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THE DISTRIBUTION OF NOR-α-AMINOACIDS IN LIQUIDTWO-PHASE TERNARY SYSTEMS FORMED BY SALTING-OUT OF LOW ALIPHATIC ALCOHOLS FROM AQUEOUSSOLUTIONS.E.S. VAISERMAN a , V.Yu. RYASHENTSEV* a & S.V. ROGOZHIN aa A.N. Nesmeyanov Institute of Organoelement Compounds, Academy of Sciences of USSR ,Vavilov st., 28, Moscow, 117813, USSRPublished online: 27 Apr 2007.

To cite this article: E.S. VAISERMAN , V.Yu. RYASHENTSEV* & S.V. ROGOZHIN (1990) THE DISTRIBUTION OF NOR-α-AMINOACIDSIN LIQUID TWO-PHASE TERNARY SYSTEMS FORMED BY SALTING-OUT OF LOW ALIPHATIC ALCOHOLS FROM AQUEOUS SOLUTIONS.,Solvent Extraction and Ion Exchange, 8:1, 35-47, DOI: 10.1080/07366299008917985

To link to this article: http://dx.doi.org/10.1080/07366299008917985

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SOLVENT EXTRACTION AND ION EXCHANGE, 8(1), 35-47 (1990)

THE DISTRIBUTION OF NOR-M-AMINOACIDS IN LIQUID TWO-PHASE TERNARY

SYSTEMS FORMED BY SALTINC-OUT OF LOW ALIPHATIC ALCOHOLS FROM

AQUEOUS SOLUTIONS.

E.S.VAIKERMAN, V.Y~.RIASHENTSEV', S.V.ROGOZHIN

il. iV. ,Vesme.vanov Ins ti tote of Organoelement Compounds, ;lcademy

of Sciences of LSSR, 11 i8l3 Moscow, L'avilov st., 28, USSR.

ABSTRACT

It is shown that the distribution of homologous series of nor-a-aminoacids in liquid two-phase three-component systems formed by salting-out of low aliphatic alcohols from their aqueous solutions by sodium chloride can be described by the folloving relation:

& I D = a + b h 9 , vhere: D is the distribution coefficient expressed as the

ratio of concentration of aminoacid in the upper and lover phases, respectively; 9 is the ratio of alcohol concentration expressed in molar percent in the same phases; a and b are constants related to activity coefficients and salting-out constants of aminoacid and alcohol by sodium chloride.

INTRODUCTION

The liquid two-phase ternary systems (TPTS) formed by

salting-out of polar organic solvents from their aqueous

Copyright 8 1990 by Marcel Dckker, Inc

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36 VAINERMAN, RYASHENTSEV, AND ROGOZHIM

solutions, the so-called Timmermans' systems, are promising with

respect to separation of various bioorganic substances (1,2). Due

to this fact the origin of fractionating action in these systems

is of primary importance.

It was shown (31, that the distribution coefficient of

3-aminophtalimide (API) in such a typical Timmermans' system as

11-PrOH - H20 - NaCl is directly dependent on the relative

concentration of alcohol in coesisting phases. Qualitatively, it

can be interpreted as the result of the difference in the

solvating strength of these phases. The experimental data on

fluorescence spectra and reversed phase HPLC enables one to assume

that the solvating strength of mised three-component solvents

comprising alcohol, salt and water out of the demising zone is

mainly determined by the water/alcohol ratio, since the salt does

not effect the solvation of API.

Due to this fact the distribution coefficient of API is

likely to depend on the ratio of alcohol concentration in the

coexisting phases. Further thorough treatment of experimental data

on distribution of API showed that the following relation is

valid:

here: D is the distribution coefficient espressed as the ra-

tio of the concentration of API (mg/ml) in the upper

and lower phases, respectively;

O is the ratio of alcohol concentration expressed in

molar percent in the same phases;

a,b are constants.

It was interesting to find out to vhat extent this relation

was applicable and to give a phisical meaning of constants a and b.

Bearing this in mind we studied esperimentally the distribution of

homologous series of nor-a-aminoacids ( Gly, Ala, .lBL', Val, Leu )

in TPTS formed by salting-out of low aliphatic alcohols

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( n-PrOH, i-PrOH, t-BuOH ) from their aqueous solutions in the

presence of sodium chloride. We also attempted to derive

theoretically the equation for this distribution.

Nor-a-aminoacids are difile molecules which are soluble in

water, alcohols and mixed aqueous organic solvents. The properties

of these aminoacids in solutions have been studied well enough.

The use of a homologous series makes it possible to compare the

results of distribution of aminoacids which differ only in the

number of CH -groups in the hydrocarbon chain. From this point of 2

view the nor-a-aminoacids are a convenient object to carry out

experiments of this kind.

EXPERIMENTAL

Materials. We used n-propanol, i-propanol, t-butanol purified

according to the method given in ( 4 ) and distilled on a rectifying

column supplied with a stainless steel spiral-prismatic nozzle

(3~3~0.2 mm.), layer height - 70 cm. The water content in alcohols was less than 0.05 X . We used deionized water having resistivity

215 &/cm, sodium chloride of hiqh purity grade ("Reakhim", USSR),

aminoacids (all racemic) of pure grade ("Reanal",C.zechoslovakia)

were used without further purification.

Methods. The distribution of aminoacids in TPTS having

different contents of equilibrium phases (Table 1) was carried out

in the following manner. A certain amount of aminoacid was

dissolved in the required amount of water. The needed amount of

salt was added to this solution on stirring, and then required

amount of alcohol was added. The formed two-phase system was

stirred for 2-3 hours at temperature 2 5 f 0.1 OC. When the

stirring was completed, the system was thermostated fir 2-3 hours

to ensure the complete separation of the layers formed. The

aliquotes of the co-existing phases *ere taken to assess the

aminoacid concentration by the reaction with o-phthalic aldehyde

described in ( 5 ) .

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TABLE 1

Composition of equilibrium phases (weight percent) at 25'~.

System : n-PrOH - H20 - NaCl

Top phase z-p-pz 1.00 17.00 82.00

1.20 21.80 77.00

2.25 32.50 65.25

2.50 37.50 60.00

System : i-PrOH - H20 - NaCl

System: t-BuOH - H20 - NaCl (6)

AC

32.73 7.19

19.37 5.48

9.23 2.99

5.37 2.30

Bottom phase

The distribution coefficient was'calculated according to the

equation: D = Clj/C2, where CI and C . are concentrations of 2 aminoacids in mg/ml in the upper and lower. phases, respectively. . In preliminary experiments we found that when the aminoacid

content is less then 2.5 mg/ml in TPTS 'the value of the '

distribution coefficient does not depend on its concentration. The

NaCl

21.10 74.00 4.90

17.00 75.70 7.30

11.75 77.00 11.25

9.75 74.00 16.25

H20 n-PrOH

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THE DISTRIBUTION OF NOR-CI-AMINOACIDS 39

value of D was determined as the average result of three or more

independent experiments. The experimental error in determination

of D was less than 5%.

The content of equilibrium phases presented in Table 1 was

determined by analysing the alcohol and salt concentration in the

coexisting phases. The alcohol content was determined by GLC and

the content of salt was obtained gravimetrically according to the

method described in (1). The experimental data on the t-BuOH - H20 - NaCl system were borrowed from paper (6).

The programmable calculator HP-9815s (USA) was used to

process experimental data.

RESULTS AND DISCUSSION

It was found in agreement with the results obtained for API

that the distribution of aminoacids in TPTS can be described by

equation [ 1 I. The experimental data on distribution of aminoacids in TPTS

comprising n-PrOH - H 0 - NaCl plotted as h D versus h @ are 2 presented in Fig.1. The constants a and b calculated by least

square fitting are given in Table 2.

The analysis of the experimental data enables one to conclude

that equation [I] has a sufficiently universal character. The

distributed substances mainly affect the value of constant b. Thus

in the series Gly - Leu the value of constant b drops aproximately 7 times and the value of constant a is almost constant and equals

zero within the experimental error. The value of constant b

correlates with the number of CH2-groups of the aminoacid

molecule. The profile of this dependence is given in Fig.2.

Qualitatively, this means that the higher hydrophobicity of

the distributed compounds (i.e. the higher degree of its alcohol

solvation), the more their trend to concentrate in the upper

organic phase.

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V A I N E M A N , RYASHENTSEV, AND ROGOZHIN

1 . 0 2 . 0 3.0 $

FIGURE 1. The distribution of nor-a-aminoacids in TFTS n-PrOH - H20 - NaCl.

TABLE 2

The least-square coefficients for aminoacids distribution.

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THE DISTRIBUTION OF NOR-a-AHINOACIDS

0 1 2 3 4 "CH,

FIGURE 2. The value of b constant versus number of CH - groups in aliphatic chain of aminoacid. 2

The same behaviour was observed for the distribution of Cly

and Val in TPTS formed by salting-out of i-propanol and t-butanol.

The corresponding calculated values of the constants of

equation [ I ] for these systems are given in Table 3 (the results

for system n-PrOH - H20 - NaCl are presented for compa-

rison).

We can deduce that the decrease in alcohol polarity in the

i-PrOH - n-PrOH - t-BuOH series results in the decrease in the distribution coefficient of the given aminoacid, which is

reflected in constant b of equation [ I ] . This fact proves that the

value of constant b is not only defined by the properties of the

distributed substance but also by the origin of alcohol.

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VAINERMAN, RYASHENTSEV, AND ROGDZHIN

TABLE 3

The least-square coefficients for Gly and Val distribution.

I aminoacid ( alcohol

It is known that the equilibrium condition for any two-phase

system occurs when the value of chemical potentials of all its

components in the coexisting phases are equal:

Here, lpi, 2pi are the chemical potentials of the i-th

component in the upper (1) and lower ( 2 ) phases, respectively.

~xpressing the chemical potentials through activity values,

one bbtains:

after rearrangement:

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THE DISTRIBUTION OF NOR-a-AMINOACIDS 43

0 0 Here lai and 2ai are the activities, lpi and 2pi the

standard chemical potentials of the i-th component in each

phase.

The equation similar to [31 can be written if both the phases

are in equilibrium with the i-th component in its saturated

solution:

Here a* and at are the activities of the i-th component in 1 1 2 i

saturated solution of each phase.

Thus, we can deduce that

If we assume that a = fC (where f is the activity

coefficient, C the concentration), and a = f * ~ (where f* is the,

activity coefficient in saturated solution, S the solubility),

the equation [ 5 ] after rearrangement can be rewritten:

Denoting h 1Ci/2Ci = Ki, where K. is the distribution coef- 1

ficient of the i-th component one obtains:

where and ?S. are the solubilities of the i-th compo- - 1

nent defined as the concentrations of its saturated solutions in

ihe upper and lower phases, respectively.

It is well known that the relation between the solubility of

a compound (e.g. alcohol) and the concentration of the salt in

aqueous-salt systems is described by Setschenov's equation (7):

Here ?and S are, respectively, the solubilities of the

compound in water and aqueous-salt solution with the salt

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44 V A I N E R M N , RYASHENTSEV, AND ROGOZHIN

concentration Cs; k is the saltig-out constant defined by the S

origin of the compound and salt.

Here one con write for each coexisting phase:

6 1 so/ S. = ki LCs 1 1 1

[91

8.n .$/?si = k; ,cs [ 101

where C and ,C: are the concentrations of salt in the upper I s - S and lower phases.

Subtracting equation (31 from [lo], one obtains:

fh lsi/2Si = ( ,ci - ,ci ) [Ill

This equation represents the relation between the solubility

of a component ill the coexisting phases and the difference of salt

concentrat ion in these phases.

Substituting the value of 1Si/2Si taken from equation [I11

into equation [ 7 ] , I;e obtain:

h K. 1 = ki s ( 2Cs - lCs ) + Pin 1f;~2fi/2f;~lfi [I21

I f we denote I C 2 s

the equation [ I 2 1

can be rewritten:

- C ) - ACs and {& f*x f./ ffx f . 1 - mi' 1 s l i 2 1 2 1 1 1

a. L

[I31

Let us consider this equation with respect to an organic sol-

vent (A]. Denoting its distribution coefficient as K = +, we A obtain:

I f one assumes that the substance ( R l introduced in the sys-

tem does not affect the content of the coexisting phases (which

seems to be true in view of its small concentrations) then,

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VAINERMN, RYASHENTSEV, AND ROGOZHIN

0.1 0.2 0.3 k: (lit.)

I r Leu 0 Val

.I 0 Ala

FIGURE 3. The comparison of calculated salting-out cons- tants of aminoacids with published data (8).

equation of the first order as a function of the difference of

salt concentration in the coexisting phases.

Table 4 represents the results of processing the experimen-

tal data on the distribution of aminoacids and n-PrOH in TPTS

comprising n-PrOH - H20 - NaCl in coordinates &% @ - PCs.

The analysis of these data proves that 'the above assumption

is valid. The comparison of the values of constants kR for S

aminoacids 'in equation [I51 which, according to our derivation,

are equal to the salting-out constants, shows that they correlate

satisfactorily with the values given in liteterature (8) (Fig. 3).

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THE DISTRIBUTION OF NOR-a-AMINOACIDS 47

This proves the validity of the proposed approach for analy-

sing the substance distribution in TPTS of this type.

It is necessary to point out that the distribution of organic

solvents as well as that of aminoacids is described by the similar

equations which put the relations between the distribution

coefficient and the difference of salt concentration in the

coexisting phases through the salting-out constant value.

It can be assumed that the established relations can be

useful both for further investigations of the nature of formation

of two-phase liquid systems and for analysis of the distribution

of various substances.

REFERENCES

(1) V.Yu Ryashentsev, M.A. Voskoboinikov, E.S. Vainerman and

S.V.Rogozhin, J.Chromatogr., 216, 346 (1981). ( 2 ) V.Yu.Ryashentsev, E.S.Vainerman and S.V.Rogozhin, in VII All-

Union Conference for Extraction Chemistrx, Nauka, Moscow,

1984, p. 50 (in Russian)

(3) V.Yu.Ryashentsev, E.S.Vainerman and S.V.Rogozhin, J.Chroma-

togr., 288, 43 (1984). ( 4 ) A . J .Gordon and R.A. Ford, The Chemist's Companion, Mir, Moscow,

1973 (in Russian).

( 5 ) V.Y.K.Shvyadas, 1.V.Galaev and I.V.Berezin, Bioorg.Chem.,i, 19

(1978) (in Russian).

(6) T.V.Kuporova and I.L.Krupatkin, in Phase Eauilibria, Kalinin-

grad university, Kaliningrad, 1974, p. 118 (in Russian).

( I ) I.Setschenov, Z. Phys. Chem., 4, 117 (1889). (8) P.K.Nnndi and D.R.Robinson, JACS, 94, 1308 (1972).

Received by Editor

May 30, 1989

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