Influence of Pore-Size/Porosity on Ion Transport and Static BSA Fouling

applied sciences

Article Inﬂuence of Pore-Size/Porosity on Ion Transport and Static BSA Fouling for TiO2-Covered Nanoporous Alumina Membranes

Lourdes Gelde 1, Ana Laura Cuevas 2 and Juana Benavente 1,*

1 Departamento de Física Aplicada I, Facultad de Ciencias, Universidad de Málaga, E-29071 Málaga, Spain; [email protected] 2 Unidad de Nanotecnología, SCBI Centro, Universidad de Málaga, E-29071 Málaga, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-952-13-1929

Abstract: The inﬂuence of geometrical parameters (pore radii and porosity) on ion transport through

two almost ideal nanoporous alumina membranes (NPAMs) coated with a thin TiO2 layer by the atomic layer deposition technique (Sf-NPAM/TiO2 and Ox-NPAM/TiO2 samples) was analyzed by membrane potential and electrochemical impedance spectroscopy measurements. The results

showed the signiﬁcant effect of pore radii (10 nm for Sf-NPAM/TiO2 and 13 nm for Ox-NPAM/TiO2) when compared with porosity (9% and 6%, respectively). Both electrochemical techniques were also used for estimation of protein (bovine serum albumin or BSA) static fouling, and the results seem to

indicate deposition of a BSA layer on the Sf-NPAM/TiO2 fouled membrane surface but pore-wall deposition in the case of the fouled Ox-NPAM/TiO sample. Moreover, a typical and simple optical 2 technique such as light transmission/reflection (wavelength ranging between 0 and 2000 nm) was also used for membrane analysis, showing only slight transmittance differences in the visible region Citation: Gelde, L.; Cuevas, A.L.; Benavente, J. Influence of when both clean membranes were compared. However, a rather significant transmittance reduction Pore-Size/Porosity on Ion Transport (~18%) was observed for the fouled Sf-NPAM/TiO2 sample compared to the fouled Ox-NPAM/TiO2 and Static BSA Fouling for sample, and was associated with BSA deposition on the membrane surface, thus supporting the

TiO2-Covered Nanoporous Alumina electrochemical analysis results. Membranes. Appl. Sci. 2021, 11, 5687. https://doi.org/10.3390/ Keywords: nanoporous TiO2-covered alumina membranes; BSA fouling, membrane potential; app11125687 impedance spectroscopy; light transmission/reﬂection

Academic Editor: Constantinos E. Salmas 1. Introduction Received: 28 April 2021 Nanoporous alumina structures obtained by electrochemical anodization of aluminum Accepted: 15 June 2021 Published: 19 June 2021 foils (two-step method), with pore radius between 10–200 nm and inter-pore distance between 65 and 500 nm (depending on anodization conditions such as electrolyte and voltage),

Publisher’s Note: MDPI stays neutral consisting of self-ordered cylindrical aligned channels with narrow pore radius distribution with regard to jurisdictional claims in and without tortuosity [1–3], were initially considered as nanofilters for the transport of published maps and institutional affil- solutions containing heavy metals ions or as templates for nanotubes and nanowires due iations. to their well-defined structure and high regularity, but other applications such as magnetic storage, solar cells, or photonic crystals have also being studied [4–8]. Besides the high structural regularity of these samples, their strong chemical and thermal resistance are also of great interest when they are used as membranes for controlled release of pharmacologic agents or in food processing due to their stability under the cleaning protocols commonly Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. used to reduce membrane fouling (adsorption/deposition of transported molecules or par- This article is an open access article ticles on the membrane structure), which commonly involves the use of chemicals covering distributed under the terms and a wide range of pH values or high/medium temperature [9–13] and is the main membrane conditions of the Creative Commons problem in such applications. Moreover, these nanoporous alumina membranes (NPAMs) Attribution (CC BY) license (https:// can also be used in microfluids or as platforms for biochemical/biological sensors and creativecommons.org/licenses/by/ medical devices [14–17], although specific properties such as surface hydrophilic character 4.0/). and biocompatibility or electrical nature are also significant depending on the particular

Appl. Sci. 2021, 11, 5687. https://doi.org/10.3390/app11125687 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 5687 2 of 16

application. Consequently, possible easy membrane surface modification able to confer the membrane optimal geometrical and material characteristics for a specific application is of great interest. Different techniques such as dip coating, grafting, chemical vapor deposition, plasma polymerization, or atomic layer deposition have already been proposed for NPAM surface functionalization/modification [18–20]. In particular, the atomic layer deposition (ALD) technique is a suitable manner of modifying both membrane pore size and surface physicochemical characteristics (charge, hydrophilicity, or biocompatibility) by covering external and internal (pore-wall) surfaces of NPAMs with a homogeneous layer of a selected material (nitrides, sulfides, or metal oxides such as SiO2, ZnO, TiO2, Fe2O3, or even Al2O3, when only geometrical parameters have to be modified), providing to these nanoporous alumina-based membranes (NPA-bMs) desired geometrical and material properties [20,21]. In fact, the ALD technique is especially appropriate for modification of membranes with high aspect ratios (that is, pore length/pore diameter > 2000), and it also allows surface modification with two or more layers from different materials [22]. As indicated above, a significant problem associated with the use of planar NPA- bMs in the separation/transport of solutions containing proteins or macromolecules is membrane fouling, because it can significantly reduce the effectiveness of the process, necessitating the development of efficient cleaning procedures [9]. Streaming potential measurements have traditionally been performed for determination and analysis of membrane fouling, although the pressure difference needed for such measurements could not be appropriate for fragile samples as is the case with the NPA-bMs indicated above. Other kinds of electrochemical experiments such as membrane potential (∆Φmbr) or electrochemical impedance spectroscopy (EIS) have also been used for membrane fouling estimation [23–27], but different and specific optical techniques (ellipsometry spectroscopy or optical coherence tomography) have also been proposed [28,29]. On the other hand, it was already reported that modification with TiO2 nanoparticles of different types of membranes for diverse applications (membrane bioreactors, oily emulsion filtration, etc.) seems to reduce the fouling effect independently of membrane material and TiO2 particle immobilization procedure (entrapped, deposited, or deep coating) [30–32]. In this context, the increase in the hydrophilic character of TiO2-modified membranes (polymeric and ceramic) has been indicated as one of the key factors for membrane fouling reduction caused by filtration of oily wastewater or BSA static fouling, although other factors such as TiO2 surface charge, which depends on solution pH and salt concentration, have also been considered [33–35]. Moreover, the biocompatibility and corrosion resistance of TiO2, of great importance for its use in dental implants, has already been reported [21]. In this work, we considered the influence of geometrical parameters on both ion transport and static protein (bovine serum-albumin or BSA) fouling for two nanoporous alumina-based membranes obtained by TiO2-layer coverage using the ALD technique, with similar thickness but different pore size and porosity, by analyzing and comparing membrane potentials and EIS diagrams determined for clean and fouled membranes in order to obtain qualitative and quantitative information on the BSA fouling process. Ac- cording to the results, the fouling mechanism seems to be strongly dependent on pore radius when compared to the porosity effect, although interfacial effects might affect particular quantification. However, due to the high transparency of the studied membranes, optical analysis using a typical and simple characterization technique such as light transmission/reflection was also used to confirm the membrane fouling mechanism established from electrochemical measurements.

2. Materials and Methods 2.1. Materials The nanoporous alumina membranes (NPAMs) used as supports in this study were synthesized by the electrochemical two-step anodization method using high purity aluminum discs (Al 99.999%, Goodfellow (UK); 0.5 mm thickness). Two different aqueous electrolyte solutions and anodization voltages were used in order to have membranes Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 17

The nanoporous alumina membranes (NPAMs) used as supports in this study were synthesized by the electrochemical two-step anodization method using high purity aluminum discs (Al 99.999%, Goodfellow (UK); 0.5 mm thickness). Two different aqueous electrolyte solutions and anodization voltages were used in order to have membranes Appl. Sci. 2021, 11, 5687 with different pore size and interpore distance: 0.3 M solution of sulfuric acid3 ofand 16 applied voltage of 25 V for the Sf-NPAM sample, and 0.3 M solution of oxalic acid and 40 V in the case of the Ox-NPAM sample; detailed information on this process is given in the litera- withture different[1,3,35].pore In Supplementary size and interpore Information, distance: 0.3 scanning M solution electron of sulfuric microscope acid and applied (SEM) pictures voltageof both of membrane 25 V for the surfaces Sf-NPAM are sample, presented and as 0.3 Figure M solution S1. of oxalic acid and 40 V in the caseModification of the Ox-NPAM of Sf-NPAM sample; and detailed Ox-NPAM information surfaces, on this both process external is given and internal in the (pore- literaturewall), with [1,3 a,35 thin]. In TiO Supplementary2 layer coated Information, by the atomic scanning layer electron deposition microscope (ALD) technique (SEM) was picturesperformed of both in a membrane Savannah surfaces 100 thermal are presented ALD reactor as Figure from S1. Cambridge Nanotech (Waltham, MA,Modification USA), using of Sf-NPAMhigh purity and Ox-NPAMargon as surfaces,carrier gas. both externalTwo precursors, and internal titanium (pore- tetrai- wall),sopropoxide with a thin (metal TiO2 precursor)layer coated at by 75 the °C atomic and water layer deposition(for substrate (ALD) functionalization technique was and as performed in a Savannah 100 thermal ALD reactor from Cambridge Nanotech (Waltham, oxidant agent) at 60 °C, were used for deposition of the conformal coating, as explained MA, USA), using high purity argon as carrier gas. Two precursors, titanium tetraiso- propoxidein a previous (metal work precursor) [20]. These at 75 ◦nanoporousC and water alumina-based (for substrate functionalization membranes will and hereafter as be oxidantcalled Sf-NPAM/TiO agent) at 60 ◦C,2 were and usedOx-NPAM/TiO for deposition2, respectively. of the conformal Almost coating, total as explainedTiO2 coverage of inboth a previous alumina work supports [20]. was These already nanoporous determined alumina-based in a previous membranes paper will[36] hereafter by the XPS tech- benique, called and Sf-NPAM/TiO the atomic 2concentrationand Ox-NPAM/TiO percentage2, respectively. (A.C. %) Almostof the different total TiO 2elementscover- found ageon the of both surfaces alumina of each supports membrane was already is shown determined in Supplementary in a previous Information paper [36] (Table by the S1); only XPSsuperficial technique, slight and differences the atomic concentrationassociated wi percentageth manufacture/enviro (A.C. %) of thenmental different contamination elements found on the surfaces of each membrane is shown in Supplementary Information (sulfur detection for Sf-NPAM/TiO2 or excess carbon in the case of the Ox-NPAM/TiO2 (Table S1); only superficial slight differences associated with manufacture/environmental sample) were determined. The thickness of the TiO2 layer was also determined (from contamination (sulfur detection for Sf-NPAM/TiO or excess carbon in the case of the depth-profile XPS analysis [36]), obtaining a value2 of ~ 6 nm, which agrees quite well with Ox-NPAM/TiO2 sample) were determined. The thickness of the TiO2 layer was also determinedpublished results (from depth-profile [21]. Figure 1 XPS shows analysis SEM [ 36surface]), obtaining micrographs a value of of both ~6 nm, NPAM/TiO which 2 sam- agreesples. The quite following well with values published for average results [ 21pore-radii]. Figure1 (r showsp), interpore SEM surface distance micrographs (Dint) and porosity 2 of(Θ both = (2 NPAM/TiOπ/√3)(rp/Dint2 )samples.) [1]), were The followingdetermined values by surface for average analysis pore-radii of SEM (rp), micrographs interpore [36]: √ 2 distance = 10 (D nm,int) and = 65 nm, (Θ =and (2π /<Θ3>)(r = p9%/D intfor) )[the1]), Sf-NPAM/TiO were determined2 sample, by surface and = 13 analysisnm, SEM= 105 micrographs nm, and <Θ [>36 =]: 6% =the 10 Ox-NPAM/TiO nm, = 652 nm,sample, and with <Θ> =both 9% membranes for thehaving Sf-NPAM/TiO a thickness2 sample, of ~ 65 and μm. = 13 into nm, = geomet 105 nm,rical and = 6% for values, the rather Ox-NPAM/TiO2 sample, with both membranes having a thickness of ~65 µm. Taking into similar theoretical hydraulic permeability was determined for both TiO2-covered mem- account geometrical parameter values, rather similar theoretical hydraulic permeability was Sf-NPAM/TiO2 −12 Ox-NPAM/TiO2 −12 branes [9]: LH = 1.92 × 10 m/s.Pa and LSf-NPAM/TiO2H = 2.17×10 −12 m/s.Pa. It should determined for both TiO2-covered membranes [9]: LH = 1.92 × 10 m/s.Pa be mentionedOx-NPAM/TiO2 that both NPAMs−12 and NPAM/TiO2 samples were obtained in the Unidad and LH = 2.17×10 m/s.Pa. It should be mentioned that both NPAMs and NPAM/TiOde Membranas2 samples Nanoporosas, were obtained Universidad in the Unidad de de Oviedo Membranas (Spain) Nanoporosas, and kindly Universi- submitted by dadProf. de V. Oviedo de la (Spain)Prida and and Dr. kindly V. Vega. submitted by Prof. V. de la Prida and Dr. V. Vega.

(a) (b)

FigureFigure 1. 1. SEMSEM micrographmicrograph of: of: ( a()a) Sf-NPAM/TiO Sf-NPAM/TiO2 membrane2 membrane surface surface and and (b )(b Ox-NPAM/TiO) Ox-NPAM/TiO2 2 mem- membranebrane surface surface (values (values and and reference reference in in text). Membranes were statically fouled with the protein bovine serum albumin (BSA), whichMembranes had the following were characteristic statically fouled parameters: with 66.5the kDaprotein molecular bovine weight serum and albumin Stokes (BSA), radiuswhich of had 3.48 the nm. following Membrane characteristic fouling was parameters: performed by 66.5 mounting kDa molecular each sample weight in the and Stokes dead-endradius of electrochemical 3.48 nm. Membrane cell shown fouling in Supplementary was performed Information by mounting (Figure each S2), ﬁllingsample in the onedead-end half-cell electrochemical with a BSA aqueous cell solutionshown in of 5Supplementary g/L and the other Information half-cell with (Figure distilled S2), filling water for 24 h, after which the cell was emptied and the membrane surface softly cleaned with distilled water. These samples will hereafter be called Sf-NPAM/TiO2(f) and Ox- NPAM/TiO2(f). Appl. Sci. 2021, 11, 5687 4 of 16

2.2. Electrochemical Characterization

Membrane potentials (∆Φmbr) and equilibrium electrical potential differences between the 2 NaCl solutions of different concentrations (Cf = 0.01 M and Cv ranging between 0.002 M and 0.1 M) at both membrane sides were determined by measurements performed in the dead-end test cell already indicated (Supplementary Information, Figure S2), with the 2 Ag/AgCl reversible electrodes (to Cl− ion) connected to a digital voltmeter (Yoko- hama 7552, 1 GΩ input resistance) and magnetic stirrers placed in the bottom of each cell working at a stirring rate of 540 rpm (to minimize concentration polarization at the membrane surfaces [37]). Because measured values (∆E) included two different contributions, electrode potential (∆Φelect) and membrane potentials (∆Φmbr), the latter values were obtained by subtracting from each ∆E value the corresponding ∆Φelect (which depended on electrolyte concentration, ∆Φelect = (RT/zF)ln(Cv/Cf), where R and F are gas and Fara- day constants, respectively, while T represents the temperature of the system); hence: ∆Φmbr = ∆E − ∆Φelect [38]. Measurements were performed at standard pH (5.8 ± 0.3) and room temperature (25 ± 2) ◦C. Electrochemical impedance spectroscopy (EIS) is an alternating current (a.c.) technique used for electrical characterization of membranes in “working condition”, that is, in contact with electrolyte solutions. EIS allows, under certain conditions, the estimation of different contributions: membrane, electrolyte solution, and membrane/solution interface, by using equivalent circuits as models [39,40]. The impedance (Z) is a complex number, with real (Zreal) and imaginary (Zimg) parts (Z = Zreal + j Zimg) that are related to both the transport of charge across the membrane and charge storage, through its electrical resistance (R) and capacitance (C) [39]. The analysis of the impedance data was performed by using the Nyquist plot in the complex plane (−Zimg versus Zreal)[39]. EIS measurements were carried out with the membranes in the electrochemical test cell by connecting the electrodes to a Frequency Response Analyzer (FRA, Solartron 1260, Hamshire, UK) using a maximum voltage of 0.01 V and an interval of frequency ranging between 1 Hz and 107 Hz, in open circuit mode. The ZView 2 data analysis program (Scribner, Southern Pines, NC, USA) was used for determination of electrical parameters, and EIS data were corrected by the inﬂuence of connecting cables and other parasite capacitances.

2.3. Optical Measurements Optical characterization of clean and fouled samples was performed by analyzing transmittance and reﬂectance spectra, which were recorded with a Varian Cary 5000 spec- trophotometer (Agilent Technologies, Santa Clara, CA, USA) provided with an integrating sphere of Spectralon for a wavelength interval of 0–2000 nm.

3. Results and Discussion 3.1. Electrochemical Characterizations Ion diffusive transport across membranes, which depends on membrane material electrical characteristics and pore size, as well as the electrolyte and solution concentrations, can be studied by analyzing membranes’ potential values. This analysis allows the estimation of membrane effective ﬁxed charge concentration (Xef) and ion transport numbers (ti), or fraction of the total current transported through the membrane by one ion (ti = Ii/IT)[38]; for an ideal positively charged membrane (anion exchanger), t− = 1 and t+ = 0, that is, only negative charges can pass through the membrane, while for cation exchangers or ideal negatively charged membranes, t+ = 1 and t− = 0. Effective ﬁxed charge controls Donnan (or interfacial) potential (∆øDon), while values of ion transport numbers in the membrane also depend on pore size and are directly related to diffusion potential (∆ϕdif ). The expressions for Donnan and diffusion potentials are [38]:

2 1/2 Donnan potential, ∆øDon = (RT/F)ln[(wXef/2C) + [(wXef/2C) + 1) ]] (1)

Diffusion potential, ∆ϕdif = − (RT/F)[(t− − t+)]ln(Cv/Cf) = (RT/F)[(1 − 2t−)]ln(Cv/Cf) (2) Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 17

Donnan (or interfacial) potential (∆øDon), while values of ion transport numbers in the

membrane also depend on pore size and are directly related to diffusion potential (∆φdif ). The expressions for Donnan and diffusion potentials are [38]:

2 1/2 Donnan potential, ∆øDon = (RT/F)ln[(wXef/2C) + [(wXef/2C) + 1) ]] (1)

Diffusion potential, ∆φdif = − (RT/F)[(t− − t+)]ln(Cv/Cf) = (RT/F)[(1 − 2t−)]ln(Cv/Cf) (2) Appl. Sci. 2021, 11, 5687 5 of 16

with w = −1/+1 for negatively/positively charged membranes, while Ci indicates solution concentration (i = v for variable solution concentration and i = f for fixed solution concen-

tration), andwith R, w F, = and−1/+1 T have for negatively/positivelybeen previously identified. charged According membranes, to while the Teorell-Meyer- Ci indicates Sieverssolution model concentration [41,42], membrane (i = v for potential variable cons solutionists concentrationof the sum of and two i =Donnan f for ﬁxed potentials solu- (one fortion each concentration), membrane/solution and R, F, andinterface) T have and been the previously diffusion identiﬁed. potential Accordingin the membrane. to the FigureTeorell-Meyer-Sievers 2 shows a scheme model of [ion41, 42transport], membrane through potential two consistsporous ofmembranes the sum of of two the Don- same materialnan (similar potentials positive (one for charge) each membrane/solution but different pore interface) size and andporosity, the diffusion as is the potential case for in the the membrane. Sf-NPAM/TiO2 and Ox-NPAM/TiO2 samples. Figure 2a shows a membrane with small Figure2 shows a scheme of ion transport through two porous membranes of the same pore size,material which (similar behaves positive as charge)an almost but differentideal anion-exchanger, pore size and porosity, avoiding as is thethe case pass for of the practi- Sf- cally allNPAM/TiO cations 2(membraneand Ox-NPAM/TiO co-ions),2 samples.but the incr Figureease2a showsof pore a membranesize favors with the small inclusion pore of electrolytesize, which solution behaves into as the an almostpores idealas shown anion-exchanger, in Figure avoiding2b, which the raises pass of the practically transport all of cationscations and increases (membrane t+ co-ions), value. Because but the increase ion transport of pore sizenumbers favors thedepend inclusion not ofonly electrolyte on mem- branesolution charge but into also the poreselectrolyte as shown electrochemical in Figure2b, which parameters, raises the membrane transport ofionic cations permselec- and increases t+ value. Because ion transport numbers depend not only on membrane charge tivity (PSi), which represents the relative variation of counter ions in the membrane with but also electrolyte electrochemical parameters, membrane ionic permselectivity (PS ), respect to their values in solution, is usually considered; then, for positively charged mem-i which represents the relative variation of counter ions in the membrane with respect to − − − o o + − branestheir and values NaCl insolutions, solution, PS is usuallyCl , is expressed considered; as then,[43]: forPSCl positively = (tCl − chargedt Cl)/t Na membranes, where tCl is the transport number of the− counter-anion in the membrane− − pores,o owhile+ toCl− and− toNa+ and NaCl solutions, PSCl , is expressed as [43]: PSCl = (tCl − t Cl)/t Na , where tCl is o − o + representthe transportthe transport number numbers of the counter-anion of the anion in and the membranecation, respectively, pores, while in t Clthe andNaCl t Na solution. represent the transport numbers of the anion and cation, respectively, in the NaCl solution.

(a) (b)

FigureFigure 2. Scheme 2. Scheme of ion of transport ion transport through through two two positi positivelyvely charged charged nanoporousnanoporous membranes membranes with with the the samesame thickness thickness and and material but but different different pore pore size size and and porosity. porosity. (a) Pore (a) charge Pore charge practically practically rejects all rejectsco-ion all co-ion favoring favoring counter-ion counter-ion transport transp in membranesort in membranes with reduced with porereduced radii; pore (b) higher radii; pore(b) higher size pore sizefavors favors co-ion co-ion inclusion, inclusion, increasing increasing co-ion transport co-ion transport number. number.

Figure3a shows the dependence of membrane potential values on solution concentra- Figure 3a shows the dependence of membrane potential values on solution concen- tion ratio for clean Sf-NPAM/TiO2 and Ox-NPAM/TiO2 membranes, but concentration trationdependence ratio for clean for solution Sf-NPAM/TiO diffusion potential2 and Ox-NPAM/TiO (dashed line) obtained2 membranes, using solution but concentration transport dependence for solution diffusiono potentiao l (dashed line) obtained using solution numbers in Equation (2) (tNa+ and tCl− [44], dashed line]; theoretical ∆Φmbr values for transportan ideal numbers anion-exchanger in Equation membrane (2) (tNa+o (t and− = 1,tCl solid−o [44], line) dashed are also line]; represented theoretical for compari- ∆Φmbr values forson. an As ideal can beanion-exchanger observed, membrane membrane potential (t values− = 1, forsolid the line) sample are with also lower represented pore radii, for comparison.Sf-NPAM/TiO As can2, be hardly observed, differ frommembrane those corresponding potential values to the for ideal the positively sample with charged lower pore radii,membrane, Sf-NPAM/TiO but reduced2, hardly∆Φmbr valuesdiffer from were obtainedthose corresp for theonding Ox-NPAM/TiO to the ideal2 membrane positively (according to Figure2 explanations), as the difference is more signiﬁcant at high concen- charged membrane, but reduced ∆Φmbr values were obtained for the Ox-NPAM/TiO2 tration ratio values as the result of a ﬁxed charge screening effect. On the other hand, dependence of ∆Φmbr values on the electrolyte solution can be observed in Figure3b, where a comparison of data points for the Sf-NPAM/TiO2 membrane using KCl and CaCl2 is shown. The combination of both membrane geometry and surface material can be observed in Supplementary Information (Figure S3), where a comparison of membrane Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 17

membrane (according to Figure 2 explanations), as the difference is more significant at high concentration ratio values as the result of a fixed charge screening effect. On the other hand, dependence of ∆Φmbr values on the electrolyte solution can be observed in Figure 3b, where a comparison of data points for the Sf-NPAM/TiO2 membrane using KCl and Appl. Sci. 2021, 11, 5687 CaCl2 is shown. The combination of both membrane geometry and surface material6 of can 16 be observed in Supplementary Information (Figure S3), where a comparison of membrane potential values measured with KCl solutions for Sf-NPAM, Ox-NPAM, Sf-NPAM/TiO2,

and Ox-NPAM/TiOpotential values2 samples measured is withpresented KCl solutions [36], while for Sf-NPAM, the effectOx-NPAM, of pore size, Sf-NPAM/TiO porosity, and2, structureand asymmetry Ox-NPAM/TiO for NPAMs2 samples has is presented already been [36], while reported the effect [35,45]. of pore size, porosity, and structure asymmetry for NPAMs has already been reported [35,45].

40 ideal ion exchanger (a) 20 (mV) solution diffusion potential mbr 0 ΔΦ

-20

-40

-60 -2 -1 0 1 2 3 Ln(C /C) v f

40 (b) 20 (mV) 0 mbr ΔΦ -20

-40

-60 -2 -1 0 1 2 3 ln(C /C) v f

Figure 3. Membrane potential as a function of solution ratio for: (a) Sf-NPAM/TiO2 membrane (),

Figure 3.Ox-NPAM/TiO Membrane potential2 membrane as a ( ♦function) solution of diffusion solution potential ratio for: (dashed (a) Sf-NPAM/TiO line) for NaCl2 membrane electrolyte, and(□), − Ox-NPAM/TiO(solid line)2 membrane ideal positively (◊) solution charged diffusion membrane potential (t = 1); (dashed (b) Sf-NPAM/TiO line) for NaCl2 membrane electrolyte, and and KCl 5 (solid line)solutions ideal (positively∆), CaCl2 solutions charged (membrane). (t− = 1); (b) Sf-NPAM/TiO2 membrane and KCl solutions (Δ), CaCl2 solutions (▽) The fit of the experimental values can be performed taking into account both Don- nan and diffusion potentials as well as membrane electroneutrality, and it allows the The fit of the experimental values can be performed taking into account both Donnan determination of the effective membrane fixed charge concentration (Xef), anion/cation and diffusiondiffusion potentials coefficient as ratio well (D asCl− membrane/DNa+, because electroneutrality, (tCl−/tNa+) = (DandCl− it/D allowsNa+) [ 44the]) ordetermi- anion nation permselectivity,of the effective asmembrane has already fixed been charge explained concentration in previous papers (Xef), anion/cation [46–48]. The following diffusion coefficientvalues ratio were (DCl determined:−/DNa+, because Xef = (t +Cl 0.015−/tNa+) M, = D (DCl−Cl−/D/DNaNa++) =[44]) 3.74, or and anion PCl− permselectivity,= 45.2% for Sf- as has alreadyNPAM/TiO been2 membrane,explained in but previous Xef = + 0.012 papers M, D[46–48].Cl-/DNa The+ = following 2.47, and P valuesCl− = 25.3% were forde- Ox-NPAM/TiO membrane. These results show the reduction in pore size increases for termined: Xef = + 0.0152 M, DCl−/DNa+ = 3.74, and PCl− = 45.2% for Sf-NPAM/TiO2 membrane, ionic diffusion coefficient ratio, which tends to a value more similar to that for NaCl solu- but Xef = + 0.012 M, DCl-/DoNa+ = 2.47, and PCl− = 25.3% for Ox-NPAM/TiO2 membrane. These tion, (DCl−/DNa+) = 1.60), and logically for anionic permselectivity, with a reduction of 44%, both associated with higher electrolyte content into the pores of the Ox-NPAM/TiO2 membrane. Figure4 shows a comparison of experimental and fitted values as well as Appl. Sci. 2021, 11, 5687 7 of 16

the separate contribution of Donnan and diffusion potentials as a function of variable concentration Cv for Sf-NPAM/TiO2 and Ox-NPAM/TiO2 membranes, where the agreement between experimental and theoretical values support the suitability of these results. On the other hand, a correlation between effective ﬁxed charge concentration (Xef) and surface charge density (σs) for cylindrical pores was already proposed by the following expression [49]: σs = (Xef.F.rp/2), where F represents Faraday constant. Surface charge 2 density values for the studied membranes were 7.2 mC/m for the Sf-NPAM/TiO2 sample 2 and 7.5 mC/m for the Ox-NPAM/TiO2 sample. Because the electrical charge exhibited by most membranes in contact with aqueous solutions (polar medium) corresponds to Appl. Sci. 2021, 11, x FOR PEER REVIEWthe adsorption of charged species from solution on the pore walls and/or dissociation8 of 17

of speciﬁc ionic groups, the similarity obtained for surface charge density values seems to be in concordance with the similitude in membrane surface material determined from XPS analysis.

40 (a) 20 (mV)

mbr mbr 0 ΔΦ

-20 Donnan potential

-40 diffusion potential

-60 0.00 0.02 0.04 0.06 0.08 0.10 C (M) v

40 (b) 20 (mV) mbr

ΔΦ 0

Donnan potential

-20 diffusion potential

-40 0,00 0,02 0,04 0,06 0,08 0,10 C (M) v

Figure 4. Membrane potential as a function of variable solution Cv.(a) Sf-NPAM/TiO2 membrane ( ) experimental values and (b) Ox-NPAM/TiO membrane ( ) experimental values, ﬁtted values Figure 4. Membrane potential as a function of variable2 solution♦ Cv. (a) Sf-NPAM/TiO2 membrane (solid line), diffusion potential contribution (dashed line) and Donnan potential contribution (dashed (□) experimental values and (b) Ox-NPAM/TiO2 membrane (◊) experimental values, fitted values (soliddot line).line), Fitteddiffusion values: potential (a)Xef = contribution +0.015 M, tCl −(dashed/tNa+ = line) 3.74; and (b)X Donnanef = +0.012 potential M, tCl−/t contributionNa+ = 2.47. (dashed dot line). Fitted values: (a) Xef = +0.015 M, tCl−/tNa+ = 3.74; (b) Xef = +0.012 M, tCl−/tNa+ = 2.47.

Another electrochemical technique used for characterization of membranes in working conditions is electrochemical impedance spectroscopy (EIS). This technique gives quantitative and/or qualitative information related to charge movement or charge adsorption by means of the electrical resistance or capacitance (equivalent capacitance in case of non-homogeneous systems [39]) respectively, which can be significantly affected by membrane structure and material characteristics [50,51]. Qualitative information is obtained by comparing impedance diagrams for membranes from the same material but different geometry, while characteristic parameter values are estimated by the fitting of experimental data to adequate equivalent circuits [39,40]. A comparison of impedance diagrams for clean Sf-NPAM/TiO2 and Ox-NPAM/TiO2 membranes measured in the system: electrode//electrolyte solution (C)/membrane/electrolyte solution (C)//electrode, with C = 0.002 M NaCl, is shown in Figure 5. The Nyquist plot (−Zimg vs. Zreal) shows significant differences between both membranes, because the impedance diagram for Sf-NPAM/TiO2 sample exhibits two differentiated relaxations that correspond to the separated contributions of membrane (m) and electrolyte solution (e) (see insert in Figure 5 where this part Appl. Sci. 2021, 11, 5687 8 of 16

Another electrochemical technique used for characterization of membranes in working conditions is electrochemical impedance spectroscopy (EIS). This technique gives quantitative and/or qualitative information related to charge movement or charge adsorption by means of the electrical resistance or capacitance (equivalent capacitance in case of non-homogeneous systems [39]) respectively, which can be signiﬁcantly affected by membrane structure and material characteristics [50,51]. Qualitative information is obtained by comparing impedance diagrams for membranes from the same material but different geometry, while characteristic parameter values are estimated by the ﬁtting of experimental data to adequate equivalent circuits [39,40]. A comparison of impedance diagrams for clean Sf-NPAM/TiO2 and Ox-NPAM/TiO2 membranes measured in the sys- Appl. Sci. 2021, 11, x FOR PEER REVIEWtem: electrode//electrolyte solution (C)/membrane/electrolyte solution (C)//electrode,9 of 17

with C = 0.002 M NaCl, is shown in Figure5. The Nyquist plot ( −Zimg vs. Zreal) shows significant differences between both membranes, because the impedance diagram for Sf-NPAM/TiO sample exhibits two differentiated relaxations that correspond to the sep- of the diagram is 2enlarged), while a unique relaxation process is observed in the diagram arated contributions of membrane (m) and electrolyte solution (e) (see insert in Figure5 for the Ox-NPAM/TiO2 sample, which corresponds to the “membrane plus electrolyte” where this part of the diagram is enlarged), while a unique relaxation process is observed contribution. As already reported, differences in impedance diagrams can depend on both in the diagram for the Ox-NPAM/TiO sample, which corresponds to the “membrane plus membrane structure/geometry and material2 hydrophilic/hydrophobic character, because electrolyte” contribution. As already reported, differences in impedance diagrams can membrane and electrolyte separated impedance contributions have been obtained for depend on both membrane structure/geometry and material hydrophilic/hydrophobic compositecharacter, reverse because osmosis membrane and even and nanofiltra electrolytetion separated membranes impedance with a two-layer contributions structure have (a denserbeen obtained thin active for layer composite and a reverseporous osmosissupport)and [48,52,53], even nanofiltration while only a relaxation membranes pro- with cessa two-layerwas observed structure for wider (a denser pore thinsize activeultrafiltration/microfiltrat layer and a porous support)ion membranes [48,52,53 [54,55]], while andonly also a for relaxation nanoporous process regenerated was observed cellulose for wider membranes pore size (2 ultrafiltration/microfiltrationkDa cut off, which should correspondmembranes to a [ 54pore,55 ]size and ~ also3.0 nm) for nanoporousassociated with regenerated high water cellulose (aqueous membranes solution) (2adsorp- kDa cut tionoff, due which to the should hydrophilic correspond character to a pore of this size membrane ~ 3.0 nm) associatedmaterial [48]. with On high the water other(aqueous hand, thesolution) analysis adsorptionof impedance due diagrams to the hydrophilic allows us characterto obtain the of this following membrane values material for the [ 48elec-]. On tricalthe resistance other hand, of theSf-NPAM/TiO analysis of2 impedanceand Ox-NPAM/TiO diagrams2 allowsmembranes: us to obtainRSf-NPAM/TiO2 the following= 4510 ohm and ROx-NPAM/TiO2 = 1665 ohm, although this latter value was determined by subtrac- values for the electrical resistance of Sf-NPAM/TiO2 and Ox-NPAM/TiO2 membranes: tionR Sf-NPAM/TiO2of electrolyte= electrical 4510 ohm resistance and ROx-NPAM/TiO2 (Re) from =that 1665 determined ohm, although for the this Ox-NPAM/TO latter value was2 membranedetermined plus by electrolyte subtraction system of electrolyte (Rme); hence: electrical (Rm = resistanceRme − Re), that (Re) is, from Rm thatwas determinedobtained fromfor two the Ox-NPAM/TOdifferent measurements2 membrane that plus may electrolyte slightly affect system the (R realme );value. hence: In (R anym = case, Rme due− R e), to thethat similar is, Rm wasmaterial obtained of both from NPAM+TO two different2 samples, measurements EIS analysis that mayfor clean slightly membranes affect the is real concordantvalue. In with any case,that duepreviously to the similarperformed material by membrane of both NPAM+TO potential2 resultssamples, with EIS respect analysis to porefor clean radii membranes influence. is concordant with that previously performed by membrane potential results with respect to pore radii influence.

2x104

6000 ) ) Ω ( Ω img ( - Z - 3000 img

m - Z

4 1x10 0 6000 9000 12000 Z (Ω) real

if ω m e 0 0 1x104 Z (Ω) 2x104 real

Figure 5. Nyquist plots for () Sf-NPAM/TiO2 membrane and ( ♦) Ox-NPAM/TiO2 membrane. (e) Electrolyte solution; (m) membrane; (if) interface; ω: angular frequency and values increase tendency. Figure 5. Nyquist plots for (□) Sf-NPAM/TiO2 membrane and (◊) Ox-NPAM/TiO2 membrane. (e) Electrolyte solution; (m) membrane; (if) interface; ω: angular frequency and values increase tendency.

The BSA fouling effect on membrane potentials for the Ox-NPAM/TiO2(f) sample is shown in Figure 6a where, for comparison, ∆Φmbr values for the clean Ox-NPAM/TiO2 are also indicated. Both membranes show rather similar values at low concentrations (interfacial or Donnan potential contribution) but differ at high concentrations, which is an indication of differences in ion diffusion between both samples that might be caused by pore size reduction due to BSA deposition on pore walls (although surface deposition might also exist). Fitted values of characteristic parameters were Xef = +0.014 M, DCl−/DNa− = 5.4, and PCl− = 58.2%, as these two latter values were much higher than those determined for the clean membrane. Figure 6b shows the comparison between experimental and fitted values depending on variable solution Cv, as well as the separate contributions of Appl. Sci. 2021, 11, 5687 9 of 16

tion might also exist). Fitted values of characteristic parameters were Xef = +0.014 M, DCl−/DNa− = 5.4, and PCl− = 58.2%, as these two latter values were much higher than those determined for the clean membrane. Figure6b shows the comparison between diffusion and Donnan potentials; this second contribution does not differ significantly experimental and ﬁtted values depending on variable solution Cv, as well as the separate fromcontributions that in Figure of diffusion 3b for andclean Donnan membrane potentials; at high this C secondv values. contribution does not differ signiﬁcantly from that in Figure3b for clean membrane at high C v values.

40 (a) 20 (mV) mbr

ΔΦ 0

-20

-40

-60 -2 -1 0 1 2 3 ln(C /C) v f

40 (b) 20

(mV) 0

mbr Donnan potential

ΔΦ -20 diffusion potential

-40

-60 0.00 0.02 0.04 0.06 0.08 0.10 C (M) v FigureFigure 6. 6. (a)( a)Comparison Comparison of of membrane potentialpotential with with solution solution concentration concentration ratio ratio for Ox- for Ox- o NPAM/TiONPAM/TiO2 (2◊)( ♦and) and Ox-NPAM/TiO Ox-NPAM/TiO2(f)2(f) (♦ ( )) membranes.membranes. ((bb)) ComparisonComparison of of experimental experimental and and cal- culatedcalculated (solid (solid line) line) values, values, Donnan Donnan (dashed-dot (dashed-dot line),line), and and diffusion diffusion (dashed (dashed line) line) potential potential contri- contri- butionsbutions for for Ox-NPAM/TiO Ox-NPAM/TiO2(f)(f) membrane.

However, the protein fouling effect or mechanism exhibited by the Sf-NPAM/TiO2(f) membrane seems to be different according to results shown in Figure 7a, where a similar concentration ratio dependence for fouled and clean samples can be observed, but the results obtained for the Sf-NPAM/TiO2(f) membrane present an almost constant shift to lower ∆Φmbr values. This result could indicate the presence of a BSA layer on the membrane surface, rather than pore-wall deposition (which could be related with a “static” fouling nature because any hydrostatic pressure is applied to the BSA solution), giving rise to a kind of “composite” or two-layer membrane, as can be deduced from results shown in Figure 6b, where ∆Φmbr values for the fouled sample after subtraction of that determined at Cv = Cf = 0.01 M are compared with those determined for the clean Sf- NPAM/TiO2 membrane, and only small differences exist between them. Figure 7b also shows the data points obtained when ∆Φmbr values for the clean membrane are subtracted from those corresponding to the fouled sample. In this context, the difficulty of obtaining quantitative information from these results should be pointed out because they do not Appl. Sci. 2021, 11, 5687 10 of 16

However, the protein fouling effect or mechanism exhibited by the Sf-NPAM/TiO2(f) membrane seems to be different according to results shown in Figure7a, where a similar concentration ratio dependence for fouled and clean samples can be observed, but the results obtained for the Sf-NPAM/TiO2(f) membrane present an almost constant shift to lower ∆Φmbr values. This result could indicate the presence of a BSA layer on the membrane surface, rather than pore-wall deposition (which could be related with a “static” fouling nature because any hydrostatic pressure is applied to the BSA solution), giving rise to a kind of “composite” or two-layer membrane, as can be deduced from results shown in Figure6b, where ∆Φmbr values for the fouled sample after subtraction of that determined at Cv = C = 0.01 M are compared with those determined for the clean Sf-NPAM/TiO2 Appl. Sci. 2021, 11, x FOR PEER REVIEW f 11 of 17 membrane, and only small differences exist between them. Figure7b also shows the data points obtained when ∆Φmbr values for the clean membrane are subtracted from those corresponding to the fouled sample. In this context, the difﬁculty of obtaining quantitative correspondinformation to fromsimilar these interfacial results shouldsituations be, pointedand a compact out because BSA theylayer do on not membrane correspond sur- to facesimilar may also interfacial affect the situations, concentration and a gradients. compact BSA layer on membrane surface may also affect the concentration gradients.

50 (a) 25 (mV) mbr

ΔΦ 0

-25

-50

-75 -2 -1 0 1 2 ln(C /C) v f

40 (b) 20 (mV)

mbr 0 ΔΦ

-20

-40

-60 -2 -1 0 1 2 3 ln(C /C) v f

Figure 7. (a) Comparison of membrane potential with solution concentration ratio for Ox- FigureNPAM/TiO 7. (a) Comparison2 (♦) and Ox-NPAM/TiO of membrane potential2(f) (o) membranes.with solution (concentrationb) Comparison ratio of for experimental Ox- and NPAM/TiOcalculated2 (solid(◊) and line) Ox-NPAM/TiO values, Donnan2(f) ( (dashed-dot♦) membranes. line), ( andb) Comparison diffusion (dashed of experimental line) potential () and contri-

calculatedbutions for(solid Ox-NPAM/TiO line) values, 2Donnan(f) membrane. (dashed-dot line), and diffusion (dashed line) potential contributions for Ox-NPAM/TiO2(f) membrane.

EIS measurements have also been used in the study of porous membrane fouling by comparing impedance plots obtained with clean and fouled samples [56,57], as shown in Figure 8 for Ox-NPAM/TiO2 and Ox-NPAM/TiO2(f) membranes. Analysis of these results has permitted the estimation of around 10% increase in the electrical resistance of the fouled sample. However, EIS measurements of the Sf-NPAM/TiO2(f) membrane have not been performed due to enormous data fluctuations, which were attributed to BSA deposited layer instability associated with a cyclic alternating current effect. Appl. Sci. 2021, 11, 5687 11 of 16

EIS measurements have also been used in the study of porous membrane fouling by comparing impedance plots obtained with clean and fouled samples [56,57], as shown in Figure8 for Ox-NPAM/TiO 2 and Ox-NPAM/TiO2(f) membranes. Analysis of these Appl. Sci. 2021, 11, x FOR PEER REVIEWresults has permitted the estimation of around 10% increase in the electrical resistance12 of 17 of the fouled sample. However, EIS measurements of the Sf-NPAM/TiO2(f) membrane have not been performed due to enormous data ﬂuctuations, which were attributed to BSA deposited layer instability associated with a cyclic alternating current effect.

1,2x104

8,0x103 ) Ω ( img

- Z - 4,0x103

0,0 0,0 4,0x103 8,0x103 1,2x104 Z (Ω) real

Figure 8. Comparison of Nyquist plot for: (♦) Ox-NPAM/TiO2 membrane and (o) Ox-NPAM/TiO2(f) membrane (C = 0.002 M NaCl). Figure 8. Comparison of Nyquist plot for: (◊) Ox-NPAM/TiO2 membrane and (♦) Ox- NPAM/TiO3.2. Optical2(f)Characterization membrane (C = 0.002 M NaCl). Optical techniques (light transmission and spectroscopic ellipsometry), which are of 3.2.great Optical interest Characterization due to their non-destructive character, have already demonstrated their suit- abilityOptical for characterization techniques (light of nanoporous transmission alumina and membranesspectroscopic with ellipsometry), different pore sizes which and are of greatporosity interest (or due even to asymmetric their non-destructive structure) or humancharacter, serum have albumin already adsorption demonstrated on alumina their suit- abilitysupports for characterization [35,58]. In this context, of nanoporous depending on alumina geometrical membranes parameters with (pore-size/porosity) different pore sizes andor porosity surface material, (or even differences asymmetric in lightstructure) transmission or human through serum nanoporous albumin alumina-basedadsorption on alu- membranes are expected. In fact, differences in transmission spectra through membranes mina supports [35,58]. In this context, depending on geometrical parameters (pore- obtained by coating Sf-NPAM or Ox-NPAM samples with a layer of different metal ox- size/porosity)ides have already or surface been analyzedmaterial, [20 differences,22]. Figure in9a showslight transmission a comparison through of transmission nanoporous alumina-based membranes are expected. In fact, differences in transmission spectra spectra for two samples obtained by coating the Sf-NPAM support with a layer of Fe2O3 throughor Al2 Omembranes3 (Sf-NPAM/Fe obtained2O3 or Sf-NPAM/Alby coating Sf-NPAM2O3 samples, or Ox-NPAM with similar samples pore-size, with porosity, a layer of differentand thickness metal oxides values thanhave Sf-NPAM/TiO already been2 ),analyzed and clear [20,22]. differences Figure in the 9a visible shows region a comparison can of betransmission observed due spectra to the for different two samples surface material obtained (although by coating both the membranes Sf-NPAM show support high with a layerand similarof Fe2O transparency3 or Al2O3 (Sf-NPAM/Fe in the near infrared2O3 or region); Sf-NPAM/Al moreover,2O3 transmissionsamples, with for similar another pore- membrane, obtained by coating the Ox-NPAM support with a SiO layer (Ox-NPAM/SiO size, porosity, and thickness values than Sf-NPAM/TiO2), and2 clear differences in the2 vis- sample), of different pore-size, porosity, and surface material than the Sf-NPAM/Fe O ible region can be observed due to the different surface material (although both2 3mem- and Sf-NPAM/Al2O3 samples, was also measured, and transmission differences in both branesvisible show and nearhigh infraredand similar regions transparency were obtained. in the These near previous infrared results region); seem moreover, to support trans- missionthe possibility for another of using membrane, light transmission obtained spectra by coating for changes the Ox-NPAM associated withsupport membrane with a SiO2 layerfouling (Ox-NPAM/SiO for this kind of2 sample), sample. of different pore-size, porosity, and surface material than the Sf-NPAM/Fe2O3 and Sf-NPAM/Al2O3 samples, was also measured, and transmission differences in both visible and near infrared regions were obtained. These previous results seem to support the possibility of using light transmission spectra for changes associated with membrane fouling for this kind of sample. Then, optical characterization of both clean membranes was firstly considered and Figure 9b shows a comparison of the transmittance spectra, where the high transparency of both membranes (>70% in the visible region) and around 88% for near infrared region (NIR) can be seen. The similarity in light transmission exhibited by both membranes is significant, taking into account that they have different geometrical parameters. This may be due to a kind of pore-size/porosity compensation, as the sample with lower pore radii presents higher porosity and the free volume of both membranes is rather similar (Ox- NPAM/TiO2/Sf-NPAM/TiO2 free volume ratio = 1.13), as indirectly established by the similarity of hydraulic permeability values already indicated. On the other hand, transmittance analysis also provides an indirect confirmation of the adequate TiO2-coverage of the nanoporous alumina support by the estimation of membrane band gap values, which are between 310 nm and 323 nm and agree with reported values for TiO2 [59], differing significantly from the band gap for Al2O3 materials (around 200 nm [60], and logically from Appl. Sci. 2021, 11, x FOR PEER REVIEW 13 of 17

Fe2O3 as can be seen in Figure 9a); this point agrees with the almost total TiO2-coverage of the alumina support determined by the XPS technique [36] already mentioned (Table S1 Appl. Sci. 2021, 11, 5687 12 of 16 in Supplementary Information).

100 100 (a) (b)

75 75

50 50 T (%)

25 Ox-NPAM+SiO2 Transmission (%) 25

Transmission (%) Transmission Sf-NPAM+Al O 70 2 3 400 600 800 1000 Sf-NPAM+Fe O wavelength (nm) 2 3 0 0 0 500 1000 1500 2000 0 500 1000 1500 2000 wavelength (nm) wavelength (nm)

Figure 9. Light transmission spectra for (a) Sf-NPAM/Al2O3 (solid gray line), Sf-NPAM+Fe2O3 (solid red line), and Ox-NPAM/SiO2 (solidFigure blue line) 9. Light samples; transmission (b) Sf-NPAM/TiO spectra2 for(solid (a) Sf-NPAM/Al black line) and2O Ox-NPAM/TiO3 (solid gray line),2 (dashed Sf-NPAM+Fe black 2O3 line) samples. (solid red line), and Ox-NPAM/SiO2 (solid blue line) samples; (b) Sf-NPAM/TiO2 (solid black line) and Ox-NPAM/TiO2 (dashed black line) samples. Then, optical characterization of both clean membranes was firstly considered and FigureThe9 effectb shows of amembrane comparison BSA of the fouling transmittance on light spectra, transmission where the spectra high transparency can be observed of both membranes (>70% in the visible region) and around 88% for near infrared region in Figure 10. In the case of the Ox-NPAM/TiO2(f) membrane (Figure 10a), a certain wave- (NIR) can be seen. The similarity in light transmission exhibited by both membranes length-dependentis significant, taking reduction into account in the that visible they haveregion different was determined geometrical (9.0% parameters. at 600 Thisnm), but almostmay beno duechange to a kindis observed of pore-size/porosity in the near infr compensation,ared region. as This the samplesmall effect with lower of BSA pore fouling on radiilight presentstransmission higher might porosity support and the protein free volume deposition of both on membranes pore walls is as rather the main similar fouling mechanism(Ox-NPAM/TiO determined2/Sf-NPAM/TiO from electrochemical2 free volume measurements, ratio = 1.13), as indirectlydue to the established reduced free by vol- umethe of similarity studied ofmembranes hydraulic permeabilityand taking into values account already the indicated. high transparency On the other exhibited hand, by theirtransmittance solid structure. analysis However, also provides an important an indirect reduction confirmation in of light the adequatetransmission TiO2-coverage for the whole of the nanoporous alumina support by the estimation of membrane band gap values, which wavelength range can be observed in Figure 10b for the Sf-NPAM/TiO2(f) sample when are between 310 nm and 323 nm and agree with reported values for TiO2 [59], differing compared with the Sf-NPAM/TiO2 sample; in particular, a reduction of around 18.0% at significantly from the band gap for Al O materials (around 200 nm [60], and logically from 600 nm and of 7.0% in the NIR (at 16002 3nm) were obtained. This reduction in transmission Fe2O3 as can be seen in Figure9a); this point agrees with the almost total TiO 2-coverage of throughthe alumina the fouled support membrane determined could by the be XPS an indica techniquetion [36 of] the already presence mentioned of a BSA (Table layer S1 in on the membraneSupplementary surface, Information). which would modify the high transparency of membrane material in- dicatedThe above; effect this of membranefact would BSA be foulingin agreem on lightent with transmission membrane spectra potential can be analysis observed previ- ouslyin Figure performed. 10. In Cons the caseequently, of the light Ox-NPAM/TiO transmission2(f) membranemeasurements (Figure for 10 membranesa), a certain made of wavelength-dependenthighly transparent materials reduction could in the be visible used as region a way was to determined determine (9.0% fouling at 600 mechanisms nm), by butcomparing almost no spectra change for is observedclean and in fouled the near samples. infrared region. This small effect of BSA fouling on light transmission might support protein deposition on pore walls as the main fouling mechanism determined from electrochemical measurements, due to the reduced free volume of studied membranes and taking into account the high transparency exhibited by their solid structure. However, an important reduction in light transmission for the whole wavelength range can be observed in Figure 10b for the Sf-NPAM/TiO2(f) sample when compared with the Sf-NPAM/TiO2 sample; in particular, a reduction of around 18.0% at 600 nm and of 7.0% in the NIR (at 1600 nm) were obtained. This reduction in transmission through the fouled membrane could be an indication of the presence of a BSA layer on the membrane surface, which would modify the high transparency of membrane material indicated above; this fact would be in agreement with membrane potential analysis previously performed. Consequently, light transmission measurements for membranes made of highly transparent materials could be used as a way to determine fouling mechanisms by comparing spectra for clean and fouled samples. Appl. Sci. 2021, 11, x FOR PEER REVIEW 14 of 17

Appl. Sci. 2021, 11, x FOR PEER REVIEW 14 of 17

Appl. Sci. 2021, 11, 5687 100 100 13 of 16 (a) (b)

75 75 100 100 (a) (b) 50 50 75 75 Transmission (%) Transmission

25 Transmission (%) 25 50 50

0 0 Transmission (%) Transmission 0 500 1000 1500 2000 0 500 1000 1500 2000

25 Transmission (%) 25 wavelength (nm) wavelength (nm)

0 0 0Figure 500 10. 1000 Comparison 1500 of 2000light transmission0 spectra 500 for ( 1000a) clean Ox-NPAM/TiO 1500 2000 2 (dashed line) and fouledwavelength Ox-NPAM/TiO (nm) 2(f) (solid line) membranes; (wavelengthb) clean Sf-NPAM/TiO (nm) 2 (dashed dot line) and fouled Sf-NPAM/TiO2(f) (solid line) membranes. Figure 10. Comparison of light transmission spectra for (a) clean Ox-NPAM/TiO2 (dashed line) and fouled Ox- NPAM/TiOFigure(f) (solid10. Comparison line) membranes; of light (b )transmission clean Sf-NPAM/TiO spectra for(dashed (a) clean dot line)Ox-NPAM/TiO and fouled Sf-NPAM/TiO2 (dashed line)(f) (solid 2 Reflection spectra (R%) for clean2 and fouled membranes were also measured,2 and the line) membranes.and fouled Ox-NPAM/TiO2(f) (solid line) membranes; (b) clean Sf-NPAM/TiO2 (dashed dot line) and fouled Sf-NPAM/TiOcorresponding2(f) (solid absorption line) membranes. curve (A%) was obtained: A (%) = 100 − T (%) − R (%). Figure 11 showsReﬂection a comparison spectra (R%) of wavelength for clean and depend fouled membranesence of absorption were also measured,spectra for and both the BSA fouledcorresponding samples; absorptionfor comparison, curve (A%) absorption was obtained: spectra A (%) for = both 100 − cleanT (%) −samplesR (%). Figure are shown 11 in Reflection spectrashows a (R%) comparison for clean of wavelengthand fouled dependence membranes of were absorption also measured, spectra for and both the BSA fouled the inlet (for wavelengths between 500 nm and 2000 nm). Differences between both fouled corresponding absorptionsamples; for curve comparison, (A%) was absorption obtained: spectra A (%) forboth = 100 clean − T samples(%) − R are (%). shown Figure in the inlet 11 shows a comparisonsamples(for wavelengths can of be wavelength observed between in 500depend the nm visible andence 2000 region, of nm). absorption with Differences a significant spectra between for adsorption bothboth fouled BSA reduction, samples but fouled samples;morecan for closely becomparison, observed similar in absorption theand visible constantregion, spectra values with for were aboth signiﬁcant obtained clean samples adsorption for both are clean reduction, shown samples. in but more the inlet (for wavelengthsclosely similar between and constant 500 nm values and 2000 were nm). obtained Differences for both between clean samples. both fouled samples can be observed in the visible region, with a significant adsorption reduction, but more closely similar20 and constant values were obtained for both clean samples.

20 10

Adsorption (%) wavelength (nm) 0 500 1000 1500 2000 10

10 (%) Adsorption

Adsorption (%) wavelength (nm) 0 500 1000 1500 2000 0

Adsorption (%) Adsorption 500 1000 1500 2000 wavelength (nm)

0 Figure 11. Comparison of light absorption spectra for Sf-NPAM/TiO 2 fouled membrane (solid line) 500 1000 1500 2000 Figureand Ox-NPAM/TiO11. Comparison2 fouledof light membrane absorption (solid spectra line). for Insert: Sf-NPAM/TiO comparison2 fouled of absorption membrane spectra (solid for line) andSf-NPAM Ox-NPAM/TiOwavelength (solid line)2 fouled and (nm) Sf-NPAM membrane (dashed (solid line) line). samples. Insert: comparison of absorption spectra for Sf- NPAM4. Conclusions (solid line) and Sf-NPAM (dashed line) samples. Figure 11. ComparisonThe of light inﬂuence absorption of pore spectra radius for on Sf-NPAM/TiO ion transport2 fouled through membrane nanoporous (solid alumina-based line) 4. Conclusions and Ox-NPAM/TiOmembranes2 fouled membrane with almost (solid ideal line). porous Insert: structure comparison (TiO of2-coated absorption alumina spectra nanoporous for Sf- sup- NPAM (solid line)ports andThe Sf-NPAM with influence different (dashed of porepore line) radii radius samples. and on porosity) ion transport was analyzed through by membranenanoporous potential alumina-based and membraneselectrochemical with impedancealmost ideal spectroscopy porous structure measurements. (TiO2-coated These resultsalumina show nanoporous the signiﬁ- sup- 4. Conclusionsports cant with effect different of pore size.pore Moreover, radii and both porosity) electrochemical was analyzed techniques by membrane can also be potential used for and quantitative/qualitative information on membrane fouling dependent on pore size: a The influenceelectrochemical of pore radius impedance on ion spectroscopy transport through measurements. nanoporous These alumina-based results show the signifi- cantBSA effect layer of on pore the membranesize. Moreover, surface both for the elec sampletrochemical with lower techniques pore radii, can but also pore-wall be used for membranes withdeposition almost ideal in the porous case of structure the membrane (TiO with2-coated wider alumina pore size. nanoporous On the other sup- hand, the quantitative/qualitative information on membrane fouling dependent on pore size: a BSA ports with differenthigh transparencypore radii and of a porosity) membrane was material analyzed enables by the membrane use of a common potential optical and technique, electrochemicallayer impedance on the membrane spectroscopy surface meas forurements. the samp Thesele with results lower show pore the radii,signifi- but pore-wall cant effect of pore size. Moreover, both electrochemical techniques can also be used for quantitative/qualitative information on membrane fouling dependent on pore size: a BSA layer on the membrane surface for the sample with lower pore radii, but pore-wall Appl. Sci. 2021, 11, 5687 14 of 16

light transmission/reflection, to obtain information on the fouling effect or mechanism. Optical results appear to confirm the findings of electrochemical analysis and also provide information on other membrane material characteristics.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/app11125687/s1. Figure S1: SEM micrograph of: (a) Sf-NPAM membrane surface and (b) Ox-NPAM membrane surface. Figure S2: Scheme of electrochemical test-cell. Figure S3: Membrane potential vs. KCl solutions concentration ratio for samples: Sf-NPAM, Ox-NPAM, Sf-NPAM+TiO2 and Ox-NPAM + TiO2 membranes, ideal anion-exchange membrane (solid line, tCl− = 1). Table S1: Atomic concentration percentages of the elements found on both surfaces of the studied samples Author Contributions: L.G. and J.B. performed electrochemical characterizations and A.L.C. was responsible for optical characterization. Experiments were coordinated by J.B. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Acknowledgments: We thank V.M. de la Prida and V. Vega, University of Oviedo, Spain, for kindly supplying studied samples. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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