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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
a r t i c l e
i n f o
Article history:
Received 18 April 2015
Received in revised form 29 July 2015
Accepted 3 August 2015
Available online 6 August 2015
Keywords:
Monosaccharide separation
Sugar beet pulp
Whole crop biorenery
Hydrolyzed pectin
Centrifugal partition chromatography
Sustainable bio-derived feedstocks
a b s t r a c t
A critical step in the bioprocessing of sustainable biomass feedstocks, such as sugar beet pulp (SBP), is the
isolation of the component sugars from the hydrolysed polysaccharides. This facilitates their subsequent
conversion into higher value chemicals and pharmaceutical intermediates. Separation methodologies such as centrifugal partition chromatography (CPC) offer an alternative to traditional resin-based
chromatographic techniques for multicomponent sugar separations. Highly polar two-phase systems
containing ethanol and aqueous ammonium sulphate are examined here for the separation of monosaccharides present in hydrolysed SBP pectin: l-rhamnose, l-arabinose, d-galactose and d-galacturonic acid.
Dimethyl sulfoxide (DMSO) was selected as an effective phase system modier improving monosaccharide separation. The best phase system identied was ethanol:DMSO:aqueous ammonium sulphate
(300 g L1 ) (0.8:0.1:1.8, v:v:v) which enabled separation of the SBP monosaccharides by CPC (200 mL column) in ascending mode (upper phase as mobile phase) with a mobile phase ow rate of 8 mL min1 . A
mixture containing all four monosaccharides (1.08 g total sugars) in the proportions found in hydrolysed
SBP was separated into three main fractions; a pure l-rhamnose fraction (>90%), a mixed l-arabinose/dgalactose fraction and a pure d-galacturonic acid fraction (>90%). The separation took less than 2 h
demonstrating that CPC is a promising technique for the separation of these sugars with potential for
application within an integrated, whole crop biorenery.
2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Over 8 million tonnes of sugar beet are grown annually in the
UK and sugar beet pulp (SBP) is a signicant waste product from
processing. Currently the SBP is pressed, dried and pelleted in an
energy intensive process before being sold as low value animal feed
[1]. SBP is a rich source of carbohydrates that consists mainly of cellulose (a polymer of d-glucose) and pectin (a co-polymer of various
hexose and pentose sugars) as shown in Fig. 1. Its abundance and
low cost suggests that SBP could provide a sustainable feedstock
for the production of chemical and pharmaceutical intermediates
within the context of a whole crop biorenery [2].
Presented at the 8th International Conference on Countercurrent Chromatography CCC 2014, 2325 July 2014, Uxbridge, United Kingdom.
Corresponding authors.
E-mail addresses: svetlana.ignatova@brunel.ac.uk (S. Ignatova), g.lye@ucl.ac.uk
(G.J. Lye).
http://dx.doi.org/10.1016/j.chroma.2015.08.006
0021-9673/ 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
85
Fig. 1. Overall composition of sugar beet pulp (SBP) (data taken from Micard et al. [5]).
L-arabinose
L-rhamnose
OH
OH
O
OH
OH
OH
CH2 OH
COOH
OH
O
OH
OH
OH
D-galacturonic acid
D-galactose
OH
OH
CH3
OH
OH
O
OH
OH
OH
86
2.1. Reagents
2.3. CPC equipment and operating conditions
The sugars l-arabinose (99%), l-rhamnose (99%), d-galacturonic
acid sodium salt (98%) and d-galactose (99%); and solvents acetonitrile, methanol, n-propanol and n-butanol were purchased
from SigmaAldrich (Gillingham, UK). Absolute ethanol, dimethyl
sulfoxide (DMSO) (99%) and ammonium sulphate (99%) were purchased from Fisher Chemicals (Loughborough, UK). The water used
was deionised in house using a Purite Select Fusion purication
system (Thame, UK).
Table 1
Summary of highly polar phase systems investigated for fractionation of hydrolysed SBP pectin. Values represent volumetric ratios of solvents used in phase system
preparation. Phase systems were formed as described in Section 2.2.
Phase system
EtOH
Sat. AS
2 M AS
Wat
DMSO
ACN
MeOH
PrOH
BuOH
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
3
3
3
0.5
0.5
1
0.8
0.8
0.5
0.5
0.6
0.8
1.2
1.2
1.2
1
1
1
1
1
1
1
1
1
4
5
6
1
1
1
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.3
0.1
0.1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.1
0.2
0.4
1
0.5
0.5
EtOH, ethanol; Sat. AS, saturated ammonium sulphate; 2 M AS, 2 M ammonium sulphate; Wat, water; DMSO, dimethyl sulfoxide; ACN, acetonitrile; MeOH, methanol; PrOH,
n-propanol; BuOH, n-butanol.
0.32
0.38
0.44
0.47
0.44
0.51
0.44
0.53
0.46
0.76
0.43
0.58
0.52
0.57
0.42
19.4
25.3
37
16.0
14.6
18.3
20.3
29.5
29.8
19.9
20.6
28.3
41.4
89.8
31.4
(%
w/
w)
50
)
w/w
25
100
0
50
0.3
0.1
1
0.3
0.4
0.4
0.5
0.4
0.5
0.4
0.5
0.5
0.8
0.4
0.7
75
ol
an
Eth
Wa
ter
(%
LP volume fraction
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
75
50
25
Phase system
Table 3
Partition coefcients (KD LP/UP ) of monosaccharides from hydrolysed SBP pectin. KD
values were determined as described in Section 2.2. Values represent one standard
deviation about the mean (n = 2).
100
75
Table 2
Lower phase (LP) volume fractions and settling times of the highly polar phase systems described in Table 1. Experiments were performed as described in Section 2.2.
Values represent one standard deviation about the mean (n = 3).
Phase system
25
87
100
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
4.3
3.0
2.2
16
7.7
10.0
4.0
2.8
2.8
5.3
4.0
3.7
2.9
1.8
2.6
0.2
0.2
N.D.
2
0.0
0.5
0.2
0.0
0.0
0.1
0.7
0.1
0.1
0.0
0.0
Gal
Rha
GA
5.2 0.1
3.5 N.D.
2.5 0.1
24 0
12 0
15 0
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
2.1 0.1
3.5 0.0
1.3 0.1
1.2 0.0
1.2 0.1
3.9 0.0
2.1 0.1
2.4 N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
1.0 0.0
1.2 0.1
13.0 1.9
8.7 0.9
4.2 N.D.
33 3
33 8
33 2
N.D.
7.2 0.0
7.3 0.1
N.D.
N.D.
N.D.
N.D.
3.6 0.1
7.2 0.2
Ara, l-arabinose; Gal, d-galactose; Rha, l-rhamnose; GA, d-galacturonic acid; N.D.,
not determined.
Concentration (g L-1)
0
0
20
40
60
80
100
120
Time (min)
Fig. 4. CPC separation of a model mixture of l-rhamnose, l-arabinose and
d-galacturonic acid using phase system XV (Table 1): ethanol:DMSO:aqueous
ammonium sulphate (300 g L1 ) (0.8:0.1:1.8, v:v:v). Experimental conditions:
50 g L1 of each solute in a 10.81 mL sample loop operating in ascending mode at a
ow rate of 8 mL min1 and a rotational speed of 1000 rpm at room temperature. ()
l-rhamnose, () l-arabinose, () d-galacturonic acid. Experiments were performed
as described in Section 2.3.
the lowest achieved partition coefcients for all sugars (KD LP/UP
Ara = 1.8). The settling time, however, is excessive at 90 s which
could lead to signicant problems with stationary phase retention
in the CPC column. As SBP is a high volume feedstock, increasing CPC
throughput is considered to be an overriding consideration and so
improvements in the settling time were sought to allow for higher
mobile phase ow rates. An increase in the ethanol proportion to
0.8 (system XV) shortened the settling time to 31 s and increased
the partition coefcient (KD LP/UP Ara = 2.6). Importantly, the separation factor for GA and Ara in system XV (2.8) is higher than both
phase system XIV (2.0) and VIII (2.6). Based on these results, phase
system XV was selected for subsequent CPC separations.
Analysis of the composition of the two phases formed showed
that the ammonium sulphate concentration was 376 g L1 in the
LP and 63 g L1 in the UP. Ethanol concentration in the LP and UP
was 97 g L1 and 333 g L1 , respectively, while DMSO concentration
was 23 g L1 and 56 g L1 , respectively. The saturated ammonium
sulphate and water (1.0:0.8, v:v) mixture gave a total aqueous
ammonium sulphate concentration of approximately 300 g L1 .
This concentration was used for larger scale preparation of phase
10
B
8
Concentration (g L-1)
88
C
4
A
2
0
0
20
40
60
80
100
120
Time (min)
Fig. 5. CPC separation of a synthetic mixture of hydrolysed SBP pectin (43% larabinose, 41% d-galacturonic acid, 5% l-rhamnose, 11% d-galactose) using phase
system XV (Table 1): ethanol:DMSO:aqueous ammonium sulphate (300 g L1 )
(0.8:0.1:1.8, v:v:v). Experimental conditions: 100 g L1 total solute concentration
in a 10.81 mL sample loop operating in ascending mode at a ow rate of 8 mL min1
and a rotational speed of 1000 rpm at room temperature. () l-rhamnose, () larabinose, () d-galactose, () d-galacturonic acid. Analytical chromatograms at
the marked points A, B and C are shown in Fig. 6. Experiments were performed as
described in Section 2.3.
systems for CPC to ensure that the concentration was kept constant
and not reliant on concentration uctuations caused by temperature in saturated ammonium sulphate.
89
Fig. 6. Analytical chromatograms of CPC fractions from the synthetic crude separation at (A) 33 min; (B) 45 min; and (C) 79.8 min; (as shown in Fig. 5). (A) and (B) show the
analysis of neutral sugars. (C) shows the analysis of GA. The two analytical methods used are described in Section 2.4.
90
91