CHJV04I02P0084

JOURNAL OF CHEMISTRY AND CHEMICAL SCIENCES
An International Open Free Access, Peer Reviewed Research Journal

www.chemistry-journal.org
ISSN
ISSN 2229-760X
0976-5727 (Print)
ISSN 2319-7625
2319-8133 (Online)
Abbr:
Abbr:J.Comp.&Math.Sci.
J. Chem. & Cheml. Sci.
2014,
Vol.5(1):
Pg.1- 4
2014,
Vol.4
(2): Pg.84-89
Partitioning of D.L. Alanine and Hexaglycine in

Dextran + Poly (ethylene glycol) + Water
Two-Phase System at 297.15K
Vijendra P. Singh
Chemistry Research Lab,
Deshbandhu College, University of Delhi,
Kalkaji, New Delhi, INDIA.
(Received on: April 4, 2014)
ABSTRACT
Partitioning of two amino acids ( DL-alanine and hexaglycine)
were measured in dextran + poly( ethylene glycol)+ water twophase system. The coefficient for partition of DL-alanine and
hexaglycine were correlated by using the osmotic viral equation.
The phase interaction or partitioning coefficientin the equation
can be calculated by hydrophilic groups parameters which gives
in fairly good agreement with the experimental data on change in
concentration at 293.15Kat room pressure.
Keywords: Partition coefficient, Hydrophilic parameter, Osmotic
Viral equation, Dextran.
INTRODUCTION
A predictive method for the partition
coefficients (Kp) of several amino acids1,15
in the dextran [DEX] poly(ethylene
glycol)[PEG] + water aqueous two phase
system has been proposed( Zhou et al. 1997)
by using the osmotic virial equation(Edmand
and Ogston, 1968; King et al.1988). Salt
effect on charged protein partitioning13 in
aqueous PEG+ Dex system by potential (non
electrostatic from electrostatic effect)
proposed by Weiyu Fan and Charles E.

Glatz (1999). The liquid-liquid interaction
also helps in salting out effects3,4,5. There
the viral coefficient needs in calculation of
Kpvalues were given by the hydrophilic
parameters9,10. The hydrophilic group
parameters helps in pharmaceutical industry
for recovery with density behavior6,7. It is of
interest to extend this procedure to larger
molecules such as peptides. In this study Kp
values for two peptides (hexaglycine and DL
alanine) in the same aqueous two phase
Journal of Chemistry and Chemical Sciences Vol. 4, Issue 2, 30 April, 2014 Pages (70-117)
85
Vijendra P. Singh, J. Chem. & Cheml. Sci. Vol.4 (2), 84-89 (2014)
system were measured to determine new

hydrophilic group parameters5,6 required for
particles behavior and their solubility and
salting out effect13 in two phase system.
Prediction performance of the present
method for Kp values of11 peptide
(hexaglycine and DL alanine) in PEG +
DEX + water is presented and discussed.
EXPERIMENTAL
Dextran (DEX) was purchased from
Merck as DEX T 40(MN=18800,
mw=38000) and DEX T 500(MN=170300,
MW=503000). Poly ethylene glycol (PEG)
standard samples with two different
molecular weights were purchased from E.
Merck Pure Chemical Ind. as 200( MN=
194, mw=194) and PEG600( MN= 550,
MW=580). DL-alanine and hexa-glycine
were purchased from Sigma Chemical
Company. All reagents were used A R grade
without further purification. Triple distilled
degased water was purified with a Millipore
Milli-Q system in acidify KMnO4 solution.
Phase interactions of DL Alanine and
Hexaglycine were carried out in thermostat
at 293.15K. For constant temperature water
was rotated by continuous starring. Reaction
flask was fitted within the thermostat.
Preparation of aqueous twophase systems
were prepared in triple distilled water with
concentration range 0.01 molL to 0.10 mol
L for stand for 24 hr. to avoid any
precipitation. The aqueous two- three
component solution were kept into
thermostat for 15min for constant
temperature. The 1X 10 M solution of
PEG`s (200and 600), Dextran T(40 and
500)were used for salting out. These
concentrations were again measured. The
liquid-liquid equilibrium for PEG`s and
Dxetran has recently been investigated. In

order to determine the partition co-efficients
of the DL-Alanine and Hexaglycine in liquid
system, two tie line were selected from each
phase diagram. Presentation of the two
phase samples were performed in 20 ml
graduated glass bottles tightly closed. The
total wt. of the component was about 7.5gm.
The initial wt. of the DL-alanine and
Hexaglycine added to system was 0.5X10
gm. The mixtures were shaken for 20 min.
and then placed in thermostatic water bath
for at least 24hr. to ensure complete
equilibrium. Syringes were used for samples
approximately 5ml carefully removed for
analysis from the top and bottom layer. The
separated solutions were centrifuge for 2
min. and the uncertainty in the amino acid
concentration for analysis of top and bottom
phase was less than 1.8 ml/L.
RESULTS AND DISCUSSION
The partition coefficients of the two
amino acids(DL-alanine andhexa-glycine) in
DEX T40 + PEG200 + water, DEX T500 +
PEG200 + water, DEX T40 + PEG600 +
water and DEX T500 +PEG600+ water
aqueous two phase systems are presented in
Table 1,and concentration ranges were 1.02.0 mili mole/ litre. The partition
coefficients values were calculated w.r.t. tie
line lengths at particular concentration.
These data were obtained from an arithmetic
average of at least three four measurements
for each condition. The some unequal
distributions of these small molecular
compounds especially for amines with lower
ratio were established despite the
experimental errors. The TLL (tie-lines
Length) data of the same aqueous two-phase
systems have been reported elsewhere
86
(Furuya et al., 1995a, 1996).

The partition coefficient, kp, and tie
line length are defined, respectively, as
follows.
amino acid solubility in the PEG phase,

which leads to a more uneven distribution of
polymers and amino acid between two
phases.
= ( 1 2 + (1 2)]/
Interaction Parameters of Osmotic Virial

Equation
.
.
mg/g
where W is the weight fraction and

subscripts 1 and 2 indicate DEX and PEG,
respectively. The accuracy of the
experimental partition coefficient data is
considered to be within 5% from the
reproducibility.
From Table 1, it can be seen that Kp
of DL-alanine and hexa-glycine decrease
with increasing tie-line length and Kp of
DL-alanine are reported to be smaller than
those
of
hexa-glycine.
At
lower
concentration of DL-alanine and hexaglycine in DEX T40 + PEG200 + water
were large difference in their Kp values due
to larger difference in TLL (Table1), it is
shown that solvent solute interaction are
stronger than that of solvent-solvent
interactions, which correlated Kp values
with the more hydrophilic groups (Table 3).
: Effect of conc. on location of binodal curve
at 293.15K (Table 2) were reported for PEG
200 and 600 with DL-alanine and
hexaglycine.. As wt% for PEG 200
decreases from 39.12-30.00, increase wt%
for DL alanine and hexahlycine were
reported to 2.55 and 1.98, for 600 decreases
from 41.23- 28.92 were 2.52 and 2.49. These
effects on salt distribution are the result of
change in solvent structure and of polymersalt (Zwitter ion) and polymer-polymer
interactions. Long chain of peptide bond will
reduce PEG solubility in the DEX phase and
To predict of the KP values, the

osmotic virial equation proposed by Edmond
and Ogston (Edmond and Ogstomn, 1968;
King et al. 1988) can be applied. The
partition coefficient in the infinite dilute
condition can be derived as:

= +

by plotting lnkp(m)/ (m2BOT-m2TOP) vs.

(m1 BOT-m1TOP)/( m2 BOT-m2TOP), the
interaction parameters a14 and a24 can be
determined. The partition coefficient,
Kp(m), expressed by the ratio of molalities
can be transformed as follows by the
experimental results of Kp,
1000 +
=
1000 +
where is the hydrophilic parameter and is

defined as follows:
=
the number of total atoms of amino acid or

peptide except hydrogen in equation, nk is
the hydrophilic parameter of group k, and
Nk is the number of group k contained in
amino acid or peptide. The authors used the
87
same values of i4 and i4 (i=1, 2) proposed

by Zhou et al. (1997) and determined the
new values of nk by the data of Kp for salts
and peptides obtained in this work (Table 2).
The values of nk determined by Zhou
et al. (1997) and obtained additionally in this

work are shown in Table 3. Table 4 shows
the deviations between experimental and
calculated K p for salts or peptides.
Table 1: Experimental. Partition coefficients of D L Alanine and Hexa-glycine in aqueous two

phase at 293.15 K.
Aqueous two phase
DEX T 40 + PEG 200 +
Water
DEX T 40 + PEG 600 +

Water
DEX T 500 + PEG 200 +

Water
DEX T 500 + PEG 600 +

Water
Concentration
Mol L
1.0X 10
1.3X 10
1.7X 10
2.0X 10
1.0X 10
1.3X 10
1.7X 10
2.0X 10
1.0X 10
1.3X 10
1.7X 10
2.0X 10
1.0X 10
1.3X 10
1.7X 10
2.0X 10
Tie- line Length

Wt %
13.33
18.16
21.98
24.84
08.53
09.15
10.48
11.37
11.94
13.33
17.00
21.98
06.62
09.56
12.48
14.48
DL Alanine
0.84
0.74
0.69
0.68
0.90
0.81
0.78
0.73
0.85
0.80
0.77
0.72
0.89
0.80
0.79
0.74
Kp
Hexa-glycine
0.83
0.69
0.65
0.63
0.84
0.87
0.63
0.62
0.84
0.81
0.76
0.65
0.86
0.78
0.76
0.69
Table 2: Effect of conc. on location of binodal curve at 293.15K

PEG200
Wt%
39.12
38.90
38.10
37.15
36.56
34.67
32.35
30.00
28.85
DL Alanine
Ws%
10.50
11.32
11.85
12.15
12.45
12.82
12.95
13.05
13.40
Hexa-glycine
Wt%
9.85
9.96
10.22
10.45
10.87
11.05
11.48
11.83
12.25
PEG600
Wt%
41.23
38.44
36.45
34.65
33.05
31.51
30.15
28.92
27.80
DL Alanine
Ws%
9.00
9.23
9.82
10.10
10.55
11.24
11.28
11.52
11.74
Hexa-glycine
Wt%
7.51
7.94
8.12
8.45
8.78
9.11
9.35
10.00
10.30
88
Table 3: Hydrophilic group parameters n at 303.15K ( Reference values)
Group
K
K
OH
Alcohol
0.949
ACH
C-
NH
CHn
CONH
COOH
NH2
O-
N-
0.104
O.O79
0.062
0.0
2.3
0.75
0.432
0.57
1.13
Table 4: % Deviations for Kp values

Systems
PEG 200+water
PEG 600+ water
DE X T-40+ Water
DEX T 500+ Water
DEX T-40+PEG 200+water
DEX T-40+PEG 600+ water
DEX T-500+ PEG 200+ Water
DEX T-500+ PEG 600+ Water
DL Alanine
2.8
3.5
4.4
4.5
4.8
4.9
6.3
5.1
Hexa glycine
2.9
2.2
3.8
4.7
7.3
3.8
3.1
1.3
CONCLUSION
REFERENCES
The partition coefficients of two

peptides hexaglycine and DL- alanine in
dextran+ poly (ethylene glycol) + water
aqueous two phase systems were measured.
The partition coefficients of two amino acids
(DL alanine and hexaglycine ) obtained in
this study was correlated by using the
osmotic virial equation. The interaction
coefficients (Kp) are estimated by using
hydrophilic group parameters and successful
correlation and results are obtained. The tie
line lengths/ composition were correlated by
hydrophilic group parameters and salting out
ability. Furthermore, by using the same
procedure, the partition coefficients of
amino acids are predicted show fairly good
agreement.
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Approach to the Study of Phase
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Phase Equilibria, 103,119-141 (1995).
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ethylene glycol)- Water Aqueous TwoPhase Systems at 20C, Fluid Phase
Equilibria, 110, 115-128 (1995b).
ACKNOWLEDGEMENT
I am thankful to all the staff of
laboratory which helps me in many ways to
contribute their valuable time, to college for
providing space.
89
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Rentsch D, Koch W, Frommer WB.
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JOURNAL OF CHEMISTRY AND CHEMICAL SCIENCES

An International Open Free Access, Peer Reviewed Research Journal

Partitioning of D.L. Alanine and Hexaglycine in

proposed by Weiyu Fan and Charles E.

system were measured to determine new

Dxetran has recently been investigated. In

(Furuya et al., 1995a, 1996).

amino acid solubility in the PEG phase,

Interaction Parameters of Osmotic Virial

where W is the weight fraction and

To predict of the KP values, the

by plotting lnkp(m)/ (m2BOT-m2TOP) vs.

where is the hydrophilic parameter and is

the number of total atoms of amino acid or

same values of i4 and i4 (i=1, 2) proposed

et al. (1997) and obtained additionally in this

Table 1: Experimental. Partition coefficients of D L Alanine and Hexa-glycine in aqueous two

DEX T 40 + PEG 600 +

DEX T 500 + PEG 200 +

DEX T 500 + PEG 600 +

Tie- line Length

Table 2: Effect of conc. on location of binodal curve at 293.15K

Table 4: % Deviations for Kp values

The partition coefficients of two

1. Edmond, E. and A.G. Ogston, An

Measurement and Correlation of

11. Wipf D, Ludewig U, Tegeder M,

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