Microwave assisted enzymatic esterification of lactic acid and ethanol in phosphonium type ionic liquids as co-solvents

Article abstract of DOI:10.1039/b820575b

Microwave heating was found to have a beneficial effect on the enzymatic esterification of lactic acid (LA) with ethanol in Cyphos 202 ionic liquid (IL) medium. Microwave irradiation caused hydrolysis of lactoyllactic acid (the linear dimer of LA) during the reaction, thus, as a result, more lactic acid was available as substrate and a higher ester yield was achieved in the esterification.

Full text of DOI:10.1039/b820575b

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Volumeꢀ11ꢀꢀ|ꢀꢀNumberꢀ5ꢀ|ꢀꢀMayꢀ2009ꢀꢀ|ꢀꢀPagesꢀ593–740
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Chiappeet al.
Gubiczaꢀet al.
Glycerylimidazoliumꢀbasedꢀionicꢀliquids Microwaveꢀassistedꢀenzymaticꢀ
esterification
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Applicationꢀofꢀwaterꢀinꢀtwo-phaseꢀ
telomerisation
Thomasꢀet al.
Dynamicꢀkineticꢀresolutionꢀofꢀ
rac-1-phenylethanolꢀinꢀscCO2
1463-9262(2009)11:3;1-B

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Microwave assisted enzymatic esterification of lactic acid and ethanol in
phosphonium type ionic liquids as co-solvents†
Brigitta Major, Ilona Kelemen-Horv a´ th, Zs o´ fia Csan a´ di, Katalin B e´ lafi-Bak o´ and L a´ szl o´ Gubicza*
Received 19th November 2008, Accepted 19th January 2009
First published as an Advance Article on the web 26th January 2009
DOI: 10.1039/b820575b
9
Microwave heating was found to have a beneficial effect
on the enzymatic esterification of lactic acid (LA) with
ethanol in Cyphos 202 ionic liquid (IL) medium. Microwave
irradiation caused hydrolysis of lactoyllactic acid (the linear
dimer of LA) during the reaction, thus, as a result, more
lactic acid was available as substrate and a higher ester yield
was achieved in the esterification.
in organic solvents. The stability of Candida antarctica lipase
10
11
B (CALB) in an organic solvent and solvent-free system
was found to be higher under microwave heating than under
conventional heating.
Although both microwave irradiation and ILs provide several
advantages in the case of esterification reactions, only one
report describes an enzymatic acylation using the two special
12
conditions simultaneously. Therefore our goal was to test the
influence of microwave energy on the enzymatic synthesis of
ethyl lactate in ILs. The reason for choosing LA to investigate
the esterification is that it has so far only been tested in organic
Introduction
The group of lactate esters is one of the most important
1
13
derivatives of LA. Lactate esters can be used in the composition
solvents, in toluene and recently in some hydrophobic ethers
14
or production of multitude products, including packaging,
paints, grease removers and cleansers. Low-cost lactate esters,
which can potentially be used to produce other chemicals
such as copolymers of biodegradable plastics, acrylates, glycol
and other specialty chemicals, are intermediate products. Ethyl
lactate is a natural flavouring compound, therefore a valuable
and ketones. Carrying out this synthesis in ILs is a novel
process itself.
Results and discussion
Several ILs were tested to find an appropriate medium for the
ethyl lactate synthesis. It was found that the reaction can be
carried out in Cyphos 104 and Cyphos 202, which consist of
the same cation with different anions (see Fig. 1) and were also
tested as extracting agents in the case of LA. Although these
ILs are somewhat more viscous than typical imidazolium salts,
when organic substrates are added the viscosity may decrease by
2
food and perfumery additive. Natural LA can be produced by
bioconversion, which is, however, not as widely used as could
be because of its costly recovery and purification steps. Several
downstream processes have been developed such as reactive
distillation, reactive extraction, electrodialysis, adsorption and
4
3
esterification. One of the latest methods is the extraction
15
an order of magnitude.
by phosphonium type ILs containing hydrophobic anions.
These ILs are proper extracting agents for LA since they
4
form complexes with it. On the other hand, ILs may be
suitable media for biocatalytic synthesis because of their enzyme
5,6
stabilization effect, reusability and negligible vapour pressure.
For that reason esterification reactions might be carried out in
the extracting agent. Avoiding an expensive separation process
would help to produce valuable products with reduced cost.
Recently, the use of microwave irradiation is spreading in
the field of organic chemistry, since it is a cleaner and more
7
efficient energy source than the traditional convective heating.
The effect of microwave irradiation was studied in the conversion
of fructose into 5-hydroxymethylfurfural (HMF)—as one of the
latest examples—and it turned out that fructose conversion and
HMF yields by microwave heating were higher than those by
8
sand bath heating. Moreover, microwave conditions are able to
increase the reaction rate of enzymatic esterification processes
Fig. 1 The structure of Cyphos 104 (trihexyl-tetradecyl-phosphonium-
bis(2,4,4-trimethylpentyl)-phosphinate) and Cyphos 202 (trihexyl-
tetradecyl-phosphonium-dodecylbenzene-sulfonate) ILs.
Research Institute of Chemical and Process Engineering, University of
Pannonia, Egyetem u 10, 8200, Veszpr e´ m, Hungary. E-mail: gubicza@
mukki.richem.hu; Fax: +36 88-624038; Tel: +36 88-624044
The investigated ILs gave a one-phase system with the
substrates and products and they were found to be a suitable
media for the enzymatic reaction.
†
This paper was published as part of the themed issue of contributions
from the Green Solvents – Progress in Science and Application
conference held in Friedrichshafen, September 2008.
6
14 | Green Chem., 2009, 11, 614–616
This journal is © The Royal Society of Chemistry 2009

In order to define the optimal reaction parameters of the
synthesis, we need to determine the minimal amount of IL
for the reaction under conventional conditions. Varying the
-
3
-3
concentration of IL in the range of 155 g dm (0.20 mol dm )
to 346 g dm- (0.44 mol dm ) the ethyl ester yield increased
extensively. Further addition of IL had no significant influence
on it. The optimal LA/ethanol molar ratio was found to be
3
-3
1
1
:7. The amount of immobilised CALB was varied between
2.5 and 50 mg mmol- LA depending on the required reaction
1
time. Using the smallest amount, the reaction was complete in
4 hours. The highest yield (95%) (correlating to the amount of
2
monomer LA) was achieved in the case of 2 w/w% initial water
content in Cyphos 104.
Fig. 2 The effect of conventional and microwave heating on the ethyl
lactate synthesis in Cyphos 202 IL using 3 and 4 w/w% initial water
content, respectively (2 mmol LA, 14 mmol ethanol, 0.77 mmol Cyphos
Control reactions were carried out to study the influence of
microwave irradiation on CALB and ILs. In these experiments,
different systems (ILs; CALB; CALB with ILs and ethanol)
2
02 IL and 12.5 mg Candida antarctica lipase B).
◦
were irradiated by microwave energy for 2 hours at 40 C. After
20
this treatment, reactions were started in a shaking incubator and
the observed yields were compared. According to the obtained
data, the incubation of ILs had no effect on the ester yield.
The irradiated pure enzyme reached only 72% of the expected
yield. The reason for this decreased activity was presumably
not the microwave irradiation but the fact that enzymes are not
really stable without a solvent. In our experiments, a mixture
of IL and ethanol was used as solvent. The microwave assisted
incubation resulted in the same yield as its control reaction under
et al. report that neither temperature change nor catalyst
additionalter the dynamic equilibriumbetween LA, lactoyllactic
acid and water, but in an esterification reaction the formation
of water causes the hydrolysis of the lactoyllactic acid. It
was found that its hydrolysis is a very slow reaction under
conventional heating and may be a rate-limiting step in ethyl
lactate formation. However, in our experiments using microwave
irradiation, an enhanced hydrolysis of lactoyllactic acid was
observed. According to the results of HPLC analysis (Fig. 3)
the lactoyllactic acid content of the reaction mixture decreases
providing fresh substrate (LA) for the reaction. The amount of
lactide, the cyclic di-ester of lactic acid, remained practically
constant during the reaction.
◦
conventional heating (2 hours, 40 C in a shaking incubator).
Although ethanol itself slightly damages the enzyme, its use in
excess was necessary to solve the mixing problems of the viscous
IL. Consequently, microwave irradiation harms neither CALB
nor Cyphos type ILs.
The esterification reaction in the mentioned ILs was tested
under microwave conditions. In the case of Cyphos 202, the
reaction rate increased while in Cyphos 104 the results remained
at the same level as in the shaking incubator. Further reactions
were carried out in Cyphos 202 because it was the only medium
where microwave irradiation had a positive effect on the reaction
rate.
Since the influence of water content is very important in
16
esterification reactions and the polar water molecules can
17
effect the energy dissipation under microwave conditions,
the most important reaction parameter is the optimal initial
water content. A constant water level in the reaction mixture
containing the ionic liquid as solvent can be maintained by
Fig. 3 Composition of the LA solution in the reaction mixture vs.
reaction time under microwave conditions (reaction conditions: see
Fig. 2).
18,19
pervaporative removal of the water formed continuously.
In this study, in both cases (conventional and microwave
heating) low (2 w/w%) initial water content resulted in low
yields. Under conventional conditions (convective heating), the
highest ethyl lactate content was achieved at 4 w/w% initial
water content, while under microwave conditions 3 w/w%. The
ester yield was 105% (Fig. 2) in both cases. Using conventional
heating, 24 hours reaction time was needed to carry out
the reaction, while under microwave conditions 7 hours were
enough.
As Fig. 2 shows, in some cases ester yield exceeded the
maximal level calculated on the initial monomer LA content
of the reaction mixture. The reason for this increased yield is
that the lactoyllactic acid (open chain dimer of LA) is able
to decompose to monomers and form ethyl lactate. Engin
While the ester yield increases to 105%, the composition of
LA/lactoyllactic acid remains stable (Fig. 4) (about 47 w/w%
LA, 43 w/w% lactoyllactic acid). In this case, the rate of
the esterification reaction is comparable with the rate of the
decomposition of lactoyllactic acid. Since microwave irradiation
accelerates the hydrolysis of the dimer, it is not a rate limiting step
any longer. Using microwave irradiation, this influence results in
shorter reaction time than under conventional conditions and
this may be the reason for the faster reaction using a higher
amount of initial water content. The application of IL, as a co-
solvent used for the extraction of lactic acid, thus increases the
ester yield.
This journal is © The Royal Society of Chemistry 2009
Green Chem., 2009, 11, 614–616 | 615

Experimental setup using microwave heating
Tests under microwave conditions were performed in commer-
cial microwave equipment (Discover series, BenchMate model,
CEM Corporation, USA) with a capacity of 4 ml. It was
equipped with a magnetic stirrer and a fibre-optic sensor
to monitor the temperature, which was set by varying the
microwave power. For the esterification reactions of LA, 10 W
◦
energy was used to hold the reaction temperature on 40 C. The
volume and the composition of the reaction mixture was the
same as under conventional conditions.
Fig. 4 Composition of the unreacted compounds in the reaction mix-
ture during reaction under microwave conditions (reaction conditions:
see Fig. 2).
Analytical methods
Water content of the substrates and the reaction mixture
was measured by a Mettler Toledo DL31 type Karl Fisher
titrator. The product samples were analysed by a HP 5890A
gas chromatograph with a HP-FFAP column and FID detector.
The HPLC analyses were carried out on MERC type equipment
with a Zorbax SB-Aq 76 column and L-7450 detector. The
monitoring wavelength was 215 nm. All reactions were carried
out in triplicate and the average error was less than 6%.
Conclusions
The enzymatic synthesis of ethyl lactate was successfully
achieved in Cyphos 104 and Cyphos 202 ILs. These media
have also been applied to extract LA from the fermentation
broth, thus the same media can be used for both extraction and
4
reaction. Further improvement of the esterification reaction
is possible using microwave heating instead of conventional
conditions. According to our results, microwave irradiation
enhanced the ethyl lactate production accelerating the hydrolysis
of lactoyllactic acid. Based on our findings, combining the
advantage of microwave treatment with the esterification in
the extracting agent as co-solvent can result in a reaction with
enhanced yields and higher rate.
Acknowledgements
This work was partly supported by the Hungarian–Slovak Sci-
ence and Technology Cooperation (Project No. SK-03/2006).
Notes and references
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Immobilized CALB (Novozym 435 ) was received from
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6
>
95 w/w%) and 202 ILs were purchased from IoLiTec GmbH,
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16 | Green Chem., 2009, 11, 614–616
This journal is © The Royal Society of Chemistry 2009