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Reactive extraction of penicillin II: Distribution coefficients and degrees of extraction
The Chemical
Engineering
Journal,
28 (1984)
Bll
- B20
Bll
Reactive Extraction of Penicillin II: Distribution Coefficients and Degrees of Extraction
M. RESCHKE Institut
and K. SCHOGERL
fiir Technische
(Received
December
Chemie,
Universittit
Hannover,
Hannover
(F.R.G.)
4,1983)
ABSTRACT
The equilibrium constants and degrees of extraction of penicillin G are investigated by using primary, secondary and tertiary amines as carriers and a range of pH values and concentrations of penicillins and amines for various solvents. In these ion pair extractions the extraction and re-extraction can be controlled by setting a suitable pH value. When secondary and tertiary amines are used, the extraction and re-ex traction of penicillin G is possible in the pH range from 5 to 7 in which penicillin G is stable enough to use common extractors at room temperature. When quaternary amines are used, the extraction is coupled with an ion exchange process. In this case the re-extraction is difficult. The reactive extraction of penicillin V and the precursors phenylacetic acid and phenoxyacetic acid is investigated and the selectivity of penicillins with regard to their precursor is determined.
1. INTRODUCTION
In Part I [ 1 J the stability of penicillin G was considered and relationships for the distribution coefficients and degrees of reactive extraction were evaluated. In this paper the measured equilibrium constants and degrees of physical and reactive extractions are presented.
with 25 ml of organic solvent for at least 1 min; if the separation of the phases took longer, a centrifuge was used. Then the penicillin content and the pH value in the aqueous phase were determined. The penicillin concentration in the organic solvent phase was evaluated from its re-extraction with a buffer solution at pH 9 and the measurement of its concentration in the buffer. The concentrations of penicillin G and penicillin V were measured with a polarimeter; at low pH values the iodometric method was used. The concentrations of precursors (phenylacetic acid and phenoxyacetic acid) were measured by photometric methods.
3. EXPERIMENTAL
3.1. Physical extraction
METHODS
The investigations were carried out in a 100 ml separator funnel at room temperature. The potassium salt of penicillin was dissolved in 25 ml of buffer solution and was shaken 0300-9467/84/$3.00
of penicillin
G
The measurements were carried out with aqueous buffer solutions at pH 4.0 and 5.0 and various organic solvents without a carrier. In Fig. 1 the degree E of extraction is shown as a function of the pH value. The highest degree E of extraction and highest partition coefficient C were achieved with n-butyl acetate. The value of 48 for the partition coefficient C of the free penicillin acid evaluated for n-butyl acetate agrees fairly well with the data given in the literature (C = 46.9 and C = 54.7 at T = 0 “C! [2] and C = 47 at T=8”C
2. EXPERIMENTAL
RESULTS
[3]).
The distribution coefficient KPh,. of penicillin G quickly decreases with increasing pH values because of the dissociation equilibrium of penicillin G; at pH 4.4, KPhy has decreased to 1.0 and at pH 5 to 0.27, which corresponds to a degree of extraction E of 21%. Hence, in spite of the high partition coefficient C no Q Elsevier
Sequoia/Printed
in The Netherlands
I312
1.0
3.
0
5.
0
pH
7-0
Fig. 1. Degree of extraction as a function of the pH value of the medium for the physical extraction of penicillin G with various solvents: curve 1, v, n-butyl acetate, C = 48;curve 2, X, isobutyl acetate, C = 37; curve 3, 0, isoamyl acetate, C = 22; curve 4, 0, chloroform, C = 12.5; curve 5, A, diisopropyl ether, C = 2.4; curve 6, 0, dioctyl ether, C = 0.4; curve 7, O, n-hexane, C = 0.2; curve 8, X, xylol, C = 0.1; curve 9, 0, kerosene, C = 0.05.
6.
0,
1
3.
m
5.
0
7.
0
9.
q
PH Fig. 3. Degree of extraction as a function of the pH value of the medium for the extraction of penicillin G with Amberlite LA-2 and n-butyl acetate (cp = 10 mm01 1-l): curve 1, A, c~ = 0; curve 2, L2, CA = 10 mm01 1-r; curve 3, V, cA = 20 mm01 l--‘; curve 4, 0, CA = 50 mm01 I-r ; curve 5,0, cA = 100 mm01 I-‘.
10’ l* mall’* (the mean of 44 measurements with a standard deviation of +2%). In Fig. 3 the degree of extraction is plotted as a function of the pH value at a constant penicillin G concentration and at various amine concentrations. The symbols indicate the measured data, and the curves were calculated from 1 _
cp(1 + 10PKs-PH)
x 100
(1)
cP, G
-2.
I
0
0
2.
lo9
0
c
Fig. 2. Relationship between the dipole moment p of the solvent and the partition coefficient of penicillin G between the solvent and the aqueous phase for the physical extraction of penicillin G: 0, data from the literature; X, measurements of the present authors.
economic extraction is possible in the pH range in which penicillin G is stable. The partition coefficient with various organic solvents obviously depends on their polarity. The relationship between the dipole moments of the solvents and the corresponding partition coefficients for penicillin G is plotted in Fig. 2. An approximately linear relationship between the dipole moment p and log C can be observed. 3.2. Reactive extraction of penicillin G with Amberlite LA-2 and n-butyl acetate The equilibrium constant K, of the reaction between 1 mol of penicillin G and 1 mol of Amberlite LA-2 was evaluated as 1.25 X
where cp is a function of C and KG (ref. 1, eqns. (6) and (7)). The agreement between the measured and calculated values is excellent. Thus the reactive extraction follows the simple stoichiometry A+P-+H+-AHP
(2)
where A is Amberlite LA-2, P- the penicillin acid anion, H+ a proton and AHP the aminepenicillin complex. (For the scheme of the reactive extraction see ref. 1, Fig. 6.) Obviously, the coextraction of buffer and chloride ions (from the aqueous HCl solution which was used to vary the pH) in some experiments does not have a great influence on the penicillin extraction. Only a slight increase in the pH of the aqueous solution is caused by the extraction. In Fig. 4, log K is plotted as a function of the pH value, again for a fixed penicillin concentration and various amine concentrations. It can be seen that the stoichiometric use of the carrier increases K significantly. E = 90% (corresponding to K = 10) can be attained at pH 3.4 without a carrier and at
B13
pH 4.5 with a stoichiometric amount of carrier. This pH shift improves the stability of penicillin G by a factor of 10. With increasing carrier concentration the pH shift increases, e.g. for a ratio of amine to penicillin G of 10 to 1 it equals 2.7. However, under these conditions the re-extraction becomes difficult. It can be seen from Fig. 3 that the optimum ratio of amine to penicillin G is about 2 to 1. Under these conditions the extraction can be carried out at pH 5.0 and the re-extraction at pH 7.5. Penicillin G is sufficiently stable in this pH range. The degree of extraction at both of these pH values is about 93%. In a two-stage equipment, 99% of penicillin G can be recovered, provided that the contact times of the phases are long enough. In Figs. 5 and 6 the influences of the concentration of amine and the concentration of penicillin on the degree of extraction are
plotted for various pH values. The influence of the amine concentration on E is more significant than that of the penicillin concentration. Thus, E can be controlled by the amount of amine and by the pH value. The influence of the concentrations of amine and penicillin (used in a stoichiometric ratio) .on E is demonstrated in Fig. 7. With increasing concentrations, E increases, as expected according to the law of mass action. The slight pH shift of buffered penicillin G solutions indicates a coextraction of the buffer components (citrate and phosphate). To investigate this effect, the degrees of extraction were calculated as a function of the pH value with various degrees of coextraction of anions (with equilibrium constants K,) and
. . 4
0
0.
02
0.
04
0.
08
cp [mol/ll 3.0
5.
0
7.
0
0.
0
PH Fig. 4. Logarithm of the partition coefficient function of the pH value of the medium (cp mm01 1-r): curve 1, a, CA = 0; curve 2, 0, c mm01 1-r ; curve 3, v, c - 20 mm01 1-l ; curvt CA = 50 mm01 1-l ; curvAe ;, 0 , c A = 100 mm01
as a = 10 - 10 4 0, l-:.
Fig. 6. Degree of extraction as a function of penicillin concentration for the extraction of penicillin G with Amberlite LA-2 and n-butyl acetate (CA = 10 mmol 1-l): curve 1, pH 4.0; curve 2, pH 5.0; curve 3, pH 6.0; curve 4, pH 7.0.
2
3.
0
5.
7.
0
0
9.
0
PH
Fig. 5. Degree of extraction as a function of amine concentration for the extraction of penicillin G with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol 1-l): curve 1, pH 4.0; curve 2, pH 5.0; curve 3, pH 6.0; curve 4, pH 7.0.
Fig. 7. Degree of extraction as a function of the pH value of the medium for stoichiometric concentrations of penicillin and carrier in the extraction of penicillin G with Amberlite LA-2: curve 1, cp = 10 mmol l-l, CA = 0; curve 2, cp = CA = 1 mm01 1-l ;
curve3,~,cp=CA=5mmolI-‘;curve4,o,cp=cA= 10 mm01 1-l; curve 5, 0, cp = CA = 50 mm01 1-l; curve 6, cp = CA = 100 mmol
1-l.
B14
E
[%I
PH 3.
0
5.
0
7.
0
9.
0
P”
Fig. 8. Calculated influence of the coextraction of an anion X in the extraction of penicillin G with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol 1-l; cx = 100 mm01 1-l): curve 1, c~ = 100 mm01 I-‘, Kx = 0;curve 2, cA = 100 mmol l-l, K, = 1 X lo7 1* rn~;In;;,~;;;~,;~ = 100 mmol:-‘, Kx = 1 X 1081* , A = 10 mmol l- ,Kx = 0;curve 5, CA = 10 mmol l-l, Kx = 1 x lo6 1’ mol-*; curve 6, cA = 10 mmol l-r, Kx = 1 X lo7 l* mol-*; curve ‘7, cA = 10 mmol l-l, Kx = 1 X lOa 1’ mol-2.
amine concentrations (Fig. 8). The equilibrium constants Kx of other anions can be estimated from the deviation of the measured curves compared with the curves which were calculated for the simple reaction (2). The equilibrium constants of other anions must be smaller than lo6 1’ mol-‘. 3.3. Extraction of penicillin G with other secondary amine-n-butyl acetate systems Dioctylamine, another secondary amine, was investigated with regard to its suitability as a carrier for penicillin extraction. The results are shown in Fig. 9 as a function of the pH value at various amine concentrations. The excellent agreement between the calculated and measured results indicates that for this amine the simple reaction (2) holds true also. The equilibrium constant KG was determined to be 7 X lo8 l2 mol-2. A comparison of the results obtained with Amberlite LA-2 and dioctylamine indicates that significantly higher distribution coefficients can be attained with dioctylamine than with LA-2, e.g. at a stoichiometric ratio of amine to penicillin G an E value of 90% can be achieved at pH 4.5 with LA-2. When dioctylamine is used, the same degree of extraction can be obtained at pH 5.0. However, the re-extraction of penicillin G with dioctylamine is difficult because of the high pH value necessary. Reextraction of penicillin with a high degree of
3.
0
s.
0
7.
0
9.
0
Fig. 9. Degree of extraction as a function of the pH value of the medium for the extraction of penicillin G with dioctylamine and n-butyl acetate (cp = 10 mmol 1-l): curve 1, CA = 0;curve 2, v, CA = 10 mmo) 1-r; curve 3, O, CA = 20 mm01 1-l; curve 4, 0, c~ = 50 mm01 1-r; curve 5, cA = 100 mm01 1-l.
.I 3.
0
5.
0
7.
0
9.
0
PH
Fig. 10. Degree of extraction as a function of the pH value for the extraction of penicillin G with secondary amines and n-butyl acetate (cP = 10 mmol 1-I ; CA = 10 mm01 l--r): curve 1, no carrier; curve 2, 0, Amberlite LA-1 ; curve 3, 0, Amberlite LA-2; curve 4, V, dioctylamine; curve 5, X, Adogen 283.
extraction in a pH range in which it is still stable would only be possible if a consecutive reaction could be used in which the penicillin concentration in the aqueous buffer solution could be kept at a low level. In Fig. 10, four secondary amine-n-butyl acetate systems are compared: Amberlite LA-1 (a technical mixture of secondary amines), Amberlite LA-2, dioctylamine and Adogen 283 (bis(tridecyl)amine). The highest degree of extraction was attained with dioctylamine and Adogen 283, and the lowest with LA-l. All these systems can be used for penicillin G extraction. The following equilibrium constants were evaluated: Kc = 4.0 X lo7 l2 molm2 for Amberlite LA-2; KG = 7.5 X lo8 l2 mole2 for Adogen 283,
B15
3.4. Extraction acetate systems
with tertiary amine-n-butyl
In Fig. 11 the degrees of extraction with different tertiary amine-n-butyl acetate systems are compared. In contrast with secondary amine systems there is a fairly small difference between them. The E uersus pH curves of all the tertiary amine-n-butyl acetate systems investigated (only a few are shown in Fig. 11) fall between the curves of Adogen 383 and dimethylpalmitylamine. All these amines are less effective than the secondary amines. However, it was possible to use them for penicillin G extraction at high amine concentrations. The following equilibrium constants were evaluated: K, = 2.0 X lo7 l* mol-* for Adogen 383; KG = 4.5 X 10’ l* mole2 for dimethylpalmitylamine; KG = 2.8 X 10’ l* mol-* for trioctylamine. (For the results for further tertiary amine-n-butyl acetate systems, see ref. 4.)
PH Fig. 12. Degree of extraction as a function of the pH value for the extraction of penicillin G with primary amines and n-butyl acetate (cp = 10 mmol 1-l ; cA = 10 mm01 I-‘): curve 1, 0, n-octylamine; curve 2,0, n-dodecylamine; curve 3, V, decylamine; curve 4,0, Amberlite LA-3; curve 5, A, Adogen 115-D.
To describe the experimental data, the following non-stoichiometric reaction model was used: mA + nH+ + nP- I
lee
E
(3)
The equilibrium constant KG of this reaction is given by
C%l
KG=
CA,W’),
(4)
CA %HnCpn
5e
3.
A,(HP),
0
5.
0
7. 0
9.
0
PH Fig. 11. Degree of extraction as a function of the pH value for the extraction of penicillin G with tertiary amine and n-butyl acetate (cp = 10 mmol 1-l): curve 1, no carrier; curve 2, V, Adogen 383, cA = 10 mmol 1-l ; curve 3, 0, trioctylamine, cA = 10 mmol 1-l ; curve 4, 0, dimethylpalmitylamine, cA = 10 mmol 1-l; curve 5, trioctylamine, cA = 20 mmol 1-l; curve 6, trioctylamine, cA = 50 mm01 1-l; curve 7, trioctylamine, cA = 100 mmol 1-r.
3.5. Extraction acetate systems
with primary
amine-n-butyl
In Fig. 12, various primary amine-n-butyl acetate systems are compared. Significant deviations are observed, compared with the simple reaction (2). The data indicate that, in accordance with the literature [ 51, it is necessary to take into account the higher complexes consisting of one penicillin G molecule and more than one amine molecule.
(For the dependence of KG on pK,, pH, c, m and n, see ref. 1.) By fitting the calculated E uersus pH curves to the measured curves, a series of formal compositions was identified: Ai.* for Amberlite LA-3; Aim4(HP)for Adogen 115-D; Ai.s(HP) for tridecylamine; A,.,(HP) for n-decylamine; A,_,(HP) for ndodecylamine; A,.a(HP) for n-octylamine. The primary amines as carriers exhibit fairly low distribution coefficients. Also, the pH dependence of E is reduced by the increasing number of amines in the complex. As a consequence, a larger pH shift is necessary for the extraction and re-extraction of penicillin G. In addition, the primary amines have a high interfacial activity and this causes fairly stable emulsions. Thus, primary amines are less suitable as carriers for penicillin G extraction than are secondary and tertiary amines. 3.6. Extraction solvents
with amines in various organic
In Fig. 13 the degree of extraction of penicillin G is plotted as a function of the pH value with Amberlite LA-2 in various solvents. For kerosene no measured values are given,
B16
since the equilibrium constant was determined at a higher carrier concentration. This was necessary because of the low degree of extraction achieved at cA = 10 mmol ll’. The E values with isoamyl acetate and n-butyl acetate are very similar. Chloroform yields high degrees of extraction at high pH values. With kerosene, low values of E are obtained in a low pH range. Again, the agreement between the measured values and the calculated values, on the assumption of the simple reaction (2), are excellent. A comparison of the physical and reactive extractions (Figs. 1 and 13) indicates an approximate proportionality between the distribution coefficients of physical and reactive extractions. In Fig. 14 the degree of extraction is plotted as a function of the pH value for penicillin G with Amberlite LA-2, Adogen 283 and dioctylamine as well as in the absence of carriers in chloroform. The large pH shift of the E uersus pH curve in the presence of these carriers is clearly apparent.
2.
0
4.
0
6.
0
e.
0
PH Fig. 13. Degree of extraction as a function of the pH value for the extraction of penicillin G with Amberlite LA-2 in various solvents (cp = 10 mmol 1-r CA = 10 mmol I-.‘): curve 1, kerosene; curve 2, A, dioctyl ether; curve 3, 0, diisopropyl ether; curve 4, n, xylol; curve 5, X, isoamyl acetate; curve 6,0, isobutyl acetate; curve 7, n-butyl acetate; curve 8, ‘J, chloroform.
E
3.7. Extraction with a quaternary aminen-butyl acetate system The reactive extraction with quaternary ammonium salts proceeds according to the following reaction: NR,+X-(org)
+ P-(aq) e
NR,‘P-(org)
+ X(5)
where X- is an anion (e.g. Cl-). The equilibrium constant of reaction (5) is given by
In Fig. 15 the degree of extraction is plotted as a function of the concentration of Adogen 464 (trioctylmethylammonium chloride) with a penicillin G concentration cr. of 10 mmol 1-l in the pH range from 6 to 7. The agreement between the measured and calculated degrees of extraction is excellent. The curve was calculated with KG = 20. For a fivefold excess of the ammonium salt a nearly quantitative extraction is possible. The pH of the medium has no influence on E in the range of interest; therefore re-extraction is difficult and a large amount of salt is required (e.g. a chloride excess of 200 times is necessary to attain E = 90% for the back extraction).
3.
0
5.
0
7.
0
9.
0
PH Fig. 14. Degree of extraction as a function of the pH value for the extraction of penicillin G with secondary amines and chloroform (cp = 10 mmol 1-l ; CA = 10 mm01 1-r): curve 1, V, no carrier; curve 2, 0, Amberlite LA-2; curve 3, A, Adogen 283; curve 4, n, dioctylamine.
0
0.
02
0.
04
0. CA Imol/l
0e I
Fig. 15. Degree of extraction as a function of the carrier concentration for the extraction of penicillin G with Adogen 464 and n-butyl acetate (cp = 10 mmol l--l ; pH 6 - 7).
B17
3.8. Reactive E
C%’
51a
..
3.
0
5.
0
7.
0
s.
0
PH Fig. 16. Degree of extraction as a function of the pH value for the extraction of penicillin V with secondary amines and n-butyl acetate (cp = 10 mmol 1-l): curve 1, A, no carrier; curve 2, 0, Amberlite LA-2, CA = 10 mm01 1-r; curve 3, 0, dioctylamine, CA = 10 mmol 1-l ; curve 4, 0, Amberlite LA-2, CA = 50 mmol 1-r; curve 5,0, dioctylamine, CA = 50 mmol 1-r.
3.
0
5.
0
7.
0
s.
0
PH Fig. 17. Degree of extraction as a function of the pH value for the extraction of phenylacetic acid with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol 1-l): curve 1, 0, CA = 0; curve 2, 0, CA = 10 mmol 1-l; curve 3, v, CA = 100 mmol 1-r.
extraction
of penicillin
V
In Fig. 16 the degree of extraction of penicillin V is plotted as a function of the pH value with various carriers in n-butyl acetate. The pK, value of penicillin V is 2.70 (the mean value of data given in the literature [ 6 - 81). Its partition coefficient in a watern-butyl acetate system is high (C = 210). This value deviates significantly from the C value of 39 reported in ref. 9. The reaction equilibrium constants KG were found to be 3.75 X lo8 l2 mo12 for Amberlite LA-2 and 3.00 X lo9 l2 molM2for dioctylamine. Hence, penicillin V can be extracted more efficiently than penicillin G, physically as well as chemically. 3.9. Extraction of penicillin precursors The product of penicillin fermentations can be controlled by the addition of a precursor, e.g. on the addition of phenylacetic acid to the medium the fungus produces penicillin G; with phenoxyacetic acid, penicillin V is formed. It is of economic interest to recover the expensive precursor and to separate it from the penicillin because of its toxicity. In Figs. 17 and 18 the degrees of extraction of phenylacetic acid and phenoxyacetic acid with Amberlite LA-2 in n-butyl acetate are plotted as a function of the pH value of the medium. The curves were calculated using the data given in Table 1 and assuming the simple reaction (2). There is fairly good agreement between the calculated and measured degrees of extraction. Phenylacetic acid can be extracted to a higher degree than penicillin G as a result of its high pK, value (small dissociation constant) which overcompensates for its small partition coefficient C. In contrast, phenoxyacetic acid can be extracted to a lesser degree than penicillin V because of its low partition TABLE
1
Data for calculating the degrees of extraction phenylacetic acid and phenoxyacetic acid
3.
0
5.
0
7.
0
a.
PEs
0
PH Fig. 18. Degree of extraction as a function of the pH value for the extraction of phenoxyacetic acid with Amberlite LA-2 and n-butyl acetate (cp = 1 mmol 1-r): curve 1, A, cA = 0; curve 2,0, CA = 1 mmol 1-r; curve 3, q, CA = 3 mmol 1-l; curve 4, 0, CA = 10 mm01 1-r.
Partition coefficient
C
of
Equilibrium constant (12 mol+)
Phenylacetic acid Phenoxyacetic acid
4.28
25
2 x 107
3.14
30
5 x 10’
RlR
coefficient which overcompensates for its high pK, value. In the presence of carriers, both precursors have significantly lower degrees of extraction than the corresponding penicillin. Thus, a higher selectivity of penicillin can be attained by reactive extraction than by physical extraction. To prove these observations, the coextraction model developed in ref. 1 was used to calculate the degrees of extraction of penicillin G and phenylacetic acid in the pH range between 4 and 7 during physical and reactive coextraction (Fig. 19). It can be observed that the precursor is extracted better physically. With increasing carrier concentration the degree of extraction for penicillin G increases more quickly than that of the precursor. Thus, at high carrier concentrations and at pH 4 - 5, penicillin G and the precursor have the same degrees of extraction. From pH 6 to pH 7 the degree of extraction of penicillin G is higher than that of the phenylacetic acid. In Fig. 20 the ratio of the degree of extraction of penicillin G to that of its precursor is plotted as a function of the carrier concentration at various pH values. It can be seen that at a higher carrier concentration and at pH 4 this ratio equals 2. In Fig. 21 the ratio of the degree of extraction of penicillin V to that of phenoxyacetic acid is plotted as a function of the carrier concentration at various pH values with
0
0.
02
0.
04
0.
06
cA [mollll
Fig. 19. Calculated degrees of extraction as a function of the carrier concentration for the coextraction of penicillin G and phenylacetic acid (each of them with 10 mmol 1-l) with Amberlite LA-2 and n-butyl acetate: curve 1, pH 4.0, phenylacetic acid; curve 2, pH 4.0, penicillin G; curve 3, pH 5.0, phenylacetic acid; curve 4, pH 5.0, penicillin G; curve 5, pH 6.0, phenylacetic acid; curve 6, pH 6.0, penicillin G; curve 7, pH 7.0, phenylacetic acid; curve 8, pH 7.0, penicillin G.
3.
01
0
0.01
0.
02
a3
0. cA [mol/l
I
Fig. 20. Ratio of the degree Ep of extraction of penicillin G to the degree Eh,, of extraction of phenylacetic acid as a function of the carrier concentration at various pH values for the coextraction of penicillin G and phenylacetic acid with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol 1-l ; cpre, = 10 mmol 1-l): curve 1, pH 4.0; curve 2, pH 5.0; curve 3, pH 6.0; curve 4, pH 7.0.
Y
d
3
0
0.
01
0.
02
0. cA
03
[mollll
Fig. 21. Ratio of the degree Ep of extraction of penicillin V to the degree Ep,.+, of extraction of phenoxyacetic acid as a function of the carrier concentration at various pH values for the coextraction of penicillin V and phenoxyacetic acid with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol I-‘;c prec = 10 mmol 1-i): curve 1, pH 4.0; curve 2, pH 5.0; curve 3, pH 6.0; curve 4, pH 7.0.
Amberlite LA-2 in n-butyl acetate. At a high pH the ratio is high; it increases with the carrier concentration, passes a maximum and then decreases. By lowering the pH, the ratio is reduced and at pH 4.0 it attains a value of unity, because for both components the degree of extraction is unity.
4. CONCLUSIONS
Primary amines are not very suitable carriers for penicillin G because of the moderate partition coefficients of their penicillin complexes. In addition, they have a fairly high
B19
solubility in water and a high interfacial activity, which causes stable emulsions. The highest degrees of extraction can be attained by using secondary amines as carriers. When secondary amines are used, penicillin G can be extracted up to a pH value of 6. The distribution coefficients K of their amine complexes with penicillin G markedly depend on the amine structure. A disadvantage of secondary amines can be their reactivity with solvents, e.g. they may form amides with acetic acid esters during the distillative purification of the solvent. Tertiary amines are suitable as carriers for penicillin G extraction but the distribution coefficients of their complexes are significantly lower than those of secondary amine complexes. Their advantage lies in their low reactivity with solvents. Quaternary amines can be used for the reactive extraction of penicillin G. However, re-extraction is difficult and very large amounts of anions (e.g. Cl-) are needed to recover the penicillin. The results obtained agree with those found for the reactive extraction of acetic acid with various amines [lo, 111. Penicillin V can be recovered more easily by physical as well as by reactive extraction than can penicillin G. Precursors of penicillin G and V can also be coextracted with amines and the selectivity of penicillin can be improved by using reactive extraction. The reactive extraction of penicillin with secondary and tertiary amines can be described by a simple stoichiometric reaction. When primary amines are used, the nonstoichiometric reaction has to be taken into account. The relationships developed in ref. 1 for distribution coefficients and degrees of reactive extraction yield useful results which agree well with the measurements. From this work it appears that reactive extraction provides new possibilities for the recovery of primary and secondary metabolites from fermentation media.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support of the Ministry of Research and Technology, F.R.G., and the support of Hoechst AG.
REFERENCES 1 M. Reschke and K. Schiigerl, Reactive extraction of penicillin, I, Stability of penicillin G in the presence of carriers and relationships for distribution coefficients and degrees of extraction, Chem. Eng. J., 28 (1984) Bl. 2 F. C. Whitmore, R. B. Wagner, C. I. Noll, G. C. Basseler et al., Processing penicillin, Ind. Eng. Chem., 38 (1946) 942 - 948. 3 S. A. Zhukovskaya, V. N. Rusin and V. L. Pebalk, Kinetics of mass transfer during transfer of antibiotics through the interface in aqueous solutionbutyl acetate system, J. Appl. Chem. U.S.S.R., (Engl. Transl.), 49 (1976) 1142 - 1146. 4 M. Reschke, Doctoral Thesis, University of Hannover, 1983. 5 R. Modin and M. Schriider-Nielsen, Quantitative determination by ion pair extraction, Part 9, Extraction of penicillins with ion pairing and adduct-forming agents, Acta Pharm. Suet., 8 (1971) 573 - 584. 6 M. Schroder-Nielsen, Extraction of carboxylic acids as adducts with trioctylphosphine oxide, Acta Pharm. Suet., 13 (1976) 133 - 144. 7 H. Margreiter and F. Gapp, Penicilline-Chemie und Eigenschaften. In R. Brunner and G. Machek (eds.), Die Antibiotika, Vol. I, Part 1, Hans Carl, Niirnberg, 1962, p. 225. 8 A. Tsuji, E. Miyamoto, N. Hashimoto and T. Yamana, GI-absorption of @actam antibiotics, II, Deviation from pH-partition hypothesis in penicillin absorption through in situ and in vitro lipoidal barriers, J. Pharm. Sci., 67 (12) (1978) 1705 - 1711. 9 S. A. Zhukovskaja, V. N. Rusin and V. L. Pebalk, Investigation of equilibrium distribution of antibiotics in the system aqueous phase-butyl acetate, J. Appl. Chem. U.S.S.R. (Engl. Transl.), 50(1977)848-852. 10 N. L. Ricker, J. N. Michaels and C. J. King, Solvent properties of organic bases for extraction of acetic acid from water, J. Sep. Process Technol., 1 (1) (1979) 36 - 41. 11 N. L. Ricker, E. F. Pittmann and C. J. King, Solvent extraction with amines for recovery of acetic acid from dilute aqueous industrial streams, J. Sep. Process Technol., I (2) (1980) 23 - 30.
APPENDIX A: NOMENCLATURE
aq
A AHP c c E H+ K
aqueous phase amine, carrier amine-penicillin complex concentration (mm01 1-r or mol 1-l) partition coefficient of physical extraction degree of extraction (5%) proton distribution coefficient of reactive extraction
B20
KG
K Phy org P-
T X-
equilibrium constant (I2 molm2) distribution coefficient of physical traction organic phase penicillin anion temperature (“C) anion
ex-
Subscripts amine, carrier A overall G proton H penicillin or penicillin P precursor Prec anion X
anion
Engineering
Journal,
28 (1984)
Bll
- B20
Bll
Reactive Extraction of Penicillin II: Distribution Coefficients and Degrees of Extraction
M. RESCHKE Institut
and K. SCHOGERL
fiir Technische
(Received
December
Chemie,
Universittit
Hannover,
Hannover
(F.R.G.)
4,1983)
ABSTRACT
The equilibrium constants and degrees of extraction of penicillin G are investigated by using primary, secondary and tertiary amines as carriers and a range of pH values and concentrations of penicillins and amines for various solvents. In these ion pair extractions the extraction and re-extraction can be controlled by setting a suitable pH value. When secondary and tertiary amines are used, the extraction and re-ex traction of penicillin G is possible in the pH range from 5 to 7 in which penicillin G is stable enough to use common extractors at room temperature. When quaternary amines are used, the extraction is coupled with an ion exchange process. In this case the re-extraction is difficult. The reactive extraction of penicillin V and the precursors phenylacetic acid and phenoxyacetic acid is investigated and the selectivity of penicillins with regard to their precursor is determined.
1. INTRODUCTION
In Part I [ 1 J the stability of penicillin G was considered and relationships for the distribution coefficients and degrees of reactive extraction were evaluated. In this paper the measured equilibrium constants and degrees of physical and reactive extractions are presented.
with 25 ml of organic solvent for at least 1 min; if the separation of the phases took longer, a centrifuge was used. Then the penicillin content and the pH value in the aqueous phase were determined. The penicillin concentration in the organic solvent phase was evaluated from its re-extraction with a buffer solution at pH 9 and the measurement of its concentration in the buffer. The concentrations of penicillin G and penicillin V were measured with a polarimeter; at low pH values the iodometric method was used. The concentrations of precursors (phenylacetic acid and phenoxyacetic acid) were measured by photometric methods.
3. EXPERIMENTAL
3.1. Physical extraction
METHODS
The investigations were carried out in a 100 ml separator funnel at room temperature. The potassium salt of penicillin was dissolved in 25 ml of buffer solution and was shaken 0300-9467/84/$3.00
of penicillin
G
The measurements were carried out with aqueous buffer solutions at pH 4.0 and 5.0 and various organic solvents without a carrier. In Fig. 1 the degree E of extraction is shown as a function of the pH value. The highest degree E of extraction and highest partition coefficient C were achieved with n-butyl acetate. The value of 48 for the partition coefficient C of the free penicillin acid evaluated for n-butyl acetate agrees fairly well with the data given in the literature (C = 46.9 and C = 54.7 at T = 0 “C! [2] and C = 47 at T=8”C
2. EXPERIMENTAL
RESULTS
[3]).
The distribution coefficient KPh,. of penicillin G quickly decreases with increasing pH values because of the dissociation equilibrium of penicillin G; at pH 4.4, KPhy has decreased to 1.0 and at pH 5 to 0.27, which corresponds to a degree of extraction E of 21%. Hence, in spite of the high partition coefficient C no Q Elsevier
Sequoia/Printed
in The Netherlands
I312
1.0
3.
0
5.
0
pH
7-0
Fig. 1. Degree of extraction as a function of the pH value of the medium for the physical extraction of penicillin G with various solvents: curve 1, v, n-butyl acetate, C = 48;curve 2, X, isobutyl acetate, C = 37; curve 3, 0, isoamyl acetate, C = 22; curve 4, 0, chloroform, C = 12.5; curve 5, A, diisopropyl ether, C = 2.4; curve 6, 0, dioctyl ether, C = 0.4; curve 7, O, n-hexane, C = 0.2; curve 8, X, xylol, C = 0.1; curve 9, 0, kerosene, C = 0.05.
6.
0,
1
3.
m
5.
0
7.
0
9.
q
PH Fig. 3. Degree of extraction as a function of the pH value of the medium for the extraction of penicillin G with Amberlite LA-2 and n-butyl acetate (cp = 10 mm01 1-l): curve 1, A, c~ = 0; curve 2, L2, CA = 10 mm01 1-r; curve 3, V, cA = 20 mm01 l--‘; curve 4, 0, CA = 50 mm01 I-r ; curve 5,0, cA = 100 mm01 I-‘.
10’ l* mall’* (the mean of 44 measurements with a standard deviation of +2%). In Fig. 3 the degree of extraction is plotted as a function of the pH value at a constant penicillin G concentration and at various amine concentrations. The symbols indicate the measured data, and the curves were calculated from 1 _
cp(1 + 10PKs-PH)
x 100
(1)
cP, G
-2.
I
0
0
2.
lo9
0
c
Fig. 2. Relationship between the dipole moment p of the solvent and the partition coefficient of penicillin G between the solvent and the aqueous phase for the physical extraction of penicillin G: 0, data from the literature; X, measurements of the present authors.
economic extraction is possible in the pH range in which penicillin G is stable. The partition coefficient with various organic solvents obviously depends on their polarity. The relationship between the dipole moments of the solvents and the corresponding partition coefficients for penicillin G is plotted in Fig. 2. An approximately linear relationship between the dipole moment p and log C can be observed. 3.2. Reactive extraction of penicillin G with Amberlite LA-2 and n-butyl acetate The equilibrium constant K, of the reaction between 1 mol of penicillin G and 1 mol of Amberlite LA-2 was evaluated as 1.25 X
where cp is a function of C and KG (ref. 1, eqns. (6) and (7)). The agreement between the measured and calculated values is excellent. Thus the reactive extraction follows the simple stoichiometry A+P-+H+-AHP
(2)
where A is Amberlite LA-2, P- the penicillin acid anion, H+ a proton and AHP the aminepenicillin complex. (For the scheme of the reactive extraction see ref. 1, Fig. 6.) Obviously, the coextraction of buffer and chloride ions (from the aqueous HCl solution which was used to vary the pH) in some experiments does not have a great influence on the penicillin extraction. Only a slight increase in the pH of the aqueous solution is caused by the extraction. In Fig. 4, log K is plotted as a function of the pH value, again for a fixed penicillin concentration and various amine concentrations. It can be seen that the stoichiometric use of the carrier increases K significantly. E = 90% (corresponding to K = 10) can be attained at pH 3.4 without a carrier and at
B13
pH 4.5 with a stoichiometric amount of carrier. This pH shift improves the stability of penicillin G by a factor of 10. With increasing carrier concentration the pH shift increases, e.g. for a ratio of amine to penicillin G of 10 to 1 it equals 2.7. However, under these conditions the re-extraction becomes difficult. It can be seen from Fig. 3 that the optimum ratio of amine to penicillin G is about 2 to 1. Under these conditions the extraction can be carried out at pH 5.0 and the re-extraction at pH 7.5. Penicillin G is sufficiently stable in this pH range. The degree of extraction at both of these pH values is about 93%. In a two-stage equipment, 99% of penicillin G can be recovered, provided that the contact times of the phases are long enough. In Figs. 5 and 6 the influences of the concentration of amine and the concentration of penicillin on the degree of extraction are
plotted for various pH values. The influence of the amine concentration on E is more significant than that of the penicillin concentration. Thus, E can be controlled by the amount of amine and by the pH value. The influence of the concentrations of amine and penicillin (used in a stoichiometric ratio) .on E is demonstrated in Fig. 7. With increasing concentrations, E increases, as expected according to the law of mass action. The slight pH shift of buffered penicillin G solutions indicates a coextraction of the buffer components (citrate and phosphate). To investigate this effect, the degrees of extraction were calculated as a function of the pH value with various degrees of coextraction of anions (with equilibrium constants K,) and
. . 4
0
0.
02
0.
04
0.
08
cp [mol/ll 3.0
5.
0
7.
0
0.
0
PH Fig. 4. Logarithm of the partition coefficient function of the pH value of the medium (cp mm01 1-r): curve 1, a, CA = 0; curve 2, 0, c mm01 1-r ; curve 3, v, c - 20 mm01 1-l ; curvt CA = 50 mm01 1-l ; curvAe ;, 0 , c A = 100 mm01
as a = 10 - 10 4 0, l-:.
Fig. 6. Degree of extraction as a function of penicillin concentration for the extraction of penicillin G with Amberlite LA-2 and n-butyl acetate (CA = 10 mmol 1-l): curve 1, pH 4.0; curve 2, pH 5.0; curve 3, pH 6.0; curve 4, pH 7.0.
2
3.
0
5.
7.
0
0
9.
0
PH
Fig. 5. Degree of extraction as a function of amine concentration for the extraction of penicillin G with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol 1-l): curve 1, pH 4.0; curve 2, pH 5.0; curve 3, pH 6.0; curve 4, pH 7.0.
Fig. 7. Degree of extraction as a function of the pH value of the medium for stoichiometric concentrations of penicillin and carrier in the extraction of penicillin G with Amberlite LA-2: curve 1, cp = 10 mmol l-l, CA = 0; curve 2, cp = CA = 1 mm01 1-l ;
curve3,~,cp=CA=5mmolI-‘;curve4,o,cp=cA= 10 mm01 1-l; curve 5, 0, cp = CA = 50 mm01 1-l; curve 6, cp = CA = 100 mmol
1-l.
B14
E
[%I
PH 3.
0
5.
0
7.
0
9.
0
P”
Fig. 8. Calculated influence of the coextraction of an anion X in the extraction of penicillin G with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol 1-l; cx = 100 mm01 1-l): curve 1, c~ = 100 mm01 I-‘, Kx = 0;curve 2, cA = 100 mmol l-l, K, = 1 X lo7 1* rn~;In;;,~;;;~,;~ = 100 mmol:-‘, Kx = 1 X 1081* , A = 10 mmol l- ,Kx = 0;curve 5, CA = 10 mmol l-l, Kx = 1 x lo6 1’ mol-*; curve 6, cA = 10 mmol l-r, Kx = 1 X lo7 l* mol-*; curve ‘7, cA = 10 mmol l-l, Kx = 1 X lOa 1’ mol-2.
amine concentrations (Fig. 8). The equilibrium constants Kx of other anions can be estimated from the deviation of the measured curves compared with the curves which were calculated for the simple reaction (2). The equilibrium constants of other anions must be smaller than lo6 1’ mol-‘. 3.3. Extraction of penicillin G with other secondary amine-n-butyl acetate systems Dioctylamine, another secondary amine, was investigated with regard to its suitability as a carrier for penicillin extraction. The results are shown in Fig. 9 as a function of the pH value at various amine concentrations. The excellent agreement between the calculated and measured results indicates that for this amine the simple reaction (2) holds true also. The equilibrium constant KG was determined to be 7 X lo8 l2 mol-2. A comparison of the results obtained with Amberlite LA-2 and dioctylamine indicates that significantly higher distribution coefficients can be attained with dioctylamine than with LA-2, e.g. at a stoichiometric ratio of amine to penicillin G an E value of 90% can be achieved at pH 4.5 with LA-2. When dioctylamine is used, the same degree of extraction can be obtained at pH 5.0. However, the re-extraction of penicillin G with dioctylamine is difficult because of the high pH value necessary. Reextraction of penicillin with a high degree of
3.
0
s.
0
7.
0
9.
0
Fig. 9. Degree of extraction as a function of the pH value of the medium for the extraction of penicillin G with dioctylamine and n-butyl acetate (cp = 10 mmol 1-l): curve 1, CA = 0;curve 2, v, CA = 10 mmo) 1-r; curve 3, O, CA = 20 mm01 1-l; curve 4, 0, c~ = 50 mm01 1-r; curve 5, cA = 100 mm01 1-l.
.I 3.
0
5.
0
7.
0
9.
0
PH
Fig. 10. Degree of extraction as a function of the pH value for the extraction of penicillin G with secondary amines and n-butyl acetate (cP = 10 mmol 1-I ; CA = 10 mm01 l--r): curve 1, no carrier; curve 2, 0, Amberlite LA-1 ; curve 3, 0, Amberlite LA-2; curve 4, V, dioctylamine; curve 5, X, Adogen 283.
extraction in a pH range in which it is still stable would only be possible if a consecutive reaction could be used in which the penicillin concentration in the aqueous buffer solution could be kept at a low level. In Fig. 10, four secondary amine-n-butyl acetate systems are compared: Amberlite LA-1 (a technical mixture of secondary amines), Amberlite LA-2, dioctylamine and Adogen 283 (bis(tridecyl)amine). The highest degree of extraction was attained with dioctylamine and Adogen 283, and the lowest with LA-l. All these systems can be used for penicillin G extraction. The following equilibrium constants were evaluated: Kc = 4.0 X lo7 l2 molm2 for Amberlite LA-2; KG = 7.5 X lo8 l2 mole2 for Adogen 283,
B15
3.4. Extraction acetate systems
with tertiary amine-n-butyl
In Fig. 11 the degrees of extraction with different tertiary amine-n-butyl acetate systems are compared. In contrast with secondary amine systems there is a fairly small difference between them. The E uersus pH curves of all the tertiary amine-n-butyl acetate systems investigated (only a few are shown in Fig. 11) fall between the curves of Adogen 383 and dimethylpalmitylamine. All these amines are less effective than the secondary amines. However, it was possible to use them for penicillin G extraction at high amine concentrations. The following equilibrium constants were evaluated: K, = 2.0 X lo7 l* mol-* for Adogen 383; KG = 4.5 X 10’ l* mole2 for dimethylpalmitylamine; KG = 2.8 X 10’ l* mol-* for trioctylamine. (For the results for further tertiary amine-n-butyl acetate systems, see ref. 4.)
PH Fig. 12. Degree of extraction as a function of the pH value for the extraction of penicillin G with primary amines and n-butyl acetate (cp = 10 mmol 1-l ; cA = 10 mm01 I-‘): curve 1, 0, n-octylamine; curve 2,0, n-dodecylamine; curve 3, V, decylamine; curve 4,0, Amberlite LA-3; curve 5, A, Adogen 115-D.
To describe the experimental data, the following non-stoichiometric reaction model was used: mA + nH+ + nP- I
lee
E
(3)
The equilibrium constant KG of this reaction is given by
C%l
KG=
CA,W’),
(4)
CA %HnCpn
5e
3.
A,(HP),
0
5.
0
7. 0
9.
0
PH Fig. 11. Degree of extraction as a function of the pH value for the extraction of penicillin G with tertiary amine and n-butyl acetate (cp = 10 mmol 1-l): curve 1, no carrier; curve 2, V, Adogen 383, cA = 10 mmol 1-l ; curve 3, 0, trioctylamine, cA = 10 mmol 1-l ; curve 4, 0, dimethylpalmitylamine, cA = 10 mmol 1-l; curve 5, trioctylamine, cA = 20 mmol 1-l; curve 6, trioctylamine, cA = 50 mm01 1-l; curve 7, trioctylamine, cA = 100 mmol 1-r.
3.5. Extraction acetate systems
with primary
amine-n-butyl
In Fig. 12, various primary amine-n-butyl acetate systems are compared. Significant deviations are observed, compared with the simple reaction (2). The data indicate that, in accordance with the literature [ 51, it is necessary to take into account the higher complexes consisting of one penicillin G molecule and more than one amine molecule.
(For the dependence of KG on pK,, pH, c, m and n, see ref. 1.) By fitting the calculated E uersus pH curves to the measured curves, a series of formal compositions was identified: Ai.* for Amberlite LA-3; Aim4(HP)for Adogen 115-D; Ai.s(HP) for tridecylamine; A,.,(HP) for n-decylamine; A,_,(HP) for ndodecylamine; A,.a(HP) for n-octylamine. The primary amines as carriers exhibit fairly low distribution coefficients. Also, the pH dependence of E is reduced by the increasing number of amines in the complex. As a consequence, a larger pH shift is necessary for the extraction and re-extraction of penicillin G. In addition, the primary amines have a high interfacial activity and this causes fairly stable emulsions. Thus, primary amines are less suitable as carriers for penicillin G extraction than are secondary and tertiary amines. 3.6. Extraction solvents
with amines in various organic
In Fig. 13 the degree of extraction of penicillin G is plotted as a function of the pH value with Amberlite LA-2 in various solvents. For kerosene no measured values are given,
B16
since the equilibrium constant was determined at a higher carrier concentration. This was necessary because of the low degree of extraction achieved at cA = 10 mmol ll’. The E values with isoamyl acetate and n-butyl acetate are very similar. Chloroform yields high degrees of extraction at high pH values. With kerosene, low values of E are obtained in a low pH range. Again, the agreement between the measured values and the calculated values, on the assumption of the simple reaction (2), are excellent. A comparison of the physical and reactive extractions (Figs. 1 and 13) indicates an approximate proportionality between the distribution coefficients of physical and reactive extractions. In Fig. 14 the degree of extraction is plotted as a function of the pH value for penicillin G with Amberlite LA-2, Adogen 283 and dioctylamine as well as in the absence of carriers in chloroform. The large pH shift of the E uersus pH curve in the presence of these carriers is clearly apparent.
2.
0
4.
0
6.
0
e.
0
PH Fig. 13. Degree of extraction as a function of the pH value for the extraction of penicillin G with Amberlite LA-2 in various solvents (cp = 10 mmol 1-r CA = 10 mmol I-.‘): curve 1, kerosene; curve 2, A, dioctyl ether; curve 3, 0, diisopropyl ether; curve 4, n, xylol; curve 5, X, isoamyl acetate; curve 6,0, isobutyl acetate; curve 7, n-butyl acetate; curve 8, ‘J, chloroform.
E
3.7. Extraction with a quaternary aminen-butyl acetate system The reactive extraction with quaternary ammonium salts proceeds according to the following reaction: NR,+X-(org)
+ P-(aq) e
NR,‘P-(org)
+ X(5)
where X- is an anion (e.g. Cl-). The equilibrium constant of reaction (5) is given by
In Fig. 15 the degree of extraction is plotted as a function of the concentration of Adogen 464 (trioctylmethylammonium chloride) with a penicillin G concentration cr. of 10 mmol 1-l in the pH range from 6 to 7. The agreement between the measured and calculated degrees of extraction is excellent. The curve was calculated with KG = 20. For a fivefold excess of the ammonium salt a nearly quantitative extraction is possible. The pH of the medium has no influence on E in the range of interest; therefore re-extraction is difficult and a large amount of salt is required (e.g. a chloride excess of 200 times is necessary to attain E = 90% for the back extraction).
3.
0
5.
0
7.
0
9.
0
PH Fig. 14. Degree of extraction as a function of the pH value for the extraction of penicillin G with secondary amines and chloroform (cp = 10 mmol 1-l ; CA = 10 mm01 1-r): curve 1, V, no carrier; curve 2, 0, Amberlite LA-2; curve 3, A, Adogen 283; curve 4, n, dioctylamine.
0
0.
02
0.
04
0. CA Imol/l
0e I
Fig. 15. Degree of extraction as a function of the carrier concentration for the extraction of penicillin G with Adogen 464 and n-butyl acetate (cp = 10 mmol l--l ; pH 6 - 7).
B17
3.8. Reactive E
C%’
51a
..
3.
0
5.
0
7.
0
s.
0
PH Fig. 16. Degree of extraction as a function of the pH value for the extraction of penicillin V with secondary amines and n-butyl acetate (cp = 10 mmol 1-l): curve 1, A, no carrier; curve 2, 0, Amberlite LA-2, CA = 10 mm01 1-r; curve 3, 0, dioctylamine, CA = 10 mmol 1-l ; curve 4, 0, Amberlite LA-2, CA = 50 mmol 1-r; curve 5,0, dioctylamine, CA = 50 mmol 1-r.
3.
0
5.
0
7.
0
s.
0
PH Fig. 17. Degree of extraction as a function of the pH value for the extraction of phenylacetic acid with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol 1-l): curve 1, 0, CA = 0; curve 2, 0, CA = 10 mmol 1-l; curve 3, v, CA = 100 mmol 1-r.
extraction
of penicillin
V
In Fig. 16 the degree of extraction of penicillin V is plotted as a function of the pH value with various carriers in n-butyl acetate. The pK, value of penicillin V is 2.70 (the mean value of data given in the literature [ 6 - 81). Its partition coefficient in a watern-butyl acetate system is high (C = 210). This value deviates significantly from the C value of 39 reported in ref. 9. The reaction equilibrium constants KG were found to be 3.75 X lo8 l2 mo12 for Amberlite LA-2 and 3.00 X lo9 l2 molM2for dioctylamine. Hence, penicillin V can be extracted more efficiently than penicillin G, physically as well as chemically. 3.9. Extraction of penicillin precursors The product of penicillin fermentations can be controlled by the addition of a precursor, e.g. on the addition of phenylacetic acid to the medium the fungus produces penicillin G; with phenoxyacetic acid, penicillin V is formed. It is of economic interest to recover the expensive precursor and to separate it from the penicillin because of its toxicity. In Figs. 17 and 18 the degrees of extraction of phenylacetic acid and phenoxyacetic acid with Amberlite LA-2 in n-butyl acetate are plotted as a function of the pH value of the medium. The curves were calculated using the data given in Table 1 and assuming the simple reaction (2). There is fairly good agreement between the calculated and measured degrees of extraction. Phenylacetic acid can be extracted to a higher degree than penicillin G as a result of its high pK, value (small dissociation constant) which overcompensates for its small partition coefficient C. In contrast, phenoxyacetic acid can be extracted to a lesser degree than penicillin V because of its low partition TABLE
1
Data for calculating the degrees of extraction phenylacetic acid and phenoxyacetic acid
3.
0
5.
0
7.
0
a.
PEs
0
PH Fig. 18. Degree of extraction as a function of the pH value for the extraction of phenoxyacetic acid with Amberlite LA-2 and n-butyl acetate (cp = 1 mmol 1-r): curve 1, A, cA = 0; curve 2,0, CA = 1 mmol 1-r; curve 3, q, CA = 3 mmol 1-l; curve 4, 0, CA = 10 mm01 1-r.
Partition coefficient
C
of
Equilibrium constant (12 mol+)
Phenylacetic acid Phenoxyacetic acid
4.28
25
2 x 107
3.14
30
5 x 10’
RlR
coefficient which overcompensates for its high pK, value. In the presence of carriers, both precursors have significantly lower degrees of extraction than the corresponding penicillin. Thus, a higher selectivity of penicillin can be attained by reactive extraction than by physical extraction. To prove these observations, the coextraction model developed in ref. 1 was used to calculate the degrees of extraction of penicillin G and phenylacetic acid in the pH range between 4 and 7 during physical and reactive coextraction (Fig. 19). It can be observed that the precursor is extracted better physically. With increasing carrier concentration the degree of extraction for penicillin G increases more quickly than that of the precursor. Thus, at high carrier concentrations and at pH 4 - 5, penicillin G and the precursor have the same degrees of extraction. From pH 6 to pH 7 the degree of extraction of penicillin G is higher than that of the phenylacetic acid. In Fig. 20 the ratio of the degree of extraction of penicillin G to that of its precursor is plotted as a function of the carrier concentration at various pH values. It can be seen that at a higher carrier concentration and at pH 4 this ratio equals 2. In Fig. 21 the ratio of the degree of extraction of penicillin V to that of phenoxyacetic acid is plotted as a function of the carrier concentration at various pH values with
0
0.
02
0.
04
0.
06
cA [mollll
Fig. 19. Calculated degrees of extraction as a function of the carrier concentration for the coextraction of penicillin G and phenylacetic acid (each of them with 10 mmol 1-l) with Amberlite LA-2 and n-butyl acetate: curve 1, pH 4.0, phenylacetic acid; curve 2, pH 4.0, penicillin G; curve 3, pH 5.0, phenylacetic acid; curve 4, pH 5.0, penicillin G; curve 5, pH 6.0, phenylacetic acid; curve 6, pH 6.0, penicillin G; curve 7, pH 7.0, phenylacetic acid; curve 8, pH 7.0, penicillin G.
3.
01
0
0.01
0.
02
a3
0. cA [mol/l
I
Fig. 20. Ratio of the degree Ep of extraction of penicillin G to the degree Eh,, of extraction of phenylacetic acid as a function of the carrier concentration at various pH values for the coextraction of penicillin G and phenylacetic acid with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol 1-l ; cpre, = 10 mmol 1-l): curve 1, pH 4.0; curve 2, pH 5.0; curve 3, pH 6.0; curve 4, pH 7.0.
Y
d
3
0
0.
01
0.
02
0. cA
03
[mollll
Fig. 21. Ratio of the degree Ep of extraction of penicillin V to the degree Ep,.+, of extraction of phenoxyacetic acid as a function of the carrier concentration at various pH values for the coextraction of penicillin V and phenoxyacetic acid with Amberlite LA-2 and n-butyl acetate (cp = 10 mmol I-‘;c prec = 10 mmol 1-i): curve 1, pH 4.0; curve 2, pH 5.0; curve 3, pH 6.0; curve 4, pH 7.0.
Amberlite LA-2 in n-butyl acetate. At a high pH the ratio is high; it increases with the carrier concentration, passes a maximum and then decreases. By lowering the pH, the ratio is reduced and at pH 4.0 it attains a value of unity, because for both components the degree of extraction is unity.
4. CONCLUSIONS
Primary amines are not very suitable carriers for penicillin G because of the moderate partition coefficients of their penicillin complexes. In addition, they have a fairly high
B19
solubility in water and a high interfacial activity, which causes stable emulsions. The highest degrees of extraction can be attained by using secondary amines as carriers. When secondary amines are used, penicillin G can be extracted up to a pH value of 6. The distribution coefficients K of their amine complexes with penicillin G markedly depend on the amine structure. A disadvantage of secondary amines can be their reactivity with solvents, e.g. they may form amides with acetic acid esters during the distillative purification of the solvent. Tertiary amines are suitable as carriers for penicillin G extraction but the distribution coefficients of their complexes are significantly lower than those of secondary amine complexes. Their advantage lies in their low reactivity with solvents. Quaternary amines can be used for the reactive extraction of penicillin G. However, re-extraction is difficult and very large amounts of anions (e.g. Cl-) are needed to recover the penicillin. The results obtained agree with those found for the reactive extraction of acetic acid with various amines [lo, 111. Penicillin V can be recovered more easily by physical as well as by reactive extraction than can penicillin G. Precursors of penicillin G and V can also be coextracted with amines and the selectivity of penicillin can be improved by using reactive extraction. The reactive extraction of penicillin with secondary and tertiary amines can be described by a simple stoichiometric reaction. When primary amines are used, the nonstoichiometric reaction has to be taken into account. The relationships developed in ref. 1 for distribution coefficients and degrees of reactive extraction yield useful results which agree well with the measurements. From this work it appears that reactive extraction provides new possibilities for the recovery of primary and secondary metabolites from fermentation media.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support of the Ministry of Research and Technology, F.R.G., and the support of Hoechst AG.
REFERENCES 1 M. Reschke and K. Schiigerl, Reactive extraction of penicillin, I, Stability of penicillin G in the presence of carriers and relationships for distribution coefficients and degrees of extraction, Chem. Eng. J., 28 (1984) Bl. 2 F. C. Whitmore, R. B. Wagner, C. I. Noll, G. C. Basseler et al., Processing penicillin, Ind. Eng. Chem., 38 (1946) 942 - 948. 3 S. A. Zhukovskaya, V. N. Rusin and V. L. Pebalk, Kinetics of mass transfer during transfer of antibiotics through the interface in aqueous solutionbutyl acetate system, J. Appl. Chem. U.S.S.R., (Engl. Transl.), 49 (1976) 1142 - 1146. 4 M. Reschke, Doctoral Thesis, University of Hannover, 1983. 5 R. Modin and M. Schriider-Nielsen, Quantitative determination by ion pair extraction, Part 9, Extraction of penicillins with ion pairing and adduct-forming agents, Acta Pharm. Suet., 8 (1971) 573 - 584. 6 M. Schroder-Nielsen, Extraction of carboxylic acids as adducts with trioctylphosphine oxide, Acta Pharm. Suet., 13 (1976) 133 - 144. 7 H. Margreiter and F. Gapp, Penicilline-Chemie und Eigenschaften. In R. Brunner and G. Machek (eds.), Die Antibiotika, Vol. I, Part 1, Hans Carl, Niirnberg, 1962, p. 225. 8 A. Tsuji, E. Miyamoto, N. Hashimoto and T. Yamana, GI-absorption of @actam antibiotics, II, Deviation from pH-partition hypothesis in penicillin absorption through in situ and in vitro lipoidal barriers, J. Pharm. Sci., 67 (12) (1978) 1705 - 1711. 9 S. A. Zhukovskaja, V. N. Rusin and V. L. Pebalk, Investigation of equilibrium distribution of antibiotics in the system aqueous phase-butyl acetate, J. Appl. Chem. U.S.S.R. (Engl. Transl.), 50(1977)848-852. 10 N. L. Ricker, J. N. Michaels and C. J. King, Solvent properties of organic bases for extraction of acetic acid from water, J. Sep. Process Technol., 1 (1) (1979) 36 - 41. 11 N. L. Ricker, E. F. Pittmann and C. J. King, Solvent extraction with amines for recovery of acetic acid from dilute aqueous industrial streams, J. Sep. Process Technol., I (2) (1980) 23 - 30.
APPENDIX A: NOMENCLATURE
aq
A AHP c c E H+ K
aqueous phase amine, carrier amine-penicillin complex concentration (mm01 1-r or mol 1-l) partition coefficient of physical extraction degree of extraction (5%) proton distribution coefficient of reactive extraction
B20
KG
K Phy org P-
T X-
equilibrium constant (I2 molm2) distribution coefficient of physical traction organic phase penicillin anion temperature (“C) anion
ex-
Subscripts amine, carrier A overall G proton H penicillin or penicillin P precursor Prec anion X
anion