is one of the most essential amino acids.

2. Experimental

The phenylalanine and solvents used, ethanol (Et), dimethylsulphoxide (DMSO), acetonitrile (AN), methanol (Me), acetone (Ac), 1-4 dioxane (Di) and dimethylformamide (DMF) were supplied from Merck. The saturated solutions of phenylalanine were prepared by dissolving it in the solvents used. The solutions were saturated with N2 gas in closed test tubes. The tubes were placed in a shaking water bath of the type assistant for a period of four days, followed by another two days without shaking to reach the necessary equilibrium.

The solubility of phenylalanine in each solution was determined gravimetrically by taking 1 ml of the saturated solution and subjecting it to complete evaporation using small aluminium disks heated by an infrared lamp.

The pH readings of the saturated solutions were measured using a pH-meter of the type Tacussel /Minis 5000. The densities were measured by using weighing bottle 1 ml and analytical balance (4 digits) of the type Mettler Toledo DA.

3. Results and Discussion

Table 1. Molal solubilities (m), densities (d) and pH values for saturated phenylalanine solution in different solvents at 298.15 K Solvent m/(g.mol/kg solvent) d pH H2O 0.1535 1.0246 6.760 Et ( Ethanol) 2.4470x10-3 0.8255 7.352 DMSO (Dimethyl sulphoxide) 0.0071 1.1136 9.496 AN (Acetonitrile) 7.0530x10-5 0.7785 10.816 Me (Methanol) 2.3210x10-2 0.8167 7.240 Di (Dioxane) 1.08780x10-3 1.0296 5.616 Ac (Acetone) 1.4490x10-3 0.8008 8.210 DMF (Dimethylformamide) 2.6581x10-3 0.9529 10.048

Amino acid contents have been studied in a variety of biological samples. Of these, plasma, urine, cerebrospinal fluid, which are accessible and clinically significant, have received most attention of these samples investigated include amniotic fluid, blood cells, fees, sweat, saliva, hair, fingernails and fingerprints. Even small amounts of protein in biological samples interfere in chromatographic analysis and have been removed.Therefore solubility measurements are very important to solve this problem[1].

The calculated molal solubilities (m) phenylalanine saturated solution was given in Table (1) from at least three average measurements. Also the measured densities and pH values of the amino acid used are also listed in Table (1).

The molar volumes (V_M) of phenylalanine were obtained by dividing the molar mass by the densities and their values are listed in Table (2). The packing density as reported by Kim et al.[10] and El-Khouly et. al.[11], i.e. the relation between Van der Waals volume (V_W) and the molar volume (V_M) of relatively large molecules (above 40) was found to be a constant value and equal to 0.661.

(1)

The electrostriction volume (V_e) which is the volume compressed by the solvent, was calculated by using equation (2) after El-Khouly[12].

(2)

The molar, Van der Waals and electrostriction volumes of phenylalanine in various solvents at 298.15 K were tabulated in Tables (2).

The different association, dissociation parameters were calculated using model explained before by us concentrating phenylalanine zwitter ion has effective hydrogen atom and that is clear from the pH values which are in weak organic acid range. Various analytical methods have been described in literature, only a few were developed for clinical use. For a good route analytical procedure, several factors have to be considered. Cost (both time and money), choice of simple reagent, simplicity of the procedure, and chromatographic properties of derivatives[2]. In this work simple and cheap methods for analysis can be used.

Table 2. Molar volumes (VM), Van der Waals volumes (VW), electrostriction volumes (Ve) and apparent molar volumes (V) for saturated solutions of phenylalanine in different solvents at 298.15 K (in cm3/mole) Solvent VM VW -Ve V H2O 161.2191 106.5658 54.6530 161.0436 Et 200.0993 132.2650 67.8337 174.6008 DMSO 148.3373 98.0510 50.2863 146.2907 AN 212.1846 140.2540 71.9300 171.8580 Me 202.2670 133.6980 68.5690 200.2480 Di 160.4360 106.0490 54.3880 158.0711 Ac 206.2780 136.3500 68.9280 188.9220 DMF 173.3550 114.5870 58.7680 169.7592

The apparent molar volumes V[13-15] were calculated using equation (3)[16]

(3)

Where M is the molar mass of benzoic acid, m is the concentration; d and d_o are the densities of saturated solution and pure solvents, respectively.

The values of V for phenylalanine in various solvents at 298.15 K are presented in Table (2).

The activity coefficient was calculated by using the relation log γ± = -0.5062 [17,18].

K_ass values were calculated[14] from the ratios of association constant to dissociation constant (i.e., K₁/K₂) for the dimmers of phenylalanine which form a complex ion (() and hydrogen ion (H⁺), and the values of K` (where K<sup>`</sup> is the dissociation constant of the associated acid complex, H₂A₂) are given by the following equations

(4)

(5)

(6)

Where a is the activity. The values obtained K₁/K₂, K` and K_ass are reported in Table (3). The maximum value of K_ass was found to be by using DMF where water is the least association parameters.

The free energies of dissociation (ΔG_d), free energies of association (ΔG_A), difference free energies (ΔΔG) and free energies of solvation (ΔG_s) for phenylalanine saturated solutions in various solvents were calculated by using the following equations and tabulated in Table (4).

(7)

(8)

(9)

(10)

(11)

The solvation volumes for phenylalanine were evaluated from the difference between Van der Waals of phenylalanine in absence and given before in various solvents. The Van der Waals volume of phenylalanine was calculated from Bondi method[19] and found to be 222.648 cm³/mole. Subtracting this value from V_w in solvents and divide the results by the molar volumes of the solvents taken from ref.[18], n (the solvation number) was obtained and given in Table (5).

It was concluded that the solute-solvent interaction increased by increasing ΔΔG and ΔG_s due mainly to the greatest of the association parameters in the corresponding solvents.

Also it was observed that the association, dissociation and microscopic interactions between solute and solvent is important for any solvent individually, whether it is polar, aprotic or amphiprotic.Increasing of the solvation numbers favour more solute- solvent interactions between phenylalanine and solvents. Also small solvation numbers favour more solute solute interaction or ion pair formation resulting in the decrease of the solute – solvent interactions in the solvent under discussion. Big positive values for ΔΔG and big negative. This work gives a lot of data which help any analyst to use suitable solvent for phenylalanine in chromatographic analysis.

Table 3. Log activity coefficients (logγ±), dissociation constants (K) and association constants for phenylalanine saturated solutions in different solvents at 298.15 K Solvent log γ± Pa K` Kass H2O -0.1980 6.9586 2.0610x103 7.8521x1012 3.8091x109 Et -0.0282 7.3802 9.1000x106 3.4101x108 37.3622 DMSO -0.0041 9.5001 1.7112x105 5.0800x1014 2.9900x107 AN -4.8100x10-4 10.8160 2.3501x1010 2.1321x1013 907.2601 Me -0.0860 5.6320 1.3002x103 3.6001x10-4 2.7801x10-7 Di -0.0164 7.3260 2.6002x107 2.1010x105 8.0711x10-3 Ac -0.0217 7.3737 2.5801x106 1.1702x109 453.4880 DMF -0.0268 10.0748 14.3001x105 9.9300x1014 6.9100x107

Table 4. Free energies of dissociation (ΔGd), free energies of association (ΔGA), difference free energies (ΔΔG) and free energies of solvation (ΔGs) for phenylalanine saturated solutions in different solvents at 298.15 K (in kJoule/mole) Solvent ΔGd ΔGA ΔΔG ΔGs H2O -18.919 -54.694 -35.775 -10.163 Et -39.727 -9.000 -12.817 -26.507 DMSO -29.859 -42.676 -5.56464 -21.312 AN -59.205 -16.884 42.321 -58.740 Me -17.776 37.426 55.202 -17.279 Di -42.330 11.949 54.279 -29.927 Ac -36.602 -15.165 21.437 -28.931 DMF -35.139 -44.749 -9.610 -26.134

Table 5. Molar volumes of solvents (Vs), difference in different Van der Waals volumes (ΔVw) and solvation numbers (ns) for phenylalanine a saturated solutions in different solvents at 298.15 K