Extraction of lactoferrin with hydrophobic ionic liquids

Separation and Purification Technology 98 (2012) 432–440

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Extraction of lactoferrin with hydrophobic ionic liquids Enrique Alvarez-Guerra ⇑, Angel Irabien Departamento de Ingeniería Química y Química Inorgánica, Universidad de Cantabria, Avenida de los Castros s/n, 39005 Santander, Cantabria, Spain

a r t i c l e

i n f o

Article history: Received 22 March 2012 Received in revised form 16 July 2012 Accepted 4 August 2012 Available online 13 August 2012 Keywords: Lactoferrin Whey Liquid–liquid extraction Ionic liquid

a b s t r a c t Lactoferrin is a high-added value protein that is contained in bovine whey. This work presents the study of lactoferrin extraction with imidazolium-based ionic liquids as a novel separation process that can overcome constraints associated with conventional techniques used to separate and purify this protein (e.g. chromatography). A bulk membrane configuration based on U-shaped tubes is used as an experimental set-up. Lactoferrin extraction efficiencies of up to 20% are achieved. Despite the lower efficiencies obtained with 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide (BmimNTf2), this ionic liquid is preferred to 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6) because the former is more stable and less soluble in water. Low protein concentration (100 mg L1), neutral pH (6.4–8.2) and low ionic strength (0.03 M) are the experimental conditions with highest extraction efficiencies. BmimNTf2 is selective towards lactoferrin with respect to bovine serum albumin (BSA), since the amount of BSA extracted is almost one order of magnitude lower. However, back-extraction of protein from the ionic liquid was not observed. In conclusion, hydrophobic, imidazolium-based ionic liquids are able to extract lactoferrin selectively, which may be due to their interaction with iron atoms of this protein. The distribution coefficient between the organic and aqueous phase depends mainly on the concentration of protein in the organic phase (exponent 2.8), showing a slight influence on the aqueous phase concentration. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Bovine whey is a by-product from cheese manufacture that is considered an industrial wastewater pollutant due to its high biological oxygen demand (BOD) [1]. However, bovine whey contains proteins, such as a-lactalbumin, b-lactoglobulin, bovine serum albumin (BSA) or lactoferrin, which are used in the food industry because of their wide range of chemical, physical and functional properties [2]. These proteins are present in whey in low concentrations, which may vary from 0.1 (lactoferrin) to 2.7 g L1 (b-lactoglobulin), since the main components of whey are water (93.5%) and lactose (4.5–5.0%) [3]. Therefore, these low concentrations make purification of whey proteins by means of traditional separation and purification methods very difficult [4]. For this reason, the separation of proteins from complex mixtures like bovine whey usually represents between 50 and 80% of total production costs [5], which constitute the major bottleneck for the valorization of whey valuable components. Among whey proteins, lactoferrin stands out due to its important biological properties: antimicrobial, anti-inflammatory, anticarcinogenic and immuno-modulatory [6]. Therefore, this protein is used to supplement food (i.e. infant formula, yoghurt, skim milk) and in skin and oral care products [7]. Lactoferrin is a 80 kDa glyco⇑ Corresponding author. Tel.: +34 942 20 67 49; fax: +34 942 20 15 91. E-mail address: [email protected] (E. Alvarez-Guerra). 1383-5866/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2012.08.010

protein which contains up to two iron atoms per molecule, even though its iron saturation does not usually exceed 10% in total [8]. Nowadays, it is obtained with a high degree of purity by means of cation-exchange chromatography, but this technology is limited by its high costs and relatively low yield [9]. Billakanti and Fee [10] obtained higher yields using a cryogel chromatographic column, but this monolith exhibited dynamic binding capacities of more than one magnitude lower than commercial beads. Other technologies, like gel filtration chromatography, immobilized monoclonal antibodies, cation exchange membranes or semi-batch foaming process, are also subject to high processing costs [11]. Pressure-driven membrane processes appear as an interesting alternative to chromatography, but their main disadvantages are their poor selectivity and fouling. In this way, electrically-enhanced membrane filtration has been applied, but the migration of other whey proteins reduces the purity of the obtained lactoferrin [6,9]. As a result, an innovative separation technology which can overcome the previously mentioned constraints is required to obtain lactoferrin. Liquid–liquid extraction seems to have a great potential to isolate the desired proteins, because it presents higher capacity, better selectivity and integration between recovery and purification, with the consequent higher yields, purities and lower costs [12– 14]. The main disadvantage of this technique is related to the use of volatile organic compounds as extracting solvents [15], which are immiscible with aqueous medium and, many of them, toxic. In the last decade, ionic liquids have replaced, in some cases, these

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volatile organic solvents to overcome environmental, operational and efficiency problems associated with the conventional application of liquid–liquid extraction [16–19]. Ionic liquids, formerly known as molten salts, are defined as organic salts composed entirely of ions that are liquids below 100 °C. Ionic liquids preserve the properties of conventional organic solvents (excellent solvation qualities, variable viscosity range, wide liquid temperature range), and they also have other advantages, such as negligible vapor pressure, high thermal and chemical stabilities, nonflammability, highionic conductivity and wide electrochemical potential window [20–24]. In fact, among the applications in which ionic liquids have been used (reaction media in catalysis, electrochemistry, etc.), separation and extraction is one of the most significant [25,26]. Separation of biomolecules by means of liquid–liquid extraction with ionic liquids is carried out in three main ways: direct extraction, ILATPS (ionic liquid-based aqueous two-phase systems) and using additional separation agents besides ionic liquids [27]. ILAPTS are considered the common alternative to extracting biomolecules with ionic liquids [12,28], and they have been used to separate proteins [5,27,29,30], enzymes [31–34], amino acids [18,19,35] and other organic compounds [22,36–40]. This technique, which was proposed by Gutowski et al. [41] in 2003, is based on a mutually incompatible hydrophilic ionic liquid/kosmotropic salt system (though both are miscible with water) so that an ionic liquid-rich aqueous phase and a salt-rich aqueous phase can be formed [19,36,39]. ILATPS provide several advantages: a gentle biocompatible environment, high extraction efficiency, quick phase separation and relatively low viscosity [22,38]. However, its main drawback is the mutual ionic liquid–water solubility, and ion exchange between salt and ionic liquid can complicate the separation procedure and the recyclability of ionic liquid [12,36,39]. In general, proteins are insoluble in ionic liquids or their solubility is very low. Consequently, very few reports concerning this issue can be found in the literature, and direct extraction of proteins with hydrophobic ionic liquids has been considered to be impractical [27,29,32,42], even though direct extraction has been applied by several research groups to smaller organic compounds, like amino acids [14,23,43,44] or antibiotics [17,24]. For this reason, proper extractants or additives have been used to enhance the solubility of biomolecules, like crown ethers [45–47]. In other cases, ionic liquids are functionalized with these crown ethers [48] or affinitydyes [49] to achieve higher extraction efficiencies. There are many approaches to select the ionic liquid used in an extraction process for a specific purpose [50–52]. From a process engineering point of view, direct extraction of proteins with highly hydrophobic ionic liquids should be sought, since it combines the advantages of liquid–liquid extraction and ionic liquids, without the phase-mixing problem associated with ILATPS or the requirement of additional chemicals. In this way, despite the previously mentioned difficulty related to the negligible solubility of proteins in ionic liquids, Cheng et al. have successfully extracted hemoglobin [53] and cytochrome C [54] by means of a non-commercial imidazolium-based ionic liquid: 1-butyl-3-trimethylsilylimidazolium hexafluorophosphate (BtmsimPF6). The extraction is achieved by the interaction/coordination reaction between the iron atoms of these proteins and the cationic imidazole-based moiety of the ionic liquid. The aim of the present work is the study of lactoferrin extraction with hydrophobic ionic liquids. To the best of our knowledge, there is no previous report in which ionic liquids are used to extract this protein. Moreover, works related to selective lactoferrin separation by means of liquid–liquid extraction have not been found. Taking into account the similarities between hemoglobin and lactoferrin (both contain iron atoms and close molecular weights), commercial imidazolium-based ionic liquids are selected

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to carry out the separation. Therefore, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide (BmimNTf2) is used as the extracting phase, replacing the anion PF6 by NTf2, because NTf2 is more hydrophobic [52,55] and it is more stable when it is in contact with water (PF6 may suffer from hydrolysis [56]). A comparison of extraction with both ionic liquids has been carried out. The influence of pH, ionic strength and lactoferrin concentration on extraction efficiency has also been analyzed. Moreover, the selectivity of the process has been studied by means of extraction of another whey protein (BSA), since this protein has a molecular weight very close to lactoferrin, which makes their separation using other techniques more difficult [57]. 2. Experimental 2.1. Materials The bovine lactoferrin (protein: 98.6%) used in this study (COA ID: LF0002-P01-03) was generously provided by The Tatua Cooperative Dairy Company Limited (New Zealand). Its iron saturation is equal to 8%, which was measured by means of a 7500ce inductively coupled plasma mass spectrometer, ICP-MS (Agilent Technologies, Japan). The BSA (protein:P98%, code: A7906) and sodium dodecyl sulfate, SDS (05030), were purchased from Sigma–Aldrich (Germany). The ionic liquid 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide (BmimNTf2) was bought from IoLiTec (Germany), whereas the 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6) was kindly provided by Solchemar (Portugal). The reagents required to prepare buffer solutions were purchased from Panreac Quimica S.A.U. (Spain): potassium hydroxide, KOH (121515), L(+)-lactic acid, LA (121034) and ortho-phosphoric acid, H3PO4 (131032). Buffer solutions at pH 3.2, 6.4 and 8.2 were prepared with H3PO4 and KOH, whereas LA and KOH were required to prepare buffer solution at pH 4.6. The 18.2 MX cm deionized water (Milli-Q, Millipore) was used throughout. 2.2. Methods The study of the protein extraction and back-extraction has been carried out in U-shaped tubes with an inner diameter of 11.5 mm. Ionic liquid acts as a bulk liquid membrane which separates both sides of the tube, as can be seen in Fig. 1. This configuration makes it possible to assess the feasibility of both the protein extraction and back-extraction only considering the influence of ionic liquids and experimental conditions on extraction and avoiding the influence of additional variables (e.g. material support when supported liquid membranes are used) [58]. One side (A) is

Fig. 1. Scheme of a U-shaped tube where the protein extraction is carried out. (A) Aqueous feed phase, containing the protein to be extracted. (B) Ionic liquid (bulk liquid membrane). (C) Aqueous stripping phase.

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filled with the feed solution prepared with the protein under study, whereas the other side (C) contains the stripping solution. The volume of each phase was 7.5 mL. The ionic liquid (B) is continuously stirred at 300 rpm to assure its homogeneity. Samples of 2.5 mL were taken at regular intervals to measure the protein concentration of aqueous solutions (A and C); once they were measured, samples were returned to the U-tube [44]. For this reason, and due to the hydrophobicity of ionic liquids, it was considered that the volume of phases remained constant, which was confirmed experimentally. All the experiments were carried out at room temperature. The protein content of lactoferrin [59,60] or BSA [5,30] in aqueous phases was determined by measuring the absorbance at 280 nm using a Hach Lange DR 5000 UV–Vis spectrofotometer. Despite the very low solubility of both ionic liquids in water, for each experiment another U-shaped tube filled with feed solution without any protein was used as blank in order to be sure that ionic liquids do not interfere in measurement. Protein concentration in the ionic liquid was calculated by mass balance. 3. Results and discussion The performance of extraction is widely measured by means of the extraction efficiency (E) [37–39,53], which represents the percentage of the solute removed from the feed phase. Considering that volumes of each phase were equal to the volume of other phases and remained constant during experiments, E is calculated according to:

E ð%Þ ¼

C A0  C At

min

C A0

100

ð1Þ

where C A0 represents the initial protein concentration in the feed phase (A) and C At jmin is the minimum protein concentration measured in A during experiments. As can be seen, E depends on a single point for each experiment, so this parameter may be very sensitive to the experimental error. For this reason, data were adjusted by means of a pseudo first-order kinetics with respect to driving force:

the difference between protein concentration at certain times, C At , and protein concentration when equilibrium is reached, C A1 . With this first-order kinetics, the following concentration profile in feed phase (A) is obtained:

C A t ¼ r þ ð1  rÞ expðk  tÞ

ð2Þ

where C A t is dimensionless protein concentration, k is the kinetic constant and r is defined as follows:

r¼

C A1

ð3Þ

C A0

In this way, r represents the protein fraction that remains at the feed phase, according to adjusted data. Consequently, (1  r) expressed as a percentage can be identified as E, with the exception that the former is calculated adjusting all the points of experiments, whereas E only depends on a single point. As a previous step to lactoferrin extraction study, two experiments were carried out to select the ionic liquid that should be used in the analysis. The ionic liquids studied are BmimPF6 and BmimNTf2, since the former is the commercial ionic liquid used by Cheng et al. [53] and the latter is characterized by its higher stability when it is in contact with water. For this purpose, lactoferrin extraction was carried out with a feed solution containing 0.1 g L1 of lactoferrin at pH = 6.4 (ionic strength: 0.01 M) and a stripping solution at pH = 4.6 (ionic strength: 0.06 M). Each experiment was replicated. Fig. 2 shows the time evolution of dimensionless lactoferrin concentration, C A t , with two different ionic liquids. As can be seen in Table 1, BmimPF6 leads to an extraction efficiency of 21%, notably higher than the 12% obtained with BmimNTf2. This difference is also observed with the parameter r, since there is a difference of 5% between values of r in both experiments. However, extraction with both ionic liquids is carried out at similar rates, since no significant difference is observed between the kinetic constants. This behavior can be explained considering that BmimNTf2 is more hydrophobic than BmimPF6: the former has lower solubility in water (0.7 % in weight fraction) than BmimPF6 (2 %) [52]. These results are consistent with the behavior exhibited by other proteins [46,61] and

1.05

1.00

0.95

A*

Ct (-)

0.90

BmimNTf 2

0.85

BmimPF6

0.80

0.75

0.70

0.65

0

1000

2000

3000

4000

5000

6000

t (min) Fig. 2. Time evolution of dimensionless lactoferrin concentration, C A t , to study the influence of ionic liquids. (s) BmimNTf2; (h) BmimPF6; (–) fitting curve of each experiment.

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E (%)

Adjusting parameters

BmimNTf2 BmimPF6

12.4 20.9

R2

k (min1)

r 0.882 ± 0.007 0.832 ± 0.021

3

2.24 ± 0.55  10 2.68 ± 1.63  103

0.956 0.798

experiments was used for each point in the analysis. The mean absolute deviation of all data points between the two replicas was ±0.4%. The influence of mentioned variables on the performance of lactoferrin extraction is stated below. It should be noted that lactoferrin was not detected in the stripping phase of any experiment. Consequently, the study of back-extraction process is required in future steps of the research. 3.1. Influence of pH and ionic strength

Table 2 Experimental conditions used to study the extraction of lactoferrin with BmimNTf2. Exp. code

1 2 3 4 5 6 7 8 9 10a 11

Lactoferrin concentration (g L1)

0.10 0.10 0.10 0.10 0.35 1.0 2.0 0.10 0.10 0.10 0.10

Feed phase (A)

Stripping phase (C)

pH

Ionic strength (M)

pH

3.2 6.4 8.2 8.2 8.2 8.2 8.2 6.4 6.4 8.2 4.6

0.03 0.03 0.03 0.30 0.03 0.03 0.03 0.01 0.01 0.03 0.06

3.2 0.03 3.2 0.03 3.2 0.03 3.2 0.03 3.2 0.03 3.2 0.03 3.2 0.03 4.6 0.06 (Deionized water) 3.2 0.03 6.4 0.01

Ionic strength (M)

a The ionic liquid, BmimNTf2, was in contact with sodium dodecyl sulfate (SDS) during one week before the experiment.

amino acids [23,43], for which an increase in hydrophobicity of ionic liquids reduces their solubility and, therefore, their extraction efficiencies. Despite the higher E achieved by BmimPF6, its use is not advisable when in contact with water because it may suffer from hydrolysis [56]. Nevertheless, due to the mild conditions of both temperature and pH, no significant changes in pH were measured in the experiment, so hydrolysis of BmimPF6 was not detected. In this way, Freire et al. [62] also reported the chemical and thermal stability of BmimPF6 under moderate experimental conditions. However, the experimental deviation with respect to the curve described by Eq. (2) and the consequent low value of R2, which are obtained when BmimPF6 is used, may be explained by considering the lower stability of this ionic liquid when it is in contact with aqueous solutions. As a result, BmimNTf2 is chosen for the rest of the experiments in order to reduce the solubility of ionic liquid in water and to avoid the risk of hydrolysis of BmimPF6. Table 2 shows the set of experiments carried out in this work to study the lactoferrin extraction with the mentioned ionic liquid, BmimNTf2. Different pH (between 3.2 and 8.2), ionic strength (up to 0.30 M), and lactoferrin concentration (between 100 mg L1 and 2.0 g L1) were applied in the experiments. The lactoferrin concentration used in most of the experiments is equal to the average concentration of this protein in whey (0.1 g L1). All the experiments were replicated once, and the mean value between the two

Table 3 summarizes the results of experiments in which the influence of pH and ionic strength are studied. The analysis of pH was carried out by means of the assessment of extraction at three different values of pH: 3.2 (exp. 1), 6.4 (exp. 2) and 8.2 (exp. 3). These pH values were selected because lactoferrin remains stable in this range at room temperature [63,64]. Moreover, all buffer solutions at the mentioned pH can be prepared with the same reagents (H3PO4 and KOH), so salt nature does not interfere in the analysis. Time evolutions of dimensionless lactoferrin concentration (C A t vs. t) of these experiments (1–3) are plotted in Fig. S-1 and provided as Supplementary Information. Extraction kinetics is not influenced by pH significantly, because confidence intervals of kinetic constant contain the values of k of the other experiments. However, extraction efficiency depends moderately on pH: acidic conditions (pHA = 3.2) lead to 9% of E [(1  r) = 0.078], whereas efficiencies higher than 12% [(1  r) = 0.112–0.115] were obtained when pH is close to neutrality (6.4 and 8.2). The influence of pH on protein extraction is associated with the charge of these molecules and, therefore, with electrostatic interactions [5,27]. In the range of pH studied, the positive charge that exhibits lactoferrin at pH = 3.2 decreases continuously and almost disappears at pH = 8.2 [65], which is very close to its isoelectric point (8–9) [66,67]. For this reason, if the influence of pH is attributed to electrostatic interactions, a higher E should be achieved at pH = 8.2, obtaining a difference between exp. 2 and 3, similar to the difference observed between exp. 1 and 2. Nevertheless, results of experiments at pH = 6.4 and 8.2 are very similar, so electrostatic interactions between lactoferrin and the ionic liquid do not seem to be the main driving force in the process. It should be stood out that both feed and stripping solutions are identical in exp. 1 but the lactoferrin back-extraction was not detected. As a result, even though measurements of the lactoferrin structure have not been carried out, it is very likely that this protein suffered from structural changes that make its back-extraction unfeasible when it is contained in the ionic liquid. Otherwise, the feed and stripping phases would have contained the same lactoferrin concentration at equilibrium. Cheng et al. [53] explained that the extraction of hemoglobin into imidazolium-based ionic liquids is caused by the covalent bond established between the cationic ionic liquid moiety and the iron atom of the heme group. In this way, lactoferrin is a protein that contains up to two iron atoms per molecule and it is usually partly saturated with this element. The lactoferrin used in the experiments had an iron saturation equal to 8%. However, the

Table 3 Extraction efficiency, E, and adjusting parameters, r and k, of experiments used to study the influence of feed phase pH, pHA, and feed phase ionic strength, IA. Exp. code

pHA

IA (M)

E (%)

Adjusting parameters r

1 2 3 4

3.2 6.4 8.2 8.2

0.03 0.03 0.03 0.30

9.1 12.6 12.4 4.0

0.922 ± 0.006 0.885 ± 0.006 0.888 ± 0.008 0.966 ± 0.004

R2

k (min1) 3

7.27 ± 3.25  10 6.25 ± 1.60  103 5.75 ± 1.95  103 1.28 ± 0.65  103

0.899 0.946 0.928 0.835

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amount of iron bound to lactoferrin varies notably with pH, so a large amount of iron is released in acidic conditions, whereas the maximum amount of iron is retained at neutral pH [64]. At pH = 6.4 and 8.2, there is a similar amount of iron bound to the protein. Consequently, a similar trend between iron content and lactoferrin extraction efficiency is observed. It can be concluded that, in the same way as hemoglobin, iron atoms play an important role in lactoferrin extraction with imidazolium-based ionic liquids. Furthermore, if iron saturation equals 8%, a maximum of 16% of lactoferrin molecules will contain an iron atom, so experimental values of E could correspond to high extraction levels of protein molecules containing iron. With respect to ionic strength, a difference of one order of magnitude in the feed phase (A) ionic strength, IA, has been considered in exp. 3 and 4: 0.03 M and 0.30 M, respectively. It should be noted that 0.30 M is chosen as an example of relatively high ionic strength because it doubles the value that corresponds to the isotonic salt concentrations of many living organisms (0.15 M), which is considered the limit of low ionic strengths [68]. Fig. S-2 graphs the time evolution of lactoferrin in these experiments. As can be seen, high ionic strengths make the extraction more difficult. On the one hand, kinetic constant is almost four times lower when ionic strength is increased by one order of magnitude. On the other hand, only one third of lactoferrin is extracted at IA = 0.30 M with respect to the experiment at IA = 0.03 M. In the same way as pH, not only can the influence of ionic strength be attributed to conformational changes in protein, but it also has influence on lactoferrin iron-binding ability: even at I = 0.10 M, a notable decrease in the iron-binding ability was reported by Kawakami et al. [69]. 3.2. Influence of lactoferrin concentration The influence of lactoferrin concentration in feed solution (A) on extraction was studied in the range 0.10–2.0 g L1 (exp. 3 and 5–7). The results of this analysis are summarized in Table 4 and plotted in Fig. S-3. The higher the lactoferrin concentration, the lower the E: whereas an extraction efficiency of 12% is achieved with 0.10 g L1, a value slightly higher than 1% is obtained when a solution of 2.0 g L1 is used. In the whole range of concentrations analyzed, the total amount of lactoferrin extracted increases by a factor of 2, but the concentration of feed solution is multiplied by 20, so E and (1  r) decrease considerably. This reduction in E with solute concentration is also reported in some works in the field of extraction with ionic liquids [54,70]. At equilibrium, the following expression is satisfied:

 a¼a

ð4Þ

 are solute activities in the aqueous and organic where a and a phase, respectively. Considering the definition of activity when molarity is used to measure concentration, C:

a¼cC

ð5Þ

where c is the activity coefficient, this definition of distribution coefficient at relatively low pressure (e.g. atmospheric) is concluded: Table 4 Extraction efficiency, E, and adjusting parameters, r and k, of experiments used to study the influence of lactoferrin concentration in feed phase, C A0 . Exp. code

C A0 (g L1)

E (%)

Adjusting parameters r

3 5 6 7

0.10 0.35 1.0 2.0

12.4 4.7 2.1 1.5

0.888 ± 0.008 0.955 ± 0.002 0.981 ± 0.002 0.987 ± 0.001

R2

k (min1) 3

5.75 ± 1.95  10 3.22 ± 0.91  103 2.51 ± 0.98  103 1.88 ± 0.82  102

0.928 0.927 0.874 0.908

D¼

C c ¼  C c

ð6Þ

Lines above variables refer to the organic phase. If this definition is applied to the separation under study, distribution coefficient is equal to the ratio between lactoferrin concentration in the ionic liquid at equilibrium, C B1 , and its concentration in the feed phase, C A1 :

D¼

C B1 C A1



1r r

ð7Þ

Fig. 3 represents distribution coefficient, D, vs. lactoferrin concentration in the feed phase, C A1 , and the organic phase at equilibrium, C B1 , expressed in logarithmic coordinates. As was previously stated, D decreases notably with concentration. This noticeable reduction in D with concentration makes it possible to conclude that extraction is limited by lactoferrin solubility, so the influence of variables such as pH or ionic strength on extraction efficiency may be explained by changes in activity coefficients (Eq. (6)), besides the role played by iron atoms of lactoferrin and the iron-binding ability of this protein. However, as can be seen in Fig. 3, the influence of lactoferrin concentration in the organic phase on D is greater than the concentration of this protein in the feed phase at equilibrium. In this way, the slope of log(D) vs. logðC B1 Þ is almost 4 times higher than the slope of log(D) vs. logðC A1 Þ. This fact implies that concentration produces more pronounced changes in the activity coefficient of lactoferrin in the organic phase. Furthermore, the way in which D depends on lactoferrin concentration (the higher the concentration, the lower the D) implies that this extraction is especially suitable for low concentrations of this protein (e.g. lactoferrin content in bovine whey). As a consequence, this process may be useful for preconcentration purposes from low concentrations of lactoferrin. 3.3. Additional influences on extraction and selectivity In this section, the influence of the stripping phase, contacting ionic liquid with sodium dodecyl sulfate (SDS) and simultaneous, moderate changes of pH and ionic strength on extraction performance are assessed. Results of experiments carried out for this study are stated in Table 5. The effect of the stripping phase has been studied by means of the use of a buffer solution at pH = 4.6 (exp. 8) as the stripping phase instead of deionized water (exp. 9). Fig. S-4 shows the results of these experiments. It can be concluded that the stripping phase does not affect the extraction kinetics, since very similar values of k are obtained in both experiments. However, the addition of salts in the stripping phase reduces the amount of lactoferrin extracted: both E and (1  r) decrease 3% approximately. As a result, the influence of the ionic strength concluded in the previous section for the feed phase is also observed when this variable is increased in the stripping phase. The effect of stripping solution on extraction can be explained by the influence this solution has on lactoferrin coefficient activity in the organic phase. In this case, the lower decrease of E can be explained considering that the increase of ionic strength is notably smaller. Cheng et al. [53] identified sodium dodecyl sulfate (SDS) as the unique stripping reagent that achieved the back-extraction of hemoglobin from BtmsimPF6. However, not only did the same stripping aqueous solution based on SDS solutions fail to back-extract lactoferrin from BmimNTf2, but this solution also decreased E (results not shown). This fact can be explained with the same reasons that were stated previously. For this reason, contacting BmimNTf2 with SDS during one week before the experiment (exp. 10) was tried to facilitate back-extraction without decreasing

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-0.6

-0.8

-1.0

( )

log(D ) = 0.550 − 0.744·log C∞A

log [D(-)]

R 2 = 0.998

-1.2

-1.4

( )

log(D ) = 2.06 − 2.83·log C∞B R 2 = 0.966

-1.6

-1.8

-2.0 1.0

1.5

2.0

2.5

3.0

3.5

-1

log [C(mg L )] Fig. 3. Distribution coefficient, D, vs. lactoferrin concentration in feed, C A1 , and organic phase at equilibrium, C B1 , expressed in logarithmic coordinates. (s) D as a function of C A1 ; (h) D as a function of C B1 ; (–) fitting curve of each function.

Table 5 Extraction efficiency, E, and adjusting parameters, r and k, of experiments used to study additional influences on lactoferrin extraction. Exp. code

3 8 9 10a 11 a

Stripping phase (A)

Stripping phase (C)

pHA

pHC

8.2 6.4 6.4 8.2 4.6

IA(M) 0.03 0.01 0.01 0.03 0.06

E (%)

IC(M)

3.2 0.03 4.6 0.06 (Deionized water) 3.2 0.03 6.4 0.01

Adjusting parameters r

12.4 12.4 15.8 12.9 5.7

0.888 ± 0.008 0.882 ± 0.007 0.857 ± 0.012 0.886 ± 0.012 0.954 ± 0.008

R2

k (min1) 3

5.75 ± 1.95  10 2.24 ± 0.55  103 2.25 ± 0.79  103 2.30 ± 0.94  103 1.40 ± 0.85  103

0.928 0.956 0.924 0.882 0.813

BmimNTf2 was in contact with sodium dodecyl sulfate (SDS) during one week before the experiment.

E. Nevertheless, the same extraction efficiency is reached at equilibrium and lactoferrin was not back-extracted in any experiment (see Fig. S-5). SDS only modifies the kinetics of the process, since the value of k is 60% lower than that obtained in exp. 3, used as reference. Furthermore, the difficulties of back-extracting proteins from ionic liquids can be attributed to the severe denaturation they can suffer in this organic phase [30,45]. Conformation changes prevent lactoferrin from being back-extracted by many different types of aqueous solutions. In previous sections, very significant changes in the variables analyzed were applied. The dependence of lactoferrin extraction on moderate changes of pH and ionic strength carried out simultaneously is considered by means of exp. 8 and 11. On the one hand, pH is fixed at 6.4 and 4.6, which correspond to the pH of sweet and acid whey, respectively [71]. On the other hand, ionic strengths are associated with the amount of phosphate (1.0 g L1) and lactic acid (6.4 g L1) contained in sweet and acid whey to prepare buffer solutions at pH = 6.4 and 4.6, respectively. In this way, exp. 8 combines favorable conditions (neutral pH and low ionic strength), in contrast to exp. 11. Experiments 8 and 11 are plotted in Fig. S-6 (C A t vs. t). All the moderate changes in the mentioned variables, under unfavorable conditions (exp. 11), cause less than 50% of the lactoferrin extracted in exp. 8 to be obtained. In addition, conditions of neutral pH and low ionic strength lead to a slightly faster extraction. These results are coherent with those stated in previous sections and

make it possible to conclude that moderate changes in several variables have the same effect on extraction as a big change in a single variable. Considering all the experiments analyzed, up to 20% of lactoferrin can be extracted with hydrophobic, imidazolium-based ionic liquids. Nevertheless, due to the non-volatility and high hydrophobicity of the extracting phase, even low partition coefficients could result in an economical recovery process [43]. Due to the low value of bovine whey, the key variable to assess the feasibility of lactoferrin extraction is selectivity rather than extraction efficiency. The whey protein selected to measure the selectivity is BSA, since its molecular weight is similar to lactoferrin, which makes their separation with other techniques more difficult [57]. For this purpose, two experiments with these proteins (in the same conditions of exp. 8) are compared and plotted in Fig. 4 (C A t vs. t), and their results are summarized in Table 6. As can be seen, the amount of BSA extracted is negligible, since 98.7% of this protein remains in the feed aqueous phase (A) at equilibrium. The wide confidence interval and low value for R2 in this experiment is explained by the very low solubility, which is very close to the order of mean experimental error. Selectivity of lactoferrin extraction with respect to BSA is 9, calculated as the ratio between (1  r) for lactoferrin and for BSA. One important structural difference between lactoferrin and BSA is the absence of iron atoms in the latter [57], which seems to be the cause of the selectivity exhibited by the imidazolium-based ionic liquid like BmimNTf2, in the same way as Cheng et al. reported on heme-proteins [53].

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1.05 1.00

BSA

0.95

A*

Ct (-)

0.90

Lactoferrin

0.85 0.80 0.75 0.70 0.65 0

1000

2000

3000 t (min)

4000

5000

6000

Fig. 4. Time evolution of dimensionless protein concentration, C A t , to study the selectivity towards lactoferrin with respect to BSA. (s) Lactoferrin; (h) BSA; (–) fitting curve of each experiment.

Table 6 Extraction efficiency, E, and adjusting parameters, r and k, of experiments used to study the selectivity of BmimNTf2 towards lactoferrin with respect to BSA. Protein

E (%)

Adjusting parameters r

Lactoferrin BSA

12.4 2.1

components (salts, carbohydrates, etc.) on the extraction efficiency and selectivity of the separation process should also be studied in further steps of the research.

0.882 ± 0.007 0.987 ± 0.010

R2

k (min1) 3

2.24 ± 0.55  10 1.49 ± 4.21  103

0.956 0.348

Results of this work are competitive with respect to other emerging technologies presented as alternatives to chromatographic techniques. For instance, electrodialysis with an ultrafiltration membrane, which Ndiaye et al. [9] proposed to overcome the disadvantages of other membrane processes, shows lower migration rates since its highest value is 15% (this variable may be compared with extraction efficiency) and a very significant lower selectivity, because other whey proteins permeate simultaneously with lactoferrin. As a result, the main challenge that has to be overcome to assure the feasibility of this process is the achievement of an efficient back-extraction. In this way, other imidazolium-based ionic liquids, which preserve selectivity and water stability and do not denaturate lactoferrin irreversibly so that back-extraction is feasible, are required as the extracting phase. There are several ways to recover lactoferrin: for instance, once lactoferrin has been extracted, an organic co-solvent that can make the extracting phase (ionic liquid) less polar so that protein could be released to the aqueous phase. Another approach involves more hydrophilic ionic liquids so that protein is extracted to an ionic liquid-rich aqueous phase. This hydrophilic ionic liquid will be removed from this phase by means of other hydrophobic solvents (which can be ionic liquids) [36,72]. In addition, even though the ionic liquids used in this work are less toxic than toluene, which is considered a reference of conventional solvents [20], other less toxic, cheaper imidazolium-based ionic liquids (modifying both cation or anion), which provide gentle biocompatible environment that prevent protein denaturation, can be assessed as future work. For this purpose, a screening among the ionic liquids with the mentioned characteristics should be carried out. The influence of other whey

4. Conclusions Extraction with hydrophobic ionic liquids is considered an alternative to separating lactoferrin from bovine whey. Selectivity of imidazolium-based ionic liquids (BmimNTf2 and BmimPF6) towards lactoferrin has been proved, which makes its separation from other proteins possible. Extraction efficiency is enhanced at neutral pH, low ionic strength and low concentration, although efficiencies higher than 20% were not obtained in any experiment. Nevertheless, relatively low extraction efficiencies are not as relevant as selectivity, due to the very low value of bovine whey. Results are explained in terms of the interaction between iron atoms of lactoferrin and ionic liquids, and changes in activity coefficients of solute with studied variables. The variables that produce more marked changes in activity coefficients are lactoferrin concentration and ionic strength. Consequently, distribution coefficient is highly dependent on lactoferrin concentration and ionic strength. As future work, the back-extraction process must be studied so that lactoferrin could be recovered in a stripping solution. Acknowledgements The authors gratefully acknowledge the financial support of the Spanish Ministry of Economy and Competitiveness through the projects CTM2006-00317 and EUI2008-03857. The authors also thank The Tatua Co-operative Dairy Company Limited for providing the lactoferrin used in experiments. Enrique Alvarez-Guerra would like to thank the Spanish Ministry of Education, Culture and Sports for an FPU research grant (AP2010-4942). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.seppur.2012. 08.010.

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