Sugaring out for separation of acetonitrile and extraction of proteins and antibiotics
Pradip B. Dhamolea,b, Prafulla Mahajana, Hao Feng a,c
a
Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA
([email protected])
b
Department of Biotechnology, Sinhgad College of Engineering, Pune, India
c
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana,
IL, USA
ABSTRACT
In this study we report a novel method for separation of acetonitrile (ACN) and extraction of proteins and
antibiotics from ACN-water mixtures. It was observed that addition of a monomeric saccharide or
disaccharide into an ACN-water mixture results in phase separation with the upper phase rich in ACN. The
extraction of selected proteins (bovine serum albumin, trypsin, and pepsin) and antibiotics (erythromycin,
streptomycin, and nalidixic acid) was studied. High glucose concentration (16.5 %) and low temperature (6
o
C) resulted in better separation and improved ACN recovery (up to 62 % w/w). The distribution of proteins
and antibiotics in the upper phase and lower phase was determined by their hydrophobic/hydrophilic nature.
After a sugaring-out phase partition, 95-100 % (w/w) of the hydrophilic proteins (bovine serum albumin,
trypsin, and pepsin) were retained in the lower aqueous phase. Hydrophobic erythromycin was extracted in
the ACN-rich phase with a concentration of up to 65% (w/w). High concentrations of streptomycin (80−90
%, w/w) and nalidixic acid (91−94 %, w/w) were retained in the aqueous phase. The effective separation of
proteins and antibiotics from ACN-water mixtures at a temperature close to room temperature and with
potentially minimal damage to the products makes this method promising for large number of applications in
the pharmaceutical and biotechnology industries
Keywords: sugaring out;, two phase separation; acetonitrile; liquid-liquid extraction
INTRODUCTION
Separation and purification of biological products involves a sequence of separation processes. Liquid-liquid
extraction, salting out and reversed phase-high pressure liquid chromatography are typical processes in the
separation of biomolecules especially proteins and biomolecules. Amongst these methods acetonitrile is
preferred as a solvent or mobile phase because of its physicochemical properties like low viscosity, high
resolution, and low boiling point, etc [1]. However, acetonitrile is miscible in all proportions with water and
separating it from aqueous phase is a major problem. Its recovery is accomplished traditionally by distillation
[2-3], cooling below subzero temperatures (-20 oC) [4-5], or salting out [6-8]. Distillation under vacuum is
an energy intensive process [3] while the high temperatures encountered in atmospheric pressure distillations
(>100 oC) pose problems to temperature sensitive biomolecules. In an effort to remove ACN from the
effluent fraction of RP-HPLC, Gu et al [4] observed a phase separation at a subzero temperature of -17 oC
where the top phase was rich in ACN (88 % v/v), with most of proteins (99 %) remained in the lower phase.
Pence and Gu [9] studied the liquid-liquid equilibrium of ACN-water systems and obtained an ACN-rich
upper phase containing 0.738 mass fraction of ACN at -18.6 oC. Other researchers have utilized salting out
for the separation of ACN from a water mixture at a higher temperature. Le et al., [7] added a salt (NaCl,
Na2SO4, or NH4Cl) to separate ACN and erythromycin from an aqueous solution at room temperature and the
solvent-rich phase had an ACN concentration of 90 % (v/v) at NaCl of 10 %. Gu and Shih [8] tested the use
of different salts (K2HPO4, KH2PO4, NaCl, NH4HCO3, and KCl) for the separation of ACN from an aqueous
phase containing proteins (bovine serum albumin and myoglobin) at 4oC and found that the best separation
was achieved with K2HPO4. The limitations of salting out are high concentrations of salt, unwanted reactions
[10], and destructive effect to some biological products [11]. Another major problem in application of salting
out is the corrosion of equipment [12]. In addition, some salts (e.g., K2HPO4) may alter the pH and damage
the product [8].
Sugaring out is a new phase separation method which uses sugar as a mass separating agent. It has been
observed that the addition of a monomeric sugar or a disaccharide to an acetonitrile (ACN)-water mixture
created two phases, one solvent-rich and the other aqueous [13-14]. This method does not require a subzero
temperature for phase partition. Moreover, the use of sugar as a phase separating agent does not alter
environment conditions (e.g., pH). In this study we have explored this method for the separation of ACN
from an aqueous phase and for the extraction and recovery of biomolecules (proteins: BSA, trypsin, pepsin
and antibiotics: erythromycin, streptomycin, nallidixic acid). Our earlier experiments showed a phase
separation at 1 oC for relatively low sugar concentrations (15-50 g/l) [13-14] and best phase separation results
were obtained with glucose as a phase separating agent. In this study we have studied the effect of
temperature and glucose concentration on sugaring out.
MATERIALS & METHODS
Chemicals
Glucose (certified ACS), HPLC-grade ACN (purity 99.9 %), and 1-butanol (99.9 %) were purchased from
Fisher Scientific Co. (Pittsburgh, PA). Erythromycin (98 %) was purchased from Acros organics Pvt LTD.
(Geel, Belgium) and biomolecules BSA (>96 %), trypsin, pepsin, streptomycin, and nalidixic acids of
analytical grade were procured from Sigma Aldrich Chemicals (St. Louis, MO).
Sugaring-out phase separation and extraction of biomolecules
Glucose at a concentration of 105-165 g/l with a 15 g/l increment was dissolved in DI water at room
temperature. The hydrophilic biomolecules (BSA, trypsin, pepsin, streptomycin, and nalidixic acid) were
dissolved in the above sugar solutions while the erythromycin molecules were dissolved in ACN at a
concentration of 10 mg/ml before adding sugar solution. Sugar solution (5 ml) containing the biomolecules
(hydrophilic molecules) were mixed with 5 ml of ACN at a ratio of 1:1 (v/v) in 15 mL scale test tubes. For
hydrophobic compound, 5 ml ACN containing erythromycin was mixed with 5 mL sugar solution. After
gentle mixing of the components, tubes were placed in a water bath filled with DI water (Poly Science
Digital Temperature Controller). Temperature of the water bath was controlled to ± 0.2 oC. Tubes were
incubated at a fixed temperature (ranging from 6 oC to 18 oC with a 3 oC increment) for 24 h to reach the
equilibrium. The upper phase and lower phase volumes were recorded after phase separation. Samples were
collected using a disposable syringe and analyzed for solute (protein/antibiotic) concentration.
The results were expressed in terms of Phase ratio (PR) defined as the ratio of the upper phase volume to the
lower phase volume and extraction (%) defined as the ratio of the amount of solute in the phase to the amount
of solute added.
Analysis
Acetonitrile concentration in the upper phase was determined by a gas chromatograph unit (GC Hewlett
Packard 5890 Series II, Avondale, PA) using DB-WAX (30 m × 0.250 mm × 0.25 µm) fused silica capillary
column (J & W scientific, Agilent Technologies, Germany). The GC was equipped with a flame ionization
detector (FID) and an auto sample injector (HP 7673A Automatic Injector). The oven temperature was
programmed from 40 °C to 190 °C at 20°C min-1. The injector and detector temperature was set to 220 °C
and 250 °C, respectively. n-butanol was chosen as an internal standard. The carrier gas was He at a flow rate
of 0.72 ml min-1.
Protein concentrations in the ACN-rich phase were determined by a standard protein assay following the
manufacturer’s protocol (Bio-Rad, CA, USA). The concentration of the antibiotics was estimated by taking
the absorbance of ACN-rich phase at different wavelengths for each antibiotic. For erythromycin,
streptomycin, and nalidixic acid, the λmax was 288 nm, 328 nm, and 326 nm, respectively. Standard solutions
with concentrations of 2-10 mg/ml were prepared for each biomolecule for calibration in the UV detection.
RESULTS & DISCUSSION
Effect of temperature and glucose concentration on phase separation and acetonitrile recovery
Increasing temperature from 6 oC to 18 oC was accompanied by a gradual reduction in the upper phase
volume and hence a reduction in the phase ratio (Fig. 1a). When the temperature was fixed, the upper phase
volume and phase ratio increased with an increase in the glucose concentration. The effect of temperature on
phase separation was more pronounced at low glucose concentration (105 g/l) than at high glucose
concentration (165 g/l). The glucose concentration had little effect on phase separation at low temperature (6
o
C). No phase separation was observed for a glucose concentration of 105 g/l above 15 oC. Under the
experimental conditions used in this study, the highest phase ratio (0.59 ± 0.017) was obtained at the highest
glucose concentration (165 g/l) and lowest temperature (6 oC).
Although the phase ratio is an indicator of separation efficiency, it is not a quantitative measure to estimate
the ACN recovery in the upper phase. Hence, the upper phase samples were analyzed for ACN
concentration. A trend similar to the phase ratio was observed for ACN extraction. Increasing temperature
lowered the ACN extraction efficiency (Fig. 1b). As the sugar concentration was increased, the amount of
ACN in the upper phase increased. The maximum ACN recovery in the upper phase was 62 % which was
achieved at the conditions for the highest phase ratio. At high sugar concentrations (16.5 %), the effect of
temperature (6-18 oC) on phase separation and ACN recovery was less evident (53 -62 %). However, the
effect of temperature at low sugar concentrations (10.5 %) is significant on ACN recovery (0 – 48 %). High
sugar concentration and low temperature are desirable for maximum ACN recovery and better separation.
Similar results were reported for salting out [7-8].
To study the relation between the phase ratio and ACN recovery, the phase ratio was plotted against the ACN
recovered (Fig. 1c). As can be seen from Fig. 1c, the ACN recovery increases linearly (R2 = 0.99) with phase
ratio. Thus one can estimate the amount of ACN extraction from the phase ratio.
Phenomena of sugaring-out can be explained on the basis of interaction between glucose, acetonitrile and
water molecules. Takamuku et al [15] reported that ACN molecules form three dimensional clusters and
these clusters are surrounded by water molecule through hydrogen bonding and dipole-dipole interactions.
Hydrogen bond network between ACN and water is enhanced when the mass fraction of ACN is less than
0.6. In the present study the mass fraction was always less than 0.6. It is thus proposed that sugar molecules
may have replaced the hydrogen bonds between ACN and water molecules. This process may force ACN
molecules out of the mixture resulting into a two phase formation. As a result, when sugar concentration is
increased it results in an increase in upper phase (ACN-rich) volume as more ACN molecules are forced out.
At low temperatures, the ACN extraction increased for a fixed glucose concentration (Fig. 1b). This could be
attributed to water structure at low temperatures. The degree of hydrogen bonding depends on temperature.
As the temperature is lowered, the distance between nearest neighboring water molecules is decreased [16].
This means that the solute (ACN) will have a less chance to replace a water molecule at a lower temperature
than at a higher temperature. Further, in an ACN-water-glucose mixture, glucose may replace the hydrogen
bonds between ACN and water molecule. An outcome of this may be translated to a better phase separation
in an ACN-water system when sugar is added at a low temperature.
Effect of temperature and glucose concentration on extraction of proteins and antibiotics
BSA, trypsin and pepsin were selected as model proteins in this study. All these proteins are hydrophilic in
nature. It can be seen from Table 1 that more than 95 % of proteins are retained in the aqueous phase. An
increase in the glucose concentration and temperature did not affect the protein extraction. No phase
separation was observed at 18 oC and 105 g/l glucose. This shows that almost all the protein is retained in
aqueous phase and very little protein (less than 2 %) is lost during the ACN separation process. Thus
sugaring-out can be used for an effective recovery or purification of proteins from the ACN-containing
effluent of a RP-HPLC process.
In the case of antibiotics, streptomycin, erythromycin and nalidixic acid were selected as model antibiotics
for the study. The distribution of antibiotics was decided by its hydrophobic/hydrophilic nature.
Erythromycin is hydrophobic whereas streptomycin and nalidixic acid are hydrophilic molecules. Table 2
shows the extraction of erythromycin in the ACN-rich phase and that of streptomycin and nalidixic acid in
aqueous phase. No phase separation was observed at 18 oC and 105 g/l glucose. For erythromycin, a phase
separation did not occur even at 120 g/l glucose at 18 oC. 80-90 % of the streptomycin was retained in the
aqueous phase. Similarly, 91-94 % nalidixic acid was retained in the water-rich phase (Table 2). The glucose
concentration and temperature had insignificant effect on the extraction of streptomycin and nalidixic acid
(less than 10 %). On the other hand, 48-65 % of erythromycin was extracted in the ACN-rich phase. The
erythromycin extraction increased with an increase in the glucose concentration at both temperatures. It
increased from 48 % to 65 % when the glucose concentration was increased from 105 g/l to 165 g/l at 6 oC.
Similarly, extraction increased from 0 % to 59 % when the glucose concentration was increased from 105 g/l
to 165 g/l at 18 oC.
(1a)
Phase Ratio
0.6
0.5
0.4
10 5 g / l
12 0 g / l
0.3
13 5 g / l
150 g / l
0.2
16 5 g / l
0.1
(1b)
70
ACN Extraction (% w/w)
0.7
60
50
40
105 g/ l
120 g/ l
30
135 g/ l
150 g/ l
20
165 g/ l
10
0
0
4
8
12
16
4
20
8
12
16
20
Te m pe rature , oC
o
Te m pe rature , C
(1c)
% ACN Extraction
60
50
120 g/l
40
105 g/l
30
Figure 1. The effects of temperature and glucose
concentration on sugaring out (1a); The effects
of temperature and glucose concentration on
acetonitrile recovery (1b and 1c).
20
y = 110.43x
R 2 = 0.9907
10
0
0
0.2
0.4
0.6
Phase Ratio
Table 1. Effect of temperature and glucose concentration on extraction of proteins (BSA, Pepsin, and
Trypsin) (ACN: water =1:1).
Protein Recovery in Aqueous Phase (%, w/w)
Protein
Pepsin
Trypsin
BSA
Glucose
18 oC
6 oC
18 oC
6 oC
18 oC
Conc. g/l
6 oC
105
98.69± 1.03
98.13 ± 0.5
98.13 ± 0.5
*
98.87± 0.02
*
120
98.88 ±0.2
97.98 ±0.98
97.98 ±0.98
97.16 ± 0.77
98.69 ± 1.1
98.18±0.24
135
99.08 ±0.27
98.42 ±0.03
98.42 ±0.03
98.18 ±0.37
98.93± 0.27
98.28±0.11
150
98.91 ±0.19
98.38 ±0.41
98.38 ±0.41
98.09 ±1.02
99.71± 0.86
99.07±0.25
165
99.56 ±0.23
98.59 ±0.63
98.59 ±0.63
98.9 ±0.63
99.45 ±0.05
98.68±0.52
*- No phase separation
Table 2. Effect of temperature and glucose concentration on extraction of
erythromycin, and nalidixic acid) (ACN: water =1:1).
Average Extraction (%, w/w)
Streptomycin (in aqueous
Erythromycin (in ACN-rich
Antibiotic
phase)
phase)
Glucose, g/l
6 oC
18 oC
6 oC
18 oC
105
80.64 ±0.27
*
48.71 ±3.5
*
120
77.80 ±0.28
87.97 ±0.46
58.52 ±1.54
*
135
77.98 ±0.0
89.17 ±2.4
57.76 ±3.59
42.23 ±0.41
150
80.12 ±0.31
89.05 ±0.04
66.62 ±3.61
50.57 ±0.97
165
78.96 ±0.31
89.59 ±1.16
64.45 ±2.9
59.42 ±0.2
* - No phase separation
antibiotics (Streptomycin,
Nalidixic Acid (in aqueous
phase)
6 oC
18 oC
94.78 ±0.1
*
94.86 ±0.17
93.78 ±0.32
94.98 ±0.17
91.44 ±0.46
94.84 ±0.06
91.44 ±0.13
94.69 ±0.0
92.14 0.25
CONCLUSIONS
Addition of glucose into an ACN-water mixture resulted into phase separation at relatively low temperature
(6-18 oC).
1. Phase ratio and ACN extraction increased with an increase in glucose concentration (105-165 g/l)
and with a decrease in temperature (from 18 oC to 6 oC).
2. More than 95 % (w/w) proteins were retained in the aqueous phase after sugaring-out separation of
ACN.
3. Hydrophobic erythromycin (65 % w/w) was extracted in the ACN –rich phase and 80-90 % (w/w)
streptomycin and 91-94 % (w/w) nalidixic acid were retained in the aqueous phase.
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