Carbohydrate Polymers 136 (2016) 930–935

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Deep eutectic solvents as efficient solvent system for the extraction of

␬-carrageenan from Kappaphycus alvarezii
Arun Kumar Das a , Mukesh Sharma b,c , Dibyendu Mondal b,c , Kamalesh Prasad b,c,∗
a
Analytical Division and Centralized Instrument Facility, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg,
Bhavnagar 364 002, India
b
Marine Biotechnology & Ecology Division, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar 364 002, India
c
Academy of Scientific and Innovative Research (AcSIR), Central Salt and Marine Chemicals Research Institute, G. B. Marg, Bhavnagar 364 002, India

a r t i c l e i n f o a b s t r a c t

Article history: Three different deep eutectic solvents (DESs) prepared by the complexation of choline chloride with
Received 16 June 2015 urea, ethylene glycol and glycerol along with their hydrated counterparts were used for the selective
Received in revised form extraction of ␬-carrageenan from Kappaphycus alvarezii. Upon comparison of the quality of the polysac-
29 September 2015
charide with the one obtained using water as extraction media as well as the one extracted using widely
Accepted 30 September 2015
practiced conventional method, it was found that, the physicochemical as well as rheological properties
Available online 9 October 2015
of ␬-carrageenan obtained using DESs as solvents was at par to the one obtained using conventional
method and was superior in quality when compared to ␬-carrageenan obtained using water as solvent.
Keywords:
Deep eutectic solvents
Considering the tedious nature of the extraction method employed in conventional extraction process,
Hydrated deep eutectic solvent the DESs can be considered as suitable alternative solvents for the facile extraction of the polysaccharide
Extraction directly from the seaweed. However, among the hydrated and non-hydrated DESs, the hydrated ones
␬-Carrageenan were found to be more effective in comparison to their non-hydrated counterparts.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction Due to unique properties such as negligible vapour pressure,

Carrageenan is a commercially important phycocolloid
extracted from red seaweeds and is used mostly as stabili-
zers and texture providers in food and ice-cream industries (Van
de Velde & Ruiter, 2005). Chemically, it consists of alternating
1,3-linked ␣-d-galactopyranose (Gal) and 1,4-linked ␤-(3,6-
anhydro-) d-galactopyranose [(3,6-A)Gal] (Fig. 1). Carrageenan is
mostly present in three varieties known as ␬-, ␫-, and ␭- and they
differ from each other in the number and position of sulphate
groups on the repeating galactopyranose units. ␬-Carrageenan
has one negative charge per the disaccharide units, while ␫- and
␭-carrageenans have 2 and 3 negative charges per the disaccharide
units, respectively. Carrageenan, alone find application in food and
beverage industries and their functional hybrids find applications
as matrices for the slow release of drug and volatile flavoured com-
pounds (Bylaite, Ilgunaite, Meyer, & Adler-Nissen, 2004; Siepmann
et al., 2007; Tapia, Corbalan, Costa, Gai, & Yazdani-Pedram, 2005).
∗ Corresponding author at: Marine Biotechnology & Ecology Division, CSIR-Central
Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar
364 002, India.
E-mail addresses: kamlesh@csmcri.org, drkamaleshp@gmail.com (K. Prasad).
high boiling point, large electrochemical window, recyclability, etc.,
ionic liquids (ILs) are much preferred choice for various applications
such as in materials design (Lodge, 2008), to dissolve and extract
various biopolymers and as extraction media for polysaccharides
(Swatloski, Spear, Holbrey, & Rogers, 2002). The unique character-
istics of ILs made them useful in many processes in carbohydrate
chemistry (Murugesan & Linhardt, 2005). ILs having the imidaz-
olium structure, such as 1-butyl-3-methylimidazolium chloride
(BMIMCl), 1-allyl-3-methylimidazolium chloride (AMIMCl) are
used to dissolve cellulose and chitin, respectively (Prasad et al.,
2009b; Swatloski et al., 2002; Wu et al., 2004; Zhang, Wu, Zhang, &
He, 2005).
Although, there were no attempts made so far to extract ␬-
carrageenan directly from seaweeds using ILs but formation of
strong ion gels of different carrageenans and their composites with
cellulose were observed in BMIMCl (Prasad, Kaneko, & Kadokawa,
2009), which envisaged the possibility of extraction of the polysac-
charide in ILs. Moreover, recently we have reported selective
precipitation of agarose from seaweed (Gracilaria dura) extractives
using bio-based ionic liquids (Sharma, Chaudhary, Mondal, Meena,
& Prasad, 2015; Sharma, Mondal, Singh, & Prasad, 2015). It should be
noted that, ␬-carrageenan is conventionally extracted using alkali
following the method reported by Craigie and Leigh (1978).

http://dx.doi.org/10.1016/j.carbpol.2015.09.114
0144-8617/© 2015 Elsevier Ltd. All rights reserved.
 A.K. Das et al. / Carbohydrate Polymers 136 (2016) 930–935
Fig. 1. Repeating units in ␬-carrageenan.
After the discovery of deep eutectic solvents (DESs) by Abbott,
Capper, Davies, Rasheed, and Tambyrajah (2003) as an alterna-
tive to conventional ILs, the application of the DESs is increasing
exponentially in various applications. Further the properties of
DESs such as non-toxicity, cheaper cost, ease of syntheses, etc.,
make them lucrative for number of applications e.g., as reaction
media, reactants, and catalysts (Zhang, Vigier, Royer, & Jérôme,
2012) and even preferable over ILs in number of applications. In
general, DESs can be obtained by the complexation of the halide
salts of quaternary ammonium or phosphonium cations (as the
hydrogen bond acceptor, HBA) with hydrogen bond donor (HBD)
moieties such as urea, glycerol, ethylene glycol, mannitol, recorci-
nol, etc., (Sirviö, Visanko, & Liimatainen, 2015). There are reports
on the applications of DESs for the dissolution of biopolymers
(Mondal, Sharma, Mukesh, Gupta, & Prasad, 2013; Sharma, Mukesh,
Mondal, & Prasad, 2013), extraction of phenolic compounds from
plants (Dai, Spronsen, Witkamp, Verpoorte, & Choi, 2013), extrac-
tion of proteins (Zeng et al., 2014), extraction of glaucarubinone
from Simaruba glauca (Kholiya, Bhatt, Rathod, Meena, & Prasad,
2015) and many more applications of the novel liquid are con-
tinuously being explored. We have shown the excellent capability
of certain bio-based DESs for the dissolution and morphological
transformation of DNA as well as material preparation using this
bio-macromolecule as building blocks employing DESs as disso-
lution media (Bhatt, Mondal, Bhojani, Chatterjee, & Prasad, 2015;
Mondal, Bhatt, Sharma, Chatterjee, & Prasad, 2014; Mukesh &
Prasad, 2015). Although the DESs are turned to be good alternate
solvents for biopolymers but extraction of biopolymers especially
any seaweed based are not attempted so far.
Herein we have employed DESs obtained by the complexation
of choline chloride with urea, ethylene glycol and glycerol as well as
their hydrated forms for the efficient extraction of ␬-carrageenan
from Kappaphycus alvarezii. The seaweed belongs to the family
Rhodophycea and commercially exploited for ␬-carrageenan.
2. Experimental
2.1. Materials
Choline chloride (AR grade) and Urea was purchased from SD
Fine chemicals, Mumbai, India. Ethylene Glycol and glycerol was
procured from Merck Chemicals, Mumbai. HPLC grade isopropyl
alcohol (IPA) was purchased from Loba Chemie, Mumbai. All chem-
icals were used as received.
Fresh K. alvarezii was collected from the cultivation sites of west
coast of India, Simar (20◦ 42 N; 71◦ 01 E) during February, 2015.
The seaweed was sun dried and was washed several times with
water to remove epiphytes and kept under shadow for drying.
After drying, the seaweed was crushed into very fine powder using
mortar and pestle in the presence of appropriate amount of liquid
nitrogen.
2.2. Synthesis of deep eutectic solvents
ChoCl. Urea 1:2, ChoCl-EG 1:2 and ChoCl-Gly 1:2 were syn-
thesized and characterized as described previously (Abbott, Bell,
931
Handa, & Stoddart, 2005). In a typical reaction, choline chloride
(hydrogen bond acceptor) and ethylene glycol, glycerol or urea
(hydrogen bond donors) were mixed separately in the optimized
molar ratio of 1:2 and were heated at 80 ◦ C until a transparent
solution was obtained.
2.3. Extraction of -carrageenan from seaweed using DES
All the three DESs were used separately to extract ␬-carrageenan
from K. alvarezii. Experiment I: In a typical experiment, 500 mg of
powdered seaweed was taken separately in 10 g of DES in a beaker.
The mixture was heated at 85/95 ◦ C for 1 h. Experiment II: 500 mg of
powdered seaweed was heated with 10 g of DES having 10% water
for 1 h.
The mixtures thus obtained from both the experiments were
centrifuged separately and the mass obtained at the bottom of cen-
trifuge tube was separated, washed several times using IPA and
dried under vacuum. A control experiment was carried out by mix-
ing 500 mg of seaweed powder and 10 ml of water keeping the
temperature and time same as that of the previous set of reactions.
The supernatant was precipitated in IPA (1:3, v/v). The precipitated
␬-carrageenan was vacuum dried.
2.4. Extraction of -carrageenan employing conventional method
␬-Carrageenan was extracted from K. alvarezii following the
method reported by Craigie and Leigh with minor modifications
(Craigie & Leigh, 1978). In a typical experiment, 10 g of dried K.
alvarezii was soaked in 200 ml of 0.5% calcium hydroxide solution
for 2 h. The soaked seaweed was autoclaved at 107 ◦ C for 1.5 h after
the addition of 200 ml water. After autoclaving, the hot mixture
was ground in a kitchen blender and centrifuged. The supernatant
was slowly added in to IPA under constant stirring (1:3, v/v). The
precipitated ␬-carrageenan was vacuum dried.
2.5. Preparation of -carrageenan gel
For the preparation of gel, 10 mg sample of each extracted ␬-
carrageenan was dissolved in 1% 1 ml KCl solution by heating at
90 ◦ C for 5 min in beakers. The gel obtained at room temperature
was further kept in refrigerator at 5 ◦ C for 12 h for curing.
2.6. Characterization
FTIR analyses of the ␬-carrageenan samples were carried out
on a Perkin-Elmer FTIR spectrometer instrument (Spectrum GX,
USA) using KBr disc in the range 400–4000 cm−1 . The 1 H NMR was
recorded on a Bruker Avance-II, 500 MHz spectrometer. Rheologi-
cal measurements were performed on an Anton Paar, Physica MCR
301 rheometer USA, using parallel plate PP50/P-PTD200 geometry
(49.971 mm diameter; 0.75 mm gap). Temperature was maintained
by Anton Paar, Viscotherm VT2 circulating water bath. The dynamic
viscosities were determined varying the shear rate at 25 ◦ C e.g.,
0.1–50 s−1 followed by measurement of steady shear viscosity in
the range of 10–50 s−1 . Frequency sweep was performed to calcu-
late storage modulus, G and the loss (or viscous) modulus, G where
angular frequency was set from 0.05 to 1. Time dependant G and
G measured at amplitude 0.1% and frequency 0.01 Hz by setting 20
measuring points of 2 s duration each in the time frame of 0–200 s.
The time and frequency dependence of the storage and loss moduli
(G and G ) for the gels were measured at 25 ◦ C.
3. Results and discussion
Well ground K. alvarezii powder was soaked separately in ChoCl-
Urea 1:2; ChoCl-EG 1:2 and ChoCl-Gly 1:2 for 10 min at 1:20 (w/w)
932 A.K. Das et al. / Carbohydrate Polymers 136 (2016) 930–935

Fig. 2. Photographic demonstration for the extraction of ␬-carrageenan from Kappaphycus alvarezii using deep eutectic solvents.

Table 1 seen from Fig. 1, ␬-carrageenan is sulphated at C-4 position and

Yield of ␬-carrageenan obtained from Kappaphycus alvarezii using different solvent
normally potassium cation present as counter ion. During the inter-
systems.
action process, the choline cations replace the potassium cations
S. no. Solvent Yield % (±S.D) Viscosity (cP) Sample simply by ion exchange. Such ion exchange was also observed dur-
system/method of solvent at code
ing reaction of ␬-carrageenan with a polymerizable ionic liquid
25 ◦ C
(Prasad & Kadokawa, 2010). Since choline has very high solubility in
1 Choline-Chloride- 37.60 ± 1.20 289.10 A water and perhaps the reason behind the higher extraction ability
Urea 1:2
of the hydrated DESs. Although in the case of hydrated ChoCl-EG,
2 Choline-Chloride- 50.66 ± 1.78 35.49 B
Ethylene glycol 1:2 the hydration has reduced the viscosity by 92% over the original
3 Choline-Chloride- 30.93 ± 0.90 266.60 C but the extraction of the polysaccharide did not improve even got
Glycerol 1:2 marginally lower. So, lower viscosity is not only the case of higher
4 10% Hydrated choline- 53.64 ± 1.25 34.49 D
extraction efficiency. Hence, the interaction of the polysaccharide
Chloride-Urea 1:2
5 10% Hydrated 46.02 ± 2.30 16.50 E with the DES must be playing crucial role in the extraction process.
choline-Chloride- The marginally higher yield in the case of the hydrated DESs in com-
Ethylene glycol 1:2 parison to water may be due to the higher affinity of the charges
6 10% Hydrated choline- 60.25 ± 1.10 52.64 F (due to choline) towards ␬-carrageenan present in the seaweed,
Chloride-Glycerol 1:2
which is absent in water. The electrostatic interactions between
7 Water 46.87 ± 2.00 0.90 G
8 Conventional methoda 36.58 ± 1.90 NA H the solvent and carrageenan in the hydrated DESs as well as their
lower viscosity are perhaps responsible for the observed higher
NA: not applicable.
a
Craigie and Leigh (1978).
yield of ␬-carrageenan. Such interaction was found to be respon-
sible for selective agarose extraction from G. dura, an agarophyte
(Sharma, Chaudhary, et al., 2015; Sharma, Mondal, et al., 2015).
ratio followed by heating at 85 ◦ C for 1 h. The mixture thus obtained The indication of the interaction between the sulphate group of
was centrifuged, which showed formation of two layers as shown ␬-carrageenan and the DESs are also evident from the elemental
in Fig. 2. The isolated bottom layer after several washings with IPA analyses data (supporting Table S1). In Table S1, the % S for the
and vacuum dry yielded ␬-carrageenan. ␬-Carrageenan was also ␬-carrageenan extracted using hydrated ChoCl-Urea 1:2 was the
extracted using 10% hydrated DESs, pure water and by conven- lowest in comparison to the rest of the samples, which showed
tional method for comparison of the physicochemical properties. the removal of some portion of the anion due to the strong inter-
The upper layer upon addition in IPA yielded a white precipi- action with the DES. However the % N was more in the samples
tate (0.5–1% with respect to the DESs), which turned hard upon extracted using DESs or their hydrated counterparts in comparison
drying and the FT-IR of the precipitate showed the presence of to the sample extracted using water as solvent. After detailed eval-
traces of the polysaccharide (supporting Fig. S1). The result con- uation of the elemental analyses data, a plausible mechanism for
firms the presence of majority of the polysaccharide in the bottom the extraction of ␬-carrageenan using different DESs is proposed
layer. as shown in Scheme 1. The calculated %N and %S in the proposed
The yields of ␬-carrageenan (with respect to dry seaweed) structure of A is 4.10 and 4.60%, respectively, while the values were
obtained using all the above solvent systems are depicted in Table 1. 4.77 and 3.36 in B, which nearly matches with the measured values.
It can be observed from Table 1 that, the yield of ␬-carrageenan Similarly in the case of extraction using hydrated ChoCl-Urea 1:2
has increased substantially when hydrated DESs were used for (pH 9.76), the alkaline hydrolysis has removed the sulphate group
extraction in comparison to the one extracted using pure DESs, of ␬-carrageenan and the elemental analysis suggested the struc-
water and conventional method. The yield was highest when 10% ture D. The elemental analyses also suggested the structures of E
hydrated ChoCl-Gly 1:2 was used as solvent for the extraction and and F for the ␬-carrageenan extracted using hydrated ChoCl-EG 1:2
lowest for the ␬-carrageenan extracted using ChoCl-Gly 1:2. Almost and ChoCl-Gly 1:2, respectively.
two fold increases in the yield was observed in the case of hydrated In order to find out quality as well as preservation of chemi-
DESs mediated extractions in comparison to the extractions car- cal structures of ␬-carrageenan extracted using various solvents,
ried out using the pure ones. Extraction of ␬-carrageenan from K. the FT-IR spectra was recorded for all the samples and is shown
alvarezii involve breaking of the seaweed cell walls first followed in Fig. 3 and Fig. S2. In all the ␬-carrageenan samples extracted
by interaction of the polysaccharide with the solvents. As can be using various solvents systems, the characteristic bands between
 A.K. Das et al. / Carbohydrate Polymers 136 (2016) 930–935
Scheme 1. Plausible mechanism showing structure of ␬-carrageenan extracted using different DESs and their hydrated counterparts.
Fig. 3. FT-IR spectra of ␬-carrageenan extracted using different solvent systems.
845 and 848 cm−1 due to d-galactose-4-sulphate was visible. How-
ever the intensity of the band was relatively better in the case of the
samples extracted using hydrated ChoCl-EG 1:2 (E) and pure ChoCl-
gly 1:2 (C). The characteristic bands due to 3,6 anhydrogalactose
was also observed between 924 and 927 cm−1 in all the samples
extracted using various solvents. However the intensity of the band
was very low in the case of ␬-carrageenan extracted using both pure
and hydrated ChoCl-urea 1:2 (A and D), which may be due to the
strong interaction of sulphate of carrageenan with the DESs even-
tually resulting modification in the structure of the polysaccharide
(Scheme 1). The presence of characteristic bands of the polysac-
charide indicates that the solvent systems employed are suitable
to extract the polysaccharide directly from the seaweed.
However, among all the solvent systems, 10% hydrated ChoCl-
EG 1:2 and 10% hydrated ChoCl-Gly 1:2 were emerged as the
superior solvent systems among all the solvents used in this study
for the extraction of the polysaccharide.
1 H NMR was recorded for the ␬-carrageenan extracted using
10% hydrated ChoCl-Gly 1:2 (which yield the highest amount of
␬-carrageenan) and was compared with the one extracted using
933
Fig. 4. Steady shear viscosity of ␬-carrageenan gel prepared from the sample
extracted using various solvent systems.
conventional method (supporting Figs. S3 and S4). The spectra were
same for both the samples with the chemical shifts (␦) of the pro-
tons comparable to the reported values for ␬-carrageenan (Van de
Velde, Knutsen, Usov, Rollema, & Cerezo, 2002).
Since ␬-carrageenan is used mostly in the gel form and hence
detail rheological investigation was carried out to study the nature
of the ␬-carrageenan gels prepared from the samples obtained
using different solvent systems and to compare their rheological
properties with the one extracted using water as solvent and the
sample extracted using conventional method.
All the ␬C gels shown in Fig. 4 have revealed different flow
behaviour with pseudoplastic nature in all the cases. The gel
prepared from ␬C by conventional method (H) had showed lower
degree of flow in comparison to the rest of the samples with
highest flowable property for the sample obtained with hydrated
ChoCl-Gly 1:2. ␬-Carrageenan is known to form strong gels with
KCl. The stiffer behaviour of the ␬C gel prepared using conventional
method and water as solvent in comparison to the ␬C gel extracted
using the DES or their hydrated counterparts indicated some sort
934 A.K. Das et al. / Carbohydrate Polymers 136 (2016) 930–935
Fig. 5. (a): Time dependent viscoelasticity profile of ␬-carrageenan gel prepared
from the samples extracted using various solvent systems. (b) Time dependence
of G and G for ␬-carrageenan gel prepared from ␬-carrageenan extracted using
different solvents.
of hindrance in the gel formation in the ␬C samples extracted
using the DESs. Furthermore the ␬C extracted using hydrated DESs
found to form weaker gels in comparison to those extracted using
their pure counterparts. Perhaps this is due to the presence of
little amount of the DESs in the ␬C (indicated in the elemental
analyses) responsible for the disturbance in the formation of three
dimensional helices during the gel formation.
Time dependent viscoelasticity measurements showed gel like
behaviour with G > > G for all the ␬C gels irrespective of the sol-
vents used for extraction in the entire time period of investigation.
However, the differences in the magnitude of G and G was max-
imum in the case of the ␬C gels extracted using ChoCl-gly 1:2 and
hydrated ChoCl-EG 1:2 in comparison to the rest of the samples. It
should be noted that, the difference between G and G was mini-
mum for the ␬C samples extracted using conventional method or
by water (Fig. 5a and b). From the flow behaviour and viscoelas-
ticity profile it can be inferred that, although the ␬C gels obtained
in the presence of DES are less stiff in nature but possess better
viscoelasticity in comparison to their counter parts extracted using
conventional method or water as solvent.
In all the ␬C gels, the crossover of G and G was observed with
increasing frequency indicating frequency dependence signature of
weak gels. However the value of frequency at which they crossover
was different for different gels. It was interesting to note that in all
the cases the loss modulus predominant over the storage modu-
lus in a wide frequency range indicating the presence of more flow
components in the gels making the gels behave like viscous liq-
uid at lower frequencies. The ␬C gels obtained using water and all
Fig. 6. Frequency dependence of G and G for ␬-carrageenan gel prepared from
␬-carrageenan extracted using different solvents.
the hydrated DESs showed liquid viscous behaviour in the angular
frequency range 0.05–1000 s−1 , however the gels obtained by con-
ventional methods as well as using pure DESs as solvents showed
viscous liquid behaviour in the angular frequency range above
100 s−1 . After the crossover of the moduli, the storage modulus
started to become predominant over the loss modulus indicating
elastic nature of the gels at higher frequency range (Fig. 6).
4. Conclusions
Three different deep eutectic solvents prepared by the complex-
ation of choline chloride with urea, ethylene glycol and glycerol
as well as their hydrated counterparts were used for the selec-
tive extraction of ␬-carrageenan from the carrageenophyte K.
alvarezii. The quality of thus obtained ␬-carrageenan was com-
pared with the quality obtained using water as extraction media
as well ␬-carrageenan extracted using well practiced conventional
method. However, traces of the DESs were found in the polysac-
charide samples. Considering the non toxic nature of choCl-EG
and ChoCl-Gly, the impurities may not pose any difficulty in the
application of ␬-carrageenan. However the impurity of DES con-
sisting of Choline chloride and urea may pose some problem. A
plausible mechanism for the modification of chemical structure
of the polysaccharide during the extraction process is proposed.
The detail rheological investigation of the carrageenan gels was
studied. It was inferred from the studies that, the physicochemi-
cal as well as rheological properties of the polysaccharide obtained
using DESs as solvents was at par to the one obtained using
conventional method and was superior in comparison to the ␬-
carrageenan obtained using water as solvent. Considering the
tedious nature of the carrageenan extraction method employed
in conventional extraction method, the DESs can be considered
as suitable alternative solvents for the facile extraction of the
polysaccharide directly from seaweed. However, among the
hydrated and non-hydrated DESs, the hydrated ones were found
to extract ␬-carrageenan more efficiently in comparison to their
non-hydrated counterparts.
Acknowledgments
KP thanks Council of Scientific and Industrial Research, New
Delhi for granting CSIR-Young Scientist Awardees Project (CSIR-
YSP/2011-12), CSIR Network project CSC10130 and OLP0080 for
overall financial support. MS and DM are grateful to UGC and CSIR
for senior research fellowships and to AcSIR for PhD registration.
Analytical Division & Centralized Instrument Facility of the Institute
 A.K. Das et al. / Carbohydrate Polymers 136 (2016) 930–935
is gratefully acknowledged for providing all necessary analytical
facilities for the work. This is CSIR-CSMCRI communication No.
090/2015.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carbpol.2015.09.114.
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