Characterization of isolated polyamide thin films of RO and NF membranes using novel TEM techniques

Journal of Membrane Science 358 (2010) 51–59

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Characterization of isolated polyamide thin films of RO and NF membranes using novel TEM techniques Federico A. Pacheco a,∗ , Ingo Pinnau b , Martin Reinhard a , James O. Leckie a a Department of Civil and Environmental Engineering, Stanford University, Jerry Yang & Akiko Yamazaki Environment & Energy Building Room 261, 473 Via Ortega, Stanford, CA 94305-4020, United States b Membranes Research Center, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 10 December 2009 Received in revised form 17 April 2010 Accepted 19 April 2010 Available online 18 May 2010 Keywords: Polyamide thin film Electron microscopy TEM Projected area TEM Reverse osmosis

a b s t r a c t Achieving a better understanding of transport and rejection mechanisms in RO and NF membranes requires more detailed information of the nanostructure of polyamide thin films. This study reports on two novel transmission electron microscopy (TEM) techniques for characterizing polyamide nanostructure. The first technique produces cross-sectional images of isolated polyamide thin films by removing the polysulfone support from regular TEM cross-sections. In the second technique called “projected area” TEM (PA-TEM), isolated polyamide thin films are placed with their surface perpendicular to the electron beam. The resulting images capture the thickness, morphology and mass density of the entire thin film. In combination, these new techniques provide information on polyamide nanostructure that is not evident using conventional methods. For the commercial RO membrane ESPA3, the cross-sectional view of the isolated polyamide thin film shows a 30–60 nm thick base of nodular polyamide (presumably the separation barrier) that forms a relatively smooth interface with the polysulfone support. Above this, a more open structure of loose polyamide extends outward giving rise to the ridge-and-valley surface structure. In PA-TEM images, the ridges and valleys correspond to the dark and bright regions, respectively; the polyamide nodular base appears as round features forming an irregular honeycomb pattern throughout the images. Membrane cross-sections were prepared with a simple resin embedding protocol using the acrylic resin LR White. The protocol did not require dehydration steps, and was applicable to both dry and wet membrane samples. Artifacts that may be produced during sample preparation were also documented. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Reverse osmosis (RO) and nanofiltration (NF) membrane performance is determined by the properties of polyamide thin films. These films are less than 500 nm thick and formed directly at the surface of a polysulfone support layer by interfacial polymerization (IP) of monomeric acid chlorides and amines [1]. The search for membranes with improved separation and better foulingresistance requires detailed understanding of the function of the separation-active layer. Consequently, there is a critical need for methods that reveal the connection between the nanostructure of polyamide thin films and their transport and rejection properties [2]. Advances in the characterization of polyamide thin films have been primarily hampered by the difficulty of inspecting thin films in isolation (i.e., separated from the polysulfone support).

∗ Corresponding author. Tel.: +1 650 723 2524; fax: +1 650 725 3164. E-mail address: [email protected] (F.A. Pacheco). 0376-7388/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2010.04.032

Transmission electron microscopy (TEM) is considered the best available imaging technique for studying the nanostructure of thin films in RO and NF membranes [2]. Although TEM has been utilized in membrane research for over 25 years, its use has been mostly limited to imaging membrane cross-sections and for estimating thin film thickness [3–7]. Few studies have relied on TEM for more advanced structural investigations of membrane thin films. Bartels et al. obtained unique information about the physical and chemical composition of the polyurea thin film of an RO membrane using heavy metal staining on TEM cross-sections [8,9]. Similarly, Freger employed selective staining methods to identify different polyamide regions within RO and NF membrane thin films [10]. Tang et al. examined cross-sections of humic acid fouled RO membranes and correlated flux decline with the density and thickness of the fouling layers [11]. The contrast between the fouling layers and the membrane components revealed the presence of surface coatings on some commercial membranes [12]. Other studies used TEM to inspect polyamide thin films modified by polymer grafting [13], strong acid treatment [14] and the incorporation of nanoparticles [15]. The pore microstructure of the separation layers in

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non-polyamide based RO and NF membranes has also been characterized with TEM [16,17]. TEM has been used almost exclusively to examine membrane cross-sections. Sarada et al. studied surface micropores in a polypropylene microfiltration membrane by sectioning the membrane from the surface rather than from the edge [18]. However, this approach is only suitable for smooth symmetric membranes because sectioning RO and NF thin films from the surface is not possible. Sundet used a metal-shadowing procedure to create a platinum/carbon replica of the polyamide thin film surface of an RO membrane that could be examined with TEM [19]. Although this complicated technique may offer good resolution of surface details [20], scanning electron microscopy (SEM) provides better results and is widely preferred for surface analysis. Lack of simple and straightforward sample preparation methods has hindered a more widespread use of TEM. To prepare TEM cross-sections, thin film composite RO and NF membranes must be embedded in a resin first. Epoxy resins are the most commonly used embedding media [20–22], and virtually all membrane crosssections reported in the literature were prepared with various epoxy resins: Epon 812 and variations thereof (e.g., Polybed 812, EMbed812, Eponate 12) [6,7,15–17], Spurr resin [18,23], Araldite 502 [10,13,14] and a combination of Epon 812 and Araldite 502 [8,9]. Notably, epoxy resins are water insoluble and specimens must be completely dehydrated before embedding. This is a significant disadvantage because additional preparation steps are inevitably required to dehydrate wet membranes used in filtration applications. Typical dehydration methods used include vacuum drying [10,13,16], and sequentially immersing membrane samples in increasingly concentrated ethanol–resin mixtures (from 0 to 100% resin) [4,11,23]. Here, we report on an alternative TEM preparation protocol that is based on the water-miscible acrylic resin LR White. Acrylic resins are preferred in biological applications where the goal is to preserve enzymatic activity and antigenicity of cells in their live aqueous environment [21]. Because LR White tolerates up to 12% of water by volume during polymerization [21], the protocol was applied identically to both dry unused and wet membrane samples without dehydration steps. Tang et al. prepared membrane cross-sections using LR White, but their protocols always included thorough dehydration sequences [4,11]. Besides simplifying sample preparation, eliminating dehydration steps reduces the chances of creating artifacts due to sample shrinkage. Dehydration can cause 5–70% shrinkage in biological specimens depending on the specimen, resin and dehydration method used [21,22]. Although the high cross-linking density of polyamide is expected to make RO and NF thin films less susceptible to shrinkage, dehydration effects are possible. Louie et al. measured significant changes in the gas permeation of RO membranes after drying samples previously exposed to water or butanol [24]. Similarly, Patterson et al. noticed differences in the microstructure of selective layers in solvent-resistant NF membranes after comparing dry and ethanol-soaked samples [17]. The extent of dehydration effects on polyamide thin films and whether these would be visually noticeable in TEM images is currently unknown. In this study we also present two novel TEM techniques aimed at examining the nanostructure of RO and NF polyamide thin films without the interference of the membrane support layers. Earlier studies have characterized isolated polyamide thin films using small angle X-ray scattering [19], atomic force microscopy [25] and various spectroscopy methods [26,27], but until now, electron microscopy techniques have not been reported. The goal of the first technique was to produce TEM crosssections of isolated polyamide thin films to accurately inspect the polyamide side that forms the interface with the polysulfone sup-

port. Because polyamide thin films are exceptionally difficult to handle after removing the polysulfone, cross-sections could not be obtained by applying a standard resin embedding protocol to isolated thin films. Instead, the polysulfone was removed after regular cross-sections had been prepared. TEM embedding resins are relatively permeable, and specimens are often stained with metal ions after embedding [20–22,28]. Therefore, we hypothesized that exposing regular TEM cross-sections to chloroform would dissolve the polysulfone support rendering cross-sections of the polyamide thin film only. The second technique is called “projected area TEM” (PA-TEM), and is based on the fact that in isolation, RO and NF polyamide thin films are thin enough to transmit electrons and therefore, visible in TEM. By placing isolated polyamide thin films perpendicular to the electron beam, the whole three-dimensional structure of the film is projected onto the image plane. Consequently, the resulting images capture the relative thickness, morphology and mass density of the entire thin film, making PA-TEM an ideal technique for investigating polyamide nanostructure. Notably, sample preparation for PA-TEM does not require embedding in a resin. Preparation consists of removing the polysulfone support with chloroform and mounting the specimen directly on the TEM grid. The motivation for this study was to develop, test and document new techniques for examining the polyamide nanostructure of RO and NF membranes using TEM. These characterization techniques may contribute to better understand how polyamide thin films determine the transport and rejection properties of RO and NF membranes. 2. Experimental 2.1. Chemicals and membranes LR White resin and ACS grade chloroform were obtained from Polysciences (Warrington, PA) and Mallinckrodt Baker (Phillipsburg, NJ), respectively. Two types of commercial thin film composite membranes were studied: ESPA3 (RO) from Hydranautics (Oceanside, CA) and NF270 (NF) from Dow FilmTec (Minneapolis, MN). The thin film of ESPA3 consists of a fully aromatic polyamide formed from the IP reaction of m-phenyldiamine (MPD) and trimesoyl chloride (TMC), while NF270 has a semiaromatic polyamide thin film made from piperazine and TMC. Both membranes have a polysulfone support and are backed by a nonwoven polyester fabric. Electron microscopy images were obtained from dry unused ESPA3 and NF270 samples, as well as from wet ESPA3 samples containing gold nanoparticles in the polyamide thin film. To prepare the wet ESPA3 samples, membrane coupons with an active area of about 1 cm2 were soaked in DI water before filtration for at least 24 h. Gold nanoparticles were deposited by filtering small volumes (3–5 mL) of either 10 nm or 30 nm gold nanoparticle suspensions in deionized (DI) water (Ted Pella, Redding, CA) at 5 bar in a dead-end Amicon 8003 filtration cell (Millipore, Billerica, MA) without stirring. 2.2. Transmission electron microscopy (TEM) Except where specified, all images were acquired with a Jeol 1230 TEM at an accelerating voltage of 80 kV using Gatan CCD cameras, either a cooled 967 slow-scan or a high-resolution Orius SC1000A. Polyamide film thickness and the size of nanostructure film features were measured directly from the images using the scale bars and the ruler tool in Adobe Photoshop. Reported values correspond to the averages of multiple measurements obtained from various images.

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2.2.1. TEM cross-sections of RO and NF membranes The polyester backing was carefully peeled off with a scalpel before immersing membrane samples in two changes of LR White resin, the first for 2 h and the second for at least 24 h. Samples were then placed into gelatin capsules completely filled with fresh LR White to exclude oxygen and cured overnight at 60 ◦ C. Crosssections (approximately 70–90 nm thick) were prepared using a Leica Ultracut S ultramicrotome with a diamond knife and mounted onto carbon/formvar (polyvinyl formal) coated copper grids. Several cross-sections of the same sample were placed on each grid. No heavy metal staining methods were used. This protocol was applied equally to both dry and wet membrane samples. 2.2.2. TEM cross-sections of isolated polyamide thin films A sample of ESPA3 containing 30 nm gold nanoparticles on the polyamide film was selected to prepare regular cross-sections as described in Section 2.2.1. Preliminary images were obtained only from the cross-section located at the center of the grid. After acquiring preliminary images, the grid was dipped three times in chloroform for 45 s each to remove the polysulfone support from the cross-sections. All cross-sections on the grid were examined in the microscope after chloroform treatment. Gold nanoparticles were used as reference markers allowing us to inspect exactly the same areas that were imaged in the preliminary step. Energy dispersive X-ray spectrometry (EDS) analysis of the cross-sections was performed on a Philips CM20 field-emission gun TEM equipped with an EDAX UTW detector at an accelerating voltage of 200 kV. 2.2.3. Cross-sections prepared using Spurr resin The cross-sections of ESPA3 featured in Section 3.3 (Fig. 4) were prepared and imaged at the Central Facility for Advanced Microscopy and Microanalysis of the University of California Riverside. The embedding protocol used was based on the epoxy Spurr resin (vinylcyclohexene dioxide). Membrane samples were stained with a 1% aqueous solution of osmium tetroxide (OsO4 ) for 1 h, and then dehydrated in a graded ethanol series of 30%, 50%, 70%, 90% and 100% for 10 min each, followed by three changes of 100% ethanol for 10 min each. They were then placed in 1/3 Spurr resin and 2/3 ethanol for 2 h, 2/3 Spurr resin and 1/3 ethanol for 2 h and two treatments of 100% Spurr resin for 2 h each. Samples were transferred to flat embedding molds filled with fresh resin and polymerized overnight at 70 ◦ C. Images were obtained with a Philips CM300 microscope. 2.2.4. Projected area TEM (PA-TEM) A simple laboratory apparatus was built for isolating the polyamide thin films of RO and NF membranes. Using reverse tweezers, a 1 mm single-hole copper TEM grid was placed flat on a stainless steel plate, whose slope was adjustable to any position. Chloroform to remove the polysulfone was delivered from a burette positioned above the plate. A membrane disc of about 2 mm in diameter was cut with a hole-punch after peeling off the polyester backing. The membrane disc (polyamide surface facing down) was placed onto the TEM grid, which was previously wetted with chloroform to help the polyamide stick to the grid. The polysulfone was then dissolved and washed away by dripping chloroform onto the stainless steel plate slightly above or below the position of the grid (avoiding solvent drops falling directly onto the membrane specimen). Polysulfone removal was controlled by adjusting the chloroform drip rate and the slope of the stainless steel plate to drain the solvent. Chloroform drip rates were normally 0.2–0.4 mL/min, and the plate was typically set with an angle of inclination of about 10◦ . When the white polysulfone layer was no longer visible, the grid was dipped several times in clean chloroform to remove any residual traces of polysulfone.

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2.3. Scanning electron microscopy (SEM) SEM micrographs of membrane surfaces were obtained with a FEI XL30 Sirion microscope at an accelerating voltage of 5 kV. Samples were previously sputter-coated with a uniform layer of approximately 10 nm palladium/gold to avoid charging effects. 3. Results and discussion 3.1. TEM cross-sections of RO and NF membranes Fig. 1 presents TEM cross-section micrographs of the RO membrane ESPA3 and the NF membrane NF270. The ESPA3 surface shows the relatively rough ridge-and-valley structure (Fig. 1a and b) characteristic of fully aromatic polyamides [1]. In contrast, the surface of the semi-aromatic polyamide thin film of NF270 (Fig. 1c) is relatively smooth and homogeneous. The measured average thicknesses of the polyamide thin films of ESPA3 and NF270 were approximately 250 and 40 nm, respectively, in agreement with previous reports [10,12]. Images of ESPA3 dry unused samples (Fig. 1a) and the wet samples containing 10 nm gold nanoparticles (Fig. 1b) show no morphological differences, confirming that dehydration is not necessary when using our LR White based protocol. Moreover, LR White provides enough contrast to distinguish between the different layers of the membrane without the need for heavy metal staining [4]. The polyamide is clearly discernible from the background resin and as expected, the underlying polysulfone support appears darker due to the presence of heavier sulfur atoms. Removing the polyester backing (which provides mechanical strength to the membrane) before embedding is an important step of the protocol. In a typical thin film composite membrane, the polyester backing has a thickness of about 100 ␮m, representing 70–80% of the total membrane thickness [1]. Different artifacts can occur during sample preparation depending on whether specimens are processed with or without the polyester. Although others have reported removing the polyester backing [10,14,15], preparation artifacts associated with this step have not been described in the literature. Polyester removal was essential to avoid artifacts such as wrinkling and layer detachment introduced during sectioning of intact membrane specimens with the ultramicrotome, which on average produced low-quality cross-sections. Fig. 2a shows the cross-section of an ESPA3 membrane prepared following the LR White based protocol described in Section 2.2.1 except that the polyester backing was not removed. Wrinkling of the resin next to the polyamide side of the cross-section was an artifact commonly observed in samples processed with the polyester. Wrinkles or stretch marks can result from non-uniform forces that are exerted on the section by the ultramicrotome knife during sectioning of specimens with varying density and hardness [21]. In this case wrinkling is attributed to the large difference in hardness between the stiff polyester and the relatively soft polysulfone, polyamide and LR White resin. Other artifacts that may occur during sectioning such as specimen folding (evidenced by very dark lines) and partial layer detachment are evident in Fig. 2a at the interface between the polyester and the polysulfone. Fig. 2a depicts an example of a cross-section that sustained limited damage during sectioning despite the presence of the polyester. Most cross-sections examined (not shown) exhibited more wrinkling and especially more detachment of the polyester backing. In the most severe cases, the polyamide film was completely separated from the polysulfone support in areas surrounding the wrinkles (i.e., areas of high stress), rendering those sections useless for acquiring accurate images. Fig. 2b presents the cross-section of an ESPA3 sample processed without the polyester backing. Lacking the mechanical stability provided by polyester, the remaining membrane curled up while

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Fig. 1. Regular TEM cross-section of RO membranes (a) ESPA3 unused, (b) ESPA3 previously used in a filtration experiment with 10 nm gold nanoparticles, and NF membrane (c) NF270 unused. Scale bars: (a–c) 0.5 ␮m.

being immersed in the resin, randomly changing its orientation inside the gelatin capsule during curing of the resin. Consequently, preparing a sectioning face on the resulting resin block that contained an edge of the membrane was often difficult. With respect to the sectioning artifacts described above, Fig. 2b shows that results were significantly better in the absence of the polyester backing. However, LR White swelled the polysulfone support causing additional artifacts, which included (1) an increase in thickness from approximately 20 ␮m (Fig. 2a) to 35 ␮m; (2) the formation of irregular undulations on the membrane surface; (3) the sporadic appearance of large oval-shaped holes in the more porous side of the layer.

Ultimately, the success of a preparation protocol depends on the ability to obtain accurate images of the polyamide structure. Fig. 2c and d compare the structure of polyamide thin films of ESPA3 samples that were processed with and without the polyester backing, respectively. After examining many cross-sections prepared both ways, we concluded that the dimensions and appearance of the polyamide layers were similar and consistent with images of polyamide films in the literature [4,11]. The observed morphology changes of the polysulfone support in samples prepared without the polyester backing did not show detrimental effects on the polyamide films, as long as the images were acquired from regions where the polysulfone surface was straight over several microns

Fig. 2. Regular TEM regular cross-section of ESPA3 RO membranes: (a and c) processed with the polyester backing; (b and d) processed without the polyester backing. Images (c and d) show that the polyamide films remain unaffected by the different artifacts arising from both maintaining or removing the polyester backing, provided that the images are acquired in areas where the underlying polysulfone surface is straight over several microns. Scale bars: (a and b) 10 ␮m; (c and d) 0.5 ␮m (identical).

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(e.g., Fig. 2d). For example, the vertices of the polysulfone undulations (Fig. 2b) should be avoided for imaging purposes, as the polyamide often presented abnormalities in these regions (images not shown). 3.2. Cross-sections of isolated polyamide thin films In regular TEM membrane cross-sections (Figs. 1 and 2), it is difficult to accurately discern the interface between the polyamide and the polysulfone. To study the structure of the polyamide thin film in isolation from the rest of the membrane layers, we used chloroform to remove the polysulfone support from regular ESPA3 cross-sections as described in Section 2.2.2. Chloroform is not only a good solvent for polysulfone, but can be easily removed from the specimen by evaporation. Polysulfone was successfully removed from all the cross-sections except from the one at the center of the grid that was exposed to the electron beam when acquiring preliminary images. The images of this cross-section obtained before and after chloroform treatment were identical (images not shown). A plausible explanation is that the heat of the beam further polymerized the resin of this particular cross-section making it completely impermeable to the solvent, thus effectively masking the polysulfone to chloroform. Similar beam effects are known to eliminate the ability to use staining solutions on resin embedded specimens, which is why exposing TEM samples to the electron beam before staining is not recommended [28]. Fig. 3a shows a cross-section image of the isolated polyamide thin film of ESPA3. The polyamide thin film consists of a 30–60 nm compact base of nodular polyamide (delimited by the square bracket in Fig. 3a) from which the ridge-and-valley structure extends outward. The backside of the polyamide base (i.e., the polyamide surface formed facing the polysulfone support) is relatively smooth. The carbon/formvar film of the TEM grid is now visible in the space originally occupied by polysulfone (Fig. 3a). Because it was not possible to image a cross-section beforehand without rendering it inaccessible to solvent treatment, TEM EDS analysis was used instead to confirm that chloroform successfully removed the polysulfone support. The EDS spectra presented in Fig. 3 were acquired after the cross-sections had been treated with chloroform. Fig. 3b shows the spectrum of the polysulfone support obtained about 1 ␮m below the polyamide film of the cross-section that was preliminarily exposed to the electron beam and unaffected by chloroform treatment. Sulfur is the only element allowing us to differentiate polysulfone from polyamide and the acrylic resin. Therefore, the sulfur peak confirmed that for this particular cross-section the polysulfone support was not removed. In contrast, Fig. 3c shows the corresponding spectrum from one of the cross-sections without polysulfone. In this case, sulfur was not detected and the oxygen peak was smaller confirming the absence of polysulfone and the effectiveness of chloroform treatment. The exposed carbon/formvar film of the grid is responsible for the observed carbon and oxygen peaks. Taken together, these spectra support the visual interpretation that Fig. 3a shows the cross-section of the isolated polyamide thin film following the removal of the polysulfone support. Fig. 3d presents the spectrum of polyamide taken from the cross-section without polysulfone. As expected, no sulfur was measured, but gold was clearly detected due to the presence of the nanoparticles. 3.3. Revising the accepted visual description of the structure of polyamide thin films The model developed by Freger [29] provides the most comprehensive theoretical analysis describing the formation of membrane polyamide thin films by IP. For the fully aromatic polyamide of

Fig. 3. (a) Cross-section of the isolated polyamide thin film of an ESPA3 RO membrane obtained after exposing regular TEM cross-sections to chloroform to remove the polysulfone support. Scale bar: 0.5 ␮m. (b–d) EDS spectra used to confirm the removal of polysulfone by monitoring the presence of sulfur: (b) polysulfone support, (c) exposed carbon/formvar film following polysulfone removal and (d) polyamide with gold nanoparticles.

RO membranes, the model predicts a heterogeneous thin film consisting of a dense core sub-layer of polyamide that separates two regions of relatively loose polyamide extending in opposite directions. According to this model the surface side loose polyamide contains more carboxyl groups, while the loose polyamide on the polysulfone side has more amine groups [29]. Freger used TEM to visually evaluate this prediction by selectively staining carboxyl and amine groups with uranyl and tungstate ions, respectively [10].

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Fig. 4. Regular TEM cross-section images of ESPA3 RO membranes prepared with a protocol based on the epoxy Spurr resin that included pre-staining with OsO4 and dehydration with ethanol (Section 2.2.3). The preparation protocol caused the formation of circular artifacts near the surface of the polysulfone support, which have been previously misinterpreted in the literature as part of the polyamide thin film [10]. The polyamide directly above the artifacts is similar to the isolated polyamide in Fig. 3a. Scale bars: (a) 1 ␮m; (b) 0.2 ␮m.

Based on the interpretation of the TEM images (Figs. 1 and 2 in [10]), the ridge-and-valley surface structure was identified as polyamide rich in carboxyl groups. Semicircular homogenous features (with radii as large as 0.5 ␮m) located within the polysulfone support and adjacent to the polyamide thin film were labeled carboxyl-free polyamide. Our cross-section images of isolated polyamide thin films (e.g., Fig. 3a) do not support the existence of regions of loose polyamide on the polysulfone side of the thin film. The removal of the polysul-

fone support with chloroform would have revealed these regions without requiring staining methods. However, Fig. 3a shows that the relatively smooth base of dense nodular polyamide forms the interface with the polysulfone support. Thus, it appears that the semicircular features described as “carboxyl-free polyamide” by Freger are artifacts created during the sample preparation process. These features most likely consist of pockets of embedding medium because in the images they show the same tone, texture and lack of structural details as the resin. Furthermore, the images show other

Fig. 5. Comparison of PA-TEM and surface SEM images of the RO membrane ESPA3: (a and d) PA-TEM; (b and d) surface SEM; (c and f) PA-TEM images with inverted tones. Scale bars: (a–c) 0.5 ␮m (identical); (d–f) 0.2 ␮m (identical).

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Fig. 6. Electron micrographs of a sample of ESPA3 containing 30 nm gold nanoparticles deposited during a dead-end filtration experiment: (a and c) PA-TEM; (b and d) surface SEM. Scale bars: (a and b) 0.5 ␮m (identical); (c and d) 0.2 ␮m (identical). (Note: Gold nanoparticles appear slightly larger in the SEM images due to the necessary metal coating to make the entire sample conductive).

features of similar characteristics present within the polysulfone support completely disconnected from the surface polyamide thin film [10]. The appearance of such discrete regions of polyamide would be inconsistent with the IP reaction process that forms the polyamide thin film in RO and NF membranes [1,29]. Fig. 4 provides evidence to support the claim that these features are artifacts formed during sample preparation. These crosssections of ESPA3 were prepared using a protocol based on the epoxy Spurr resin, which included pre-embedding staining with OsO4 and dehydration with ethanol (Section 2.2.3). Very similar protocols are standard for processing biological specimens [22], and have also been used with membrane samples in the past [18]. The polysulfone support in Fig. 4 shows circular and semicircular features similar to those labeled as “carboxyl-free polyamide” by Freger [10]. However, in this case the appearance of these features cannot be attributed to selective staining because OsO4 is a general stain that would not exclusively bind negative functional groups as uranyl ions typically do [21]. Moreover, OsO4 has strong affinity for amines [22], which means that these circular features should have been strongly stained if in fact they were polyamide rich in amine groups. Instead, they show the same image tone and uniformity as the resin. The formation of these artifacts severely disrupted the polysulfone surface causing partial detachment of the polyamide thin film from the support. Notably, the detached polyamide film next to these artifacts looks very similar to the isolated polyamide film in Fig. 3a. A structure of polysulfone with such large circular features

is not consistent with the characteristics of polysulfone observed in all the images that we have obtained using LR White as the embedding medium as well as those published by others [3,6–9,11,12]. As seen in Fig. 2a and b the polysulfone support is asymmetric with significantly decreasing porosity towards the surface, and micronsize pores are not observed directly adjacent to the polyamide thin film (Fig. 1 and Fig. 2c and d). It is also noteworthy that none of the pores in the support are perfectly circular like they appear in Fig. 4a and b and in Freger’s work [10]. The exact causes leading to these artifacts remain unknown and are difficult to evaluate because the preparation protocols employ different dehydration and staining methods. The only similarity is the use of epoxy resins (Spurr and Araldite 502), yet the densities and components of these resins are also quite different [21]. 3.4. Projected area TEM (PA-TEM) Fig. 5 presents PA-TEM and surface SEM images of the RO membrane ESPA3. To facilitate the comparison between the two techniques, image scale bars across each row of micrographs are identical (0.5 and 0.2 ␮m, respectively). Fig. 5a and d (first column) show PA-TEM images of the polyamide thin film. Because polyamide is composed only of light elements of similar atomic mass (H, C, N and O), image contrast originates primarily from variations of the thin film thickness caused by the ridge-and-valley structure (Fig. 2c and d). The greater the thickness of a local region of polyamide, the darker it appears on the image as less elec-

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trons are transmitted due to scattering. Therefore, in Fig. 5a and d dark features match ridge structures, while bright regions correspond to valleys where relatively more electrons pass through the polyamide film unhindered. Because PA-TEM images are generated from the electrons transmitted through the thin film, they do not depend on which side of the polyamide film is facing the electron beam. Fig. 5b and d (second column) show SEM images of the ESPA3 surface. In SEM, the outermost features on the surface (polyamide ridges) appear brighter as they generate more secondary electrons, while areas deeper within the specimen (the valleys) appear relatively dark because fewer electrons are able to escape [20]. When comparing the images obtained both from PA-TEM and surface SEM (Fig. 5a vs. b, and Fig. 5d vs. e), it is evident that they essentially constitute inverted (negative) depictions of the same polyamide structure. This result is better illustrated by Fig. 5c and f (third column), which show inverted (negative) versions of Fig. 5a and d, respectively, after applying the invert function in Adobe Photoshop. Fig. 5c and f visually support the interpretation that the darkest features in the original PA-TEM images correspond to the polyamide ridge structures that appear bright in SEM. The most important advantage over surface SEM is that PA-TEM provides morphology information from the entire thin film and not only from the outermost features on the surface. The polyamide nodular structure that we identified as the base of the thin film (Fig. 3a) cannot be observed with SEM because it is hidden underneath the polyamide ridges. In contrast, the nodular polyamide base appears throughout the entire PA-TEM image as relatively round features resembling an irregular honeycomb pattern that is especially evident in the valleys (e.g., see Fig. 5d). These circular features forming the honeycomb pattern are approximately 20–60 nm in diameter, which correlate well with the thickness of the polyamide base nodules as determined from Fig. 3a.

Fig. 6 shows PA-TEM and surface SEM images of an ESPA3 membrane containing 30 nm gold nanoparticles deposited during a dead-end filtration experiment. From inspection of the PA-TEM images (Fig. 6a and c), it is evident that the nanoparticles accumulated overwhelmingly over the dark rather than the bright regions, which correspond, respectively, to ridges and valleys. SEM images (Fig. 6b and d) are consistent with this interpretation. PA-TEM provides a more complete visualization of the particle distribution over the entire thin film, whereas SEM offers limited information beyond the polyamide ridges. For example, it is hard to evaluate the true extent of particle deposition in the polyamide valleys using SEM images alone. Furthermore, the honeycomb pattern corresponding to the nodular structure of the polyamide thin film base is clearly visible in the PA-TEM images only (Fig. 6a and c). Fig. 7 presents different TEM and SEM images of NF270. The TEM cross-sections in Fig. 7a and b differ from that in Fig. 1c in that bubble-like features appear as part of the polyamide thin film. To our knowledge, images of this membrane type with similar polyamide bubbles have not been reported in the literature. Therefore, we initially dismissed these features as artifacts caused by the embedding protocol and avoided them when acquiring crosssection images. SEM analysis of the membrane surface further supported our initial assumption, as none of the images (e.g., Fig. 7e) showed evidence of bubble-like features, in agreement with previously published images of NF270 [30,31]. However, examination of NF270 thin films using PA-TEM (Fig. 7c and d) revealed that these polyamide bubbles are common and randomly distributed over the membrane surface. Because PA-TEM sample preparation does not require the use of resins, Fig. 7c and d indicate that the polyamide bubbles cannot be artifacts from the embedding process, but are in fact real features of the thin film. It is noteworthy that the bubble cross-sectional view in Fig. 7b is clearly consistent with the inner structure of the bubbles shown in Fig. 7d. With the exception of these bubbles, the lack of contrast in the PA-TEM images confirmed

Fig. 7. Electron micrographs of NF270 obtained using different techniques: (a and b) regular TEM cross-sections; (c and d) PA-TEM; (e) surface SEM. The bubble-like features of the polyamide film occasionally encountered in TEM cross-sections, although not observed in SEM, were easily identifiable with PA-TEM, thus eliminating the possibility of them being artifacts of the resin embedding process. Scale bars: (a and b) 0.5 ␮m; (c) 2 ␮m; (d and e) 1 ␮m (identical).

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that the majority of the NF270 polyamide thin film is smooth and uniform in thickness. 4. Conclusions TEM cross-sections of RO and NF membranes were successfully obtained using a protocol that is based on the acrylic LR White resin that does not require dehydration and staining steps. Artifacts resulting from the preparation process that could produce low-quality cross-sections or inaccurate depictions of polyamide thin films were documented. A cross-sectional view of the isolated polyamide thin film of ESPA3 revealed that the thin film consists of two distinct polyamide regions: (1) a dense base of nodular polyamide that forms a relatively smooth interface with the polysulfone support; (2) a more open structure of loose polyamide extending outward from the nodular base comprising the ridge-and-valley structure. This visual characterization of the polyamide thin film nanostructure is consistent with most predictions of Freger’s theoretical model [29], with the major exception that a second region of loose polyamide does not exist on the polysulfone side of the thin film. A possible explanation for this discrepancy is that the model does not consider the physical obstruction to film growth posed by the presence of the polysulfone support. Recent work by Ghosh and Hoek showed that during the IP reaction the polysulfone support plays an important role in determining the final characteristics and performance properties of polyamide thin films [7]. PA-TEM offers a powerful new way to visually examine isolated polyamide thin films without requiring extensive sample preparation like resin embedding (cross-section TEM) or metal coatings (SEM). By capturing the entire nanostructure of thin films, PA-TEM images clearly depicted polyamide features such as the dense nodular base of ESPA3 and the surface bubble-like features of NF270, which could not be observed with surface SEM. In combination, the two new TEM techniques presented in this study provided a comprehensive visualization of polyamide nanostructure. In the case of RO membranes, the ability to accurately examine the dense base of nodular polyamide is especially crucial based on previous reports suggesting that the dense polyamide sub-layer presumably constitutes the true separation barrier [5,6,29]. Acknowledgements Funding for this work was provided by the Singapore Stanford Partnership (SSP); the STC WaterCAMPWS of the National Science Foundation under agreement #CTS-0120978; the Santa Clara Valley Water District (Agreement #A2727A); the Metropolitan Water District (Agreement 41808); and the California Department of Water Resources. Membrane samples used in this study were kindly donated by Dr. Craig Bartels of Hydranautics (ESPA3), and Dow FilmTec (NF270). The authors gratefully acknowledge John Perrino of the Cell Sciences Imaging Facility at Stanford University for his valuable contributions in developing the TEM embedding protocol and preparation of the grids, as well as Dr. Ann Marshall of the Stanford Nanocharacterization Laboratory for her generous assistance with the TEM EDS analysis. References [1] R.J. Petersen, Composite reverse osmosis and nanofiltration membranes, J. Membr. Sci. 83 (1993) 81–150. [2] D.G. Cahill, V. Freger, S.-Y. Kwak, Microscopy and microanalysis of reverse osmosis and nanofiltration membranes, MRS Bull. 33 (2008) 27–32.

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