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Purification of oligosaccharides by nanofiltration
Journal of Membrane Science 209 (2002) 321–335
Purification of oligosaccharides by nanofiltration Athanasios K. Goulas, Petros G. Kapasakalidis, Haydn R. Sinclair, Robert A. Rastall, Alistair S. Grandison∗ School of Food Biosciences, The University of Reading, P.O. Box 226, Whiteknights, Reading RG6 6AP, UK Received 16 April 2002; received in revised form 3 June 2002; accepted 25 July 2002
Abstract Nanofiltration (NF) of a model sugar solution and a commercial galacto-oligosaccharide mixture has been studied with a cross-flow filtration unit in an attempt to investigate the significance of the operating parameters and evaluate a possible purification process. The pressure, feed concentration and filtration temperature effects were studied in experiments carried out in full recycle mode of operation. The rejection factors of the sugar components present in the solutions increased with increasing pressure due to increased solvent flux and also compaction of the membranes (e.g. rejection factor increased from 0.10 to 0.42 for fructose), with this effect being greater for the low molecular weight sugars in the solutions. Increasing the total sugar concentration of the feed caused a decrease to the rejections of the sugars (e.g. from 0.58 to 0.43 for glucose). Increasing the filtration temperature caused a decrease to the observed rejections of the low molecular weight sugars of the solutions (e.g. from 0.72 to 0.48 for fructose). Continuous diafiltration (CD) purification using NF-CA-50 membranes (at 25 ◦ C) and DS-5-DL membranes (at 60 ◦ C), gave yield values of 14–18% for the monosaccharide, 59–89% for the disaccharide and 81–98% for the trisaccharide (oligosaccharide), respectively. The study clearly demonstrates the potential of cross-flow nanofiltration in the purification of oligosaccharide from mixtures containing contaminant monosaccharides. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Nanofiltration; Oligosaccharides; Monosaccharides; Diafiltration; Sugar fractionation
1. Introduction Certain oligosaccharides are considered to be functional food ingredients, combining prebiotic properties, beneficial to the human health, and physicochemical properties beneficial in food processing [1]. Prebiotic oligosaccharides, generally consisting of 2–10 linked sugar monomers, are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria ∗ Corresponding author. Tel.: +44-118-931-6724; fax: +44-118-931-0080. E-mail address: [email protected] (A.S. Grandison).
in the colon (i.e. Bifidobacterium sp. and Lactobacillus sp.) [1]. They are produced by enzymatic trans-glycosylation or controlled degradation reactions, in complex synthesis mixtures [2]. Microfiltration and ultrafiltration, are well established separation processes in the biotechnology and fermentation industry, used as a means of purifying oligosaccharides from high molecular weight enzymes and polysaccharides [2–5]. However, these commercial products often contain low molecular weight sugars that do not contribute to the beneficial properties of the higher molecular weight oligosaccharides. Nanofiltration (NF) appears to be a potential industrial scale method for purification and concentration of oligosaccharide mixtures. The potential of
0376-7388/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 0 2 ) 0 0 3 6 2 - 9
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Nomenclature A C Ci Cf CP CD JV MWCO NF R t Vc Vs VP Y
effective membrane area (m2 ) solute concentration in the feed solution (g ml−1 ) solute concentration in the initial feed solution (g ml−1 ) solute concentration in the final feed solution (g ml−1 ) solute concentration in the permeate (g ml−1 ) continuous diafiltration volumetric permeate flux through the NF membrane (l m−2 h−1 ) molecular weight cut-off (Da) nanofiltration observed fractional rejection of solute time (h) cumulative permeate volume during the CD (ml) feed solution volume during the CD (ml) permeate volume (l) percentage yield of a solute during the CD
NF to fractionate oligosaccharide mixtures has not yet been critically evaluated. Whilst there has been some patent activity, very few reports have appeared in peer-reviewed literature. According to Playne and Crittenden [2], short-chain fructo-oligosaccharides have been produced in Japan using loose reverse osmosis membranes, although chromatography is still the principal method. Lopez Leiva and Guzman [6], reported that concentration and some purification of oligosaccharide mixtures is possible using NF membranes as an alternative to more expensive chromatographic techniques. In addition, Matsubara et al. [7] reported partial concentration of oligosaccharides from steamed soybean wastewater using NF membranes. Sarney et al. [8] used NF for the fractionation of human milk oligosaccharides and produced biologically active oligosaccharide mixtures with very little contaminating lactose. The molecular weight cut-off (MWCO) of NF membranes lies in the range 200–1000 Da [9], combining ultrafiltration and reverse osmosis separation properties. However, these boundaries are not precisely
defined and there are literature reports where membranes with MWCO values out of this range are referred to as NF membranes. Due to the heterogeneity of the NF membrane structure and composition, the mass transport and segregation characteristics of this membrane process result from several mechanisms [10]. Mass transport is based on two mechanisms: sieving and charge effects [9]. Charge effects can play a very important role in electrolyte separations, because of the electrostatic forces generated between the membranes and the ions. Some sugars, like pectate oligosaccharides (which are acidic in aqueous solutions), carry an electric charge, which affects their separation properties by NF membranes [11]. However, most sugars are neutral molecules in aqueous solution, whose mass transport through NF membranes is controlled by convection and diffusion [9]. Pontalier et al. [10], reported that diffusive transport of sugars depends on the concentration gradient and remains pressure independent, whereas convective transport increases with pressure. Although literature reports (e.g. Aydogan et al. [12]) have stated that NF separations of sugars, with differences in their molecular size in the range of a few glucosyl units, are not feasible due to poor selectivity, the present study is an attempt to evaluate cross-flow NF as a possible fractionation and purification process for oligosaccharide mixtures. Pressure dependence experiments were first carried out with a model solution containing a mono-, di-, and trisaccharide, at concentrations resembling those of commercial oligosaccharide synthesis mixtures (not in all cases). The significance of the feed concentration and temperature were then studied to optimise the separation. Finally, taking account of previous experiments, continuous diafiltration purification of a model solution, and of a commercial oligosaccharide, was performed. 2. Theory The volumetric flux of permeate was expressed in (l m−2 h−1 ): VP JV = (1) At where VP is the permeate volume, A the membrane effective area, t the time necessary for the VP litres of permeate to be collected.
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The observed rejection for a given solute, based on the concentration determined from the sample analysis, was calculated from the equation: R =1−
CP C
Cf × 100% Ci
CD process, which in the case of this study requires the total sugar concentration to remain fairly constant throughout processing.
(2) 3. Experimental
where CP and C are the concentration of the solute in the permeate and the feed, respectively. The yield values for each individual component throughout the continuous diafiltration, were calculated as the fraction of the original solute concentration remaining in the feed: Y =
323
(3)
where Cf and Ci are the concentrations in the final and initial feed, respectively. Continuous diafiltration (CD) is a membrane process where water, at the appropriate pH and temperature, is added to the feed tank at the same rate as the permeate flux, thus keeping the feed volume constant during processing [3]. Mathematical models found in the literature (e.g. Pontalier et al. [15]), describe mass transfer in nanofiltration separation processes by taking into account the membrane characteristics (e.g. pore size, charge), the feed solute and solvent characteristics (e.g. solution viscosity, solute radius), and also the type of solute flux through the membrane (e.g. diffusive, convective). The purpose of this study was to estimate the extent of the CD purification possible, therefore a simple mathematical analysis of the CD process, as described by Grandison and Lewis [4], was used: when R = 0 for a given solute; and CP = C(1 − R) the concentration of that solute in the feed can be calculated using the following equation: Ci Vc ln = (1 − R) (4) Cf Vs where Vs and Vc are the feed volume and the cumulative permeate volume removed during the course of the process. In the same way, the variation in concentration of a specific solute in the permeate can be monitored from the equation: Ci (1 − R) Vc ln = (1 − R) (5) CP Vs Both Eqs. (4) and (5) are applicable when the rejection rate of a solute remains constant throughout the
3.1. Chemicals Analytical grade purity d(−)-fructose and sucrose were supplied by BDH Laboratory Chemicals (Poole, Dorset, UK), and d(+)-raffinose pentahydrate was supplied by Sigma Chemicals (Poole, Dorset, UK). These were used for the preparation of the model sugar solutions and as standards for HPLC analysis. The commercial oligosaccharide mixture used was a galacto-oligosaccharide syrup (Vivinal® GOS), a gift from Friesland Coberco Dairy Foods (Deventer, The Netherlands), with typical specifications: 73 wt.% dry matter of which, 57 wt.% were galacto-oligosaccharides, 23 wt.% lactose, 19 wt.% glucose and 0.9 wt.% galactose (values provided by the manufacturer). All solutions were prepared in demineralised water. 3.2. Membranes Two flat sheet asymmetric cellulose acetate membranes were used, NF-CA-50 and UF-CA-1, supplied by Intersep Ltd. (Wokingham, UK). According to the specifications supplied by the manufacturer, NF-CA-50 had a nominal 50% rejection of NaCl and UF-CA-1 had a molecular weight cut-off (MWCO) of 1000 Da. Both membranes had a maximum operating pressure of 35 bar and a maximum operating temperature of 30 ◦ C. Three thin film composite membranes, DS-5-DL (Class NF), DS-51-HL (Class NF) and DS-GE (Class UF) were supplied by Osmonics Desal (Le Mee sur Seine, France). According to the manufacturer, DS-5-DL and DS-51-HL completely rejected sucrose and glucose, and gave 96 and 95% rejection of MgSO4 , respectively. The DS-GE had a MWCO of 1000 Da. Further information about the properties and application range of these membranes are summarised in Table 1. Membranes were cut to size and soaked overnight in demineralised water in order to remove any preservative material such as glycerol or azide. To facilitate
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Table 1 Properties of the NF/UF membranes Type
NMWCO (Da)
Rejection NaCl or MgSO4
pH range
Maximum pressure (bar)
Maximum temperature (◦ C)
NF-CA-50 UF-CA-1 DS-5-DL DS-51-HL DS-GE
– 1000 On sucrose and glucose On sucrose and glucose 1000
50% NaCl – 96% MgSO4 95% MgSO4 –
– – 1–11 3–9 2–11
35 35 27 20 27
30 30 90 50 90
fitting of the membranes, a 25 vol.% ethanol solution in demineralised water was used to wet the effective filtration surface of the cells where the membranes were placed. Membranes were conditioned by compressing them to a steady state of compaction (determined by the flux of permeate), with demineralised water as feed, at an intermediate pressure according to their pressure limits (at 25 ◦ C). However, compaction of flat sheet membranes is not permanent, and the membranes exhibit considerable reversibility [13], thus compaction effects were not completely excluded from the results. Membranes were stored in 10 vol.% ethanol solutions at 4 ◦ C after each experimental run with sugar solutions. 3.3. High pressure cell test unit The high pressure cell test unit (Fig. 1) was supplied by Osmonics Desal (Le Mee sur Seine, France) and consisted of a tri-piston pump, a feed tank and two stainless steel high pressure cross-flow cells connected in parallel to the pump outlet. Before the separation of the feed stream from the pump to the two inlets of the NF cells, a pressure gauge was fitted to allow readings of the system pressure. The unit had a maximum operating pressure of 70 bar, a maximum operating temperature of 90 ◦ C, a pH operating range of 1–13, and the pump had a feed flow rate of 220.8 l h−1 . The feed tank (2–5 l capacity) had a vertically positioned baffle to prevent aeration and turbulence. A cooling/heating coil connected to a temperature controlled water bath, was fitted to allow temperature control of the feed. Each high pressure filtration cell consisted of a square stainless steel base, in which a stainless steel porous support disc was centrally positioned. The sup-
port disc had an effective membrane area of 81 cm2 and 0.16 cm thickness. The base underneath the support was designed to prevent back-pressure generation in the permeate side; and thus the trans-membrane pressure was assumed to be the pressure measured in the feed inlet of the cells. In order to maximise turbulence in the feed and thus minimise concentration polarisation, the feed solution inlet on the stainless steel cover was positioned towards the perimeter of the cylindrical space above the membrane, and the concentrate outlet was positioned centrally. An O-ring was also fitted on the perimeter of the top plate in order to ensure adequate sealing of the cell, and a back-pressure regulator on the concentrate outlet was used to control the pressure. Due to the parallel configuration of the two cells, a flow meter was used to ensure that the same pressure was applied to both cells. The flow meter was connected to the concentrate outlets in parallel with the concentrate outlets leading to the feed tank (see Fig. 1). Thus, by adjusting the feed flow rate of the two cells to the same level, the same trans-membrane pressure was applied to both cells. This was carried out by redirecting the flow of the concentrate from one cell to the flow meter, while maintaining the flow of concentrate from the other to the feed tank, and vice versa. In addition to the temperature control coil in the feed tank, the two filtration cells were maintained in a water bath, to ensure adequate control of the filtration temperature. The equilibrium between the water supplied (with a peristaltic pump) and the permeate removed in the CD experiments was achieved: (a) by adjusting the pump flow rate to the flow rate of the permeates measured at set time intervals; (b) by continuously controlling the amount of total cumulative permeate and water added; and (c) by controlling the feed volume in the feed tank.
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Fig. 1. Cross-flow membrane unit: (1–3) temperature controlled water bath, Pump, and cooling or heating coil for temperature control of the feed; (4) feed tank, (5 and 6) volumetric cylinder and pump for water addition, used only during the CD experiments; (7) tri-piston pump; (8) by-pass valve kept closed during the experimental runs; (9) pressure gauge; (10) nanofiltration cells; (11) back-pressure regulator valves; (12) valves used for redirecting the retentate flow of each cell towards the flow meter (one at the time) in order to equalise the pressure applied to the two cells; (13) flow meter. In the figure the permeate streams are recycled to the feed (total recycle mode), but when CD was used, the permeates from the two cells were removed.
3.4. Experimental 3.4.1. Experimental procedure After conditioning of the membranes the water flux was measured at various pressures within the pressure range for each membrane, at 25 ± 1 ◦ C. These initial water flux measurements were compared with measurements carried out under the same conditions after each experiment, to indicate any irreversible fouling or membrane damage. With the sugar solutions used, however, irreversible fouling was never observed. In experiments where the dependence of the separation on a varying condition (e.g. pressure, concentration, temperature) was studied, total recycle mode was used (both concentrates and permeates were recycled to the feed tank) in order to avoid changes in
the feed concentration. Purification of oligosaccharide mixtures was performed with CD. Sugar solutions (always 2 l), were added to the filtration system (after drainage of the existing demineralised water) and circulated for 10 min, before the initial feed concentrations were measured. All permeate concentration and flux values presented in this study are the average value of both filtration cells of the unit. For the total recycle experiments, unless otherwise stated, measurements for a set of filtration conditions were taken as follows: the pressure and temperature were adjusted to the desired levels, and the system was allowed to stabilise for 45 min. Flux measurements were taken, and the feed and permeates were sampled. In experiments where the sugar concentrations or the temperatures were varied, the pressure was
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kept constant, and the system was allowed to stabilise for 45 min (or 1 h for the temperature experiment) under the set conditions before any measurements or samples were taken. In the CD experiments, the desired pressure and temperature were set, and the system was allowed to stabilise for 1 h in total recycle mode. Permeate flux measurements and samples (zero cumulative permeate) were then taken and CD started. During the process at time intervals of 30 or 60 min, samples of the feed and permeate were taken, and the permeate flux and total cumulative permeate collected during this period, were measured. This was carried out by stopping the CD (returning to total recycle mode), in order to perform the measurements and sampling, assuming that the system was at steady state for the conditions measured. 3.4.2. Pressure and temperature dependence experiments In order to investigate the effect of pressure on the separation of sugars, four experiments were carried out using a model solution of approximately 0.07– 0.08 g ml−1 total sugars, at 25±1 ◦ C. The model solution, composed of approximately equal concentrations of fructose, sucrose, and raffinose pentahydrate, was tested at four pressures, varying for each membrane according to their pressure range. Taking in account the information provided in the literature [9], the thin film composite DS-5-DL membrane, which in preliminary experiments had given high rejections of monosaccharides, was used to perform a temperature dependence study. This membrane had a maximum operating temperature of 90 ◦ C and a high porosity, which was reflected in the high permeate fluxes obtained even at low temperatures. The experiments were carried out using a model solution (total sugar 0.055 g ml−1 ), at 13.8 bar pressure, and temperatures 25, 35, 45, 60 ± 1 ◦ C. Additionally, and based on the results from the temperature dependence experiments, the pressure effect on the separation characteristics of this membrane was tested at 25 and 60 ◦ C. 3.4.3. Concentration dependence experiments The effect of feed concentration on the separation characteristics of the sugar components in an oligosaccharide mixture was studied with varying concentrations of the commercial oligosaccharide mixture
Vivinal® GOS. The sugar composition of this oligosaccharide mixture allowed comparisons with the model sugar solution experiments, because it contained glucose and lactose, which have similar molecular weights to fructose and sucrose, respectively, and higher oligosaccharides with molecular sizes in the same range as raffinose. An approximately 0.07–0.08 g ml−1 initial feed solution of the syrup was prepared in demineralised water, placed into the feed tank, and then an intermediate pressure according to the pressure limits of each membrane was applied (at 25 ± 0.5 ◦ C). After each measurement appropriate dilutions of the feed solution with water were carried out in order to obtain a series of lower concentration measurements. All experiments were carried out total recycle mode of operation, in order to ensure constant feed concentration. 3.4.4. Continuous diafiltration The NF-CA-50 and DS-5-DL (at 60 ◦ C) membranes gave the best separation characteristics of monosaccharides from oligosaccharides with respect to the retention of the oligosaccharides, which were the target components in this study. For that reason, CD purification of a model solution and a commercial oligosaccharide mixture, was carried out using these membranes at 13.8 bar pressure, with temperature regulated at 25 ± 0.5 ◦ C for the NF-CA-50, and at 60 ± 0.5 ◦ C for the DS-5-DL. The total cumulative permeate which had to be removed in order to achieve a five-fold purification of these mixtures relative to the monosaccharide was calculated to be in the range of 6.0–6.5 l using Eq. (4), assuming that the rejection of this sugar (taken from the data of the pressure dependence experiments) remained constant throughout the CD process. Further information about the solution concentrations and the total cumulative permeate removed is shown in Table 4. 3.4.5. Sample analysis Sugar solution samples were analysed using an Aminex HPX-87C Ca2+ resin-based column (300 mm × 7.7 mm) supplied by Bio-Rad Laboratories Ltd. (Hertfordshire, UK) and an HPLC analyser coupled to a refractive index detector. The column was maintained at 85 ◦ C and HPLC grade water was used as mobile phase at a flow rate 0.6 ml min−1 .
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Calibration curves were prepared for each sugar separately, and for the galacto-oligosaccharide mixture, calibration curves were prepared from the actual mixture and also from glucose and lactose. The oligosaccharide mixture appeared as three separate peaks, the third and the second corresponding to the retention times of glucose and lactose, respectively. Thus, in the discussion and presentation of the results, the term glucose is used to refer to all the monosaccharides present in the mixture (since it is the predominant one), the term lactose stands for all the disaccharides (since it is the predominant one), and the term “oligos” for all the sugars which had a higher molecular weight than a disaccharide. Each sample was analysed twice and the average was used.
4. Results and discussion 4.1. Effect of pressure and temperature All membranes tested with sugar solutions showed a linear relationship between the permeate flux and the applied pressure (Fig. 2). No irreversible fouling occurred during the experiments since there were no differences in the demineralised water flux measurements before and after each filtration run. With cellulose acetate membranes at the higher pressures, the effect on permeate flux increased to a limiting flux value, presumably due to the resistance in the boundary layer of the membrane [12]. Table 2 summarises the rejection values for the sugars of the model solution. The observed differ-
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ences between the rejection values of the sugars present in the solution indicate the potential of some NF membranes for purifying oligosaccharides from monosaccharides. The rejection values for the three sugars were directly dependent on the total sugar concentration of the feed solution, and increased with pressure due to membrane compaction and also due to increased solvent flux. Compaction reduces the membrane thickness, and that normally would lead to an increased permeate flux. However, pore size reduction, caused also by compaction, is the predominant characteristic upon which the rejection of neutral solutes is dependent (sieving effect), hence causing an overall increase in the rejections observed. Increased pressure also caused the differences between the rejections of the three sugars to decrease, indicating a less effective separation. That is because the effect of pressure on rejection is less marked as the molecular weight of the sugar increases, with respect to the pore size of the membranes used. As pore size decreases, increasing pressure causes the convective flux of solutes to decrease to a much greater extent than the diffusive flux [10]. Thus, with NF membranes, and neutral solutes, the significance of convective solute flux becomes greater as the molecular size of the sugar decreases leading to greater changes in rejection. The increase in the rejection rate at higher pressures caused by the higher solvent flux was observed because the diffusive transport of solutes through the membranes remained the same at higher pressures, thus reducing the solute concentration in the permeate stream. The significance of convective and diffusive solute flux, with respect to the MWCO of the membranes,
Fig. 2. Permeate flux vs. pressure for the model sugar solution at 25 ◦ C during cross-flow NF.
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Table 2 Rejection values for raffinose, sucrose and fructose at a range of applied pressures during cross-flow NF of a model solution of the three sugars
Raffinose Sucrose Fructose
NF-CA-50a /0.0761 g ml−1
b
UF-CA-1/0.0754 g ml−1
6.9c
13.8c
20.7c
27.6c
6.9c
13.8c
20.7c
27.6c
0.80 0.56 0.10
0.90 0.73 0.26
0.93 0.79 0.35
0.95 0.83 0.42
0.71 0.45 0.11
0.80 0.60 0.22
0.84 0.66 0.29
0.87 0.72 0.35
DS-GE/0.0775 g ml−1
Raffinose Sucrose Fructose
Raffinose Sucrose Fructose
DS-51-HL/0.0702 g ml−1
6.9c
13.8c
20.7c
24.1c
6.9c
10.3c
13.8c
17.2c
0.70 0.41 0.08
0.79 0.56 0.18
0.80 0.61 0.24
0.80 0.62 0.25
0.84 0.77 0.51
0.92 0.87 0.67
0.95 0.92 0.76
0.96 0.93 0.78
DS-5-DL (25 ◦ C)/0.0496 g ml−1
DS-5-DL (60 ◦ C)/0.0496 g ml−1
6.9c
13.8c
20.7c
6.9c
13.8c
20.7c
1.00 0.99 0.54
1.00 0.99 0.72
1.00 0.99 0.77
0.99 0.94 0.28
1.00 0.97 0.48
1.00 0.97 0.53
a
Membrane type. Feed concentrations varied due to dilution of the hold-up volume of the system. In the DS-5-DL experiment lower concentration was used due to lack of raffinose. c Pressure in bar. b
becomes more evident by looking to the solute flux values presented in Fig. 3a–e. Solute flux is directly dependent on the pressure when permeation of the solutes is convective controlled, while it becomes independent of the pressure when permeation is diffusive controlled. The linear increase of the fructose flux with pressure, seen with the UF-CA-1, DS-GE, NF-CA-50 (at 25 ◦ C), and DS-5-DL (at temperatures higher than 25 ◦ C) membranes, shows that fructose permeation through these membranes is mainly convective controlled, with compaction leading to a limiting solute flux for the two cellulose acetate membranes. At the same time, diffusion becomes increasingly important for sugar permeation through these membranes as the molecular size of the sugars increases and the MWCO of the membranes decreases. For the lower MWCO membranes DS-51-HL and DS-5-DL (at 25 ◦ C), it appears that diffusion is the main phenomenon controlling solute permeation. Results from the DS-5-DL membrane with varying temperature (Fig. 4a) show that the flux increased with increasing temperature, as expected, and the effect of pressure was the same at low and high tem-
peratures. There are several potential explanations for this behaviour according to Tsuru et al. [9]. (A) The increased thermal energy of the solvent molecules at higher temperatures allows them to overcome the energy barrier generated in the micropores due to the pore wall frictional forces. (B) The effective pore diameter increases with temperature, because the adsorbed water layer on the hydrophilic pore surface becomes thinner. (C) Reduced viscosity at higher temperatures leads to higher fluxes. Increased temperature caused the sugar rejections to decrease (Fig. 4b), but the effect was quite different for the three sugars in the model solution. The monosaccharide (fructose) showed the greatest change in rejection values, followed by the disaccharide (sucrose), which showed a similar, but less marked, change in rejection than fructose (about 10 times smaller). The rejection of raffinose, which was totally rejected at 25 ◦ C, remained constant at elevated temperatures. These results are in agreement with the results reported from Tsuru et al. [9], for the effect of temperature on the transport performance of inorganic NF membranes. Since diffusion of molecules
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Fig. 3. Solute flux vs. pressure during NF of the model solution (total feed concentrations shown in Table 2 for (a–e), and 0.0055 g ml−1 for (f)); (a–d) at 25 ◦ C; (e) at 25 and 60 ◦ C; and (f) solute flux vs. temperature.
through pores is an activated process, because of the hydrodynamic drag forces inside the pores, higher temperatures supply thermal energy, thus increasing the diffusivity of these molecules. This increase in diffusivity, in relation to the higher permeate flux, results in a decrease in solute rejection, with the
fructose rejection being affected more, because of the higher convective flux of permeate. The constant rejection of raffinose shows that the actual pore size of the membrane was not affected by temperature, although the effective pore diameter changed due to the thinner layer of adsorbed water molecules on
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Fig. 4. (a) Permeate flux vs. temperature and, (b) rejection vs. temperature in the cross-flow NF of the model solution with the DS-5-DL membrane at 13.8 bar pressure and 0.055 g ml−1 total feed concentration.
the pore walls. The effect of pressure on rejection and permeate flux was exactly the same at elevated temperatures (Table 2). The difference in rejections between fructose (monosaccharide) and sucrose (disaccharide) is much larger, particularly with the lower MWCO membranes, than the difference between sucrose and raffinose (trisaccharide). This intensifies the argument, that the spatial configuration of the molecules and their conformation in solutions, is very important to their separation characteristics. 4.2. Effect of sugar concentration Concentration polarisation increased, as expected, with increasing sugar concentration at constant pressure and cross-flow velocity (results not shown),
having a marked effect on the permeate flux and increasing in severity with decreasing MWCO. Fig. 5a and b shows the effect of sugar concentration on solute flux with two membranes of different MWCO. Clearly solute flux depends both on the pore size and sugar concentration of each sugar present in the solution. Where the pore size is small with respect to the molecular size of the solutes (e.g. NF-CA-50 membrane), the solute flux of the lower molecular weight solutes is higher than the larger molecules, irrespective of the concentration of the solutes in the solution. However, with higher MWCO membranes (DS-GE) the sucrose appears to have a higher solute flux than glucose since it is present in higher concentration in the oligosaccharide mixture. The rejections of the three sugar components decreased as the total sugar concentration of the solution
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331
Fig. 5. (a and b) Solute flux vs. feed concentration for the DS-GE and NF-CA-50 membranes during NF with varying feed concentrations of the commercial oligosaccharide mixture at 13.8 bar pressure and 25 ◦ C.
increased (Table 3). This result is consistent with the findings of Vellenga and Tragardh [14] who found, with membranes too dense for sugars to permeate, that in combined sugar and salt solutions the salt rejection decreased as the sugar concentration increased. This was explained as a direct effect of the increased concentration polarisation layer viscosity, due to the sugar concentration, which caused back diffusion of the salt to be hindered, resulting in reduced salt rejection. In the same way the total sugar concentration affects the individual sugar rejections causing them to decrease as the total concentration increases. This effect was more intense as the molecular weight of the sugars decreased and the MWCO of the membranes increased, as a direct effect of the convective solute flux. According to this, for each individual sugar component of the mixture, total sugar concentration determines the
rejection values observed, which is also confirmed from the CD experimental results. 4.3. Continuous diafiltration The NF-CA-50 and the DS-5-DL membranes were chosen to perform CD purification using a model solution and an oligosaccharide mixture, because these membranes showed greater differences in rejection values between the monosaccharides and the higher molecular weight sugars. The choice of these membranes was also made with respect to the retention of the oligosaccharide, which was the component of interest in this study. The pressures chosen for the CD purification represented a compromise between permeate flux and rejections observed at each pressure.
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The rejection values for the UF-CA-1 and DS-GE membranes indicate the feasibility of fractionation of an oligosaccharide mixture by additionally removing the disaccharides present, but with substantial loss of the larger oligosaccharides.
CD experiments with the NF-CA-50 membrane were carried out at 25 and at 60 ◦ C with the DS-5-DL. The zero cumulative permeate rejection values were used with Eqs. (4) and (5) to predict the course of the CD purification.
Fig. 6. Model solution CD with the NF-CA-50 membrane at 25 ◦ C at 13.8 bar pressure: (a) feed concentration vs. cumulative permeate; (b) permeate concentration vs. cumulative permeate; and (c) sugar rejections and total sugar feed concentration vs. cumulative permeate.
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During the course of CD, the permeate flux increased slightly (results not shown), due to decreased concentration polarisation resulting from reduction of the feed concentration. Figs. 6a–c and 7a–c show the
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actual and predicted concentrations of the feed and permeate, and the rejection values of the sugars. The rejection of the sugars remained constant throughout the purification (Figs. 6c and 7c), with only a slight
Fig. 7. Vivinal® GOS CD with the DS-5-DL membrane at 60 ◦ C at 13.8 bar pressure: (a) feed concentration vs. cumulative permeate; (b) permeate concentration vs. cumulative permeate; and (c) sugar rejections and total sugar feed concentration vs. cumulative permeate.
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Table 3 Rejection values for oligosaccharides, lactose, and glucose at varying concentrations of Vivinal® GOS during cross-flow NF Feed concentration (g ml−1 )a 0.0160
0.0300
0.0408
0.0557
0.0682
0.0793
0.95 0.86 0.48
0.95 0.85 0.48
0.94 0.83 0.42
0.93 0.83 0.43
0.0296
0.0426
0.0557
0.0666
0.0775
0.93 0.81 0.47
0.92 0.79 0.43
0.91 0.77 0.40
0.91 0.75 0.37
0.89 0.72 0.32
0.0307
0.0435
0.0558
0.0679
0.0780
0.96 0.94 0.81
0.94 0.91 0.72
0.93 0.87 0.61
0.89 0.80 0.46
0.83 0.72 0.36
0.0294
0.0422
0.0535
0.0643
0.0736
0.87 0.67 0.33
0.85 0.62 0.28
0.85 0.61 0.26
0.83 0.58 0.23
0.83 0.58 0.23
NF-CA-50/13.8 barb Oligos 0.96 0.95 Lactose 0.89 0.87 Glucose 0.58 0.53 0.0155 UF-CA-1/13.8 bar Oligos 0.93 Lactose 0.82 Glucose 0.50 0.0159 DS-51-HL/6.9 bar Oligos 0.97 Lactose 0.95 Glucose 0.83 0.0160 DS-GE/13.8 bar Oligos 0.86 Lactose 0.64 Glucose 0.30
a From the total sugars in each solution on average 40.4% was galacto-oligosaccharides as (defined in the Section 1), 40.0% was lactose, and 19.6% was glucose. b The pressures were chosen by taking in account the separation achieved at the pressure dependence experiments and also the permeate flux at each pressure.
increase due to the decrease of the total sugar concentration of the feed. Moreover, the rejection values of the monosaccharide, which is the sugar with the greatest change in concentration, increased only slightly, confirming that the rejection of a sugar in a mixed solution is determined by the total sugar concentration (also indicated by the concentration dependence experiments). As sugar rejection values remained virtually constant, the mathematical analysis applied to predict the concentration in the feed and permeate, using the zero cumulative permeate rejection values observed, displayed an adequate fit to the experimental results (Figs. 6a and b, and 7a and b). This mathematical analysis provides a useful tool in predicting the course of similar CD purifications only in cases where the total feed concentration does not change greatly. However such a prediction will be restricted in cases where high protein content is present in the oligosaccharide synthesis mixture, since proteins have different separation characteristics from sugars. Further modelling of such a process is required to take into account the membrane, solute and solvent characteristics, together with the mass transfer mechanisms of the process. Examination of the solute flux values (Figs. 6a and 7a), which are directly dependent on the concentration of the sugar in the feed solution, allows determination of the extent of purification achievable without significant loss of the oligosaccharide content. This level is
Table 4 Yield values from the continuous diafiltration experiments with the NF-CA-50 and DS-5-DL membrane with the model solution and the commercial oligosaccharide mixture Yield (%)
Raffinose Sucrose Fructose Oligosd Lactose Glucose
NF-CA-50a /13.8 bar Feed 0.077 g ml−1 /TCPc 6562 ml
DS-5-DLb /13.8 bar Feed 0.0551 g ml−1 /TCP 906 ml
81 59 15
98 89 14
Feed 0.0821 g ml−1 /TCP 6388 ml
Feed 0.0791 g ml−1 /TCP 6623 ml
84 62 18
98 89 18
Experiments carried out at 25 ◦ C. Experiments carried out at 60 ◦ C. c Total cumulative permeate removed during the CD. d The term oligos stands for all the galacto-oligosaccharide content of the mixture used. a
b
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where the solute flux of the monosaccharide coincides with the solute flux of the oligosaccharides. The DS-5-DL membrane (at 60 ◦ C) generally performed better than the NF-CA-50, in terms of monosaccharide removal, retention of oligosaccharide (Table 4), and permeate flux.
5. Concluding remarks The separation performance of five NF membranes in a cross-flow system with a model solution containing a mono-, di-, and trisaccharide, and a commercial galacto-oligosaccharide mixture, provided valuable information about their potential, and limitations for purification or fractionation of these sugar mixtures. The following conclusions can be made: 1. CD purification of oligosaccharide mixtures gave satisfactory yield values (e.g. 98% for the oligosaccharide; 18% for glucose). 2. Increasing pressure decreases sugar rejections, due to compaction of the membranes and increased solvent flux, with the low molecular weight sugars being more affected. Thus, the optimum process for a separation process should be a compromise between the solute rejections and the permeate fluxes for a given membrane. 3. Increased temperature causes rejections to decrease, due to higher diffusivity and also higher convective flow of the sugar solutes. The effective pore diameter increases as the water layer adsorbed on the pore walls becomes thinner, affecting the convective flow of solutes and thus having a greater effect with lower molecular weight sugars. However, the actual pore diameter remains unchanged and hence the sieving effect also becomes the major parameter determining the separation of sugars elevated temperatures. 4. Concentration of the feed solutions also affects the sugar rejections. Due to the higher viscosity generated in the concentration polarisation layer at the membrane surface, the back diffusion of solutes is hindered and the solute rejections decrease. Also the total sugar concentration is very important, determining the sugar rejections irrespective of the individual sugar concentration in the feed solution.
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Acknowledgements The financial support of the State Scholarship Foundation of Greece was greatly appreciated. References [1] G.R. Gibson, M.B. Roberfroid, Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics, J. Nutr. Crit. Rev. 125 (1995) 1401. [2] M.J. Playne, R. Crittenden, Commercially available oligosaccharide, Bull. Int. Dairy Fed. 313 (1996) 10. [3] M. Cheryan, Ultrafiltration and Microfiltration Handbook, Technomic Publishing Co. Inc., Lancaster, 1998. [4] A.S. Grandison, M.J. Lewis, Separation processes in the food and biotechnology industries, Woodhead Publishing Ltd., Cambridge, 1996. [5] K.C. Mountzouris, S.G. Gilmour, A.S. Grandison, R.A. Rastall, Modelling of oligodextran production in an ultrafiltration stirred-cell membrane reactor, Enzyme Microb. Technol. 24 (1999) 24. [6] M.H. Lopez Leiva, M. Guzman, Formation of oligosaccharides during enzymatic hydrolysis of milk whey permeates, Proc. Biochem. 30 (1995) 757. [7] Y. Matsubara, K. Iwasaki, M. Nakajima, H. Nabetani, S. Nakao, Recovery of oligosaccharides from streamed soybean waste water in tofu processing by reverse osmosis and nanofiltration membranes, Biosci. Biotech. Biochem. 60 (1996) 420. [8] D.B. Sarney, C. Hale, G. Frankel, E.N. Vulfson, A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration, Biotechnol. Bioeng. 69 (2000) 461. [9] T. Tsuru, S. Izumi, T. Yoshioka, M. Asaeda, Temperature effect on transport performance by inorganic nanofiltration membranes, AIChE J. 46 (2000) 565. [10] P.-Y. Pontalier, A. Ismail, M. Ghoul, Mechanism for the selective rejection of solutes in nanofiltration membranes, Sep. Purif. Technol. 12 (1997) 175. [11] K.-I. Iwasaki, Y. Matsubara, Purification of pectate oligosaccharides showing root-growth-promoting activity in lettuce using ultrafiltration and nanofiltration membranes, J. Biosci. Bioeng. 89 (2000) 495. [12] N. Aydogan, T. Gurkan, L. Yilmaz, Effect of operating parameters on the separation of sugars by nanofiltration, Sep. Sci. Technol. 33 (1998) 1767. [13] J.A. Whu, B.C. Baltzis, K.K. Sirkar, Nanofiltration studies of larger organic microsolutes in methanol solutions, J. Membr. Sci. 170 (2000) 159. [14] E. Vellenga, G. Tragardh, Nanofiltration of combined salt and sugar solutions: coupling between retentions, Desalination 120 (1998) 211. [15] P.-Y. Pontalier, A. Ismail, M. Ghoul, Specific model for nanofiltration, J. Food Eng. 40 (1999) 145.
Purification of oligosaccharides by nanofiltration Athanasios K. Goulas, Petros G. Kapasakalidis, Haydn R. Sinclair, Robert A. Rastall, Alistair S. Grandison∗ School of Food Biosciences, The University of Reading, P.O. Box 226, Whiteknights, Reading RG6 6AP, UK Received 16 April 2002; received in revised form 3 June 2002; accepted 25 July 2002
Abstract Nanofiltration (NF) of a model sugar solution and a commercial galacto-oligosaccharide mixture has been studied with a cross-flow filtration unit in an attempt to investigate the significance of the operating parameters and evaluate a possible purification process. The pressure, feed concentration and filtration temperature effects were studied in experiments carried out in full recycle mode of operation. The rejection factors of the sugar components present in the solutions increased with increasing pressure due to increased solvent flux and also compaction of the membranes (e.g. rejection factor increased from 0.10 to 0.42 for fructose), with this effect being greater for the low molecular weight sugars in the solutions. Increasing the total sugar concentration of the feed caused a decrease to the rejections of the sugars (e.g. from 0.58 to 0.43 for glucose). Increasing the filtration temperature caused a decrease to the observed rejections of the low molecular weight sugars of the solutions (e.g. from 0.72 to 0.48 for fructose). Continuous diafiltration (CD) purification using NF-CA-50 membranes (at 25 ◦ C) and DS-5-DL membranes (at 60 ◦ C), gave yield values of 14–18% for the monosaccharide, 59–89% for the disaccharide and 81–98% for the trisaccharide (oligosaccharide), respectively. The study clearly demonstrates the potential of cross-flow nanofiltration in the purification of oligosaccharide from mixtures containing contaminant monosaccharides. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Nanofiltration; Oligosaccharides; Monosaccharides; Diafiltration; Sugar fractionation
1. Introduction Certain oligosaccharides are considered to be functional food ingredients, combining prebiotic properties, beneficial to the human health, and physicochemical properties beneficial in food processing [1]. Prebiotic oligosaccharides, generally consisting of 2–10 linked sugar monomers, are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria ∗ Corresponding author. Tel.: +44-118-931-6724; fax: +44-118-931-0080. E-mail address: [email protected] (A.S. Grandison).
in the colon (i.e. Bifidobacterium sp. and Lactobacillus sp.) [1]. They are produced by enzymatic trans-glycosylation or controlled degradation reactions, in complex synthesis mixtures [2]. Microfiltration and ultrafiltration, are well established separation processes in the biotechnology and fermentation industry, used as a means of purifying oligosaccharides from high molecular weight enzymes and polysaccharides [2–5]. However, these commercial products often contain low molecular weight sugars that do not contribute to the beneficial properties of the higher molecular weight oligosaccharides. Nanofiltration (NF) appears to be a potential industrial scale method for purification and concentration of oligosaccharide mixtures. The potential of
0376-7388/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 0 2 ) 0 0 3 6 2 - 9
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Nomenclature A C Ci Cf CP CD JV MWCO NF R t Vc Vs VP Y
effective membrane area (m2 ) solute concentration in the feed solution (g ml−1 ) solute concentration in the initial feed solution (g ml−1 ) solute concentration in the final feed solution (g ml−1 ) solute concentration in the permeate (g ml−1 ) continuous diafiltration volumetric permeate flux through the NF membrane (l m−2 h−1 ) molecular weight cut-off (Da) nanofiltration observed fractional rejection of solute time (h) cumulative permeate volume during the CD (ml) feed solution volume during the CD (ml) permeate volume (l) percentage yield of a solute during the CD
NF to fractionate oligosaccharide mixtures has not yet been critically evaluated. Whilst there has been some patent activity, very few reports have appeared in peer-reviewed literature. According to Playne and Crittenden [2], short-chain fructo-oligosaccharides have been produced in Japan using loose reverse osmosis membranes, although chromatography is still the principal method. Lopez Leiva and Guzman [6], reported that concentration and some purification of oligosaccharide mixtures is possible using NF membranes as an alternative to more expensive chromatographic techniques. In addition, Matsubara et al. [7] reported partial concentration of oligosaccharides from steamed soybean wastewater using NF membranes. Sarney et al. [8] used NF for the fractionation of human milk oligosaccharides and produced biologically active oligosaccharide mixtures with very little contaminating lactose. The molecular weight cut-off (MWCO) of NF membranes lies in the range 200–1000 Da [9], combining ultrafiltration and reverse osmosis separation properties. However, these boundaries are not precisely
defined and there are literature reports where membranes with MWCO values out of this range are referred to as NF membranes. Due to the heterogeneity of the NF membrane structure and composition, the mass transport and segregation characteristics of this membrane process result from several mechanisms [10]. Mass transport is based on two mechanisms: sieving and charge effects [9]. Charge effects can play a very important role in electrolyte separations, because of the electrostatic forces generated between the membranes and the ions. Some sugars, like pectate oligosaccharides (which are acidic in aqueous solutions), carry an electric charge, which affects their separation properties by NF membranes [11]. However, most sugars are neutral molecules in aqueous solution, whose mass transport through NF membranes is controlled by convection and diffusion [9]. Pontalier et al. [10], reported that diffusive transport of sugars depends on the concentration gradient and remains pressure independent, whereas convective transport increases with pressure. Although literature reports (e.g. Aydogan et al. [12]) have stated that NF separations of sugars, with differences in their molecular size in the range of a few glucosyl units, are not feasible due to poor selectivity, the present study is an attempt to evaluate cross-flow NF as a possible fractionation and purification process for oligosaccharide mixtures. Pressure dependence experiments were first carried out with a model solution containing a mono-, di-, and trisaccharide, at concentrations resembling those of commercial oligosaccharide synthesis mixtures (not in all cases). The significance of the feed concentration and temperature were then studied to optimise the separation. Finally, taking account of previous experiments, continuous diafiltration purification of a model solution, and of a commercial oligosaccharide, was performed. 2. Theory The volumetric flux of permeate was expressed in (l m−2 h−1 ): VP JV = (1) At where VP is the permeate volume, A the membrane effective area, t the time necessary for the VP litres of permeate to be collected.
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The observed rejection for a given solute, based on the concentration determined from the sample analysis, was calculated from the equation: R =1−
CP C
Cf × 100% Ci
CD process, which in the case of this study requires the total sugar concentration to remain fairly constant throughout processing.
(2) 3. Experimental
where CP and C are the concentration of the solute in the permeate and the feed, respectively. The yield values for each individual component throughout the continuous diafiltration, were calculated as the fraction of the original solute concentration remaining in the feed: Y =
323
(3)
where Cf and Ci are the concentrations in the final and initial feed, respectively. Continuous diafiltration (CD) is a membrane process where water, at the appropriate pH and temperature, is added to the feed tank at the same rate as the permeate flux, thus keeping the feed volume constant during processing [3]. Mathematical models found in the literature (e.g. Pontalier et al. [15]), describe mass transfer in nanofiltration separation processes by taking into account the membrane characteristics (e.g. pore size, charge), the feed solute and solvent characteristics (e.g. solution viscosity, solute radius), and also the type of solute flux through the membrane (e.g. diffusive, convective). The purpose of this study was to estimate the extent of the CD purification possible, therefore a simple mathematical analysis of the CD process, as described by Grandison and Lewis [4], was used: when R = 0 for a given solute; and CP = C(1 − R) the concentration of that solute in the feed can be calculated using the following equation: Ci Vc ln = (1 − R) (4) Cf Vs where Vs and Vc are the feed volume and the cumulative permeate volume removed during the course of the process. In the same way, the variation in concentration of a specific solute in the permeate can be monitored from the equation: Ci (1 − R) Vc ln = (1 − R) (5) CP Vs Both Eqs. (4) and (5) are applicable when the rejection rate of a solute remains constant throughout the
3.1. Chemicals Analytical grade purity d(−)-fructose and sucrose were supplied by BDH Laboratory Chemicals (Poole, Dorset, UK), and d(+)-raffinose pentahydrate was supplied by Sigma Chemicals (Poole, Dorset, UK). These were used for the preparation of the model sugar solutions and as standards for HPLC analysis. The commercial oligosaccharide mixture used was a galacto-oligosaccharide syrup (Vivinal® GOS), a gift from Friesland Coberco Dairy Foods (Deventer, The Netherlands), with typical specifications: 73 wt.% dry matter of which, 57 wt.% were galacto-oligosaccharides, 23 wt.% lactose, 19 wt.% glucose and 0.9 wt.% galactose (values provided by the manufacturer). All solutions were prepared in demineralised water. 3.2. Membranes Two flat sheet asymmetric cellulose acetate membranes were used, NF-CA-50 and UF-CA-1, supplied by Intersep Ltd. (Wokingham, UK). According to the specifications supplied by the manufacturer, NF-CA-50 had a nominal 50% rejection of NaCl and UF-CA-1 had a molecular weight cut-off (MWCO) of 1000 Da. Both membranes had a maximum operating pressure of 35 bar and a maximum operating temperature of 30 ◦ C. Three thin film composite membranes, DS-5-DL (Class NF), DS-51-HL (Class NF) and DS-GE (Class UF) were supplied by Osmonics Desal (Le Mee sur Seine, France). According to the manufacturer, DS-5-DL and DS-51-HL completely rejected sucrose and glucose, and gave 96 and 95% rejection of MgSO4 , respectively. The DS-GE had a MWCO of 1000 Da. Further information about the properties and application range of these membranes are summarised in Table 1. Membranes were cut to size and soaked overnight in demineralised water in order to remove any preservative material such as glycerol or azide. To facilitate
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Table 1 Properties of the NF/UF membranes Type
NMWCO (Da)
Rejection NaCl or MgSO4
pH range
Maximum pressure (bar)
Maximum temperature (◦ C)
NF-CA-50 UF-CA-1 DS-5-DL DS-51-HL DS-GE
– 1000 On sucrose and glucose On sucrose and glucose 1000
50% NaCl – 96% MgSO4 95% MgSO4 –
– – 1–11 3–9 2–11
35 35 27 20 27
30 30 90 50 90
fitting of the membranes, a 25 vol.% ethanol solution in demineralised water was used to wet the effective filtration surface of the cells where the membranes were placed. Membranes were conditioned by compressing them to a steady state of compaction (determined by the flux of permeate), with demineralised water as feed, at an intermediate pressure according to their pressure limits (at 25 ◦ C). However, compaction of flat sheet membranes is not permanent, and the membranes exhibit considerable reversibility [13], thus compaction effects were not completely excluded from the results. Membranes were stored in 10 vol.% ethanol solutions at 4 ◦ C after each experimental run with sugar solutions. 3.3. High pressure cell test unit The high pressure cell test unit (Fig. 1) was supplied by Osmonics Desal (Le Mee sur Seine, France) and consisted of a tri-piston pump, a feed tank and two stainless steel high pressure cross-flow cells connected in parallel to the pump outlet. Before the separation of the feed stream from the pump to the two inlets of the NF cells, a pressure gauge was fitted to allow readings of the system pressure. The unit had a maximum operating pressure of 70 bar, a maximum operating temperature of 90 ◦ C, a pH operating range of 1–13, and the pump had a feed flow rate of 220.8 l h−1 . The feed tank (2–5 l capacity) had a vertically positioned baffle to prevent aeration and turbulence. A cooling/heating coil connected to a temperature controlled water bath, was fitted to allow temperature control of the feed. Each high pressure filtration cell consisted of a square stainless steel base, in which a stainless steel porous support disc was centrally positioned. The sup-
port disc had an effective membrane area of 81 cm2 and 0.16 cm thickness. The base underneath the support was designed to prevent back-pressure generation in the permeate side; and thus the trans-membrane pressure was assumed to be the pressure measured in the feed inlet of the cells. In order to maximise turbulence in the feed and thus minimise concentration polarisation, the feed solution inlet on the stainless steel cover was positioned towards the perimeter of the cylindrical space above the membrane, and the concentrate outlet was positioned centrally. An O-ring was also fitted on the perimeter of the top plate in order to ensure adequate sealing of the cell, and a back-pressure regulator on the concentrate outlet was used to control the pressure. Due to the parallel configuration of the two cells, a flow meter was used to ensure that the same pressure was applied to both cells. The flow meter was connected to the concentrate outlets in parallel with the concentrate outlets leading to the feed tank (see Fig. 1). Thus, by adjusting the feed flow rate of the two cells to the same level, the same trans-membrane pressure was applied to both cells. This was carried out by redirecting the flow of the concentrate from one cell to the flow meter, while maintaining the flow of concentrate from the other to the feed tank, and vice versa. In addition to the temperature control coil in the feed tank, the two filtration cells were maintained in a water bath, to ensure adequate control of the filtration temperature. The equilibrium between the water supplied (with a peristaltic pump) and the permeate removed in the CD experiments was achieved: (a) by adjusting the pump flow rate to the flow rate of the permeates measured at set time intervals; (b) by continuously controlling the amount of total cumulative permeate and water added; and (c) by controlling the feed volume in the feed tank.
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325
Fig. 1. Cross-flow membrane unit: (1–3) temperature controlled water bath, Pump, and cooling or heating coil for temperature control of the feed; (4) feed tank, (5 and 6) volumetric cylinder and pump for water addition, used only during the CD experiments; (7) tri-piston pump; (8) by-pass valve kept closed during the experimental runs; (9) pressure gauge; (10) nanofiltration cells; (11) back-pressure regulator valves; (12) valves used for redirecting the retentate flow of each cell towards the flow meter (one at the time) in order to equalise the pressure applied to the two cells; (13) flow meter. In the figure the permeate streams are recycled to the feed (total recycle mode), but when CD was used, the permeates from the two cells were removed.
3.4. Experimental 3.4.1. Experimental procedure After conditioning of the membranes the water flux was measured at various pressures within the pressure range for each membrane, at 25 ± 1 ◦ C. These initial water flux measurements were compared with measurements carried out under the same conditions after each experiment, to indicate any irreversible fouling or membrane damage. With the sugar solutions used, however, irreversible fouling was never observed. In experiments where the dependence of the separation on a varying condition (e.g. pressure, concentration, temperature) was studied, total recycle mode was used (both concentrates and permeates were recycled to the feed tank) in order to avoid changes in
the feed concentration. Purification of oligosaccharide mixtures was performed with CD. Sugar solutions (always 2 l), were added to the filtration system (after drainage of the existing demineralised water) and circulated for 10 min, before the initial feed concentrations were measured. All permeate concentration and flux values presented in this study are the average value of both filtration cells of the unit. For the total recycle experiments, unless otherwise stated, measurements for a set of filtration conditions were taken as follows: the pressure and temperature were adjusted to the desired levels, and the system was allowed to stabilise for 45 min. Flux measurements were taken, and the feed and permeates were sampled. In experiments where the sugar concentrations or the temperatures were varied, the pressure was
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kept constant, and the system was allowed to stabilise for 45 min (or 1 h for the temperature experiment) under the set conditions before any measurements or samples were taken. In the CD experiments, the desired pressure and temperature were set, and the system was allowed to stabilise for 1 h in total recycle mode. Permeate flux measurements and samples (zero cumulative permeate) were then taken and CD started. During the process at time intervals of 30 or 60 min, samples of the feed and permeate were taken, and the permeate flux and total cumulative permeate collected during this period, were measured. This was carried out by stopping the CD (returning to total recycle mode), in order to perform the measurements and sampling, assuming that the system was at steady state for the conditions measured. 3.4.2. Pressure and temperature dependence experiments In order to investigate the effect of pressure on the separation of sugars, four experiments were carried out using a model solution of approximately 0.07– 0.08 g ml−1 total sugars, at 25±1 ◦ C. The model solution, composed of approximately equal concentrations of fructose, sucrose, and raffinose pentahydrate, was tested at four pressures, varying for each membrane according to their pressure range. Taking in account the information provided in the literature [9], the thin film composite DS-5-DL membrane, which in preliminary experiments had given high rejections of monosaccharides, was used to perform a temperature dependence study. This membrane had a maximum operating temperature of 90 ◦ C and a high porosity, which was reflected in the high permeate fluxes obtained even at low temperatures. The experiments were carried out using a model solution (total sugar 0.055 g ml−1 ), at 13.8 bar pressure, and temperatures 25, 35, 45, 60 ± 1 ◦ C. Additionally, and based on the results from the temperature dependence experiments, the pressure effect on the separation characteristics of this membrane was tested at 25 and 60 ◦ C. 3.4.3. Concentration dependence experiments The effect of feed concentration on the separation characteristics of the sugar components in an oligosaccharide mixture was studied with varying concentrations of the commercial oligosaccharide mixture
Vivinal® GOS. The sugar composition of this oligosaccharide mixture allowed comparisons with the model sugar solution experiments, because it contained glucose and lactose, which have similar molecular weights to fructose and sucrose, respectively, and higher oligosaccharides with molecular sizes in the same range as raffinose. An approximately 0.07–0.08 g ml−1 initial feed solution of the syrup was prepared in demineralised water, placed into the feed tank, and then an intermediate pressure according to the pressure limits of each membrane was applied (at 25 ± 0.5 ◦ C). After each measurement appropriate dilutions of the feed solution with water were carried out in order to obtain a series of lower concentration measurements. All experiments were carried out total recycle mode of operation, in order to ensure constant feed concentration. 3.4.4. Continuous diafiltration The NF-CA-50 and DS-5-DL (at 60 ◦ C) membranes gave the best separation characteristics of monosaccharides from oligosaccharides with respect to the retention of the oligosaccharides, which were the target components in this study. For that reason, CD purification of a model solution and a commercial oligosaccharide mixture, was carried out using these membranes at 13.8 bar pressure, with temperature regulated at 25 ± 0.5 ◦ C for the NF-CA-50, and at 60 ± 0.5 ◦ C for the DS-5-DL. The total cumulative permeate which had to be removed in order to achieve a five-fold purification of these mixtures relative to the monosaccharide was calculated to be in the range of 6.0–6.5 l using Eq. (4), assuming that the rejection of this sugar (taken from the data of the pressure dependence experiments) remained constant throughout the CD process. Further information about the solution concentrations and the total cumulative permeate removed is shown in Table 4. 3.4.5. Sample analysis Sugar solution samples were analysed using an Aminex HPX-87C Ca2+ resin-based column (300 mm × 7.7 mm) supplied by Bio-Rad Laboratories Ltd. (Hertfordshire, UK) and an HPLC analyser coupled to a refractive index detector. The column was maintained at 85 ◦ C and HPLC grade water was used as mobile phase at a flow rate 0.6 ml min−1 .
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Calibration curves were prepared for each sugar separately, and for the galacto-oligosaccharide mixture, calibration curves were prepared from the actual mixture and also from glucose and lactose. The oligosaccharide mixture appeared as three separate peaks, the third and the second corresponding to the retention times of glucose and lactose, respectively. Thus, in the discussion and presentation of the results, the term glucose is used to refer to all the monosaccharides present in the mixture (since it is the predominant one), the term lactose stands for all the disaccharides (since it is the predominant one), and the term “oligos” for all the sugars which had a higher molecular weight than a disaccharide. Each sample was analysed twice and the average was used.
4. Results and discussion 4.1. Effect of pressure and temperature All membranes tested with sugar solutions showed a linear relationship between the permeate flux and the applied pressure (Fig. 2). No irreversible fouling occurred during the experiments since there were no differences in the demineralised water flux measurements before and after each filtration run. With cellulose acetate membranes at the higher pressures, the effect on permeate flux increased to a limiting flux value, presumably due to the resistance in the boundary layer of the membrane [12]. Table 2 summarises the rejection values for the sugars of the model solution. The observed differ-
327
ences between the rejection values of the sugars present in the solution indicate the potential of some NF membranes for purifying oligosaccharides from monosaccharides. The rejection values for the three sugars were directly dependent on the total sugar concentration of the feed solution, and increased with pressure due to membrane compaction and also due to increased solvent flux. Compaction reduces the membrane thickness, and that normally would lead to an increased permeate flux. However, pore size reduction, caused also by compaction, is the predominant characteristic upon which the rejection of neutral solutes is dependent (sieving effect), hence causing an overall increase in the rejections observed. Increased pressure also caused the differences between the rejections of the three sugars to decrease, indicating a less effective separation. That is because the effect of pressure on rejection is less marked as the molecular weight of the sugar increases, with respect to the pore size of the membranes used. As pore size decreases, increasing pressure causes the convective flux of solutes to decrease to a much greater extent than the diffusive flux [10]. Thus, with NF membranes, and neutral solutes, the significance of convective solute flux becomes greater as the molecular size of the sugar decreases leading to greater changes in rejection. The increase in the rejection rate at higher pressures caused by the higher solvent flux was observed because the diffusive transport of solutes through the membranes remained the same at higher pressures, thus reducing the solute concentration in the permeate stream. The significance of convective and diffusive solute flux, with respect to the MWCO of the membranes,
Fig. 2. Permeate flux vs. pressure for the model sugar solution at 25 ◦ C during cross-flow NF.
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Table 2 Rejection values for raffinose, sucrose and fructose at a range of applied pressures during cross-flow NF of a model solution of the three sugars
Raffinose Sucrose Fructose
NF-CA-50a /0.0761 g ml−1
b
UF-CA-1/0.0754 g ml−1
6.9c
13.8c
20.7c
27.6c
6.9c
13.8c
20.7c
27.6c
0.80 0.56 0.10
0.90 0.73 0.26
0.93 0.79 0.35
0.95 0.83 0.42
0.71 0.45 0.11
0.80 0.60 0.22
0.84 0.66 0.29
0.87 0.72 0.35
DS-GE/0.0775 g ml−1
Raffinose Sucrose Fructose
Raffinose Sucrose Fructose
DS-51-HL/0.0702 g ml−1
6.9c
13.8c
20.7c
24.1c
6.9c
10.3c
13.8c
17.2c
0.70 0.41 0.08
0.79 0.56 0.18
0.80 0.61 0.24
0.80 0.62 0.25
0.84 0.77 0.51
0.92 0.87 0.67
0.95 0.92 0.76
0.96 0.93 0.78
DS-5-DL (25 ◦ C)/0.0496 g ml−1
DS-5-DL (60 ◦ C)/0.0496 g ml−1
6.9c
13.8c
20.7c
6.9c
13.8c
20.7c
1.00 0.99 0.54
1.00 0.99 0.72
1.00 0.99 0.77
0.99 0.94 0.28
1.00 0.97 0.48
1.00 0.97 0.53
a
Membrane type. Feed concentrations varied due to dilution of the hold-up volume of the system. In the DS-5-DL experiment lower concentration was used due to lack of raffinose. c Pressure in bar. b
becomes more evident by looking to the solute flux values presented in Fig. 3a–e. Solute flux is directly dependent on the pressure when permeation of the solutes is convective controlled, while it becomes independent of the pressure when permeation is diffusive controlled. The linear increase of the fructose flux with pressure, seen with the UF-CA-1, DS-GE, NF-CA-50 (at 25 ◦ C), and DS-5-DL (at temperatures higher than 25 ◦ C) membranes, shows that fructose permeation through these membranes is mainly convective controlled, with compaction leading to a limiting solute flux for the two cellulose acetate membranes. At the same time, diffusion becomes increasingly important for sugar permeation through these membranes as the molecular size of the sugars increases and the MWCO of the membranes decreases. For the lower MWCO membranes DS-51-HL and DS-5-DL (at 25 ◦ C), it appears that diffusion is the main phenomenon controlling solute permeation. Results from the DS-5-DL membrane with varying temperature (Fig. 4a) show that the flux increased with increasing temperature, as expected, and the effect of pressure was the same at low and high tem-
peratures. There are several potential explanations for this behaviour according to Tsuru et al. [9]. (A) The increased thermal energy of the solvent molecules at higher temperatures allows them to overcome the energy barrier generated in the micropores due to the pore wall frictional forces. (B) The effective pore diameter increases with temperature, because the adsorbed water layer on the hydrophilic pore surface becomes thinner. (C) Reduced viscosity at higher temperatures leads to higher fluxes. Increased temperature caused the sugar rejections to decrease (Fig. 4b), but the effect was quite different for the three sugars in the model solution. The monosaccharide (fructose) showed the greatest change in rejection values, followed by the disaccharide (sucrose), which showed a similar, but less marked, change in rejection than fructose (about 10 times smaller). The rejection of raffinose, which was totally rejected at 25 ◦ C, remained constant at elevated temperatures. These results are in agreement with the results reported from Tsuru et al. [9], for the effect of temperature on the transport performance of inorganic NF membranes. Since diffusion of molecules
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Fig. 3. Solute flux vs. pressure during NF of the model solution (total feed concentrations shown in Table 2 for (a–e), and 0.0055 g ml−1 for (f)); (a–d) at 25 ◦ C; (e) at 25 and 60 ◦ C; and (f) solute flux vs. temperature.
through pores is an activated process, because of the hydrodynamic drag forces inside the pores, higher temperatures supply thermal energy, thus increasing the diffusivity of these molecules. This increase in diffusivity, in relation to the higher permeate flux, results in a decrease in solute rejection, with the
fructose rejection being affected more, because of the higher convective flux of permeate. The constant rejection of raffinose shows that the actual pore size of the membrane was not affected by temperature, although the effective pore diameter changed due to the thinner layer of adsorbed water molecules on
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Fig. 4. (a) Permeate flux vs. temperature and, (b) rejection vs. temperature in the cross-flow NF of the model solution with the DS-5-DL membrane at 13.8 bar pressure and 0.055 g ml−1 total feed concentration.
the pore walls. The effect of pressure on rejection and permeate flux was exactly the same at elevated temperatures (Table 2). The difference in rejections between fructose (monosaccharide) and sucrose (disaccharide) is much larger, particularly with the lower MWCO membranes, than the difference between sucrose and raffinose (trisaccharide). This intensifies the argument, that the spatial configuration of the molecules and their conformation in solutions, is very important to their separation characteristics. 4.2. Effect of sugar concentration Concentration polarisation increased, as expected, with increasing sugar concentration at constant pressure and cross-flow velocity (results not shown),
having a marked effect on the permeate flux and increasing in severity with decreasing MWCO. Fig. 5a and b shows the effect of sugar concentration on solute flux with two membranes of different MWCO. Clearly solute flux depends both on the pore size and sugar concentration of each sugar present in the solution. Where the pore size is small with respect to the molecular size of the solutes (e.g. NF-CA-50 membrane), the solute flux of the lower molecular weight solutes is higher than the larger molecules, irrespective of the concentration of the solutes in the solution. However, with higher MWCO membranes (DS-GE) the sucrose appears to have a higher solute flux than glucose since it is present in higher concentration in the oligosaccharide mixture. The rejections of the three sugar components decreased as the total sugar concentration of the solution
A.K. Goulas et al. / Journal of Membrane Science 209 (2002) 321–335
331
Fig. 5. (a and b) Solute flux vs. feed concentration for the DS-GE and NF-CA-50 membranes during NF with varying feed concentrations of the commercial oligosaccharide mixture at 13.8 bar pressure and 25 ◦ C.
increased (Table 3). This result is consistent with the findings of Vellenga and Tragardh [14] who found, with membranes too dense for sugars to permeate, that in combined sugar and salt solutions the salt rejection decreased as the sugar concentration increased. This was explained as a direct effect of the increased concentration polarisation layer viscosity, due to the sugar concentration, which caused back diffusion of the salt to be hindered, resulting in reduced salt rejection. In the same way the total sugar concentration affects the individual sugar rejections causing them to decrease as the total concentration increases. This effect was more intense as the molecular weight of the sugars decreased and the MWCO of the membranes increased, as a direct effect of the convective solute flux. According to this, for each individual sugar component of the mixture, total sugar concentration determines the
rejection values observed, which is also confirmed from the CD experimental results. 4.3. Continuous diafiltration The NF-CA-50 and the DS-5-DL membranes were chosen to perform CD purification using a model solution and an oligosaccharide mixture, because these membranes showed greater differences in rejection values between the monosaccharides and the higher molecular weight sugars. The choice of these membranes was also made with respect to the retention of the oligosaccharide, which was the component of interest in this study. The pressures chosen for the CD purification represented a compromise between permeate flux and rejections observed at each pressure.
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The rejection values for the UF-CA-1 and DS-GE membranes indicate the feasibility of fractionation of an oligosaccharide mixture by additionally removing the disaccharides present, but with substantial loss of the larger oligosaccharides.
CD experiments with the NF-CA-50 membrane were carried out at 25 and at 60 ◦ C with the DS-5-DL. The zero cumulative permeate rejection values were used with Eqs. (4) and (5) to predict the course of the CD purification.
Fig. 6. Model solution CD with the NF-CA-50 membrane at 25 ◦ C at 13.8 bar pressure: (a) feed concentration vs. cumulative permeate; (b) permeate concentration vs. cumulative permeate; and (c) sugar rejections and total sugar feed concentration vs. cumulative permeate.
A.K. Goulas et al. / Journal of Membrane Science 209 (2002) 321–335
During the course of CD, the permeate flux increased slightly (results not shown), due to decreased concentration polarisation resulting from reduction of the feed concentration. Figs. 6a–c and 7a–c show the
333
actual and predicted concentrations of the feed and permeate, and the rejection values of the sugars. The rejection of the sugars remained constant throughout the purification (Figs. 6c and 7c), with only a slight
Fig. 7. Vivinal® GOS CD with the DS-5-DL membrane at 60 ◦ C at 13.8 bar pressure: (a) feed concentration vs. cumulative permeate; (b) permeate concentration vs. cumulative permeate; and (c) sugar rejections and total sugar feed concentration vs. cumulative permeate.
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Table 3 Rejection values for oligosaccharides, lactose, and glucose at varying concentrations of Vivinal® GOS during cross-flow NF Feed concentration (g ml−1 )a 0.0160
0.0300
0.0408
0.0557
0.0682
0.0793
0.95 0.86 0.48
0.95 0.85 0.48
0.94 0.83 0.42
0.93 0.83 0.43
0.0296
0.0426
0.0557
0.0666
0.0775
0.93 0.81 0.47
0.92 0.79 0.43
0.91 0.77 0.40
0.91 0.75 0.37
0.89 0.72 0.32
0.0307
0.0435
0.0558
0.0679
0.0780
0.96 0.94 0.81
0.94 0.91 0.72
0.93 0.87 0.61
0.89 0.80 0.46
0.83 0.72 0.36
0.0294
0.0422
0.0535
0.0643
0.0736
0.87 0.67 0.33
0.85 0.62 0.28
0.85 0.61 0.26
0.83 0.58 0.23
0.83 0.58 0.23
NF-CA-50/13.8 barb Oligos 0.96 0.95 Lactose 0.89 0.87 Glucose 0.58 0.53 0.0155 UF-CA-1/13.8 bar Oligos 0.93 Lactose 0.82 Glucose 0.50 0.0159 DS-51-HL/6.9 bar Oligos 0.97 Lactose 0.95 Glucose 0.83 0.0160 DS-GE/13.8 bar Oligos 0.86 Lactose 0.64 Glucose 0.30
a From the total sugars in each solution on average 40.4% was galacto-oligosaccharides as (defined in the Section 1), 40.0% was lactose, and 19.6% was glucose. b The pressures were chosen by taking in account the separation achieved at the pressure dependence experiments and also the permeate flux at each pressure.
increase due to the decrease of the total sugar concentration of the feed. Moreover, the rejection values of the monosaccharide, which is the sugar with the greatest change in concentration, increased only slightly, confirming that the rejection of a sugar in a mixed solution is determined by the total sugar concentration (also indicated by the concentration dependence experiments). As sugar rejection values remained virtually constant, the mathematical analysis applied to predict the concentration in the feed and permeate, using the zero cumulative permeate rejection values observed, displayed an adequate fit to the experimental results (Figs. 6a and b, and 7a and b). This mathematical analysis provides a useful tool in predicting the course of similar CD purifications only in cases where the total feed concentration does not change greatly. However such a prediction will be restricted in cases where high protein content is present in the oligosaccharide synthesis mixture, since proteins have different separation characteristics from sugars. Further modelling of such a process is required to take into account the membrane, solute and solvent characteristics, together with the mass transfer mechanisms of the process. Examination of the solute flux values (Figs. 6a and 7a), which are directly dependent on the concentration of the sugar in the feed solution, allows determination of the extent of purification achievable without significant loss of the oligosaccharide content. This level is
Table 4 Yield values from the continuous diafiltration experiments with the NF-CA-50 and DS-5-DL membrane with the model solution and the commercial oligosaccharide mixture Yield (%)
Raffinose Sucrose Fructose Oligosd Lactose Glucose
NF-CA-50a /13.8 bar Feed 0.077 g ml−1 /TCPc 6562 ml
DS-5-DLb /13.8 bar Feed 0.0551 g ml−1 /TCP 906 ml
81 59 15
98 89 14
Feed 0.0821 g ml−1 /TCP 6388 ml
Feed 0.0791 g ml−1 /TCP 6623 ml
84 62 18
98 89 18
Experiments carried out at 25 ◦ C. Experiments carried out at 60 ◦ C. c Total cumulative permeate removed during the CD. d The term oligos stands for all the galacto-oligosaccharide content of the mixture used. a
b
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where the solute flux of the monosaccharide coincides with the solute flux of the oligosaccharides. The DS-5-DL membrane (at 60 ◦ C) generally performed better than the NF-CA-50, in terms of monosaccharide removal, retention of oligosaccharide (Table 4), and permeate flux.
5. Concluding remarks The separation performance of five NF membranes in a cross-flow system with a model solution containing a mono-, di-, and trisaccharide, and a commercial galacto-oligosaccharide mixture, provided valuable information about their potential, and limitations for purification or fractionation of these sugar mixtures. The following conclusions can be made: 1. CD purification of oligosaccharide mixtures gave satisfactory yield values (e.g. 98% for the oligosaccharide; 18% for glucose). 2. Increasing pressure decreases sugar rejections, due to compaction of the membranes and increased solvent flux, with the low molecular weight sugars being more affected. Thus, the optimum process for a separation process should be a compromise between the solute rejections and the permeate fluxes for a given membrane. 3. Increased temperature causes rejections to decrease, due to higher diffusivity and also higher convective flow of the sugar solutes. The effective pore diameter increases as the water layer adsorbed on the pore walls becomes thinner, affecting the convective flow of solutes and thus having a greater effect with lower molecular weight sugars. However, the actual pore diameter remains unchanged and hence the sieving effect also becomes the major parameter determining the separation of sugars elevated temperatures. 4. Concentration of the feed solutions also affects the sugar rejections. Due to the higher viscosity generated in the concentration polarisation layer at the membrane surface, the back diffusion of solutes is hindered and the solute rejections decrease. Also the total sugar concentration is very important, determining the sugar rejections irrespective of the individual sugar concentration in the feed solution.
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Acknowledgements The financial support of the State Scholarship Foundation of Greece was greatly appreciated. References [1] G.R. Gibson, M.B. Roberfroid, Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics, J. Nutr. Crit. Rev. 125 (1995) 1401. [2] M.J. Playne, R. Crittenden, Commercially available oligosaccharide, Bull. Int. Dairy Fed. 313 (1996) 10. [3] M. Cheryan, Ultrafiltration and Microfiltration Handbook, Technomic Publishing Co. Inc., Lancaster, 1998. [4] A.S. Grandison, M.J. Lewis, Separation processes in the food and biotechnology industries, Woodhead Publishing Ltd., Cambridge, 1996. [5] K.C. Mountzouris, S.G. Gilmour, A.S. Grandison, R.A. Rastall, Modelling of oligodextran production in an ultrafiltration stirred-cell membrane reactor, Enzyme Microb. Technol. 24 (1999) 24. [6] M.H. Lopez Leiva, M. Guzman, Formation of oligosaccharides during enzymatic hydrolysis of milk whey permeates, Proc. Biochem. 30 (1995) 757. [7] Y. Matsubara, K. Iwasaki, M. Nakajima, H. Nabetani, S. Nakao, Recovery of oligosaccharides from streamed soybean waste water in tofu processing by reverse osmosis and nanofiltration membranes, Biosci. Biotech. Biochem. 60 (1996) 420. [8] D.B. Sarney, C. Hale, G. Frankel, E.N. Vulfson, A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration, Biotechnol. Bioeng. 69 (2000) 461. [9] T. Tsuru, S. Izumi, T. Yoshioka, M. Asaeda, Temperature effect on transport performance by inorganic nanofiltration membranes, AIChE J. 46 (2000) 565. [10] P.-Y. Pontalier, A. Ismail, M. Ghoul, Mechanism for the selective rejection of solutes in nanofiltration membranes, Sep. Purif. Technol. 12 (1997) 175. [11] K.-I. Iwasaki, Y. Matsubara, Purification of pectate oligosaccharides showing root-growth-promoting activity in lettuce using ultrafiltration and nanofiltration membranes, J. Biosci. Bioeng. 89 (2000) 495. [12] N. Aydogan, T. Gurkan, L. Yilmaz, Effect of operating parameters on the separation of sugars by nanofiltration, Sep. Sci. Technol. 33 (1998) 1767. [13] J.A. Whu, B.C. Baltzis, K.K. Sirkar, Nanofiltration studies of larger organic microsolutes in methanol solutions, J. Membr. Sci. 170 (2000) 159. [14] E. Vellenga, G. Tragardh, Nanofiltration of combined salt and sugar solutions: coupling between retentions, Desalination 120 (1998) 211. [15] P.-Y. Pontalier, A. Ismail, M. Ghoul, Specific model for nanofiltration, J. Food Eng. 40 (1999) 145.