1. Introduction

Knowledge of the existence of amino acids dates back over a century in many cases, as does knowledge of their extended in proteins. When amino acids were discovered, their identity was established by isolating and purifng 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 colour with amino acids, and various intermediate colours, with compounds containing an amino group and a sulphonic group.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 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 solvents were 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 5 vol of acid followed by a high speed centrifugation[3]. Plasma, cerebrospinal fluid and urine are most commonly deproteinized using 5 vol 3% sulphosalicylic acid as protein precipitate, again followed by centrifugation. The precipitated proteins can be purified by washing with hot trichloaceic 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 glycine 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 glycine is necessary.

2. Experimental

The glycine and solvents used, dimethylsulphoxide (DMSO) , ethanol (Et), 1-4 dioxane (Di) acetonitrile (AN), dimethylformamide (DMF),acetone (Ac) and methanol (Me) were supplied from Merck. The saturated solutions of glycine 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 glycine 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

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 , bloodcells,feces,sweat,saliva,hair,fingernails and fingerprints. Even small amounts of protein in biological samples interfere in chromatographic analysis and have been removed[1]. Therefore solubility measurements are very important to solve this problem[1].

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

The molar volumes (V_M) of glycine 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 Gomaa 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 Gomaa[12].

(2)

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

The apparent molar volumes V_[10,11] were calculated using equation (3)[13]

(3)

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

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

The different association, dissociation parameters were calculated using model explained before by us concentrating glycine 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 (1). Molal solubilities (m), densities (d) and pH values for saturated glycine solutions in different solvents at 298.15 K Solvent m/(g.mol/kg solvent) d pH H2O 1.8752 1.0332 5.530 DMSO 0.1482 1.1060 13.250 Et 0.7028 0.7829 8.720 Di 0.2036 1.0147 4.060 AN 0.3048 0.7766 8.070 DMF 4.7051x10-5 0.9612 9.408 Ac 1.6979x10-4 10.7864 5.464 Me 0.0409 0.8075 6.929

Table (2). Molar volumes (VM), Van der Waals volumes (VW), electrostriction volumes (Ve) and apparent molar volumes (V) for saturated solutions of glycine in different solvents at 298.15 K (in cm3/mole) Solvent VM VW -Ve V H2O 75.240 49.7336 25.5063 71.736 DMSO 68.463 45.2540 23.4043 -162.827 Et 95.557 63.1632 32.3938 -2250.5 Di 85.145 56.2824 28.8644 -178.220 AN 96.578 63.8383 32.7400 -164.168 DMF 79.447 52.5146 26.9326 -2744.67 Ac 95.569 63.1713 32.3980 -2546.5 Me 95.375 63.0428 32.3321 -4309.1

The activity coefficient was calculated by using the relation [14,15].

K_ass values were calculated[16-18] from the ratios of association constant to dissociation constant (i.e., K₁/K₂) for the dimmers of glycine 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 glycine saturated solutions in various solvents were calculated by using the following equations and tabulated in Table (4).

Amino acids are strongly polar compounds with a variety of functional groups and can not be analysed by a GC method without prior conversion into suitable volatile derivatives. The preparation of such derivatives should be simple, rapid, and reproducible and give high product yield.

(7)

(8)

(9)

(10)

(11)

The solvation volumes for glycine were evaluated from the difference between Van der Waals of benzoic acid in absence and given before in various solvents. The Van der Waals volume of glycine was calculated from Bondi method[19] and found to be 106.4361 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).

Table (3). Log activity coefficients (log γ±), dissociation constants (K) and association constants for benzoic acid saturated solutions in different solvents at 298.15 K Solvent log γ± Pa K` Kass H2O -0.6932 6.2232 1.0176x10-13 1299241 1.2766x1014 DMSO -0.1904 13.4404 5.9974x10-26 25.2222 4.2055x1026 Et -0.1342 8.8541 3.9625x10-8 7.7016 1.9434x108 Di -0.2284 4.2884 6.3163x-8 3.5973 5.6953x107 AN -0.2285 8.2985 6.0307x10-10 7.6099 1.2619x1016 DMF -3.2769x10-3 9.4113 6.7511x1011 5.0838 7.5303x1016 Ac -6.5959x10-3 5.4706 3.9719x10-4 1.7005 4.2814x103 Me -0.1024 7.0314 5.1151x1012 5.5406 1.0831x1011

Table (4). Free energies of dissociation (ΔGd), free energies of association (ΔGA), difference free energies (ΔΔG) and free energies of salvation (ΔGs) for glycine saturated solutions in different solvents at 298.15 K (in k Joule/mole) Solvent ΔGd ΔGA ΔΔG ΔGs H2O -1.5580 74.1698 -80.5279 -6.3576 DMSO -4.7342 143.9851 -151.9873 8.0023 Et -6.5822 42.25559 -47.3169 -5.0611 Di -3.9458 41.0999 -44.2739 -3.1704 AN -3.9305 86.8843 -91.9580 -5.0315 DMF -24.7009 58.0610 62.0925 -4.0314 Ac -21.5196 19.4153 -20.7316 -1.3163 Me -7.9248 64.4577 -68.7025 -4.2448

Table (5). Molar volumes of solvents (Vs), difference in different Van der Waals volumes (Vw) and solvation numbers (ns) for glycine saturated solutions in different solvents at 298.15 K Solvent Vs ΔVw ns H2O 18.0724 56.8643 3.1466 DMSO 71.2995 61.3399 0.8604 Et 58.6804 43.4308 0.7401 Di 86.3552 50.3126 0.5826 AN 52.8450 42.7557 0.8091 DMF 77.4118 54.0794 0.6985 Ac 63.0510 43.4226 0.6887 Me 40.7322 43.5511 1.0692

4. Conclustions

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.

Table (5). Molar volumes of solvents (V_s), difference in different Van der Waals volumes (ΔV_w) and solvation numbers (n_s) for glycine saturated solutions in different solvents at 298.15 K .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 glycine 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. This work give a lot of data which help any analyst to use suitable solvent for glycine in chromatographic analysis.