1. INTRODUCTION:
Protein is basically considered as one important
biomolecule because of its vast application area covering pharmaceutical, food,
chemical industries.1-6 Protein is basically composes of several
amino acid moieties. The study on solubility of simple amino acid glycine in
various solvent system and mixtures become very important for industrial as
well as research purpose.7-12 Glycine can be used as complexing
agent in many cases and also as middle material during production of large
varieties of chemical products. The solubility result of glycine is not only
important for chemical industry but also plays a crucial role in the
preparation of some food supplement containing protein as an ingredient.13-18
Glycine is also used in drug production, pet food
preparation, and fertilizer formation.19-23 Beside this qualitative
data also helps in understanding several interactions as well as various
thermodynamic grounds. The aim of current paper is to study solubility of the
said amino acid through pure qualitative way from various angles at fixed
temperature as well as many temperature ranges. This fulfil also rationalise
the solubility concept of glycine.
2. EXPERIMENTAL SECTION:
2.1 Chemicals and purification:
Our experimental amino acid i.e., glycine was
basically bought from Sigma-Aldrich containing 99.7 % (mass) purity. After
purchasing, Glycine was dried for 5 days at 350 K in desiccator. The salts Na2SO4
and KCl used here as electrolyte were purchased from E. Merck, India with 99.0
% mass purity. These were wilted at 400 K in an oven for 4 days and before
using then they were cooled for 7 days with the help of a vacuum desiccator
functioning to avoid water absorption. Ethanol and dioxane having 99 % (mass)
purity were purchased from Spectrochem, Bombay. Ethanol and dioxane were
purified by distillation under reduced pressure, and were taken in a vacuum
desiccator containing anhydrous calcium chloride to avoid water absorption. In
the entire study the experimental solution used were prepared with triple
distilled water. All solutions were created using triple-distilled water, which
had a very low conductivity throughout the entire experiment (0.9 micro
siemens/cm) A detail chemical speciation is given below in Table 1.
2.2 Experimental Procedure:
Our experimental procedure was analytical gravimetric
which basically passes through several stages. The solubility behaviour was
studied under both conditions i.e., at constant temperature and range of
temperature. A thermostat having accuracy of ± 0.10 K at
atmospheric pressure was used for controlling temperature. At a temperature
(298.15 K ± 0.10 K) saturated solution of glycine were made in EtOH and dioxane
separately having different concentrations shown in Tables 2 and 3
respectively. In order to find out equilibrium those solutions were routinely
stirred for 24 hours and before going for sampling the stirred materials were
standing at rest for 7 hours so that undissolved amino acids were simmered
down.
5mL each of solution were picked up with pre dried
pipettes from supernatant part and put them into glass vessel. After filtered
using 0.22µm HPLC disposable filter those were rapidly weighed. Then most of
the solvent molecule were taken out through evaporation with the help of
heating plate and consequently the crystal amount of the said amino acid rise
up and for complete drying finally a drying stove were employed having
temperature 400.15 K.
The entire dried sample are cooled in a
dehydrator containing silica gel for 48 hrs and weighed. The process used in
the entire study was doing with regular interval until a constant weight. The
amount of glycine the amino acids, which were determined by simple mathematical
equations as given below:
Amount of amino acid (W4) = (W/W3)
× 1000 g/kg of solvent mixture
Where, W = W2 – W1
W = Weight of dissolved glycine in grams (in the
solution).
W1 = Weight of the empty dried glass jar
W2 = Weight of glass jar after evaporation
of taken solution and complete dried solute (glycine)
W3 = evaporated mass of solvent mixture
From the amount of solute glycine in gram per kg, we
canculated the solubility in mol per kg of solvent. The variation of solubility
with pH was also reported at a temperature.
2.3 Theoretical Section for measurement of Gibbs free
energy of transfer:
Like former24,25 studies the following
equation was recognisable to measure the Gibbs free energy of solution at a
constant temperature.
Where, S = Solubility in molal scale in ethanol or
dioxane
γ = Activity co-efficient of glycine in ethanol
or dioxane.
The free energy of transfer (
) has been defined as the change in the
chemical potential of solute in going from water to any other solvent, at the
same molality. Unit should be
kilojoule/mole, but in this manuscript, we convert the unit from joule to
calorie. It is presented as,
Where, 
and 
are the corresponding Gibbs free energy of
glycine in co-solvent (ethanol/dioxane) and in pure water respectively.
Again,
Here ‘s’ stands for ethanol/water solution and ‘R’
Stands for pure aqueous solution of glycine. Experimental values for γ are
available for some of the solutes we have used, on the basis of the work of
Smith and Smith26 and of Ellerton et al.27 However,
self-interaction in aqueous ethanol and dioxane solutions has not been
measured, so that values of γ in such solutions have to be estimated by
some arbitrary procedure. We therefore ignored the self-interaction term and
reported values for the parameter.
, apparent free energy of transfer is represented as
/
So, the basic difference between these parameters is
that in calculation of transfer Gibbs Free energy self-interaction is
considered but not considered during calculation of apparent free energy of
transfer. Both are standard function because those are measured under standard
conditions. But it should be kept in mind that first one is more standard than
second one. Relative error of and lies in the range of ± (0.2 to 2.5) %.
Table 2: Solubility of glycine
in ethanol at various concentrations at 298.15 K ± 0.10 K
|
Wt % ethanol
in water
|
Solubility
(g/100g solvent)
|
Solubility (S)
(mol/kg solvent)
|
|
0
|
23.43
|
3.124
|
|
20
|
11.31
|
1.508
|
|
30
|
6.84
|
0.912
|
|
40
|
4.28
|
0.571
|
|
50
|
2.57
|
0.343
|
|
60
|
1.42
|
0.189
|
|
80
|
0.24
|
0.032
|
|
90
|
0.05
|
0.007
|
Standard uncertainty of temperature u is
u(T) = ± 0.10 K; Standard uncertainty in solubility = ± 001mol/kg
Plot 1: Solubility of glycine in ethanol at various
concentrations at (298.15 ±0.10) K
Table 3: Solubility of glycine
in dioxane at various concentrations at (298.15 ±0.10) K
|
Weight %
dioxane in water
|
Solubility
(g/100g solvent)
|
Solubility (S)
(mol/kg solvent)
|
|
0
|
23.43
|
3.124
|
|
20
|
10.92
|
1.456
|
|
30
|
6.35
|
0.847
|
|
40
|
3.30
|
0.440
|
|
60
|
0.64
|
0.085
|
|
80
|
0.06
|
0.008
|
Standard uncertainty of temperature u is u(T) = ± 0.10
K; Standard uncertainty in solubility ± 0015 mol/kg
3. RESULT AND DISCUSSION:
3.1 Observed solubility behaviour of glycine:
The following behaviour is enlisted regarding the
solubility of glycine.
a. Glycine is less soluble in ethanol and
dioxane than pure water.28
b. With increasing percentage of ethanol and
dioxane solution glycine shows lower solubility.
c. Glycine is more soluble in ethanol compare
to dioxane for a particular concentration.
d. The solvation tendency of glycine in water
increases with increasing KCl and Na2SO4 concentration
individually. (Influence of electrolyte on solubility.)
e. Variation of solubility with pH.29
f. The solubility of glycine increases with
increasing temperature.
Plot 2: Solubility of glycine in dioxane at
various concentrations at (298.15 ±0.10) K
3.2 Explanation of less solubility of
glycine in ethanol and dioxane than pure water:
The investigation on solvation capability of glycine
in water-ethanol mixture was done at (298.15 ±0.10) K. The Table-2 and Table-3
denote that the soluble capability of glycine decreases with increasing alcohol
and dioxane concentration at the said temperature. These Tables and plots (1
and 2) also expel that glycine is more soluble in pure water than in
water-ethanol and water-dioxane solvent system for a particular temperature.
Plot 2 denotes the variation of degree or extent of glycine solubility in water
in presence of dioxane i.e., how much glycine interacts with water in presence
of dioxane at a particular temperature. This also signifies greater
hydrophobicity of glycine in dioxane environment than in pure aqueous solution.
This observation can be explained simply on the basis
of interaction present between amino acid and said solvent system. Glycine
basically exists as zwitterionic form in aqueous solution.30 Thus
ion (zwitterionic form of glycine)-dipole (water) interactions become sole
reason behind the solubility of glycine in water. Beside this zwitterionic form
of glycine has a capability of forming H-bond with water molecule31
and this result produced a strong interaction between these two and thus
glycine becomes soluble in water.
When ethanol is mixed in water, the soluble capability
of glycine in that solvent system gradually decreases. Basically, two major
forces viz. electrostatic and hydrophobic interactions affect the solubility
behaviour.32 With the enhancement of alcohol concentration, the
hydrophobic interaction between glycine and ethanol could be promoted leading
to decreasing solubility. Beside this electrostatic repulsion among glycine
molecules also contribute to solubility and it is offset by the hydrophobicity
by glycine molecules and result a decrease of the solubility. In dioxane-water
system similar types of behaviour is shown by glycine molecule and give support
to the tabular data.
3.3 Explanation of rising solubility of glycine with
temperature:
The solubility of glycine increases with the
enhancement of temperature as shown in Table 4 and Plot 3. Temperature is one
of important variable of any experiment. This plot 3 signifies
interrelationship between temperature and aqueous solubility variation i.e.,
how solubility changes with temperature hike. This plot denotes proportional
relation between temperature and solubility. We consider temperature range
(293.15 ± 0.10) K to (318.15 ± 0.10) K and the proceeding method is
gravimetric. This conclusion might
be owing to the fact that the kinetic energy of the solvent molecule increases
with the rising of temperature. This helps the solvent molecule to distort the
association of solute molecule held by strong intermolecular force. Therefore,
glycine can now effectively make interaction with solvent molecules and
consequently solubility increases.
Table 4: Solubility of glycine
at different temperature in pure water
|
Temperature
(K) ± 0.10 K
|
Solubility (S)
(g/100g solvent)
|
Solubility (S)
(mol/kg solvent)
|
|
293.15
|
22.55
|
3.007
|
|
298.15
|
23.43
|
3.124
|
|
303.15
|
24.09
|
3.212
|
|
308.15
|
25.09
|
3.345
|
|
313.15
|
25.34
|
3.379
|
|
318.15
|
26.89
|
3.585
|
Standard uncertainty of temperature u is
u(T) = ± 0.10 K; Standard uncertainty in solubility ± 005 mol/kg
Plot 3: Solubility of glycine at different temperatures
(K) in pure water
3.4 Effect of pH on solubility of glycine:
The pH data and effect of pH on solubility were taken
from previous studied.29 It was found that the effect of pH on the
solubility of glycine is quite interesting because up to pH 7 its solubility
goes down with increasing the value of pH. But after pH 7, the value rises up
as shown in Table 5 and plot 4 and it looks a U-shaped curve.29,33
It basically based on existence of zwitterionic form. Lesser the zwitterionic
form lesser will be the solubility. With increasing concentration of OH- ion
the existence of anionic form rises up and consequently solubility decreases.
But after reaching some limit the existence of zwitterionic form enhanced as a
result solubility increases.
Table 5: Solubilities of
glycine in various pH values at (298.15 ±0.10) K
|
pH
|
Solubility (S)
(g/100g solvent)
|
Solubility (S)
(mol/kg solvent)
|
|
2.45
|
36.47
|
4.863
|
|
2.98
|
28.82
|
3.843
|
|
3.27
|
25.17
|
3.356
|
|
3.67
|
23.36
|
3.115
|
|
4.44
|
22.26
|
2.968
|
|
5.42
|
21.14
|
2.819
|
|
6.14
|
20.60
|
2.747
|
|
6.79
|
21.79
|
2.905
|
|
7.58
|
23.27
|
3.103
|
|
8.90
|
27.76
|
3.701
|
|
9.88
|
35.14
|
4.685
|
|
10.32
|
44.46
|
4.863
|
Standard uncertainty of temperature u is u(T) = ± 0.10
K; Standard uncertainty in solubility ± 006 mol/kg
Plot 4: Solubilities at various pH values at (298.15 ±
0.10) K
3.5 Effect of electrolytes on solubility of glycine:
Glycine is an example of biomolecules, a part of
protein and therefore several forces namely electrostatic, Vander Waals,
hydrophobic must come into consideration when we look toward the solubility of
glycine in presence of electrolyte like KCl, Na2SO4,
etc., [Plot 5].34-36 An interaction between biomolecules and the
electrolyte becomes the sole parameter during the consideration of solubility
of glycine in that particular electrolyte.
When glycine (+A-)
dissolve in aqueous solution of KCl/ Na2SO4 (B+C-) the
following reaction takes place:
(+A-) + (B+C-) = B+(-A+) C-
The obtained complex has a capability of shielding
hydrophobic interaction and consequently the solubility of glycine enhance in
present of Na2SO4. But with the increasing concentration
of electrolytes repulsive force rise up leading to appearing glycine molecule
closer and for this solubility of glycine decreases. This supports the tabular
data (Table 6) that is the difference in solubility in presence of Na2SO4/KCl.
Table 6: Solubility of glycine
in various electrolytic media at (298.15 ± 0.10) K
|
Electrolyte molality (m) (mol/kg)
|
Solubility in
KCl (g/100g solvent)
|
Solubility
(mol/kg solvent)
|
Solubility in
Na2SO4 (g/100g solvent)
|
Solubility
(mol/kg solvent)
|
|
0.00
|
23.57
|
3.143
|
23.57
|
3.143
|
|
0.10
|
23.80
|
3.173
|
-
|
-
|
|
0.30
|
24.00
|
3.200
|
-
|
-
|
|
0.50
|
24.20
|
3.227
|
26.11
|
3.481
|
|
0.70
|
24.36
|
3.248
|
-
|
-
|
|
1.00
|
24.60
|
3.280
|
27.02
|
3.603
|
|
1.50
|
24.83
|
3.311
|
27.28
|
3.637
|
Standard uncertainty of molality of electrolyte = ±
0.02 mol/kg; Standard uncertainty of temperature u is u(T) = ± 0.10 K
Plot 5: Solubility of glycine in KCl and Na2SO4
electrolytic media at (298.15 ±0.10) K
3.6 The thermodynamic points of views on solubility of
glycine:
For better understanding of the solvation process as
well as solubility of glycine in terms of both free energies of transfer and
apparent free energy of transfer from water to aqueous ethanol and water to
aqueous dioxane play a crucial role. The values for said thermodynamic
parameter are given in Tables 7 and 8 and plots 6 and 7. Those data reveal that
the value become more and more positive with increasing concentration of
ethanol as well as dioxane and its leads to destabilization. These also suggest
that glycine become more soluble in aqueous medium than in aqueous ethanol or
dioxane solution. This thermodynamic fact can be explained on the ground of
dipole-dipole interaction. The dipole moments of glycine, water, ethanol and
dioxane are 5.76D, 1.85D, 1.66D, 0.45D respectively.36 Therefore, it
can easily be said that better dipole-dipole interaction exists between
water-glycine compare to mentioned binary system that is glycine-ethanol and
glycine-dioxane. As a result, with ethanol and dioxane concentration
dipole-dipole interaction become weak leading to destabilization in those
binary media and also in turn result in the positive increment of free energies
data.
Table 7: Energy values with variation of
ethanol weight % concentration
|
Weight %
ethanol concentration
|
Transfer Gibbs
Free Energy (cal/mol)
|
Apparent free
energetics of transfer (cal/mol)
|
|
20
|
360
|
395
|
|
30
|
585
|
650
|
|
40
|
805
|
890
|
|
50
|
1035
|
1145
|
|
60
|
1330
|
1450
|
|
80
|
2215
|
2360
|
|
90
|
3060
|
3240
|
Relative error lies in the range of ± (0.2
to 2.5) %.
Plot 6: Variation of Gibbs free energy values with
variation of ethanol concentration
Solvent-solvent and solute-solute interaction here
also plays a great role. Glycine has an ability to form H-bonding with water
molecule but when ethanol is added to the solution, and then the mentioned
H-bonding present between solvent-solute breakdowns with the enhancement of
concentration of ethanol, water molecule produces H-bonding with ethanol in a
better way rather with glycine. Due to lower dielectric constant value of
ethanol (24.55 at 25°C) it shows lower affinity towards glycine compare to water.
These also support the solubility data.
Table 8: Energy values variation with
dioxane concentration
|
Wt % of
dioxane concentration
|
Transfer Gibbs
Free Energy (cal/mole)
|
Apparent free
energetics of transfer (cal/mole)
|
|
20
|
350
|
375
|
|
30
|
580
|
630
|
|
40
|
865
|
945
|
|
60
|
1635
|
1765
|
|
80
|
2785
|
2930
|
Relative error lies in the range of ± (0.2
to 2.5) %.
Plot 7: Energy values variation
with dioxane concentration
3.7 Explanation of more solubility of glycine in
ethanol than dioxane:
From the previous tabular forms (2 and 3), it is also
clear that glycine has lower solubility in dioxane-water system than
ethanol-water system for a particular concentration. For example- in 20%
dioxane-water system glycine shows solubility value 10.9 gm/100 gm (solvent)
whereas for the same the solubility value for ethanol-water system is 11.30 gm/100gm(solvent).
This can be explained by cavity interaction. Ethanol produces better
interaction with glycine due to availability of oxygen and also glycine fit
into the cavity created by ethanol through intermolecular H-bonding. But in
dioxane has chair type conformation as shown below. Due to its structural
rigidity, it cannot able to interact with glycine and also fails to produce
cavity like ethanol. Here a point to be mentioned that our discussion basically
focused on 1,4-Dioxane.
1,4 -dioxane
4. CONCLUSION:
This study concludes on the solubility of glycine from
different aspect such as presence of electrolyte,27-31 temperature
variation, and pH variation. Various interactions are the key factor for
showing versatile behaviour regarding solubility of glycine. This present study
becomes an effective tool towards such application areas where glycine is
employed as solute. This also makes a bridge between solubility behaviour of
amino acid and electrolytes. The present study is helpful for studying on
solvation thermodynamics of glycine in pure water, in solution or in
electrolytic solution.
5. ACKNOWLEDGEMENT:
Dr. Roy is grateful to Netaji Subhas Open University
for supporting the through minor Project (Project Number: Reg/0518 dated
07/06/24).
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