J . Chem. Tech. Biotechnol. 1992, 53, 97-103
The Extraction of Penicillin G with Aliphatic Amines in Organic Solvents of Different Polarities Zhi-Fa Yang, Shu-Qiu Yu & Jia-Yong (Chia-yung) Chen Institute of Chemical Metallurgy, Academia Sinica, Beijing, 100080, People's Republic of China (Received 19 November 1990; revised version received 4 June 1991; accepted 8 July 1991)
Abstract: The equilibrium constants of the extraction, the molar ratios of amines to penicillin G in the extract and the bonding structures of the extracted species were studied with chemical analysis and the absorbance shift of FT-IR spectra of the functional groups in penicillin G. Extraction of penicillin G from the filtrate of fermentation broth indicates that amines will be difficult to use as the industrial solvent for the production of penicillin G because of the poor quality of the final crystalline product obtained and the difficulties involved in the stripping and solvent recovery. Key words: solvent extraction, penicillin G, amines, FT-IR spectrum.
been published in the last 10 years or so on the mechanism and chemistry of solvent extraction and the process improvement on its downstream isolation from fermentation broth.' Penicillin G is a weak acid (pK, = 2.75). The pH of the medium during extraction is set at 1.8-2.5 to bring the acid anion into an undissociated free state. Because of its instability, the cell-free medium is cooled to 5°C and is usually extracted by n-butyl acetate in centrifugal extractor (for example, Podbielniak, Luwesta and Alfa-Laval) to keep the contact time as short as possible. Quaternary ammonium salts are generally used as additives to avoid the formation of stable emulsions due to the proteins in the medium flocculating at low pH values. Stripping is carried out at pH 6.7-7.2 by a carbonate or phosphate buffer. After the extraction of penicillin G from the buffer with n-butyl acetate, the potassium salt of penicillin G is precipitated from the organic solution by addition of potassium acetate.'-3 Since the losses in penicillin G are high (10-15 YO),^,^ it is worthwhile to investigate how these can be reduced. Reschke and Schugerl"." investigated the extraction of penicillin G with aliphatic amines in butyl acetate as solvent. The mechanism of extraction and the possible bonding structures of the extracted species with aliphatic amines have not been reported. The extraction mechanism and the FT-IR spectra of the extracted
NOTATION Penicillin G anion Solvent Free concentration of solvent at equilibrium (mol drn-.") Initial concentration of solvent (mol dm-3) Concentration of penicillin G in aqueous solution at equilibrium (mol dm-3) Initial concentration of penicillin G in aqueous solution (mol dm-3) Distribution coefficient Degree of extraction (YO) Concentration of penicillin G in solvent at equilibrium (mol drn-.") Equilibrium constant of extraction Dissociation constant of penicillanic G acid, pK, = 2.75 The numbers of moles of penicillin G and solvent in the extracted species, respectively
1 INTRODUCTION
Penicillin G has been the most widely used antibiotic for many years. However, only a few research papers have 97
J . Chem. Tech. Biotechnol. 0268-2575/92/$05.00 0 1992 SCI. Printed in Great Britain
Z.-F. Yung, S.-Q. Yu, J.-Y. Chen
98
species of penicillin G with different types of aliphatic amines in solvents o r diluents of different polarities are reported in this paper.
80
K
'0. -0-
70
-0
2 EXPERIMENTAL 40
2.1 Method and apparatus Portions (25 ml) of various solvents and 50 ml of a solution of potassium salt of penicillin G (or filtrate of fermentation broth) were added into a 150 ml beaker and stirred with a magnetic stirrer for 1 min. During stirring 5 % (w/v) sulphuric acid was added slowly into the solutions to control the pH. The concentrations of penicillin G in the organic and aqueous phases and the pH of the aqueous solution were measured with a polarimctcr4,z and a pH meter respectively. The concentration of penicillin G in fermentation solution and the organic phase were determined by high-pressure liquid chromatography (HPLC)'. The FT-IR spectrum of the extracted species was measured with a Nicolet 7199B FT-IR Spectrometer.
30
2o 10
I
ot
I
2
1
I
I
3
5
4
6
Fig. 1. Effect of pH and primary amine concentration on penicillin G extraction by primary amine N,,,, in benzene. (+) 4 % (v/v) N,,,, in benzene, (A) 3 YO(v/v) N,,,, in benzene, (0) 2Y0 (v/v) N,,,, in benzene, (+) I % (v/v) N,,,, in benzene. O / A = 1/2, T = 12"C, T = 1 min. 0.041 mol dm-3 penicillin G.
2.2 Chemicals Primary amine N,,PB,average molecular weight 3 12, with the formula RR'CHNH, with total number of C atoms 19 23, reagent grade, was supplied by Shanghai Institute of Organic Chemistry, Academia Sinica. Secondary amine 720 I , average molecular weight 467, with the formula (RR'CH),NH, (R, R' = C,-C,) technical grade, was supplied by the Uranium Ore Processing Institute, Ministry of Nuclear Industry. Tertiary amine tri-noctylamine (TOA), molecular weight 353.7, reagent grade, was produced by Fluka. All other chemical reagents used in the experiments are commercial products of chemical o r analytical pure grades.
7
PH
4
401
,
,
,
3
4
..'..; ..-,,,. . , ~ . 4...,
30
1
20 10
0 2
5
6
7
PH
Fig. 2. Effect of pH and amine concentration on penicillin G extraction by sccondary amine 7201 in benzene. ( + ) 8 YO(v/v) 7201 in benzene, (A)6 % (v/v) 7201 in benzene, (0) 4 % (v/v) 7201 in benzene, (+) 2 % (v/v) 7201 in benzene. O / A = 1/2, T = 12"C, t : 1 min, 0.0343 mol dm-3penicillin G.
3 RESULTS AND DISCUSSION 3.1 Extraction of penicillin G with arnines in benzene For the extraction with amines, the use of diluents can increase the extraction selectivity, and decrease the dehydration energy of the ions.' When penicillin G is extracted from the weak acid solution, the solubility of the extracted species in aliphatic hydrocarbons as diluents is very low, probably due to polymerisation and hydration of the amine-penicillin G ion-pairs. The solubility and dehydration of ion-pairs are increased in polar solvents. Aromatic hydrocarbons have a strong solvation effect to the highly polar amine salts and the addition of benzene to the amines has a similar effect as using polar solvents such as alcohols and esters. The effects of concentration of amines in benzene as
2
3
4
5
6
7
PH
Fig. 3. Effect of pH and arnine concentration on penicillin G extraction by TOA in benzene. ( ) 7 YO(v/v) TOA in benzene, (A)5 YO(v/v) TOA in benzene, (0)3 YO(v/v) TOA in benzene, (+) 1 YO(v/v) TOA in benzene. O / A = 1/2, T = 12"C, t = I min, 0.0343 mol dm-3 penicillin G.
+
The extraction of penicillin G
40 30
10
0
99
i I
2
I
3
4
5
.
,
6
that the use of additives for the extraction of penicillin G with amines is to increase the solubility of the extracted species in hydrocarbons. The polarity of hydrocarbons (such as heptane) will be increased with the addition of either donor or acceptor solvents. If polar solvents are added to the solvent systems, the extraction capability of different kinds of amines for the solute will be decreased to some extent. The effect of addition of 15% (v/v) hexanol-I to amines in heptane on the extraction of penicillin G is shown in Figs 5-7 from which it can be seen that the hexanol-1 has a retarding effect on the extraction capability of the different amines. The extraction of penicillin G is improved by the addition of polar additives in amines of low concentration. Polar additives, however, have a negative effect on the extraction with high concentrations of the amines. In comparison with tributyl phosphate (TBP) and butyl acetate (BA), hexanol- 1 has the strongest inhibition effect on the extraction of penicillin G, which may be due to the molecular interaction between amines and hexanol1 (Figs 5-7). The acceptor properties of active hydrogens attached to nitrogen and donor properties of nitrogen atoms in
.
7
8
PH
Fig. 4. Effect of pH on penicillin G extraction by amines of different classes in benzene. (+) N,,,, in benzene, (A)7201, (0) TOA. O / A = 1/2, T = 12"C, T = 1 min, 0.038 mol dm-, penicillin G, 0.09 mol dm-3 amines.
solvent and equilibrium pH on the extraction of penicillin G are shown in Figs 1-3. Results show that the primary, secondary, and tertiary amines are able to extract penicillin G effectively at low pH and the extent of extraction increases with increasing concentration of amines. Differences between the extraction capabilities of different kinds of amines on penicillin G at high pH are observed. The retarding effect of increasing pH on the extraction of penicillin G increases in the order of tertiary, secondary and primary amines, as given in Fig. 4. The extraction of penicillin G is increased with increasing concentration of amines as shown in Table 1. It can be observed from the table that, at higher concentrations of amines, the effect of pH on the extraction is smaller. 3.2 Extraction with amines in solvents or diluents of different polarities
0
("a)
3
0.060
0109
0120 __
5.0
Primary N,,,,
-
Secondary 720 1
-
-
89.5
45
Tertiary TOA O / A = 1/2, T = 12"C, diluent:
~
pH 3.5
5.0
p H 3.5
5.0
83.6
69 66.3
__
-
-
-
91
68
90
-
6
Fig. 5. Effect of concentration of primary amine in different diluents on penicillin G extraction. Diluents or diluent with additive-( +) benzene, (A)15 Ya (v/v) hexanol-1 in heptane. O / A = 1/2, T = 15"C, 5 = 1 min, 0.034 rnol dm-3 penicillin G, pH 4.0.
TABLE 1 of Penicillin G with Different Kinds of Amines in Benzene"
0.0663 p H 3.5
5
4
Primary n m i n e , %
The extent of extraction of penicillin G by amines is higher in solution with benzene as diluent than with other types of diluent; extraction of penicillin G with benzene or other aliphatic hydrocarbons alone is negligible in the pH values examined. It can be considered
The Extraction
2
I
benzene, concentration of penicillin G : 0.0378 mol dm-'.
pH3.5 96
._
5.0
96.6
96 92.8
-
-
Z.-F. Yang, S.-Q..Yu, J.-Y. Chen
I 00
0
1
2
3
4
5
6
7
8
9
Sec. amine, %
Fig. 6. Effect of Concentration of secondary amine 7201 in diffcrent diluents on penicillin G extraction. Diluent or diluent with additives--( + ) benzene, 1 5 % (v/v) hexanol in heplane, (0) 5% (v/v) TBP in heptane. O / A = 1/2, T = 12°C. T = I min, 0.035 mol dm-3 penicillin G, pH 4.0.
(a)
amines.' The extraction with amines as solvent is usiially carried out in a weak acid solution. There are two main types of mechanisms for the extraction with amines in acid solution, that is anion exchange and adduct formation reaction.','" A solution of potassium penicillin G was prepared from crystalline penicillin G for this part of the study. The organic phase was not acidified beforehand and dilute sulphuric acid was added during mixing of the two phases to adjust the pH of the aqueous solution. The relationships among the distribution constants of the extraction, pH, and concentration of the solvent are expressed below based on the following assumptions : ( I ) the polymerisation of the amine molecules can be neglected; (2) the ionic strength of the aqueous solution is considered to be constant during the extraction and (3) the activity coeficients of penicillin G in the aqueous and organic phases are considered to be equal to unity because of the low concentration used.
;I
Aqueous phase HA (aq)
nHA (aq)
!+
Organic phase K Bq ( H A ) n (org) 4 B (0%)-
~:LL 10
00
1
2
3
4
5
6
7
8
(3)
TOA, %
Fig. 7. Effect of concentration of tertiary m i n e TOA in different solvents on penicillin G extraction. Diluent or diluent with additives-( +) benzene, (A) 30% (v/v) BA in kerosene, (0) 7.5 Yo (v/v) TRP in kerosene, (+) 30% (v/v) hexanol in
kerosene. amines can form hydrogen bonding with solvents having acceptor or donor atoms such as alcohols and esters. The reactivity of amines during extraction will be decreased by the formation of the hydrogen bonding between the polar solvents and amines. The distribution constants of extraction of penicillin G with amines decrease in the order of primary amine N,,,,, secondary amine 7201, and tertiary amine TOA, which probably indicates the effects of steric hindrance and the active hydrogen atoms attached to the nitrogen atom of the amines. As penicillin G is a large organic acid molecule, the ion-pairs formed between tertiary amine salt and the penicillin G anion are probably unstable owing to the strong steric effect.
3.3 The extraction mechanism of penicillin G with amines Thc cxtraction behaviour or different classes of amines is different due to the dil'ferences of the structures of the
(4) log D
= (log K +
log n) - log c,,
+ n{log C,, -pH
-
log (1 + l O'2'75-pH'
11 ( 5 )
The data given in Figs 1-3 were treated by linear regression analysis with eqns (5) and (6) with the results shown in Table 2. The data indicate that the ratios of penicillin G to amines (primary, secondary, and tertiary) are 1, and the extraction equilibrium constants Kincrease in the order of tertiary, secondary, and primary. The IR spectra of the extracted species with primary, secondary, and tertiary amines as solvent indicate that, first, the absorbance values of the p-lactam carboxyl, amide carboxyl, and the amide N-H undergo practically no chemical shift, suggesting that these groups do not participate in hydrogen bond formation during extraction. Secondly, the absorbance of the carboxyl group in penicillin G shifts towards low wave number of 1601 cm-I (primary), 1605 cm (secondary), and 1606 cm ' (tertiary). Finally, there are absorbances at 3253 cm-' and
The extraction of penicillin G
101 TABLE 2
The Extraction Equilibrium Constant K and the Coefficients in Eqns ( 5 ) and (6)” Co
Y
n
4
log K
0.04 1 0.034 0.038
0.970 0.980 0.999
0.80 0.90 0.999
1.20 0.99 0.999
8.15 7.16 6.73
- -~
Primary amine Secondary amine Tertiary amine
a O / A = 1/2, T = 12°C pH 5.0, diluent: heptane, C,,,,, = 0.05-0.15 mol dm-3, n, q are the coefficients of eqns ( 5 ) and (6), y is correlative coefficient of the experimental data and the equations.
3265 cm-’ (of NH,) for primary amine, 2251 cm-l (of NH,) for secondary amine and 2703 cm-’ (of NH) for tertiary amine, which show that amines have reacted with penicillin G and the extracted species show a type of salt formation (Figs 8-10, Table 3). From above, the extraction equilibrium of penicillin G with amines, such as primary amines, can be considered as HA
+ RNH, e RNH; A-
The structure of the extracted species could be expressed as
1.4
Q
I
0.6
0.4 0.2
.
04 4 0 0 0 3600 3200 2 8 0 0 2 4 0 0 2 0 0 0 1600 1200 800 Wavenumber (cm-’)
3.4 Extraction of penicillin G from the filtrate of fermentation broth
7
Fig. 9. IR spectra of 3 YO(v/v) 720 1 in benzene after extraction of penicillin G. 0.066 mol dm-3 penicillin G, pH 5.0, 0.2 mm CaF,.
From the work described above, amines are different from neutral oxygen-donating solvents since they react with penicillin G to form adducts during extraction.
al
;0.8‘ ra
13
b
0.6-
Q . 1 4 0 0 0 3600 3200 2 8 0 0 2400 2000 1600 1200 800
044
Waven um ber (cm-’)
I
I
02? , 0 7 1 4 0 0 0 3600 3200 2800 2400 2000 1600 1 2 0 0 8 0 0
Fig. 10. IR spectra of 3 % (v/v) TOA in benzene after extraction of penicillin G. 0.068 mol dm-3 penicillin G, pH 5.0, 0.2 mm CaF,.
T---
Wavenumber (crn-’1
Fig. 8. IR spectra of 3 % (v/v) N L g Zin3 benzene after extraction of penicillin G. 0.085 mol dm-3 penicillin G, pH 5.0, 0.2 mm CaF,.
Stripping of the extracted penicillin G might be difficult within the range pH 6.5-7.2. The effects of pH and the concentration of tertiary amine on the extraction of penicillin G are given in Figs 3 and 11, from which it is
Z.-F. Yung, S.-Q. Yu, J.-Y. Chrn
102 TABLE 3
kerosene as solvent (Table 4)showed that some pigments and impurities in the fermentation solution were extracted into the solvent. After extraction, the solvent is dark brown as compared to light yellow in industrial production with butyl acetate as solvent; and the crystalline product is yellow which is far from the specifications given for penicillin G. The solvent after crystallisation cannot be reused without purification. One advantage of using TOA as solvent is that significantly less demulsifier is required for the phase separatioq6 indicating that tertiary amine can suppress emulsion formation during extraction.
The IR Shift of the Main Functional Groups of Penicillin G in the Extracted Species" C=Oin
C,
C=Oin
(mol dmP) 8-luctum (ern-')
Standard
1787
-
Primary
0.085
Secondary
0.066
Tertiary
0.066
C=Oin umide
curhoxyl (em- ')
(em -')
1658 1668 1689 I688
1736 1601
1780 1787 I782
1605 I606
Specifications of the spectra are based on Refs 11-13
4 CONCLUSIONS
1
ot 0
1
2
3
4
5
6
7
0
9
TOA, W
Fig. 11. Effect of pH on penicillin G cxtraction by adding long chain amines in 30Y0 (v/v) hexanol-l in heptane as diluents. ( + ) pH 3.0, (A) 3.5, (0) 4.0. O / A = 1/2. T = 14°C. 5 = 1 min, 0.037 mot dm-' penicillin G.
From the data presented, a number of conclusions may be drawn. The ratios of amines to penicillin G in the organic phase are determined by chemical analysis and the possible bonding structure of the extracted species is obtained by means of the absorbance shift of IR spectra of functional groups in penicillin G. Amines react with penicillin G during extraction, which is different from the hydrogen bonding formation between neutral oxygendonating solvents and penicillin G. Finally, the extent of extraction of penicillin G is higher by using amines with benzene as diluent than with polar solvents as diluents. However, the amines probably could not be used as an industrial solvent for the extraction of penicillin G because of the poor quality of product obtained and the problems involved in stripping and solvent recovery.
ACKNOWLEDGEMENTS shown that the higher the concentration of TOA, the less the influence of pH on the extraction is. This means that the stripping of penicillin G extracted by changing of pH shift is difficult if the concentration of TOA is higher than 5 % (v/v). The results of extraction of penicillin from the filtrate of fermentation broth with tertiary TOA in TBP-
The authors gratefully acknowledge the kind supply of potassium penicillin G and the filtrate of fermentation broth by North China Pharmaceutical Corporation. FT-IR analysis work was generously supported by the Zhongguancun Analytical & Testing Funding Foundation, Beijing.
TABLE 4 The Extraction of Penicillin G from the Filtrate of Fermentation Broth with Tertiary Amine" Colour of
solvent
Colour of the product -~
10 Yo TOA-90 % RA
10% TOA-10% TBP 1O0/a
TOA-10% TBP
1 5 % TOA-10% TBP 15 o/' TOA- 10 Yo TBP "
C,, = 0421 mol dm-:3,O / A
=
4.0 4.4 3.8 3.4 3. I
1/2, T = IO'C, BA-butyl
93.0 67.0 92.0 * 100
-
100
Brown Yellow
-
Dark yellow Brown Dark brown
-
acetate, diluent: kcroscnc.
Yellow Yellow
The extraction ojpenicillin G
REFERENCES 1, Swartz, R. W., In Cumprehensive Biuiechnology (Yol. 31, ed. M. Moo-young, Pergamon Press, New York, USA, 1985, section 2. 2. Reschke, M. & Schugerl, K., Reactive extraction of penicillin G : I. Stability of penicillin G in the presence of carriers and relationship for distribution coefficients and degrees of extraction. Chem. Engng J., 28 (1984) B1-9. 3. Reschke, M. & Schugerl, K., Reactive extraction of penicillin G : 11. Distribution coefficients and degrees of extraction. Chem. Engng J., 28 (1984) B9-19. 4. Chen, Z. Z. & Zhang, S. L., The Analysis and Determination of Antibiotics. People’s Health and Hygiene Press (China), Beijing, People’s Republic of China, 1959. 5. British Pharmacopoeia, Pharmpress, London, UK, 1968. 6. Yang, Z. F., Studies on new solvent systems for extraction of penicillin G. Doctoral dissertation, Institute of Chemical Metallurgy, Academia Sinica, Beijing, People’s Republic of China. 1989.
103 7. Blumberg, R. & Gai, J. E., “Strong” diluent effects. Proc. Znt. Solvent Extraction ConJ, 1 (1977) 9-16. 8. Yu, S. Q., Meng, X. S . & Chen, C . Y., Extraction of vanadium (V) from aqueous solutions with aliphatic amines. Scientia Sinica (B), 1 (1982) 11-18. 9. Yu, S. Q. & Chen, C. Y., Studies on extraction of Cr, Mo, W, and V with aliphatic amines as solvents. Acta Metallurgica Sinica, 2 (1982) 187-200. 10. Yu, S. Q., Effect of hydrogen bond on extraction. Rare Metals (China), 5 (1986) 28-36. 11. Clarke, H. T., Johnson, J. R. & Robinson, S. R., The Chemistry of Penicillin. Princeton University Press, New Jersey, USA, 1949, pp. 39W15. 12. Xie, J. X., The Application of IR Spectrum in Organic & Pharmaceutical Chemistry. Academic Press (China), Beijing, People’s Republic of China, 1987. 13. Nakanshi, K. & Solomon, P. H., Znfi.ared Absorption Spectroscopy. Holden-Day Inc., San Francisco, USA, 1977.
The Extraction of Penicillin G with Aliphatic Amines in Organic Solvents of Different Polarities Zhi-Fa Yang, Shu-Qiu Yu & Jia-Yong (Chia-yung) Chen Institute of Chemical Metallurgy, Academia Sinica, Beijing, 100080, People's Republic of China (Received 19 November 1990; revised version received 4 June 1991; accepted 8 July 1991)
Abstract: The equilibrium constants of the extraction, the molar ratios of amines to penicillin G in the extract and the bonding structures of the extracted species were studied with chemical analysis and the absorbance shift of FT-IR spectra of the functional groups in penicillin G. Extraction of penicillin G from the filtrate of fermentation broth indicates that amines will be difficult to use as the industrial solvent for the production of penicillin G because of the poor quality of the final crystalline product obtained and the difficulties involved in the stripping and solvent recovery. Key words: solvent extraction, penicillin G, amines, FT-IR spectrum.
been published in the last 10 years or so on the mechanism and chemistry of solvent extraction and the process improvement on its downstream isolation from fermentation broth.' Penicillin G is a weak acid (pK, = 2.75). The pH of the medium during extraction is set at 1.8-2.5 to bring the acid anion into an undissociated free state. Because of its instability, the cell-free medium is cooled to 5°C and is usually extracted by n-butyl acetate in centrifugal extractor (for example, Podbielniak, Luwesta and Alfa-Laval) to keep the contact time as short as possible. Quaternary ammonium salts are generally used as additives to avoid the formation of stable emulsions due to the proteins in the medium flocculating at low pH values. Stripping is carried out at pH 6.7-7.2 by a carbonate or phosphate buffer. After the extraction of penicillin G from the buffer with n-butyl acetate, the potassium salt of penicillin G is precipitated from the organic solution by addition of potassium acetate.'-3 Since the losses in penicillin G are high (10-15 YO),^,^ it is worthwhile to investigate how these can be reduced. Reschke and Schugerl"." investigated the extraction of penicillin G with aliphatic amines in butyl acetate as solvent. The mechanism of extraction and the possible bonding structures of the extracted species with aliphatic amines have not been reported. The extraction mechanism and the FT-IR spectra of the extracted
NOTATION Penicillin G anion Solvent Free concentration of solvent at equilibrium (mol drn-.") Initial concentration of solvent (mol dm-3) Concentration of penicillin G in aqueous solution at equilibrium (mol dm-3) Initial concentration of penicillin G in aqueous solution (mol dm-3) Distribution coefficient Degree of extraction (YO) Concentration of penicillin G in solvent at equilibrium (mol drn-.") Equilibrium constant of extraction Dissociation constant of penicillanic G acid, pK, = 2.75 The numbers of moles of penicillin G and solvent in the extracted species, respectively
1 INTRODUCTION
Penicillin G has been the most widely used antibiotic for many years. However, only a few research papers have 97
J . Chem. Tech. Biotechnol. 0268-2575/92/$05.00 0 1992 SCI. Printed in Great Britain
Z.-F. Yung, S.-Q. Yu, J.-Y. Chen
98
species of penicillin G with different types of aliphatic amines in solvents o r diluents of different polarities are reported in this paper.
80
K
'0. -0-
70
-0
2 EXPERIMENTAL 40
2.1 Method and apparatus Portions (25 ml) of various solvents and 50 ml of a solution of potassium salt of penicillin G (or filtrate of fermentation broth) were added into a 150 ml beaker and stirred with a magnetic stirrer for 1 min. During stirring 5 % (w/v) sulphuric acid was added slowly into the solutions to control the pH. The concentrations of penicillin G in the organic and aqueous phases and the pH of the aqueous solution were measured with a polarimctcr4,z and a pH meter respectively. The concentration of penicillin G in fermentation solution and the organic phase were determined by high-pressure liquid chromatography (HPLC)'. The FT-IR spectrum of the extracted species was measured with a Nicolet 7199B FT-IR Spectrometer.
30
2o 10
I
ot
I
2
1
I
I
3
5
4
6
Fig. 1. Effect of pH and primary amine concentration on penicillin G extraction by primary amine N,,,, in benzene. (+) 4 % (v/v) N,,,, in benzene, (A) 3 YO(v/v) N,,,, in benzene, (0) 2Y0 (v/v) N,,,, in benzene, (+) I % (v/v) N,,,, in benzene. O / A = 1/2, T = 12"C, T = 1 min. 0.041 mol dm-3 penicillin G.
2.2 Chemicals Primary amine N,,PB,average molecular weight 3 12, with the formula RR'CHNH, with total number of C atoms 19 23, reagent grade, was supplied by Shanghai Institute of Organic Chemistry, Academia Sinica. Secondary amine 720 I , average molecular weight 467, with the formula (RR'CH),NH, (R, R' = C,-C,) technical grade, was supplied by the Uranium Ore Processing Institute, Ministry of Nuclear Industry. Tertiary amine tri-noctylamine (TOA), molecular weight 353.7, reagent grade, was produced by Fluka. All other chemical reagents used in the experiments are commercial products of chemical o r analytical pure grades.
7
PH
4
401
,
,
,
3
4
..'..; ..-,,,. . , ~ . 4...,
30
1
20 10
0 2
5
6
7
PH
Fig. 2. Effect of pH and amine concentration on penicillin G extraction by sccondary amine 7201 in benzene. ( + ) 8 YO(v/v) 7201 in benzene, (A)6 % (v/v) 7201 in benzene, (0) 4 % (v/v) 7201 in benzene, (+) 2 % (v/v) 7201 in benzene. O / A = 1/2, T = 12"C, t : 1 min, 0.0343 mol dm-3penicillin G.
3 RESULTS AND DISCUSSION 3.1 Extraction of penicillin G with arnines in benzene For the extraction with amines, the use of diluents can increase the extraction selectivity, and decrease the dehydration energy of the ions.' When penicillin G is extracted from the weak acid solution, the solubility of the extracted species in aliphatic hydrocarbons as diluents is very low, probably due to polymerisation and hydration of the amine-penicillin G ion-pairs. The solubility and dehydration of ion-pairs are increased in polar solvents. Aromatic hydrocarbons have a strong solvation effect to the highly polar amine salts and the addition of benzene to the amines has a similar effect as using polar solvents such as alcohols and esters. The effects of concentration of amines in benzene as
2
3
4
5
6
7
PH
Fig. 3. Effect of pH and arnine concentration on penicillin G extraction by TOA in benzene. ( ) 7 YO(v/v) TOA in benzene, (A)5 YO(v/v) TOA in benzene, (0)3 YO(v/v) TOA in benzene, (+) 1 YO(v/v) TOA in benzene. O / A = 1/2, T = 12"C, t = I min, 0.0343 mol dm-3 penicillin G.
+
The extraction of penicillin G
40 30
10
0
99
i I
2
I
3
4
5
.
,
6
that the use of additives for the extraction of penicillin G with amines is to increase the solubility of the extracted species in hydrocarbons. The polarity of hydrocarbons (such as heptane) will be increased with the addition of either donor or acceptor solvents. If polar solvents are added to the solvent systems, the extraction capability of different kinds of amines for the solute will be decreased to some extent. The effect of addition of 15% (v/v) hexanol-I to amines in heptane on the extraction of penicillin G is shown in Figs 5-7 from which it can be seen that the hexanol-1 has a retarding effect on the extraction capability of the different amines. The extraction of penicillin G is improved by the addition of polar additives in amines of low concentration. Polar additives, however, have a negative effect on the extraction with high concentrations of the amines. In comparison with tributyl phosphate (TBP) and butyl acetate (BA), hexanol- 1 has the strongest inhibition effect on the extraction of penicillin G, which may be due to the molecular interaction between amines and hexanol1 (Figs 5-7). The acceptor properties of active hydrogens attached to nitrogen and donor properties of nitrogen atoms in
.
7
8
PH
Fig. 4. Effect of pH on penicillin G extraction by amines of different classes in benzene. (+) N,,,, in benzene, (A)7201, (0) TOA. O / A = 1/2, T = 12"C, T = 1 min, 0.038 mol dm-, penicillin G, 0.09 mol dm-3 amines.
solvent and equilibrium pH on the extraction of penicillin G are shown in Figs 1-3. Results show that the primary, secondary, and tertiary amines are able to extract penicillin G effectively at low pH and the extent of extraction increases with increasing concentration of amines. Differences between the extraction capabilities of different kinds of amines on penicillin G at high pH are observed. The retarding effect of increasing pH on the extraction of penicillin G increases in the order of tertiary, secondary and primary amines, as given in Fig. 4. The extraction of penicillin G is increased with increasing concentration of amines as shown in Table 1. It can be observed from the table that, at higher concentrations of amines, the effect of pH on the extraction is smaller. 3.2 Extraction with amines in solvents or diluents of different polarities
0
("a)
3
0.060
0109
0120 __
5.0
Primary N,,,,
-
Secondary 720 1
-
-
89.5
45
Tertiary TOA O / A = 1/2, T = 12"C, diluent:
~
pH 3.5
5.0
p H 3.5
5.0
83.6
69 66.3
__
-
-
-
91
68
90
-
6
Fig. 5. Effect of concentration of primary amine in different diluents on penicillin G extraction. Diluents or diluent with additive-( +) benzene, (A)15 Ya (v/v) hexanol-1 in heptane. O / A = 1/2, T = 15"C, 5 = 1 min, 0.034 rnol dm-3 penicillin G, pH 4.0.
TABLE 1 of Penicillin G with Different Kinds of Amines in Benzene"
0.0663 p H 3.5
5
4
Primary n m i n e , %
The extent of extraction of penicillin G by amines is higher in solution with benzene as diluent than with other types of diluent; extraction of penicillin G with benzene or other aliphatic hydrocarbons alone is negligible in the pH values examined. It can be considered
The Extraction
2
I
benzene, concentration of penicillin G : 0.0378 mol dm-'.
pH3.5 96
._
5.0
96.6
96 92.8
-
-
Z.-F. Yang, S.-Q..Yu, J.-Y. Chen
I 00
0
1
2
3
4
5
6
7
8
9
Sec. amine, %
Fig. 6. Effect of Concentration of secondary amine 7201 in diffcrent diluents on penicillin G extraction. Diluent or diluent with additives--( + ) benzene, 1 5 % (v/v) hexanol in heplane, (0) 5% (v/v) TBP in heptane. O / A = 1/2, T = 12°C. T = I min, 0.035 mol dm-3 penicillin G, pH 4.0.
(a)
amines.' The extraction with amines as solvent is usiially carried out in a weak acid solution. There are two main types of mechanisms for the extraction with amines in acid solution, that is anion exchange and adduct formation reaction.','" A solution of potassium penicillin G was prepared from crystalline penicillin G for this part of the study. The organic phase was not acidified beforehand and dilute sulphuric acid was added during mixing of the two phases to adjust the pH of the aqueous solution. The relationships among the distribution constants of the extraction, pH, and concentration of the solvent are expressed below based on the following assumptions : ( I ) the polymerisation of the amine molecules can be neglected; (2) the ionic strength of the aqueous solution is considered to be constant during the extraction and (3) the activity coeficients of penicillin G in the aqueous and organic phases are considered to be equal to unity because of the low concentration used.
;I
Aqueous phase HA (aq)
nHA (aq)
!+
Organic phase K Bq ( H A ) n (org) 4 B (0%)-
~:LL 10
00
1
2
3
4
5
6
7
8
(3)
TOA, %
Fig. 7. Effect of concentration of tertiary m i n e TOA in different solvents on penicillin G extraction. Diluent or diluent with additives-( +) benzene, (A) 30% (v/v) BA in kerosene, (0) 7.5 Yo (v/v) TRP in kerosene, (+) 30% (v/v) hexanol in
kerosene. amines can form hydrogen bonding with solvents having acceptor or donor atoms such as alcohols and esters. The reactivity of amines during extraction will be decreased by the formation of the hydrogen bonding between the polar solvents and amines. The distribution constants of extraction of penicillin G with amines decrease in the order of primary amine N,,,,, secondary amine 7201, and tertiary amine TOA, which probably indicates the effects of steric hindrance and the active hydrogen atoms attached to the nitrogen atom of the amines. As penicillin G is a large organic acid molecule, the ion-pairs formed between tertiary amine salt and the penicillin G anion are probably unstable owing to the strong steric effect.
3.3 The extraction mechanism of penicillin G with amines Thc cxtraction behaviour or different classes of amines is different due to the dil'ferences of the structures of the
(4) log D
= (log K +
log n) - log c,,
+ n{log C,, -pH
-
log (1 + l O'2'75-pH'
11 ( 5 )
The data given in Figs 1-3 were treated by linear regression analysis with eqns (5) and (6) with the results shown in Table 2. The data indicate that the ratios of penicillin G to amines (primary, secondary, and tertiary) are 1, and the extraction equilibrium constants Kincrease in the order of tertiary, secondary, and primary. The IR spectra of the extracted species with primary, secondary, and tertiary amines as solvent indicate that, first, the absorbance values of the p-lactam carboxyl, amide carboxyl, and the amide N-H undergo practically no chemical shift, suggesting that these groups do not participate in hydrogen bond formation during extraction. Secondly, the absorbance of the carboxyl group in penicillin G shifts towards low wave number of 1601 cm-I (primary), 1605 cm (secondary), and 1606 cm ' (tertiary). Finally, there are absorbances at 3253 cm-' and
The extraction of penicillin G
101 TABLE 2
The Extraction Equilibrium Constant K and the Coefficients in Eqns ( 5 ) and (6)” Co
Y
n
4
log K
0.04 1 0.034 0.038
0.970 0.980 0.999
0.80 0.90 0.999
1.20 0.99 0.999
8.15 7.16 6.73
- -~
Primary amine Secondary amine Tertiary amine
a O / A = 1/2, T = 12°C pH 5.0, diluent: heptane, C,,,,, = 0.05-0.15 mol dm-3, n, q are the coefficients of eqns ( 5 ) and (6), y is correlative coefficient of the experimental data and the equations.
3265 cm-’ (of NH,) for primary amine, 2251 cm-l (of NH,) for secondary amine and 2703 cm-’ (of NH) for tertiary amine, which show that amines have reacted with penicillin G and the extracted species show a type of salt formation (Figs 8-10, Table 3). From above, the extraction equilibrium of penicillin G with amines, such as primary amines, can be considered as HA
+ RNH, e RNH; A-
The structure of the extracted species could be expressed as
1.4
Q
I
0.6
0.4 0.2
.
04 4 0 0 0 3600 3200 2 8 0 0 2 4 0 0 2 0 0 0 1600 1200 800 Wavenumber (cm-’)
3.4 Extraction of penicillin G from the filtrate of fermentation broth
7
Fig. 9. IR spectra of 3 YO(v/v) 720 1 in benzene after extraction of penicillin G. 0.066 mol dm-3 penicillin G, pH 5.0, 0.2 mm CaF,.
From the work described above, amines are different from neutral oxygen-donating solvents since they react with penicillin G to form adducts during extraction.
al
;0.8‘ ra
13
b
0.6-
Q . 1 4 0 0 0 3600 3200 2 8 0 0 2400 2000 1600 1200 800
044
Waven um ber (cm-’)
I
I
02? , 0 7 1 4 0 0 0 3600 3200 2800 2400 2000 1600 1 2 0 0 8 0 0
Fig. 10. IR spectra of 3 % (v/v) TOA in benzene after extraction of penicillin G. 0.068 mol dm-3 penicillin G, pH 5.0, 0.2 mm CaF,.
T---
Wavenumber (crn-’1
Fig. 8. IR spectra of 3 % (v/v) N L g Zin3 benzene after extraction of penicillin G. 0.085 mol dm-3 penicillin G, pH 5.0, 0.2 mm CaF,.
Stripping of the extracted penicillin G might be difficult within the range pH 6.5-7.2. The effects of pH and the concentration of tertiary amine on the extraction of penicillin G are given in Figs 3 and 11, from which it is
Z.-F. Yung, S.-Q. Yu, J.-Y. Chrn
102 TABLE 3
kerosene as solvent (Table 4)showed that some pigments and impurities in the fermentation solution were extracted into the solvent. After extraction, the solvent is dark brown as compared to light yellow in industrial production with butyl acetate as solvent; and the crystalline product is yellow which is far from the specifications given for penicillin G. The solvent after crystallisation cannot be reused without purification. One advantage of using TOA as solvent is that significantly less demulsifier is required for the phase separatioq6 indicating that tertiary amine can suppress emulsion formation during extraction.
The IR Shift of the Main Functional Groups of Penicillin G in the Extracted Species" C=Oin
C,
C=Oin
(mol dmP) 8-luctum (ern-')
Standard
1787
-
Primary
0.085
Secondary
0.066
Tertiary
0.066
C=Oin umide
curhoxyl (em- ')
(em -')
1658 1668 1689 I688
1736 1601
1780 1787 I782
1605 I606
Specifications of the spectra are based on Refs 11-13
4 CONCLUSIONS
1
ot 0
1
2
3
4
5
6
7
0
9
TOA, W
Fig. 11. Effect of pH on penicillin G cxtraction by adding long chain amines in 30Y0 (v/v) hexanol-l in heptane as diluents. ( + ) pH 3.0, (A) 3.5, (0) 4.0. O / A = 1/2. T = 14°C. 5 = 1 min, 0.037 mot dm-' penicillin G.
From the data presented, a number of conclusions may be drawn. The ratios of amines to penicillin G in the organic phase are determined by chemical analysis and the possible bonding structure of the extracted species is obtained by means of the absorbance shift of IR spectra of functional groups in penicillin G. Amines react with penicillin G during extraction, which is different from the hydrogen bonding formation between neutral oxygendonating solvents and penicillin G. Finally, the extent of extraction of penicillin G is higher by using amines with benzene as diluent than with polar solvents as diluents. However, the amines probably could not be used as an industrial solvent for the extraction of penicillin G because of the poor quality of product obtained and the problems involved in stripping and solvent recovery.
ACKNOWLEDGEMENTS shown that the higher the concentration of TOA, the less the influence of pH on the extraction is. This means that the stripping of penicillin G extracted by changing of pH shift is difficult if the concentration of TOA is higher than 5 % (v/v). The results of extraction of penicillin from the filtrate of fermentation broth with tertiary TOA in TBP-
The authors gratefully acknowledge the kind supply of potassium penicillin G and the filtrate of fermentation broth by North China Pharmaceutical Corporation. FT-IR analysis work was generously supported by the Zhongguancun Analytical & Testing Funding Foundation, Beijing.
TABLE 4 The Extraction of Penicillin G from the Filtrate of Fermentation Broth with Tertiary Amine" Colour of
solvent
Colour of the product -~
10 Yo TOA-90 % RA
10% TOA-10% TBP 1O0/a
TOA-10% TBP
1 5 % TOA-10% TBP 15 o/' TOA- 10 Yo TBP "
C,, = 0421 mol dm-:3,O / A
=
4.0 4.4 3.8 3.4 3. I
1/2, T = IO'C, BA-butyl
93.0 67.0 92.0 * 100
-
100
Brown Yellow
-
Dark yellow Brown Dark brown
-
acetate, diluent: kcroscnc.
Yellow Yellow
The extraction ojpenicillin G
REFERENCES 1, Swartz, R. W., In Cumprehensive Biuiechnology (Yol. 31, ed. M. Moo-young, Pergamon Press, New York, USA, 1985, section 2. 2. Reschke, M. & Schugerl, K., Reactive extraction of penicillin G : I. Stability of penicillin G in the presence of carriers and relationship for distribution coefficients and degrees of extraction. Chem. Engng J., 28 (1984) B1-9. 3. Reschke, M. & Schugerl, K., Reactive extraction of penicillin G : 11. Distribution coefficients and degrees of extraction. Chem. Engng J., 28 (1984) B9-19. 4. Chen, Z. Z. & Zhang, S. L., The Analysis and Determination of Antibiotics. People’s Health and Hygiene Press (China), Beijing, People’s Republic of China, 1959. 5. British Pharmacopoeia, Pharmpress, London, UK, 1968. 6. Yang, Z. F., Studies on new solvent systems for extraction of penicillin G. Doctoral dissertation, Institute of Chemical Metallurgy, Academia Sinica, Beijing, People’s Republic of China. 1989.
103 7. Blumberg, R. & Gai, J. E., “Strong” diluent effects. Proc. Znt. Solvent Extraction ConJ, 1 (1977) 9-16. 8. Yu, S. Q., Meng, X. S . & Chen, C . Y., Extraction of vanadium (V) from aqueous solutions with aliphatic amines. Scientia Sinica (B), 1 (1982) 11-18. 9. Yu, S. Q. & Chen, C. Y., Studies on extraction of Cr, Mo, W, and V with aliphatic amines as solvents. Acta Metallurgica Sinica, 2 (1982) 187-200. 10. Yu, S. Q., Effect of hydrogen bond on extraction. Rare Metals (China), 5 (1986) 28-36. 11. Clarke, H. T., Johnson, J. R. & Robinson, S. R., The Chemistry of Penicillin. Princeton University Press, New Jersey, USA, 1949, pp. 39W15. 12. Xie, J. X., The Application of IR Spectrum in Organic & Pharmaceutical Chemistry. Academic Press (China), Beijing, People’s Republic of China, 1987. 13. Nakanshi, K. & Solomon, P. H., Znfi.ared Absorption Spectroscopy. Holden-Day Inc., San Francisco, USA, 1977.
