Journal of Crystal Growth 198/199 (1999) 1320—1325

Separation through crystallization and hydrotropy:

the 6-aminopenicillanic acid (6-APA) and phenoxyacetic acid
(PAA) system
Narayan S. Tavare*, Vivek K. Jadhav
Department of Chemical Engineering, University of Bradford, West Yorkshire BD7 1 DP, UK

Abstract

The specificity of hydrotropes in solubilization has been successfully employed for the separation of close-boiling
isomeric components from their binary mixtures forming simple eutectics, in some cases even at the eutectic composi-
tions. The purpose of this paper is to propose a novel technique of crystallizing 6-aminopenicillanic acid (6-APA) in
a good quality and pure crystalline form from product reaction mixture in a hydrotrope environment. The remaining
mother liquor may be used to recover crystalline phenoxyacetic acid (PAA) by dilution with water and subsequently to
concentrate hydrotrope solution for recycle. Semibatch crystallization experiments for 6-APA in a 2 l laboratory-scale
agitated crystallizer with controlled acid addition from product reaction mixture in hydrotrope solution yield better
quality crystalline product than that in absence of hydrotrope. The present study demonstrates the potential efficacy of
sodium butyl monoglycol sulphate (NaBMGS) hydrotrope in the separation of 6-APA in pure crystalline form from the
product reaction mixture.  1999 Elsevier Science B.V. All rights reserved.

Keywords: Semibatch crystallization; Hydrotropy; Separations; Precipitation; 6-aminopenicillanic acid (6-APA)

1. Introduction methods based on the molecular size, the separ-

A variety of isomeric or closely related mixtures
of organic compounds having similar molecular
sizes and very close chemical and physical proper-
ties often require their separation into nearly pure
components. As the constituents are difficult to
separate in most cases by conventional separation
* Corresponding author. Fax: #44 1274 235700; e-mail:
n.s.tavare@bradford.ac.uk.
ation methods exploiting the differences in molecu-
lar structure of the components are likely to be
effective. Several possible techniques are available;
most depend on the addition of a third component
which augments the ease of separation as a result of
the increased differences in physical properties such
as boiling points, melting points and solubilities.
Extractive or adductive crystallization, reactive dis-
tillation or extraction and extraction with super-
critical fluids have been advocated. The present
research was motivated to look systematically at

0022-0248/99/$ — see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 0 2 4 8 ( 9 8 ) 0 1 0 1 7 - 3
 N.S. Tavare, V.K. Jadhav / Journal of Crystal Growth 198/199 (1999) 1320–1325
the use of the phenomenon of hydrotropy for the
separation of isomeric component from their mix-
tures and demonstrate how it can be approached
using sound analysis based on crystallization
principles.
Hydrotropes, a class of organic compounds, sig-
nificantly increase the aqueous solubility of certain
sparingly soluble compounds and can be used for
a variety of applications. Sharma and his co-
workers [1—3] used hydrotropes to enhance the
rates of multiphase chemical reactions as well as to
separate selectively close-boiling compounds either
by extractive distillation or liquid-liquid extraction.
Tavare and co-workers [4—9] proposed to use the
phenomenon of hydrotropy for the separation of
isomeric mixtures. For several binary mixtures,
they found that the enhancement in the solubility
with the hydrotrope concentration was signifi-
cantly higher for one of the components and used
this observation to selectively precipitate one of the
components by controlled dilution with water.
They demonstrated its successful application for
the separation of o- and p-chloronitrobenzenes, 1-
and 2-naphthols, m- and p-aminoacetophenones, in
some cases even at eutectic compositions. The spe-
cificity in solubilization has been successfully em-
ployed not only for the separation of close boiling
and both close boiling and close melting compo-
nents from binary mixtures forming simple eutec-
tics but also for achieving improved quality and
purity of pharmaceutical intermediates [10]. The
purpose of this article is to illustrate the efficacy of
a novel crystallization (or precipitation) technique
for the separation of good quality pharmaceutical
intermediate from its reaction product mixture in
hydrotrope solution.
6-Aminopenicellanic acid (6-APA), an important
intermediate in the production of semisynthetic
antibiotics, is basically the nucleus of the original
penicillin molecule. In the manufacture of 6-APA,
enzymatic hydrolysis reaction of potassium penicil-
lin V yields 6-APA and Phenoxyacetic acid (PAA)
in stoichiometric amount containing equimolar
(0.25 mol/l) at near neutral pH (pH"7.5). Conven-
tionally 6-APA is separated from the product reac-
tion mixture by crystallizing it with a controlled
addition of aqueous nitric acid solution up to pH
&3.7. Purity and recovery of 6-APA in a good
1321
quality crystalline form are important consider-
ations in the process. Frequently, the purity speci-
fication of 6-APA is rather difficult to attain in such
a crystallization process as PAA may cocrystallize
as an impurity. Solubilities of 6-APA and PAA
in water are low (&0.0025 and &0.011 g/g water,
respectively). Only the solubility of PAA is aug-
mented rapidly with hydrotrope concentration to
&0.39 g/g water in neat sodium butyl mono-
glycol sulphate solution (NaBMGS 50%) while
that of 6-APA remains practically constant
(&0.003—0.004 g/g water). The purpose of this
study was to propose a novel technique of crystal-
lizing 6-APA in a good quality and pure crystalline
form from product reaction mixture in a hydro-
trope environment. Should the crystallization be
carried out from the reaction product in the pres-
ence of hydrotrope PAA would remain in the solu-
tion phase and 6-APA will precipitate in a good
quality crystalline form under controlled condi-
tions. The remaining mother liquor may be used to
recover crystalline PAA by dilution with water and
subsequently to concentrate hydrotrope solution
for recycle. The initial experimental study con-
cerned the determination of the relevant solubility
data of 6-APA in reaction product mixtures with or
without hydrotrope environment [10]. An addi-
tional experimental programme was undertaken to
perform a series of precipitation experiments with
composition corresponding to the hydrolysis reac-
tion product and also in the presence of the
NaBMGS hydrotrope solution. Solution-phase
supersaturation profiles and crystal size distribu-
tion statistics from the population density plots at
different sample times for the experiments with and
without hydrotrope environment were evaluated.
2. Precipitation experiments
The crystallizer used was a 2 l jacketed glass tank
equipped with polished stainless steel four full
baffles and six bladed disc turbine impeller. The
vessel dimensions were well defined to conform to
the standard vessel geometry as shown in Fig. 1.
A 160 W DC electrical motor with controller
providing a digital speed display over the range
&1—25 Hz and an analogue electric power meter
1322 N.S. Tavare, V.K. Jadhav / Journal of Crystal Growth 198/199 (1999) 1320–1325
Fig. 1. The crystallizer configuration.
was linked to a shaft drive of 10 mm diameter via
a rapid coupling system. The shaft assembly design
allowed easy access to, and removal of, the drive
shaft stainless ball bearings and standard lipseals
located within the top plate for cleaning or replace-
ment. Temperature measurement in the crystallizer
was carried out by a 2 wire PRT probe sensor while
pH measurement was effected by a combined elec-
trode pH probe with possible manual or automatic
temperature compensation. Both temperature and
pH probes were interfaced to a computer perform-
ing datalogging, display and control functions.
Thus a control scheme incorporating a PC in a
supervisory/management capacity interfaced to the
crystallizer assembly to effect control over the ex-
perimental conditions of pH, temperature, stirrer
speed, rate of acid addition and facilitating auto-
mated acquisition and graphical presentation of
pH, temperature and acid dosage. Temperature of
the crystallizer and acid reservoir was controlled to
20°C (within $0.2) by the circulation of coolant
at sufficiently high rates to minimize response time
lag to the changes in temperature created by the
heat of mixing and crystallization and the rate of
acid addition was controlled to a specified value.
Effective stirring was facilitated by a motor driven
six bladed stainless steel disc turbine impeller
driven at rotational speed varying from 4.2 to
12.5 Hz. Nitric acid was fed to the crystallizer using
a computer controlled gear pump (Isamatec 240)
fitted with a graphite pump head (Micropump)
which was attached to a 1.0 mm diameter stainless
steel feed pipe using acid resistant 1.6 mm diameter
tubing (Marprene). The acid feed point was located
at a point close to impeller in the discharge stream
within the vessel (Fig. 1).
In a typical run, 2 l of twice filtered solution
having desired composition of 6-APA and PAA
(250 mM each, made up by dissolving of 108.15 g
6-APA and 76.08 g PAA in 4 M ammonia and
distilled water at room temperature) through two
0.7 lm glass microfibre filters was charged, temper-
ature, stirrer speed and pH were adjusted to 20°C,
8.3 Hz and 7.5, respectively. For the runs with hy-
drotrope the initial solution was prepared by
adding commercially available neat (NaBMGS
50%) hydrotrope solution in such an amount that
it would have 30% NaBMGS by mass in the final
reaction mixture. Known amount of nitric acid was
added at a constant flowrate over a predetermined
period in the agitated reaction mixture, while the
same amount of acid being added in all cases. For
seeded experiments a predetermined amount of
seed crystals, produced from sieving commercial
6-APA powder between 55 and 65 lm mesh sieves,
was suspended in 10 g filtered distilled water pre-
viously saturated with respect to 6-APA. This seed
suspension was then added to the crystallizer when
acid dosed had changed the solution pH 7.5 to
&pH 6. Previous solubility investigations had
shown that at this pH the solution was super-
saturated with respect to 6-APA, thus providing
stable environment for the seed crystals to grow.
The liquid level in the tank was 132 mm at the
beginning of each experiment and 149 mm at the
end when all the acid had been added. At the end of
the acid addition (corresponding pH &4.3) the
pump was automatically switched off and the sus-
pension was stirred for another hour. At the end the
slurry was filtered under vacuum through a 0.7 lm
pore glass microfibre filter, the crystals washed with
100 ml ice-cold water followed by 100 ml ice-cold
acetone, vacuum dried overnight at 45°C, weighed
and the yield calculated.
 N.S. Tavare, V.K. Jadhav / Journal of Crystal Growth 198/199 (1999) 1320–1325
After the onset of sufficient nucleation or im-
mediately after seeding, crystal suspension samples
were periodically withdrawn from the centre of the
crystallizer using a 10 ml glass pipette for con-
centration and crystal size distribution analyses.
The sample was then immediately vacuum filtered
through a 0.7 lm pore glass microfibre filter
housed within 25 mm diameter Büchner filter
holder. The filtrate was weighed and quenched with
a known amount phosphate buffer to avoid the
occurrence of further nucleation in already super-
saturated filtrate. The sample was then repeatedly
diluted between 2 and 30 times until the concentra-
tion lay within the HPLC calibrated range of 1.0 to
2 mg/g solution. 100 ll samples were injected three
times into the HPLC (Varian 5000) and analysed at
215 and 218 nm UV wave length to determine the
6-APA concentration. The crystals deposited on
the microfibre filter were carefully resuspended in
20—80 ml 30% glycerol solution in Isoton electro-
lyte saturated with 6-APA. A 1 ml sample of homo-
geneous suspension of all the crystals on the filter
paper was used for particle size analysis (Coulter
counter, Multisizer II) using 128 size channels
over the size range from 8.8 to 267.1 lm.
Initial exploratory experimental studies relating
the influence of a wide range of process variables on
product crystal size distribution, yield, impurity
content and end-user product quality indicated
that not only do individual process variables exert
differing influences on the precipitated product but
in some cases interact significantly with other vari-
ables. Seeding, impeller speed and type, acid feed
rate and precipitation medium were found to be sig-
nificant but not the feed point location and rate
profile over the range of variables used for the
present recipe. Thirteen successful experiments
were performed varying acid addition flowrate, stir-
rer speed, seed loading and medium. Out of thirteen
successful runs five unseeded and four seeded and
the rest with hydrotrope medium were performed.
3. Solubility data
In order to obtain solubility data that are perti-
nent to the semibatch precipitation process used in
the plant and laboratory, the solubility was experi-
1323
mentally determined by approaching the equili-
bration from supersaturated conditions which
simulate the precipitation process in stages. In this
way the influence of time-varying solution ionic
strength as a result of semibatch nature of the pre-
cipitations was inherently accounted for. The
solubilities of 6-APA in an aqueous standard reac-
tion product solution containing 250 mM 6-APA
(54.075 g/l solution) and 250 mM PAA (38.04 g/l
solution) buffered at pH"7.5 used in the precipita-
tion studies were evaluated at 20°C with or without
hydrotrope environment. For a hydrotrope envi-
ronment the initial standard solution was prepared
in such a way that the final hydrotrope concentra-
tion in the solution was 30% NaBMGS on mass
basis. Solubility variations were determined as
a function of the added acid concentration and pH
of the equilibrated solution [10]. All 6-APA con-
centrations were evaluated on the basis of the total
inert solvent, i.e. water, so as to be consistent with
the time-varying solvent capacity in the subsequent
semibatch precipitation experiments. Clearly the
6-APA solubilities in the standard reaction product
solution were much higher than those in water and
hydrotrope solutions. Over the majority of the ad-
ded acid concentration range the decrease in solu-
bility was approximately proportional to the added
acid concentration indicating that an equivalent
reduction in the equilibrium concentration of 6-
APA in solution was produced due to reaction. The
6-APA solubility in the reaction product solution
was strongly influenced with pH over the pH range
(up to &4) used in the precipitation process. 6-
APA displayed a minimum solubility at isoelectric
pH (&3.6) and increasing solubility with lower or
higher pH levels. The zwitterionic species is the
most stable and least soluble in water. At any
temperature, the observed variation in solubility
with pH is due to the equilibrium concentration of
the cation and anion. In the presence of H> in acid
solution (or OH\ in alkaline solutions) the sparing-
ly soluble zwitterion was converted into the more
soluble anion (or cation), respectively. In the pres-
ent study the solubility measurements and sub-
sequent precipitation of 6-APA at pH greater than
isoelectric point were relevant. In this pH range the
solubility of 6-APA is mainly contributed by the
anion and zwitterion species and 6-APA acts as
1324 N.S. Tavare, V.K. Jadhav / Journal of Crystal Growth 198/199 (1999) 1320–1325

a weak acid. The added acid appears to react with

6-APA anions in solution to produce a relatively
insoluble product in the form of the zwitterion on
an equimolar basis. The amount of acid added
therefore controls the residual anion concentration
and hence the equilibrium hydrogen ion concentra-
tion (i.e., pH). The presence of hydrotrope in the
standard reaction product solution increases the
solubility of 6-APA and consequently the amount
of acid that would be required to attain the min-
imum solubility and the corresponding pH range is
more than that required without hydrotrope. The
approach towards the isoelectric point is slightly
slower than that without hydrotrope with the same Fig. 2. Representation of three process runs on a ternary dia-
gram.
amount of acid added.

4. Results and discussion

In this paper the results from three typical runs

(H3F4S5, C4F4S5, L1F4S5) performed at acid ad-
dition rate of 4.42 g/min and stirrer speed of 8.3 Hz
are reported. Run H3F4S5 was carried out in
NaBMGS hydrotrope environment (with the final
composition &30% by mass, i.e., &42 g/g water).
Run C4F4S5 used conventional production route,
while in Run L1F4S5 1.08 g of seed crystals ob-
tained from manual sieving of commercial 6-APA
between 55 and 65 lm resulting in a seed loading of
0.58 kg/m was charged when acid dosed had
changed the solution pH from 7.5 to 6. Composi-
tions of vessel contents in the semibatch precipita-
tion process along with corresponding equilibrium
state for these three runs are represented on a ter-
nary diagram as shown in Fig. 2, depicting relative
changes in composition due to acid additions. The
difference between the measured 6-APA concentra-
tion in the solution phase of a sample and corre-
sponding empirically determined 6-APA solubility
was used as supersaturation at any sample time.
Measured supersaturation profiles and variations
of the total specific crystal population count (no./kg
water) and population mean crystal size (lm) in
these three runs are reported in Figs. 3 and 4,
respectively. Acid addition rate has a signifi-
cant influence on both supersaturation profiles
and population density plots and consequently on
the product size distribution obtained from the
Fig. 3. Supersaturation profiles.
precipitator in any run. For a given acid addition
rate supersaturation passes through a maximum as
a result of competitive character between genera-
tion and depletion of supersaturation. In the pres-
ence of hydrotrope the peaks are generally sharper
and earlier than those without hydrotrope. Max-
imum level of supersaturation attainable is usually
high, thus providing a better controllability of the
particulate system in the presence of hydrotrope.
Seed loading provides surface area on which solids
will be deposited by relieving the supersaturation.
Larger product mean size with low total specific
crystal population count resulted in seeded experi-
ments. In general both product mean size and total
specific population count increase rapidly during
the initial period of run and then levels off. The
 N.S. Tavare, V.K. Jadhav / Journal of Crystal Growth 198/199 (1999) 1320–1325
Fig. 4. Variations of population mean size and total crystal
number concentration.
supersaturation profiles induced this behaviour
and also reflected in the variation of population
density with time and crystal size. These results are
reported here only in a qualitative way and require
further quantitative analysis in terms of the process
identification (i.e., characterization of crystalliza-
tion and reaction kinetics) and simulation (i.e.,
mathematical modelling using characterized rate
processes).
1325
Acknowledgements
The experimental work reported in this paper
was performed at Department of Chemical Engin-
eering, UMIST, Manchester, M60 1QD, England.
The partial financial support from SERC, UK is
gratefully acknowledged. Free supply of hydro-
tropes from Hüls (UK) Ltd. and Hickson Manro Ltd.
is appreciated. Thanks are due to Dr S. Mwangi for
his help in the experimental programme.
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