Introduction


 

 

      
Al-Khwarizmi 
Engineering   

Journal 
Al-Khwarizmi Engineering Journal, Vol. 8, No. 2, PP 77 - 83 (2012) 

 

 

Extraction of Penicillin V from Simulated Fermentation  

Broth by Liquid-Liquid Membrane Technique 
 

Khalid W. Hameed 
Department of Biochemical Engineering/Al-Khwarizmi College of Engineering/University of Baghdad  

Email:Kwhameed74@yahoo.com 

 
(Received 25 December 2011; accepted 22 February 2012) 

 

 

Abstract 

 
Liquid-liquid membrane extraction technique, pertraction, using three types of solvents (methyl isobutyl ketone, n-

butyl acetate, and n-amyl acetate) was used for recovery of penicillin V from simulated fermentation broth under 

various operating conditions of pH value (4-6) for feed and (6-8) for receiver phase, time (0-40 min), and agitation 

speed (300-500 rpm) in a batch laboratory unit system. The optimum conditions for extraction were at pH of 4 for feed, 

and 8 for receiver phase, rotation speed of 500 rpm, time of 40 min, and solvent of MIBK as membrane, where more 

than 98% of penicillin was extracted.   
 

Keywords: Liquid-liquid membrane, Penicillin V, pertraction, extraction.  
 

 

1. Introduction 
 

Liquid-liquid membranes extraction technique 

combines extraction and stripping into one step, 
rather than the two separate steps required in 

conventional processes such as solvent 

extractions. A one-step liquid membrane process 
provides the maximum driving force for the 

separation of a targeted species, leading to the 

best clean-up and recovery of the species
(1)

. 

Liquid membrane process, called also pertraction 
process, have gained increased attention due to its 

ambient temperature operation, relatively low 

capital cost, high separation efficiencies and 
modular construction. The process is inherently 

low-energy, continuous, and can be made highly-

automated. The amount of organic solvent 
required are generally very small, and thus the 

technology is environmentally benign
(2, 3)

. Two 

aqueous solutions, feed solution F, and receiver 

solution R, are separated by a third, organic liquid 
M, representing the “liquid membrane” which is 

insoluble in the other two liquids. The solute is 

transferred from the feed to the acceptor solution 
under the effect of appropriately chosen 

equilibrium conditions at the two interface F/M 

and M/R. In liquid membranes, facilitated 

transport is the mass transfer mechanism for the 

target species to go from the feed solution to the 
receiver solution.

(4, 5)
. Extraction using liquid 

membranes has been studied since the 1980s and 

is one of the most advantageous techniques of 
separation at the present. This separation method 

consists in the transfer of a solute between two 

aqueous phases of different pH which are 

separated by a solvent and carrier layer. The 
claimed advantages are as follows: the quantity of 

solvent used is small because of its continuous 

regeneration, the loss of solvent is small during 
extraction process provided the pH gradient 

between the two aqueous phases is maintained, 

there is a possibility of solute transport through 
liquid membranes that have been used for the 

separation of some biosynthetic products, namely 

carboxylic acids, amino acids and antibiotics
(6, 

7)
.The membrane interposed between two miscible 

aqueous solution, at one side (feed phase) in 

which the solute to be transport is extracted, while 

at the other side (strip phase), re-extraction 
occurs. Since in each of the aqueous phase some 

specific, and different for each of them, 

thermodynamic conditions exist, the extraction 
and re-extraction occur simultaneously

(8, 9)
. 

mailto:Kwhameed74@yahoo.com


 Khalid W. Hameed                              Al-Khwarizmi Engineering Journal, Vol. 8, No. 2, PP 77- 83 (2012) 

78 

 

     The steps of transport of solute in the 
pertraction system are described as: diffusion 

through the boundary layer in the feed solution, 

sorption on the feed solution/liquid membrane 
interface, diffusion through boundary layer on the 

feed side, transport in the membrane, diffusion 

through boundary layer on the receiving side, 

desorption on the membrane/receiving solution 
interface and diffusion through the boundary layer 

in the receiving solution
 (10)

.    

     The incessant stripping of solute of the liquid 
membrane keeps low concentration of solute in 

this phase and therefore provides its complete 

recovery from the feed solution. One of the 

principal advantages of pertraction process is the 
practically complete removal of the valuable 

component from the source material using, in 

most cases, not sophisticated, friendly solvents, in 
particular-water. As far as the membrane liquid is 

considered, it is noteworthy to mention that the 

requirements to the liquid membrane are not the 
same as to the conventional solvents used in a 

solvent extraction process, because in pertraction, 

priority is given to the membrane selectivity, 

rather than to the capacity and the solute 
distribution coefficient 

(11)
.       

Penicillin V is a secondary metabolite 

produced at low growth rates and its syntheses 
have been described extensively in the literature. 

Penicillin formation starts from three activated 

amino acids, and involves several enzymes and 
isopenicillin N as a major intermediate. Penicillin 

V (phenoxymethylpenicillin) is the commercially 

most important penicillin. It is mainly converted 

to 6-aminopenicillanic acid (6-APA), which in 
turn is used to make amoxicillin and ampicillin. 

Penicillin V is a weak acid and it is extracted with 

n-butyl acetate at pH 2-3. In this pH range 
penicillin V is unstable and decomposes, therefore 

the aqueous medium in fermentation broth is 

cooled to 0
o
C and extracted in centrifugal 

extractor to keep contact time as short as 
possible

(12)
.   

In the present work the fermentation broth was 

simulated by dissolving penicillin V sodium salt 
in distilled water. Liquid-liquid pertraction 

technique was conducted for the recovery of 

penicillin V in a batch pertraction laboratory unit. 
Methyl isobutyl ketone (MIBK), n-amyl acetate, 

and n-butyl acetate  were proposed as membranes 

for penicillin V pertaction at 25
o
C. The effect of 

speed of agitation, time, and pH were studied. 
 

 

 
 

2. Experimental Work 
 

2.1.  Material 
  
Feed Phase (Donor Phase):  

The feed phase was prepared by dissolving 1 g 
of penicillin V sodium salt in 1 L of distilled 

water, the pH of solution is adjusted by [4% 

H2SO4 (BDH)] and [5% Na2CO3 (BDH)]
(6)

. 
 

Receiver Phase (Stripping Phase): 

A sodium carbonate solution was used as a 
receiver phase. 

 

Membrane Phase: 

In the present study, methyl isobutyl ketone 
(BDH), n-amyl acetate (BDH), and n-butyl 

acetate (BDH) were used as liquid membrane. 

 

2.2.  Pertraction Lab Unit 
 

Pertraction experiments were carried out in 1 

liter laboratory pertractor as shown in Fig. 1. The 

pertractor consists of two coaxial Pyrex beakers 
and baffles where placed in each beaker as shown 

in Fig. 1. The outer beaker is 1 liter and the inner 

is 250 ml. The two beakers were arranged as 
shown in Fig. 1 and placed on a magnetic stirrer 

with heater in order to control the temperature and 

the speed. The membrane, feed, and receiver 

phases were stirred by using two Teflon-coated 
magnetic bars. 

 

2.3.  Experimental Setup 
 

500 ml of feed phase was placed in the annular 

space between the two beakers, and 200 ml of 

receiver phase was placed in the inner beaker. 

After that 300 ml of membrane phase was added 
to cover the other two phases as shown in Fig.1. 

The outer beaker was covered with a thin plastic 

layer to prevent evaporation of membrane phase. 
In the present study the effect of speed of 

agitation using the three proposed membranes was 

studied in the range of 300-500 rpm. The speed of 
agitation and temperature were adjusted and 

controlled by using hotplate and magnetic stirrer. 

The pertraction time was continuing up to 40 min 

and during this period of time samples were taken 
at a specified time interval from the feed and 

receiver phases for penicillin V analysis by 

HPLC. HPLC type Shimadzu model LC20AD 
was used in this analysis using column 100 RP-18 

(5 μm). The penicillin V in the membrane organic 

phase was evaluated by material balance.  



 Khalid W. Hameed                              Al-Khwarizmi Engineering Journal, Vol. 8, No. 2, PP 77- 83 (2012) 

79 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

Fig. 1.Schematic Diagram of Pertraction Laboratory Unit. 

 

 

3. Results and Discussion  
 
     In the present work, the batch pertraction of 
penicillin V using the three proposed liquid 

membranes  was studied, the agitation speed, and 

liquid membrane type was conducted in this work. 

The efficiency of penicillin V extracted, E, was 
calculated as follows: 

  %100
ffo

rr

VC

VC
E                                   …(1) 

Where Cr is the penicillin V concentration in 

the receiver phase, Cfo is the initial concentration 
in the feed phase, Vr is the volume of receiver 

phase, and Vf is the volume of feed phase. 

 

3.1.  Effect of pH 
  

 Figures 2 and 3 show the relationship between 

the penicillin transport from feed and to the 

receiver phases respectively with pH at different 
liquids membrane and at agitation speed of 400 

rpm and time of extraction of 40 min. The range 
of pH for feed phase is taken between (4-6) and 

for receiver phase between (6-8) because the 

penicillin V is unstable and decomposes for pH < 

4 and pH > 8
(13)

. From Figure 2, it can be seen that 
the extraction of penicillin V from feed is 

increasing with the decrease of pH value and also 

from Figure 3 the extraction of penicillin V by the 
receiver is increasing by increasing of pH value 

because the over all mass transfer coefficient 

increases when the difference in the pH value 

between two phases is high as possible
(14)

.  In the 
Figure 2, it seems that the best value of pH for 

extraction of penicillin V from feed is 4 where 

about 98% of penicillin V is extracted by MIBK, 
while from Figure 3, the best value of pH for 

extraction of penicillin V by the receiver phase is 

8 although the pH value of 7.5 has little effect on 
the extraction.   

 

 

 
 

 

1 Baffles 7 Membrane-Receiver interface 

2 Magnetic Stirrer  8 Membrane phase 

3 Magnetic bars 9 Receiver phase 

4 Membrane-Feed interface 10 Feed Phase 

5 Feed-baker 11 Plastic cover 

6 Receiver Baker   

 

2 

4 

5 

7 

6 

8 1 

1 

3 

9 

10 

11 



 Khalid W. Hameed                              Al-Khwarizmi Engineering Journal, Vol. 8, No. 2, PP 77- 83 (2012) 

80 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 2. pH Effect on Penicillin Extraction from Feed 

Phase after 40 min and Agitation Speed of 400 rpm. 

 
 

 

Fig. 3. pH Effect on Penicillin Extraction by 

Stripping Phase after 40 min and Agitation Speed of 

400 rpm. 

 

 

3.2.   Effect of Membrane type  

 
From figures 2 and 3, it can be seen that is a 

better solvent as a membrane and it can give  

better extraction efficiency methyl isobutyl ketone 

(MIBK) with respect to other solvents (n-amyl 
acetate and n-butyl acetate). The extraction 

efficiency of penicillin V is evaluated by using 

equation 1 for three types of membranes at 

temperature of 25
o
C, rotation speed of 400 rpm 

and pH for feed 4 and for receiver phase 8 as 

shown in Table 1, where the difference in 

extraction efficiency for three types of membrane 
are very little; i.e., the effect of membrane type on 

extraction is little.    

 
Table 1, 

 Extraction Efficiency, E, of Penicillin V for 3 Types 

of  Membrane at 25
o
C, 400 rpm and pH of Feed 4 

and of Receiver 8. 

Membrane type E 

MIBK 90.8 
n-amyl acetate 90 
n-butyl acetate 89.6 

 

 

Figure 4 shows the penicillin V content in the feed 

(Rf = 
fo

f

C

C
), membrane, MIBK, (Rm = 

fo

m

C

C
) and 

receiver phases (Rr = 
fo

r

C

C
) during the extraction 

at temperature of 25
o
C, agitation speed of 400 

rpm and pH of 4 for feed and 8 for receiver 

phases.  It seems that about 80% of penicillin V is 

extracted during 15 min; i.e., the extraction 
process during this time is fast, while after 30 min 

the extraction is stable and there is no effect of 

time on extraction. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 4. Penicillin V Content in Feed, Membrane 

(MIBK), and Receiver Phases with Time at 25
o
C 

and 400 rpm . 

 

 

 

pH

C
o

n
c
e

n
tr

a
ti
o

n
 g

/l

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

3.5 4.0 4.5 5.0 5.5 6.0 6.5

MIBK

Am yl acetate

Butyl acetat

time (min)

R
 (

-)

0.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 20 25 30 35 40 45 50

Feed pH=4

Membrane

Receiv er pH=8

pH

C
o

n
c
e

n
tr

a
ti
o

n
 g

/l

2.02

2.06

2.10

2.14

2.18

2.22

2.26

2.30

5.5 6.0 6.5 7.0 7.5 8.0 8.5

n-butyl acetate

n-amyl acetate

MIBK



 Khalid W. Hameed                              Al-Khwarizmi Engineering Journal, Vol. 8, No. 2, PP 77- 83 (2012) 

81 

 

3.2. Effect of Agitation Speed  
 

Figure 5 shows the effect of speed of agitation 
on the extraction efficiency, E. In order to explore 
the effects of stirring rate, the extraction 

experiment were carried out at three different 
stirring rates, 300, 400, and 500 rpm. E value 

increases with increasing speed of agitation, 

which means that the extraction efficiency of 

penicillin V from feed phase to the receiver phase 
through liquid membrane improved with 

increasing the speed of agitation; this is because 

the higher stirring rate leads to much severer 
mixing between the aqueous solution and organic 

phase, which could accelerate the transport of 

penicillin V and enhance the mass transfer area 

between the aqueous solution and liquid 
membrane solution and reduce the mass transfer 

resistances of penicillin V from feed to liquid 

membrane in the extraction process, and from 
liquid membrane to the receiver phase in the 

stripping process.  This variation in the efficiency 
indicates a diffusion control of the extraction 
process. According to previously published 

literature, although the mass transfer improved 

with higher speed of agitation, it was not applied 

because of increased risk of droplet formation 
which causes phase intermixing and deterioration 

of the process 
(10)

.  

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 5. Effect of Rotation Speed on the Extraction 

Efficiency Using MIBK as Membrane, at 25
o
C, and 

pH of 4 for Feed and 8 for Receiver Phases. 

 

 
 

4. Conclusion 
 
The liquid-liquid membrane extraction, 

pertraction, of biosynthetic products constitutes 

advantageous alternatives to conventional 

separation methods because it reduces the number 
of stages required for an efficient separation and, 

therefore, for the corresponding energy and 

material consumption. It can be concluded that the 

separation of Penicillin V from simulated broth 
could be enhanced by decreasing the pH value for 

feed up to 4 and increasing pH value up to 8 for 

receiver phase and increasing rotation speed up to 
500 rpm. The type of solvent as a membrane has 

little effect on the extraction efficiency. 

 

 

5. Reference 
 

[1] James N. Parker, M.D. and Philip M. Parker, 
Penicillin, 2004,  “A medical Dictionary, 

Bibliography”, and Annotation Research 
Guide to internet References, copyright by 

Icon group international, Inc. 

[2] Bradley D. Smith et al, 1998, “Facilitated of 
transport of carbohydrates, catecholamines, 

and amino acids though liquid and plasticized 

organic membranes”, Journal of inclusion 

phenomena and molecular recognition in 
chemistry 32: 121-131. 

[3] Mona M. Naima, Abir A. Monir, 2002, 
“Desalination using supported liquid 
membranes”, Desalination 153,  361–369. 

[4] Norman N. Li, Anthony G. Fane,W. S. 
Winston Ho, and T. Matsuura, 2008,  
“Advance membrane Technology and 

Application”,  John Wiley & Sons. 

[5] Anil K. Pabby, Syed S. H. Rizvi, Ana Maria 
Sastre, 2009, “Handbook of membrane 
separation”,  Taylor & Francis Group. 

[6] Cascavala D.,  Oniscua C., Cascavalb C., 
2000, “Selective separation of penicillin V 
from phenoxyacetic acid using liquid 

membranes” , Biochemical Engineering 

Journal 5, 45-50. 

[7] Zainuddin Abdul Manan, Mohamed 
Mahmoud Nasef, and Siti Hamidah Mohd, 

2007, “Advances in separation process”, First 

edition.  
[8] Richard W. Baker, 2004, “Membrane 

Technology and applications”, John Willy 

and Sons.  2nd Ed.   
[9] Kamniski W., 2000, "Applicability of liquid 

membrane in the environmental protection", 

rpm

85

86

87

88

89

90

91

92

93

94

95

280 320 360 400 440 480 520

 E 

    rpm 



 Khalid W. Hameed                              Al-Khwarizmi Engineering Journal, Vol. 8, No. 2, PP 77- 83 (2012) 

82 

 

Polish Journal of environmental studies Vol. 
9, No. 1, 37-43. 

[10] Boyadzhiev L., K. Dimitrov, D. Metcheva, 
2006, "Integration of solvent extraction and 
liquid membrane separation: An efficient 

tool for recovery of bio-active substances 

from botantial", Elsevier 20 March. 

[11] Nabil N. Ahmed Al-Hadithi, 2007, 
“Determination of drug and metabolites in 

the water by use of liquid membrane 

systems and HPLC-Method development 
and application” M.Sc. in Chemistry, Al-

Anbar-Iraq.   

[12] Elmar Heinzle, Arno P. Biwer, and Charles 
L. Coony, 2006, “Development of 

Sustainable Bioprocesses Modeling and 
Assessment”, Copyright John Wiley & 

Sons Ltd. 

[13] Benedict, RG., Schmidt, WH., Coghill, 
RD., Oleson, AP.(1945) “The stability of 

penicillin in aqueous solution”, J. Bact. 49: 

85-95.  

[14] Kheirolomoon A., Sayfkordi A. A., 
Kazemi-Vaysari A., Ardjmand M., and 

Baradar-Khoshfetrat A. July 2001, “Mass 

transfer analysis of penicillin extraction”, 
Scientia Iranica vol. 8, No. 3 PP 179-184. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 (2012 )77 - 83 ، صفحة2، العذد 8 مجلة الخىارزمي الهنذسية المجلذخالذ وليذ حميذ                                                                 

 

83 

 

 

 

من ناتج التخمير المصطنع بأستخذام تقنية الغشاء السائل V استخالص البنسلين 
 

خالذ وليذ حميذ 
جاهؼت بغذاد / كل٘ت الٌِذست الخْاسصهٖ/ قسن الٌِذست الك٘و٘ائ٘ت االح٘ائ٘ت 

Kwhameed74@yahoo.com :البشٗذ االلكخشًّٖ  

 
 

 

الخالصة 

 
اٗضّبْ٘حاٗل هثل ك٘خْى، اس٘خاث البْ٘حاٗل األػخ٘ادٕ، اس٘خاث )حن اسخخذام حقٌ٘ت األسخخالص بطشٗقت الغشاء السائل لثالد اًْاع هي الوزٗباث الؼضْٗت 

( 6-4) هي ًاحج الخخو٘ش الوصطٌغ ححج ظشّف حشغ٘ل هخباٌٗت هي اط ُ٘ذسّجٌٖ٘ بوذٓ Vّالخٖ أسخخذهج لغشض اسخخالص البٌس٘ل٘ي  (األهاٗل األػخ٘ادٕ

. فٖ هٌظْهت هخخبشٗت راث الٌظام الذفؼٖ (دق٘قت/ دّسة500-300)، ّسشػت خلط بوذٓ ( دق٘قت40-0)للطْس الوسخلن، صهي اسخخالص بوذٓ  (8-6)للق٘ن ّ 

 دق٘قت ّللوزٗب 40دق٘قت، صهي اسخخالص / دّسة500 للطْس الوسخلن، سشػت خلط 8 للق٘ن ّ 4الظشّف الوثلٔ لألسخخالص كاًج ػٌذ اط ُ٘ذسّجٌٖ٘ 

  .   هي البٌسل٘ي حن اسخخالصَ ضوي ُزٍ الظشّف% 98الؼضْٕ اٗضّبْ٘حاٗل هثل ك٘خْى، ح٘ذ اكثش هي 

  

mailto:Kwhameed74@yahoo.com������
mailto:Kwhameed74@yahoo.com������