M. Cvjetko et al. / Process Biochemistry 47 (2012) 1344–1350
2.3. GC analysis
1345
have successfully demonstrated a relatively cheap, reproducible
and reusable miniaturized fixed-bed reactor for the esterification
of acetic acid and n-butanol employing Amberlyst-15 as a cata-
lyst. Esterification of various fatty acids was accomplished in a
microreactor packed with Novozym 435 [14], where higher pro-
ductivity of the miniaturized flow reactor was attributed to the
tor, unachievable in batch reactors. A continuous-flow mezzo-scale
reactor packed with various immobilized lipases was significantly
more favorable also for the kinetic resolution of 2-methylene-
substituted cycloalkanols, when compared to its batch counterpart
[15].
Isoamyl acetate, isoamyl alcohol and acetic anhydride concentrations in the n-
heptane phase were determined by a gas chromatograph HP 6890 (Hewlett-Packard,
Palo Alto, USA) equipped with a hydrogen flame ionization detector and a HP-
INNOWAX column (30 m × 0.25 mm i.d. × 0.25 mm). Nitrogen was used as a carrier
gas at a flow rate of 29 ml/min. The temperature of the oven at the injection was
100 ◦C and was kept constant for 1 min. The linear increase in temperature to 200 ◦
C
was set by 358 min−1, and was kept at 200 ◦C till the end of the analysis. Injector and
detector temperatures were set at 250 ◦C [18]. Quantification of data was done by
the calibration with standard samples. The retention time for isoamyl acetate and
acetic anhydride was 2.5 min.
The aim of this work was to take advantages of ILs as biotransfor-
mation media and to harness the benefits of miniaturized packed
bed reactors as an efficient tool for the development of a sustainable
and environmentally friendly process of enzymatic ester synthesis.
For this purpose, a series of alkyl-, alkenyl-, alkynyl-, benzyl- and N-
alkoxyl-substituted imidazolium-based ILs were synthesized and
screened together with some commercial imidazolium-based ILs
for lipase-catalyzed esterification of isoamyl alcohol. Furthermore,
the effect of temperature and flow rate of reactants on the reaction
yield was investigated within a continuously operated miniatur-
ized bioreactor packed with C. antarctica lipase B (Novozym 435),
which was used to attain the optimization of process parameters
in a very short time with low reagent consumption.
2.4. Determination of partitioning coefficients for isoamyl acetate
According to our previous findings [7] the equilibrium concentration of isoamyl
acetate within n-heptane/IL system was achieved in less than a minute using a vortex
mixer, 1 ml of IL containing 0.5 M isoamyl acetate was incubated for 3 min with
1 ml of n-heptane at 1500 rpm at 25 ◦C. The concentration of isoamyl acetate in
the n-heptane phase was determined by gas chromatography as stated above. The
partitioning coefficient was evaluated as the ratio of isoamyl acetate concentration
in n-heptane phase to its concentration in IL, where the latter was calculated from
the initial isoamyl acetate concentration.
2.5. Determination of viscosities of ionic liquids
A rotational viscosimeter (Rotovisco RV 20, Haake, Karlsruhe, Germany) ther-
mostated at 25 ◦C was used for determination of viscosities of [C4mim][Tf2N] and
[C7mmim][Tf2N].
2. Materials and methods
2.1. Materials
2.6. Screening of ionic liquids for enzymatic synthesis of isoamyl acetate
Novozym 435 (lipase B from C. antarctica; immobilized on macro-porous poly-
acrylic resin beads, specific activity 10,000 propyl laurate unit (PLU)/g; water
content 1–2%, w/w) was kindly provided by Novozymes, Bagsværd, Denmark. The six
tested commercial ILs [C2mim][Tf2N], [C2mim][BF4], [C4mim][PF6], [C4mim][BF4],
[C4mim][Tf2N], [C5mim][Tf2N] and [C7mim][Tf2N] were purchased from Ionic Liq-
uid Technologies GmbH & Co. KG, Denzlingen, Germany (purity > 98%). Isoamyl
alcohol, acetic anhydride, isoamyl acetate and n-heptane were purchased from
Sigma–Aldrich Chemie GmbH (Steinheim, Germany) and were of analytical grade.
All chemicals for ILs syntheses were purchased from Acros Organics. 1H NMR and
13C NMR spectra were recorded in a DMSO-d6 on a VARIAN XL-GEM-300 MHz spec-
trometer. Chemical shifts were expressed in ppm values using TMS as an internal
standard.
All experiments were carried out in thermostated test tubes at 25 ◦C placed on
a vortex mixer at 2000 rpm. The reaction started by adding 20 mg Novozym 435 to
600 l of IL or n-heptane containing 0.8 M acetic anhydride and 2.4 M isoamyl alco-
hol. Reactions without the enzyme were also performed. When monitoring reactions
in ILs, isoamyl acetate was recovered at specified time intervals via liquid extraction
using n-heptane until no further raise in yield was detected. The biphasic mixture
containing 50 l of IL-phase aliquots and 50 l of n-heptane was strongly shaken
for 3 min and analyzed by a gas chromatograph as stated above. Isoamyl acetate
concentration was calculated by taking into account the partitioning coefficient for
isoamyl acetate in the n-heptane/IL system. For reactions performed in n-heptane,
aliquots of 20 l were directly analyzed by a gas chromatograph. All experiments
were carried out in duplicates and the average values were calculated from the
results.
2.2. General procedure for the preparation of ionic liquids
2.7. Enzyme stability in [C7mmim][Tf2N]
formed according to standard procedures [16] or their modifications. Aliphatic
or aromatic halide was added in 10% excess to the stirred solution of 1-
methylimidazole or 1,2-dimethylimidazole in toluene at 0 ◦C and the reaction
mixture was stirred for 24–48 h at 70 ◦C. The sedimented product was washed thor-
oughly with ethyl-acetate, dried under reduced pressure at 70 ◦C, and characterized
by 1H NMR and 13C NMR (see ESI†) as specified above.
Novozym 435 (2 mg) was incubated in [C7mmim][Tf2N] (393 l) at 25 ◦C. At
selected intervals within one month of incubation, enzyme reactions were initiated
by adding acetic anhydride isoamyl alcohol into the lipase–IL mixture in amounts
to yield final concentrations 0.5 M and 1.5 M, respectively and the reactions were
monitored as stated above. The relative activity (%) was calculated from the initial
reaction rate obtained by the enzyme after incubation in IL, compared to the one
obtained without previous exposure to IL.
1-(3-Aminopropyl)-imidazole was used for the preparation of 1-(3-N,N-
dimethylaminopropyl)-3-methylimidazolium iodide and 1-(3-aminopropyl)-3-
ethylimidazolium iodide, and the preparation proceeded as described above.
For the synthesis of 1-methyl-3-pentoxyimidazolium bromide and 1-methyl-
3-heptoxyimidazolium bromide, hydrogen peroxide (0.6 mol) was added dropwise
to the stirred solution of 1-methylimidazole (0.2 mol). The reaction mixture was
stirred for 5 h at room temperature, and the excess of reactants was removed under
reduced pressure. The obtained yellow viscous oil that partially crystallized was
bromoheptane (R-X, 0.22 mol) was added dropwise and the reaction mixture was
stirred for 48 h at 60 ◦C. Acetonitrile was evaporated under reduced pressure and the
under reduced pressure at 70 ◦C, and characterized by 1H NMR and 13C NMR (see
ESI†) as specified above. All ILs were vacuum-dried prior to use.
A miniaturized continuous flow reactor made of PMMA and an olefin-based
laboratory film assembled in a sandwich-like structure providing microchannel
structure was used for achieving continuous esterification (Fig. 1). One layer of
Novozym 435 beads (70 mg) was incorporated into the channel of 1 cm width,
450 m height and 75 mm length [19]. [C7mmim][Tf2N] containing 0.5 M of acetic
anhydride and 1.5 M of isoamyl alcohol was pump-driven through the packed bed
microreactor by means of a high pressure syringe pump (Harvard Apparatus, Hol-
liston, USA) at flow rates between 5 and 90 l min−1. The reaction was carried out
at 25, 35, 45 and 55 ◦C. The reaction temperature was controlled by embedding the
enzyme microreactor into a thermostated bath. After reaching a steady state, the
outflow of the microreactor was collected and analyzed at selected time intervals
by gas chromatography as stated above. The developed miniaturized reactor was
repeatedly used every day over consecutive 14 days in order to evaluate system sta-
bility. After each continuous-flow experiment performed at 25 ◦C as stated above,
tion of quaternary imidazolium halides with a slight excess (10%) of LiTf2N and
stirred for approximately 2 h [17]. The upper aqueous phase was decanted and the
lower product portion was washed with water until chloride free, as determined by
the silver nitrate test.
The IL was dried under high vacuum at 70 ◦C and characterized by 1H NMR and
13C NMR (see ESI†) as specified above.
the flow through the microreactor was stopped and the system was stored at 25 ◦
till further use.
C