A green and sustainable approach to utilize bio-ionic liquids for the selective precipitation of high purity agarose from an agarophyte extract

Article abstract of DOI:10.1039/c4gc02498b

A few choline based bio-ionic liquids (bio-ILs) were employed for the first time for the selective precipitation of agarose from the hot seaweed extract of Gracilaria dura (an agarophyte) under ambient conditions. The method thus developed is much greener and economical in comparison with the methods widely practiced for agarose production. Among the bio-ILs, choline laurate was found to be the most effective for the isolation of agarose with a lower usage level (4.0%, w/w) with the yield of 14.0 ± 0.5% w/w. Agarose obtained by this process had the desired properties required for molecular biological applications and gel electrophoresis. Furthermore the bio-ILs were recycled and reused for subsequent batches of agarose isolation without compromising the yield and quality of biopolymers.

Full text of DOI:10.1039/c4gc02498b

View Article Online
View Journal
Green
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. Sharma, J. P.
Chaudhary, D. Mondal, R. Meena and K. Prasad, Green Chem., 2015, DOI: 10.1039/C4GC02498B.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/greenchem

Page 1 of 7
Green Chemistry
View Article Online
DOI: 10.1039/C4GC02498B
Green Chemistry
RSCPublishing
ARTICLE
A green and sustainable approach to utilize bio-ionic
liquids for the selective precipitation of high purity
agarose from an agarophyte extract
Cite this: DOI: 10.1039/x0xx00000x
Mukesh Sharma,b,c Jai Prakash Chaudhary,a,c Dibyendu Mondal,b,c Ramavatar
Meena a,c * and Kamalesh Prasad b,c *
Received 00th January 2015,
Accepted 00th February 2015
Few choline based bioꢀionic liquids (bioꢀILs) were employed for the first time for the selective
precipitation of agarose from the hot seaweed extract of Gracilaria dura (an agarophyte) under
ambient conditions. The method thus developed is much “greener” and economical in
comparison to the methods widely practiced for agarose production. Among the bioꢀILs,
choline laurate was found to be the most effective for the isolation of agarose with lower usage
DOI: 10.1039/x0xx00000x
www.rsc.org/
level (4.0%, w/w) with the yield of (14.0
± 0.5) % w/w. Agarose obtained by this process had
the desired properties required for molecular biological applications and gel electrophoresis.
Further the bioꢀILs were recycled and reused for subsequent batches of agarose isolation
without compromising with the yield and quality of biopolymer.
Keywords: Agarose; Gracilaria dura
; bioꢀionic liquid; selective precipitation; gelꢀ
electrophoresis.
various biopolymers8 and as extraction media for
polysaccharides. 9
1. Introduction
Agarose is a hydrophilic linear galactan, composed of repeating
Although we have recently demonstrated the ability of few
synthetic surfactants for the selective precipitation of agarose4
but a more “greener” route for the isolation of the biopolymer
was explored in the present work, where nonꢀtoxic and
biodegradable ionic liquids of bio origin (Fig. 1b) were
employed for the job. Further during the downstream
processing, the bioꢀIL was successfully recovered from the
spent solutions and was reused for the subsequent batches
without compromising with the purity and yield of agarose.
disaccharide units (1,3) linked βꢀDꢀgalactopyranose (
G) and
(1,4) linked αꢀ3,6ꢀanhydroꢀLꢀgalactopyranose (
A
) (Fig. 1a).1 It
forms weak to strong hydrogels depending on its concentration,
which is ideal for diffusion and electroꢀkinetic movement of
biopolymers such as DNA. The quality of agarose mainly
depends on the quantity of sulphate present in it and is the basis
of its commercial grading. Red seaweeds such as Gelidium
spp., Gracilaria spp., Acanthopeltis spp., Ceramium spp.,
Pterocladia spp., and Campylaephora spp. are mainly used for
the production of agar and agarose.2 Native and cultivated
Gracilaria dura (G. dura) of Indian seawater has been recently
reported as a promising bioꢀresource for the preparation of
agarose through alkali treatment and surfactantꢀinduced
selective precipitation processes.3,4 In addition to these, there
are number of methods available in the literature describing
isolation of agar such as repeated freezeꢀthaw, pressure
syneresis, solvent precipitation etc..5 Agarose (Figure 1a),
which is almost charge less is normally obtained by the
purification of agar by chromatographic fractionations.6
Due to unique properties such as negligible vapour pressure,
high boiling point, large electrochemical window, recyclability
etc., ionic liquids (ILs) are much preferred choice for various
applications such as in materials design7, to dissolve and extract
Figure 1: Structure of (a) repeating unit of agarose and (b) choline
based bioꢀionic liquids used in this study.
2. Experimental Section
2.1 Materials
The cultivated Gracilaria dura seaweed was collected from the
south east coast of India, Kruisudai Island (9.28°N; 79.12°E)
This journal is © The Royal Society of Chemistry 2015
Green Chemistry., 2015, 00, 1-3 | 1

Green Chemistry
Page 2 of 7
View Article Online
ARTICLE
DOI: 10.1G03r9e/eCn4GCCh0e2m49i8sBtry
for the extraction of agarose. Harvested seaweed was brought to seawater bittern was used as draw solution for the FO process.
13
the laboratory, air dried and stored in plastic bags before use.
The permeate stream was found to be free from Choline
Choline bicarbonate and agarose (Cat. No. A0576) was laurate (ESI, Fig. S7 NMR of permeate stream showed absence
purchased from Sigma Aldrich, USA. Analytical grade formic of impurities). Formation of white precipitate was observed in
acid, acetic acid, caproic acid, caprylic acid, lauric acid and the concentrated solution obtained after FO upon addition of
isopropyl alcohol (IPA) were purchased from S.D. Fine IPA. The precipitated impurities was filtered through Whatman
Chemicals, Mumbai, India. All chemicals were used as filter paper to obtain purified bioꢀILꢀIPA mixture and after the
received.
removal of IPA, pure IL was obtained and reused for successive
cycles.
2.2 Synthesis of bio-ionic liquids
2.5 Characterizations
All the five bioꢀionic liquids namely choline formate, choline
acetate, choline caproate, choline caprylate, choline laurate Fourier transformꢀinfra red (FTꢀIR) spectra were measured on a
were synthesized following method reported in the PerkinꢀElmer FTꢀIR machine (Spectrum GX, USA) using KBr
a
1
literature.10 In briefly, drop wise addition of corresponding acid pellets in the range 4000ꢀ400 cmꢀ1. H and 13C NMR spectra
to the aqueous choline bicarbonate (80 % solution) in 1:1 molar were recorded on a Bruker AvanceꢀII 500 (Ultra shield)
ratio under stirring at room temperature. The reaction mixture Spectrometer, Switzerland. For the measurements, agarose
thus obtained was refluxed at 100 °C for 24h with continuous samples were dissolved in D2O (40 mg/mL) and the NMR
stirring. The ILs thus prepared were finally washed with ethyl spectra were recorded at 70 °C, using d6ꢀDMSO as internal
acetate and dried under reduced pressure using rotary standard (ca. δ 39.5 ppm). The dynamic light scattering (DLS)
1
evaporator (e.g. 60 °C, 2h) and characterized by H NMR, ESIꢀ measurements were carried out at 25 °C using a Zetasizer Nano
MS, and TGA (ESI Fig. S1ꢀS6, Table S1).
ZS light scattering apparatus (Malvern instrument, UK) at a
scattered angle of 90° with a HeꢀNe laser light (633 nm, 4 Mw).
Appropriately diluted solutions were filtered directly into
quartz cell using a membrane filter of 0.45 µm pore size. Prior
to measurements, the quartz cell was rinsed several times with
2.3 Selective precipitation of agarose
Alkali treated G. dura seaweed (ATS) was prepared following a milliꢀQ water and then filled with the filtered sample solutions.
method described earlier.11,12 Briefly, 10 g of G. dura seaweed Sulphate contents and other inorganic ions were analyzed using
(with 13 % moisture) yielded 3 g of ATS (with 18 % moisture). a PerkinꢀElmer ICPꢀOES Optima 2000DV machine. Viscosities
This ATS was used for the preparation of alkali treated seaweed of agarose solution (0.75% w/v) were carried out on a
extractives (ATSEs) by the partial modification of a process Brookfield DVꢀII+ Pro viscometer at 60 rpm and 80 °C.
reported earlier.11,12 In brief, ATS was autoclaved with water Electrospray ionizationꢀmass spectra (ESIꢀMS) measurements
(1:20 w/v) followed by centrifugation to obtain an aqueous were carried out on a QꢀTOF micro mass spectrometer (USA).
agarose rich seaweed extract (ATSEs). The extracts thus Mass fragmentation patterns were recorded in both ESI positive
obtained were further treated with 2ꢀ10 wt % bioꢀILs (with mode (ESI+) and negative mode (ESIꢀ). Mass spectra were
respect to the aqueous extracts) at 80 °C under constant stirring recorded in the mass range of m/z 50ꢀ500 and data were
to optimise the concentration of ILs required for an efficient processed using Masslynx 4.0 software (Waters corp., UK).
precipitation of agarose. The treated hot seaweed extract was Elemental analyses were done on Elementor, Vario Microcube
gradually cooled to room temperature for 4 h until formation of instrument.
white coloured precipitates of agarose. The precipitate was
recovered by centrifugation whereas the spent IL stream was 3. Result and discussion
utilised for the recovery and reuse of IL. The recovered agarose
after centrifugation was purified by stepwise washing with 50 Five different bioꢀILs namely choline formate, choline acetate,
%, 75 % and 90 % IPA/water mixture respectively and finally choline caproate, choline caprylate and choline laurate were
washed with pure IPA. The powdered agarose product (1.2 g found to have affinity towards agarose present in the hot
with 5 % moisture) was obtained by vacuum drying at 60 °C for seaweed extracts, which resulted eventual selective
2 h. The yields of agarose were expressed as % w/w on the dry precipitation of the biopolymer as depicted photographically in
basis of G. dura seaweed taken.
Fig. 2. However the rate of affinity towards selective binding
with agarose was varied with carbon chain length and followed
2.4 Recovery of bio-ILs
the
order
CH3(CH2)10COOꢀ
>
CH3(CH2)6COOꢀ
>
The aqueous bioꢀIL after the recovery of crude agarose CH3(CH2)4COOꢀ > CH3COOꢀ > HCOOꢀ (Table 1). It was
contained some inorganic salts and other water soluble observed from Table 1 that, the optimum concentration
impurities. Ca. 85 % of the water content was removed from required for the precipitation of agarose was varied with the
the spent liquor by forward osmosis (FO) process reported nature of the bioꢀILs. The optimized concentration required for
earlier.13 Thin film composites (TFC) polyamide membrane and the precipitation of agarose was 8 % w/w for choline formate,
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 3 of 7
Green Chemistry
Green Chemistry
View Article Online
ARTICLE
DOI: 10.1039/C4GC02498B
inferior properties such as gel strength: 650 ± 50 g cmꢀ2, yield:
10 %, sulphate content: 0.41 ± 0.01(Table 2).
while 4 % w/w for choline laurate was adequate for the desired
precipitation. Furthermore, the yield of agarose with 4 % w/w
choline laurate within a short time (4 h) was maximum and was
considered as the best IL for the isolation of agarose.
Table 2: Physicochemical properties of agarose isolated using various bioꢀ
ILs under optimized reaction conditions
b
c
BioꢀIonic liquid
(Cholineꢀ)
Yield
(%)a
Gel
strength
(g cmꢀ2)
GT
MT
Viscosity
(cP) at
80 ºC
Sulphate
(%)
(oC)
(oC)
Formate
Acetate
11 ± 0.5 650 ± 50
11 ± 0.5 800 ± 50
12 ± 0.5 800 ± 50
13 ± 0.5 850 ± 50
14 ± 0.5 1250 ± 50
32 ± 1
33 ± 1
34 ± 1
34 ± 1
34 ± 1
80 ± 1
84 ± 1
83 ± 1
83 ± 1
91 ± 1
6 ± 1
7 ± 1
7 ± 1
8 ± 1
9 ± 1
0.41 ± 0.01
0.36 ± 0.01
0.35 ± 0.01
0.30 ± 0.01
0.21 ± 0.01
Caproate
Caprylate
Laurate
a
Note: Yield of agarose calculated on the dry basis of seaweed with nil
moisture; bGelling Temperature; cMelting temperature. All the measurements
were taken in 0.75 % (w/v) agarose aqueous gel and viscosity was measured
at 60 rpm.
Table 3 lists the comparison of the properties of agarose
obtained using choline laurate and the agarose obtained using
synthetic nonionic surfactant4 as well as a commercially
available agarose. The gel strength of the agarose obtained in
this study was almost comparable to that obtained earlier and
substantially higher than that sold as Sigma agarose (A0576)
[employed for the separation of high molecular weight nucleic
acids]. All the other crucial parameters required for high end
applications were comparable with commercial agarose.
Figure 2: Photographic demonstration of the complete process of
agarose production using bioꢀionic liquids.
For carrying out the laboratory scale experiment, in a typical
batch G. dura (100 g) was treated with aqueous NaOH (10%
w/v) at elevated temperature to obtain alkali modified seaweed,
which was then autoclaved at 120 oC for 2 h followed by
centrifugation. Optimised amounts of bioꢀILs were added to the
clear extract as shown in Table 1 at 80 oC resulting formation of
white precipitates. The white mass thus obtained was washed
with IPA and water as shown in Fig. 2 to isolate pure agarose.
The ILs mixed with water and IPA was finally recovered and
reused for the process. It can be observed that the yield and
purity of agarose was the highest for the one obtained using
choline laurate at 4 % w/w demonstrating maximum affinity of
the IL towards agarose.
Table 3: Comparison of various parameters of agarose obtained using
choline laurate with agarose obtained using synthetic surfactant (Triton
X100) and sigma agarose.
Parameters
G. dura agarose
(present study)
G. dura agarose4
Sigma agarose
(Cat. No. A0576)c
Gel strength (g/cm2)a
1250 ± 50
1200 ± 50
0.20 ± 0.01
0.13 ± 0.01
89 ± 1
≥ 860 ± 50
≤ 0.12
Sulphate content (%; w/w) 0.21 ± 0.01
EEOb
0.14 ± 0.01
Melting temperature (ºC) 91 ± 1
≤ 0.12
86 ± 2
Table 1: Optimization of the concentration of bioꢀILs for the selective
precipitation of agarose from 100 mL 2 wt % alkali treated G. dura seaweed
extracts (ATSEs).
Gelling temperature (ºC)
34 ± 1
35 ± 1
36 ± 1.5
a
b
Note: gel strength taken in 0.75 % (w/v) agarose gel; ≤0.13 considered as
low electroendooesmosis (EEO); cavailable from the product catalogue
Concentration
of bioꢀILs
Name of bioꢀILs (cholineꢀ) / Yield (% w/w)
formate
Acetate
caproate
caprylate
laurate
(% w/w)
20
3
4
5
6
7
8
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
Yield of agarose (wt%)
Gel strength
Sulphate content (wt%)
( )
Kg cmꢀ2
P / 14.0 ± 0.5
P / 14.1 ± 0.5
15
10
5
P / 12.0 ± 0.5 ND
P / 11.0 ± 0.5 P / 11.0 ± 0.5 P / 12.0 ± 0.5 P / 12.2 ± 0.5 ND
P / 10.8 ± 0.5 P / 11.1 ± 0.5 P / 12.1 ± 0.5 ND ND
Note: NP = No precipitation; P = Precipitation; ND = Not done
1.0
0.5
0.0
Table 2 summarizes the properties of agarose obtained above.
The best agarose sample with gel strength: 1250 ± 50 g cmꢀ2,
yield: 13 %, sulphate content: 0.21
±
0.01 and
electroendosmosis (EEO): 0.14 ± 0.01 was obtained, when
choline laurate (4 % w/w) was used for precipitation. The
properties of this agarose were comparable with those available
commercially as well as agarose products obtained through
precipitation using surfactants of synthetic origin.4 On the other
hand the agarose obtained by using choline formate has the
1
2
3
Number of cycle
Figure 3: Effect of recovered choline laurate on quality of agarose isolated in
three subsequent batches.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

Green Chemistry
Page 4 of 7
View Article Online
ARTICLE
DOI: 10.1G03r9e/eCn4GCCh0e2m49i8sBtry
Fig. 3 presents recyclability and reusability profile of choline Result of elemental analysis further revealed that the resulting
laurate for three consecutive cycles of agarose isolation. The agarose is free from residual IL (Table S3).
1
purity of recovered choline laurate was confirmed by H NMR
(ESI Fig. S8). Agarose of desired quality was obtained in the
range of 10ꢀ13 % yield with recovered BioꢀIL. IPA was also
recycled and reused for purification of agarose.
The FTꢀIR spectra of the G. dura and Sigma agarose were
recorded and depicted in Fig. 4. The characteristic IR bands for
G. dura agarose obtained using choline laurate and Sigma
agarose were found identical and are in good agreement with
the previous report.12,14 The main characteristic absorption
bands of agarose were detected at 3430 cmꢀ1 (stretching band of
hydroxyl group), 1072 cmꢀ1 (vibration of CꢀOꢀC bridge of
glycosidic linkage) and 932 cmꢀ1 (vibration of CꢀOꢀC bridge of
3,6ꢀanhydroglactose unit).12,14ꢀ15 G. dura agarose obtained from
choline laurate did not show any signal at 1258 cmꢀ1
(asymmetric stretching of S=O, ester sulphate) and in the range
Figure 5: 13C NMR spectra of G. dura agarose of present study precipitated
using choline laureate.
of 845 cmꢀ1 to 805 cmꢀ1 corresponding to CꢀOꢀS stretching was
indicating the absence of sulphation on the C2, C4 and C6 of the
galactose unit and on C2 of the 3,6ꢀanhydroꢀLꢀgalactose unit.14
The very low sulphate content was also confirmed by ICP data,
the agarose obtained using choline laurate show 0.21 ± 0.01 %
w/w and sigma agarose shows 0.12 ± 0.01 % w/w (Table 3).
In order to check suitability of the agarose isolated using
choline laurate for gel electrophoresis, DNA was eluted in the
gel prepared using this agarose and reference agarose (Fig. 6).
The results revealed that resolutions of DNA were identical in
both the gels confirming identical quality of both the agarose
products. Gel electrophoresis of DNA was carried out several
times during process development and it was consistently
observed that DNA does not degrade in the gel, confirming
absence of DNAse activity. These further confirm applicability
of this agarose for molecular biology applications.
65
60
(b)
55
50
(a)
45
40
CꢀOꢀC
(Glycosidic linkage)
35
CꢀOH
(Hydroxy)
CꢀOꢀC
(3,6ꢀanhydrogalactose)
30
25
4000 3600 3200 2800 2400 2000 1600 1200 800 400
Wavenumber ( )
cmꢀ1
Figure 4: FTꢀIR spectra of Sigma agarose (a) and G. dura agarose obtained
using choline laurate (b).
13CꢀNMR spectra of the agarose are presented in Fig. 5. The
chemical shifts of the 12 carbon atoms of the disaccharide
repeating units of agarose were comparable with those reported
in the literature.12,16ꢀ17 The 13C NMR spectra exhibited twelve
Figure 6: Gel electrophoresis (gel was prepared in 1x TBE buffer) and DNA
was electrophoresed at 50 V in (a) 1.0% reference agarose gel and (b) 1.0%
agarose gel prepared in the present study. Lane 1: 1Kb ladder; Lanes 2ꢀ5:
DNA bands.
peaks at
δ = 62.27, 69.59, 70.15, 70.73, 71.08, 76.16, 76.37,
78.09, 80.90, 83.00, 99.03 and 103.22 ppm for G. dura agarose
obtained from the choline laurate (Fig. 5), which was similar to
the sigma agarose and agarose of G. dura reported in the
literature.12 The carbon resonances in the 13C NMR of the
agarose showed no peak at ca. 59.0 ppm indicating absence of –
OCH3 group. In the 13C NMR spectra of these agarose, a single
peak appearing at ca. 103.1 ppm corresponding to the anomeric
carbons of G and A moieties of the agarose repeating units (Fig.
1a) was observed. Fig. S9a&b shows ESIꢀMS data of the
hydrolyzed agarose of this study, and absence of characteristic
peaks at m/z 104.12 (due to choline) and at m/z 200.75 (due to
laurate) confirm the absence of IL in the resulting agarose.
It was observed from above studies that, addition of all the
choline based ionic liquids resulted selective precipitation of
agarose from the seaweed extracts but the affinity towards
binding with agarose was different for all the bioꢀILs. The
tendency to precipitate agarose followed the trend
CH3(CH2)10COOꢀ > CH3(CH2)6COOꢀ > CH3COOꢀ > HCOOꢀ.
This has indicated role of the chain length of the ILs on the
efficiency of precipitation. Further, the seaweed extracts
contains many other impurities and salts along with agarose.
The impurities also contain lipids and proteins i.e., lesser water
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 5 of 7
Green Chemistry
Green Chemistry
View Article Online
ARTICLE
DOI: 10.1039/C4GC02498B
soluble in comparison to agarose. It has been observed that, the system again increased beyond C3 may be due to the formation
choline substituted soaps has better water solubility in of free micelles in the solution.
comparison to their alkali metal counter parts and choline has
the lowest affinity towards carboxylate when compared with
alkali cations such as Na+ and K+ (present in the seaweed
extract along with agarose).18 When choline based ILs were
added in the seaweed extract, the choline cation got replaced by
the mono valent alkali metals and interacted with the lipids
present in the extract leaving Agarose to present predominantly
in the extract as shown in Scheme 1. In the contrary, choline
became free for interaction with other anions present in the
extract. The agarose which was earlier attached with Na+ and
K+ in the extract got attached to choline (choline–agarose
complex) and due to bulky structure it got precipitated out.
Since the Kraft point rises with increasing chain length of
hydrophobic chain resulting increase in its solubility power.
The choline laurate having the lengthiest chain among the bioꢀ
ILs in the study has the maximum solubility in water.19 This
results higher mobility of the choline cation to bind with
agarose for precipitation in comparison to the rest of the ILs.
On the other hand to bind choline cation to agarose, it required
more amounts of choline formate and due to improper
interaction with agarose the yield and quality of agarose was
inferior in comparison to that obtained using choline laurate.
Figure 7: CMC of choline laureate in aqueous solution (a) and in 0.02%
(w/v) agarose aqueous solution (b).
Fig. 8 shows the DLS profiles of choline laurate (64 mM) in
water and in G. dura aqueous extract at 4% (w/v). In water the
IL showed two different hydrodynamic sizes (Rh) of about 1 nm
and 60 nm. The size of 60 nm in water may be ascribed to the
micelle formation since the concentration of the surfactant was
far in excess to cmc.4 When the IL was added into the seaweed
extract, the peak at 1 nm disappeared, while peak at 60 nm
shifted markedly to 195 nm with broaden shape due to
formation of a fine suspension.
Choline laurate
1.0
AgaroseꢀCholine laurate
0.8
0.6
0.4
0.2
0.0
Scheme 1: A plausible mechanism showing selective precipitation of agarose
from seaweed extract.
In addition to the plausible mechanism, it should be noted that
choline laurate has much low Kraft point and hence the
micelles of the ILs might have played a role in agarose
precipitation at room temperature. Critical micelle
concentration (cmc) for choline laurate was found to be about
21.7 mM (Fig. 7a & ESI, Table S1). Choline laurate was added
gradually into 0.02 % (w/v) aqueous agarose solution and the
conductivity profile was monitored. As shown in Fig. 7b the
conductivity of the binary system increased up to critical
aggregation concentration C1 (cac) of choline laurate (the
conductivity was ca. 410 µS/cm). Above C1 the conductivity
increased linearly up to saturation concentration C2 (sc). The
conductivity at C2 (the conductivity was ca. 700 µS/cm)
indicating interaction of agarose with choline laurate below its
cmc. The interaction of choline laurate and agarose is due to the
strong ion–dipole interactions of the choline cation with oxygen
functionalities of agarose, which formed hydrogen bond bridges
to aggregate agarose perhaps responsible for the precipitation.
1n
10n
1ꢁ
10ꢁ
RH /1(0m0n)
Figure 8: DLS profile of bioꢀIL choline laurate (red) and choline laurate with
alkali treated seaweed extracts (ATSE) of G. dura (blue).
4. Conclusions
We reports an ecoꢀfriendly “green” method for the preparation
of agarose by the selective precipitation using bio based ionic
liquids from aqueous seaweed extract. Unlike the time intensive
traditional methods of agarose preparation, the present method
is capable of isolating agarose selectively from seaweed extract
at room temperature. Among the four different bio based ionic
liquids, choline laurate was found to be the most effective one
to bind with agarose, while choline formate was the least
effective. The ionic liquids used in the process could be
recycled and reused for three consecutive batches of agarose
precipitation without compromising with the agarose yield and
quality. The quality of agarose was comparable to that available
commercially and has the desired purity required for molecular
biology applications.
On increasing the concentration of IL in the system beyond C2
,
it began to aggregate again resulting in an increase in
conductivity, which reached a second maximum at critical
micelle concentration (cmc) C3. The enhanced conductivity at
C3 is ca. 980 µS/cm may be due to the saturated interaction of
IL micelles with agarose. The conductivity of the agaroseꢀIL
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

Green Chemistry
Page 6 of 7
View Article Online
ARTICLE
DOI: 10.1G03r9e/eCn4GCCh0e2m49i8sBtry
11 A. K. Siddhanta, R. Meena, K. Prasad, B. K. Ramavat, P. K. Ghosh,
K. Eswaran, S. Thiruppathi and V. A. Mantri, Costꢀeffective process
Acknowledgements
for preparing agarose from Gracilaria spp.
20050267296, 2005.
12 R. Meena, A. K. Siddhanta, K. Prasad, B. K. Ramavat, K. Eswaran,
S. Thiruppathi, M. Ganesan, V. A. Mantri and P. V. S. Rao,
Carbohydrate Polymers, 2007, 69, 179.
13 (a) P. K. Ghosh, D. Mondal, S. K. Nataraj, A. V. R. Reddy and S. C.
Upadhyay, New use of salt bitterns as draw solution in forward
osmosis for energy efficient dewatering and process thereof.
Provisional patent application has been filed. Indian application Noꢀ
0735/DEL/2014 dated 13 March, 2014; (b) D. Mondal, S. K.
Nataraj, A. V. R. Reddy, K. K. Ghara, P. Maiti, S. C. Upadhyay and
P. K. Ghosh, RSC Advances, 2015, DOI: 10.1039/C5RA00617A.
14 F. C. N. Barros, D. C. da Silva, V. G. Sombra, J. S. Maciel, J. P. A.
Feitosa, A. L. P. Freitas and R. C. M. de Paula, Carbohydrate
Polymers, 2013, 92, 598.
, US Pat. No.:
CSIRꢀCSMCRI communication No.: 219/2014. We are
gratefully acknowledged to DST (GAP1086) and CSIR
(CSC0130) for financial support. KP thanks CSIR, New Delhi
for the grant of CSIRꢀYoung Scientist Awardees Project and
overall financial support. MS and DM thanks UGC and CSIR
for senior research fellowships respectively. JPC thanks DST
for project SRF. Analytical and centralized instrument facility
department of the institute is acknowledged for over all
analytical support. We also acknowledge to Dr S. K. Nataraj for
his kind support.
15 (a) J. C. Mollet, A. Rahaoui and Y. Lemoine, Journal of Applied
Phycology, 1998, 10, 59; (b) M. R. S. Melo, J. P. A. Feitosa, A. L.
Notes and references
a
*
Process Design & Engineering Cell, CSIRꢀCentral Salt and Marine
Chemicals Research Institute, G. B. Marg, Bhavnagarꢀ364002 (Gujarat),
P. Freitas and R. C. M. de Paula, Carbohydrate Polymers, 2002, 49,
491.
India. Email: rmeena@csmcri.org; Phone No.: +91ꢀ278ꢀ2567760. Fax: 16 M. Lahaye, W. Yaphe, M. T. P. Viet and C. Rochas, Carbohydrate
+91ꢀ278ꢀ2567562.
Research, 1989, 190, 249.
17 A. I. Usov, S. V. Yarotsky and A. S. Shashkov, Biopolymers, 1980,
19, 977.
b
*
Marine Biotechnology & Ecology Discipline, CSIRꢀCentral Salt and
Marine Chemicals Research Institute, G. B. Marg, Bhavnagarꢀ364002 18 R. Klein, M. Kellermeier, M. Drechsler, D. Touraud and W. Kunz,
(Gujarat), India. Email: kamlesh@csmcri.org; drkamaleshp@gmail.com;
Phone No.: +91ꢀ278ꢀ2567760. Fax: +91ꢀ278ꢀ2567562.
Colloids and surfaces A: Physicochemical and engineering aspects
2009, 338, 129.
,
19 W. C. Preston, J. Phys. Chem., 1948, 52, 84.
c
AcSIRꢀCentral Salt & Marine Chemicals Research Institute, G. B.
Marg, Bhavnagarꢀ364002 (Gujarat), India.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
1
C. Araki, Some recent studies on the polysaccharides of
agarophytes, In Proceeding of the fifth International Seaweed
Symposum, Halifax, 1966, pp 3.
2
3
K. M. Cole and R. G. Sheath, In Biology of the red algae,
Cambridge Univercity Press, 1990, pp 238.
R. Meena, K. Prasad, A. K. Siddhanta, P. K. Ghosh, G. K. Mehta, B.
K. Ramavat, M. S. Ganesan, B. Jha, A. Mishra and M. R. Gandhi,
Process for the preparation of agarose polymer from seaweed
extractive, Pat. No.: 2010109289, 2010.
4
5
R. Meena, J. P. Chaudhary, P. K. Agarwal, P. Maiti, S. Chatterjee,
H. D. Raval, P. Agarwal, A. K. Siddhanta, K. Prasad and P. K.
Ghosh, RSC Advances, 2014, 4, 28093.
G. HernándezꢀCarmona, Y. FreileꢀPelegrín and E. Hernándezꢀ
Garibay, 14ꢀConventional and alternative technologies for the
extraction of algal polysaccharides. In Functional Ingredients from
Algae for Foods and Nutraceuticals (Ed.: H. Dominguez).
Woodhead Publishing Series in Food Science, Technology &
Nutrition, 2013, pp 475.
6
G. Balkan, B. Coban and K. C. Güven, Acta Pharmaceutica
Turcica, 2005, 47, 93.
7
8
T. P. Lodge, Science, 2008, 321, 50.
(a) R. D. Rogers and K. R. Seddon, Science, 2003, 302, 792; (b) K.
Prasad, Y. Kaneko and J. Kadokawa, J Macromol. Biosci., 2009, 9,
376; (c) C. Mukesh, D. Mondal, M. Sharma and K. Prasad, Chem.
Commun., 2013, 49, 6849; (d) D. Mondal, M. Sharma, C. Mukesh,
V. Gupta and K. Prasad, Chem. Commun., 2013, 49, 9606; (e)
Tushar J. Trivedi and Arvind Kumar, Green and Sustainable
Chemistry, 2014, 4, 190; (f) H. Monteillet, M. Workamp, X. Li, B.
Schuur, J. M. Kleijn, F. A. M. Leermakersa and J. Sprakel, Chem.
Commun., 2014, 50, 12197.
9
(a) M. Abe, Y. Fukaya and H. Ohno, Green. Chem., 2010, 12, 1274;
(b) D. Fu, G. Mazza and Y. Tamaki, J. Agric. Food Chem., 2010,
58, 2915.
10 M. Petkovic, J. L. Ferguson, H. Q. N. Gunaratne, R. Ferreira, M. C.
Leitão, K. R. Seddon, L. P. Rebelo and C. S. Pereira, Green Chem.,
2010, 12, 643.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 7 of 7
Green Chemistry
View Article Online
DOI: 10.1039/C4GC02498B
TOC
A green and sustainable approach to utilize bio-ionic liquids for the selective
precipitation of high purity agarose from an agarophyte extract
M. Sharma, J. P. Chaudhary, D. Mondal, R. Meena* and K. Prasad *
Preferential precipitation of agarose was observed upon addition of choline based bio-ionic
liquids in Gracilaria dura seaweed extract. Choline laurate was found to be the most
effective for the work.