Indian Journal o f Chemica l Techno logy Vol. 9. May 2002, pp. 2 12-2 17

Articles

Thermodynamic dissociation constants of glycine and dl-alanine in water- ethanol mixtures

M Ashfaqur Rahman*, A R Chowdh ury & M R Zaman**

Department of Applied Chemi stry and Chemical Technology, Raj shah i Univers ity, Raj shah i-6205 . Bang ladesh

Received 12 Ju11 e 2001: revised received 10 Ja11uary 2002; accepted 7 February 2002

The thermodynamic dissociation constants of glycine and dl-alanine in 0, 20, 40, 60 and 80% (wlw) water- ethanol mixtures at 30°C and at a constant ionic strength, Is= 0.1 M KN03 have been measured pH-metrically using Irving and Ros~otti technique.pK1 andpK2 values in aqueous medium have been found to agree well with previously reported ones. It is observed that the pK 1 values of these acids increase regularly with the increase of ethanol contents in the mixtures while pK2 values show distinct minima ca. at 40% (wlw) ethanol content. The variation in pK values with the solvent composition is discussed in terms of the free energy transfer from water to ethanol-water mixtures. It is concluded that the specific solute-solvent interactions play a vital role in changing the p K values in ethanol-water mixtures.

In earli er communications 1•3 the thermodynamic dis­

sociation constants of some ac ids and bases of the type A 08 - and A± s· in water and in di fferent mixed solvents have been reported . These results, to some extent contribute to the understanding of acid -base equilibria in aqueous as well as in mi xed solvents. However, understanding of the regularities of the sol­vent effect requires the study of a large number of mixed systems from many aspects. Where there have been many physicochemical studies in water - ethanol mixed solven ts, relatively few data are available on the effect of these mi xed solvent systems on the di s­sociation of weak acids and bases4

·9

. The measure­ment of the free energy of transfer of amino acids from water to different mixed solvents has biological significance9

-12

• Considering the importance and use­fulness of amino acids in biological processes, a sys­tematic study on the thermodynamic dissociation con­stants of glycine and d/-alanine in water as well as in water - ethanol mi xtures (0-80 wt% ethanol) pH­metrically using Irving and Rossotti technique 13 is described herein.

Experimental Procedure For the experiments glycine (>99%) and d/-alanine

(>99%) were obtained from B.D.H Chemicals Ltd., Poole, England and E. Merck, Darmstadt, Germany

*Present address: Lecturer, Department of Chemistry, Rajshahi College, Raj shahi. **For Correspondence.

respectively and used as received . Absolute alcohol (Carew and Company Ltd. , Bangladesh) was treated with requi site amount of preheated calci um oxide, kept overn ight and distilled. It was redistilled before use and the middle fraction was utili sed . Hydrochloric ac id (B.D.H Chemicals, England) and sodium hy­droxide (Assay 97.5%, B.D.H Chemicals, England) was standardised in the usual way . Doubly distilled water from all-glass distilling sets was used as aque­ous medium as well as for the preparation of water -ethanol mix tures in a weight by weight composition.

A Corning digital pH-meter (Model 106) equipped with a combined glass and calomel electrodes was employed for measurements in water and water -ethanol mixtures at 30°C. A titration vessel was kept in a thermostat at 30 ±0.01 °C. The electrode assembly was calibrated with buffer solutions (pH = 4, 7 and 9, Riedel -de Haen, AGD-30 16, Seelze I) before each measurement. Buffer solution of pH=2 was prepared according to the formula enumerated by Yadav 14

•

Each titration was carried out using 23 mL of solution at a constant ionic strength of Is = 0.01 M KN03. The sample was titrated with hydrochloric acid dissolved in a solvent mixture having composition the same as that of the experimental acid solution to maintain the constancy of the composition of solvent mixture dur­ing titration . For each measurement a blank titration was carried out without experimental acids. For the determination of second dissociation constant of amino acids, the same procedure was followed, only

Ashfaqur Rahman er al.: Thermodynamic Di ssociaton Constants of Glycine and ell-Alanine Articles

HCI was replaced by NaOH. About 12-18 readings were recorded during the course of each titration. Be­fore changing over for measurement in a different solvent composition the glass electrode was equili­brated for at least 12 h with the new solvent mixtures.

Results and Discussion Irving and Rossotti pH-metric titration technique 13

was applied in order to obtain the pK values of gly­cine and d/-alanine in water and ethanol-water media of different compositions. pH values obtained by cali­bration of the pH meter assembly with aqueous buff­ers was converted to pH* values for the alcoholic me­dia by introducing a correction term, 8 , using the re­lationship:

pH*=pH-8 ... (1)

where '8' accounts for the residual liquid junction potential and the medium effects. Values of 8 for ethanol-water mixtures were taken from literature 15

•

The pK values of the amino acids were obtained from a stepwise calculation using the expressions:

1-n pK 1 = pH*-log--H +logfRHz•

nH

n pK 2 = pH*+ log--H- +log fR -

1-nH

(2)

... (3)

where, nH denotes the number of protons attached per molecule of acid corresponding to a given value of pH*. Following Van Uitert and Haas 16 and Irving and Rossotti 13 following expressions are obtained,

(v2 - v1)(N+E) 11H = y-

T(V + v1)

d (v2 - v1)(N+E0

)

an nH = rev+ v,)

... (4)

... (5)

Here, y is the number of replaceable hydrogen atoms

which is one for amino acids, v1 and v2 are the volu­mes of acids I alkali of strength N, required for titra­tion of the mineral and amino acids respectively , V is the initial volume of the solution and Tis the experi­mental acid concentration and T 0 are the ex perimen­tal base concentration. Values of nH have been taken between 0.30 and 0.70. logfRHZ+ and logfR- were cal­culated from the Debye-Hlickel limiting law. The De­bye- Hlickel limiting law has been used for the evaluation of -log/; in the form of

A .J Is log!;= _1

I+~ Is

where A = Temperature dependent Debye- HUcke! limiting constant.

(2nNd) 11 2 e3 I A = ---- ------::-:-:------::-c:-

100 2.302 K 312 (DT) 31 2

where N = Avogadro's number, e = electrostatic charge, d = density of the solvent, k = Boltzmann con­stant, D = Dielectric constant of the solvent, T = Ab­solute Temperature, °K

In this expression Is is the ionic strength of the so­lution and defined as follows:

c; = Molar concentration of the ionic species, Z; = Electrostatic charge of the ith ionic species, I = Sym­bol for the summation of all cl terms for each ionic species in solution .

Dissociation constants in aqueous medium The pK1 and pK2 values of glycine and d/-alanine

in water calculated at 30°C from Eqs (2) & (3) are listed in Table l. For comparison, previously reported values in water are also incorporated in the same ta­ble. It is seen from the table that the present results agree well with the previously reported values within ±0.01 pK unit in case of pK 1 values and ±0.02 pK unit in case of pK2 values. It is worth mentioning here that

Table !- Comparison of pK values of glycine and ell-alanine in water from different measurements including thi s work

Amino acids

Glycine

ell-Alanine

at 25°C Ref (6)

pK1

2.38

pK1

9.78

at 25°C Ref (6)

2.39 9.85

Values from literature at oc at 30°C Ref ( 17)

pK1

2.34

pK1

9.65

at 30°C Ref ( 19)

2.33 9.74

at 30°C Ref ( 18)

pK1

2.34

pK1

9.65

at 37.5°C Ref (20)

2.33 9.55

This work at 30°C

pK1

2.34

2.33

pK1

9.67

9.77

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Articles

6

I ~ glyci ne 6.5 '-------

* dl-alanine

I 11

10.5

5 10

45 9.5

9 N

I . :.: a.

85

8

2 5 7.5

2 ~--·------------------------~ 7 1.5 2 2.5 3 3.5

--- D-1_, 02 ---- >

Fig. !- Dependence of pK of g lyc ine and dl-alanine on inverse of dielectric constants (0-1

) of ethanol- water mixtures

the pH measurements in acid solutions are rapid whereas a long time is required for equilibration in alkali solutions, particularly in higher alcohol con­centration of solvent mixtures.

Dissociation constants in water - ethanol mixtures Variation of pK1 and pK2 values of the amino acids

used in the present investigation with water - ethanol mixtures are shown in Table 2 and graphically shown in Figs l & 2. It is evident from the figures that in the all the cases, pK 1 values increase as the concentration of ethanol increases. On the contrary, pK2 values de­crease up to 40% (w/w) and increase afterwards with the increasing concentration of ethanol in the solvent mjxtures.

It is to be recalled that solvent plays an essential role in determining the relative strength of acids and bases. Variation of solvent naturally has prominent effects on the equi librium. Likewise, there has been increasing recognition of the importance of specific solute-solvent interactions in account for the equil ib­rium and kinetic behaviour of electrolytes in solution . A comparison of the dissociation constants of the same in water and a mixture of water and an organic solvent aids in elucidating the influence of lowered dielectric constant on the dissociation process and thus sheds light on the nature of the solute-solvent interactions which are operative in the present set of

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Indian J. Chem. Techno!., May 2002

6 1.===================~ -+ d~ala ni:~

11

-+- glycine "10.5 5.5

5 10

" " 9.5

9

"' ::,: Q_

8.5

8

7.5

7 20 40 60 80

2L_ __________________________ ~.

0

----wt% Ethanol ->

Fig. 2- Variation of pK of glycine and dl-a lanine in ethanol­water mixtures

solvent mixtures. In order to observe the above facts, in Figs I & 2, pK values are plotted against the recip­rocal of the dielectric constant (D-1

) and weight per­cent of ethanol respectively . It is seen from these fig­ures that the pK 1 values in mixed solvents of all these amino acids increase with the decrease of dielectric constants of the solvent mixtures or with the increase of the ~thanol contents. However, the increase in the values has been found to be smaller than that in the case of corresponding carboxy lic acid"2

i.22 which may

be attributed to the dipolar nature of the amino acids. For pK2 values, it is found that these values decrease as the concentration of ethanol is increased up to 40% (w/w) and then increase again in the mixtures of higher ethanol contents. This anomalous dependence of pK2 values of these amino acids on composition or dielectric constant of ethanol-water mixtures can be attributed to changes in structures of these acids when they transfer from water to ethanol-water media. It may be concluded here that dielectric constant has a profound effect on the dissociation process of these amino acids in ethanol-water mixtures.

Change in pK value i.e pKrpKI in transfer from water to ethanol-water system

Table 2 includes values pKrpK 1 at various compo­sitions of ethanol-water solvents. The values of pK2-

pK1 is decreasing with dielectric constant of the sol-

Ashfaqur Rahman era/.: Thermodynamic Dissociaton Constants of Glycine and d/-Aianine Articles

Table 2- Dissociation constants of glycine & d/-alanine in water- ethanol mixtures at 303K

glycine d/-Aianine

Cone. of ethanol ( o/o wlw) pK1 pK2 pK2-PK1 pK1 pK2 pK2-pK1

0 2.34 9.67 7.33 2.33 9.77 7.44

20 2.58 9.45 6.87 2.60 9.54 6.94

40 2.82 9.20 6.38 2.85 9.36 6.5 1

60 3.13 9.35 6.22 3.25 9.57 6.32

80 3.48 9.58 6. 10 3.74 9.78 6.04

Table 3-Free energy (kJ mol" 1) of transfer of glycine from water to ethanol-water mixtures at 30°C

Terms 20

!:'J.G,o (I )diss 1.37

!:'J.G,o (W)* -4.50

!:'J.G,o (RH±) - !:'J.G," (RH/) 5.87

!:'J.G,o (2)diss -1.25

!:'J.G,0 (R.)- !:'J.G1° (RH±) 3.25

* Values of !:'J.G1° (H+) taken from literature24

vents. The decreasing values of pKrpK1 with varia­tion of solvent composition suggest that some specific interactions between charged sites in an amino acid molecule are taking place with the variation of the solvent composition . Ohtaki and Tanaka23 have evaluated the values of pK2-pK1 of aliphatic diamines in methanol-water mixtures and concluded that values of pKrpK 1 of diamines have been independent of sol­vent compositions. Free energy terms: The free energy of transfer (for the dissolution) of amino acids

In order to explore the nature of solute-solvent in­teractions, attempts have been made to determine the free energies of cations and anions of the amino acids in water and the free energies of transfer of ions from water to mixed solvents as these values provide the quantitative measure of the solute-solvent interac­tions. !'1G1° (i) di ss is the Gibbs free energy change ac­companying transfer for the dissolution of species (i) from the standard state in reference solvent water (w) to that in the solvent (s) of the process i(w)--7i(s).

Values for 1'1Gr0 (1 )cti ss and 1'1G1° (2)ctiss from pK1 and

pK2 have been calculated and are recorded in Tables 3 and 4. The evaluation of free energy of transfer from H20 (w) to EtOH-H20 (s) mixtures should be useful in assessing the relative importance of electrostatic charging effect and ion-solvent interactions on the sign and magnitude of the change in pK23

. It is im­portant to note that 1'1G1° (I )ctiss are positive, i.e. trans-

% of ethanol (w/w)

40 60

2.74 4.50

-8.30 -7.50

11.04 12.00

-2.68 -1.82

5.62 5.68

80

6.50

-0.5 i3

fer process is increasingly favourable. It is also found that the !'l.G1° (I )cti ss values increase sharply with in­creasing ethanol content. This observation is consis­tent with the effect of lowered dielectric constant of the mixed solvent in increasing the electrostatic free energies of the ions produced in the dissociation proc­ess. In changing the solvent from water to ethanol­water mixtures, the positive values of 1'1G1° (I )cti ss sug­gests that these amino acids are present predominantly in zwitterionic form in mixed solvents through a slight conversion to neutral form (NH2RCH2COOH). This fact has also been ratified by the experimental determination of the ratio of zwitterion to neutral form by other workers25-28 . The change in 1'1G1° (I )ctiss

may arise due to change from zwitterion to neutral form but contribution due to this factor is found to be small. The main contributory factors are the structural changes, the structure formation of water molecules due to the addition of alcohol on one hand and hydro­phobic interactions of amino acids and ethanol on the other hand. The change of zwitterions to neutral molecules would be great at higher concentrations of ethanol.

Basicity terms From Tables 3 and 4 , it is evident that the basicity

of the solvent mixture has profound influence on the [1'1G1° (RH±) - 1'1G1° (RH2+)] and [1'1G1° (R-) - 1'1Gt0

(RH±)] values. These results suggest that the mixed

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Table 4-Free energy (kJ mor 1) of transfer of d/-alanine from

water to ethanol-water mi xtures at 30°C

Terms % of ethanol (wlw) 20 40 60 80

tJ.G,o ( I )diss !.54 2.96 5.24 8.04

tJ.G1° (RH±) - tl.G1° (RH2 +) 6.04 11.26 12 .74

tJ.G,o (2)diss -1.31 -2.34 -1.14 -0.06

tJ.G1° (R") - tJ.G,o (RH±) 3.19 5.96 6.36

Table 5-Change in proton affinity tJ.P, (kJ mor 1) in transfer H20

-7 EtOH-H20 system. Values of tJ.P, at 3 ooc % EtOH R·=Giycinate d/-Aianinate M, c

(wlw)

20 6.28 6.45 20.5144 -0.4103

40 12.48 12.70 30.0267 -1.4431

60 13.43 14.17 28.3880 -1.4321

solvents are more basic than water but the maximum basicity appears at different percentages of the etha­nol-water mixtures. This conclusion is in accordance with the observations of Wells29·30, Popovych31 , and Paabo, Bates and Robinson et a/.32·33. However, at higher percentages of ethanol, the basicity of the sol­vent mixtures may decrease whereby the cation rather than anion formation may be favoured energetically.

pH values at the isoionic points of the amino acids in water

The isoionic point is the arithmetic mean of the two pK values

... (6)

At the isoionic point the average net electric charge of the amino acid molecule is zero. pH values at the isoionic point of the amino acids investigated in water at 30°C have been calculated. The isoionic point (PI) of glycine and dl-alanine are 6.00 & 6.05 respectively at 30°C. At their isoionic points, the amino acids will not migrate in an electric field and have a minimum solubility. At other pH values, they are more soluble.

Predictions from Born equation For formal verification of the Born equation [pKs­

pKw] needed to be plotted against [D-1] where sub­script (w) indicates solvent water and subscript (s) indicates solvent other than water. Straight line rela­tionship of the plot would prove the validity of the Born equation. The plots for these amino acids pro­duce non-linearity. Non-linearity of the plots suggests

216

Indian J. Chem. Techno!., May 2002

that the basicity of the solvent reflected in the nonelectrostatic part of the free energy change plays a dominant role in the di ssociation process of A ±B-. The deviations suggest that the effect of the ethanol-water mixtures on the acidity of amino aci ds does not de­pend solely on electrostatic features as predicted by the Born theory. This conclusion is in agreement with the earlier works 1

.4.6

.34 .

Change in proton affinity M>a of conjugate base (R) of amino acids

The change in proton affinity 1'1Pa of conjugate base (K) of these amino acids on transfer from water to ethanol-water mixtures are calculated by Eq. (7) and are arranged in Table 5.

1'1P a=1'1Gr0( 1 )diss-1'1Gr0 (H+)-C (7)

Constant C=5 .7 log(18.0ld/M5.dw) (8) (in molar scale)

where ds represents the density of solvents and dw is the density of water. The mass Ms of EtOH-H20 mixtures are calculated from Eq. (9) and are also given in Table 5 in kJ mor' .

M5=100/[(wt%Et0H/46.07)+wt%H20/18.06)]35

(9)

t.G1° (H+) are free energies of proton from water to ethanol-water mixtures and have been taken from lit­erature24.

These values show that glycine and dl-alanine are grouped together giving similar trend in variation of t.Pa with composition of EtOH-H20 mixtures. There­fore, from the results it can be said that the amino ac­ids in water are present as equilibrium of zwitterion and ions (cation and anion) where cation predomi­nates. Again, from the results, it is evident that the amino acids in ethanol-water mixture are also present as an equilibrium mixture of zwittetion and anion (predominantly). The concentration of anion increases with the concentration of ethanol. Hence, one can conclude from the results that specific solute-solvent interactions are of great importance in explaining the variation of pK values in ethanol-water mixed sol­vents.

Acknowledgement The authors wish to thank Professor M.A. Rahman

of the Department of Applied Chemistry and Chemi­cal Technology, Rajshahi University for his active interest in this work.

Ashfaqur Rahman eta/.: Thermodynamic Di ssociaton Constants of Glycine and d/-Alanine Articles

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