Microsc. Microanal. 21, 626–636, 2015 doi:10.1017/S1431927615000410

© MICROSCOPY SOCIETY OF AMERICA 2015

The Advantages of an Attenuated Total Internal Reflection Infrared Microspectroscopic Imaging Technique for the Analysis of Polymer Laminates Chen Ling, and André J. Sommer* Molecular Microspectroscopy Laboratory, Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA

Abstract: Until recently, the analysis of polymer laminates using infrared microspectroscopy involved the painstaking separation of individual layers by dissection or by obtaining micrometer thin cross-sections. The latter usually requires the expertise of an individual trained in microtomy and even then, the very structure of the laminate could affect the outcome of the spectral results. The recent development of attenuated total internal reflection (ATR) infrared microspectroscopy imaging has provided a new avenue for the analysis of these multilayer structures. This report compares ATR infrared microspectroscopy imaging with conventional transmission infrared microspectroscopy imaging. The results demonstrate that the ATR method offers improved spatial resolution, eliminates a variety of competing optical processes, and requires minimal sample preparation relative to transmission measurements. These advantages were illustrated using a polymer laminate consisting of 11 different layers whose thickness ranged in size from 4–20 μm. The spatial resolution achieved by using an ATR-FTIR (Fourier transform infrared spectroscopy) imaging technique was diffraction limited. Contrast in the ATR images was enhanced by principal component analysis. Key words: ATR, IR microscopy, laminate, transmission

I NTRODUCTION Polymer laminates are film structures consisting of two or more polymeric layers bonded together. They are one of the most inexpensive and versatile types of polymer systems utilized as food packaging materials. Food packaging systems must retard product deterioration, retain the beneficial effects of processing, extend shelf life, and maintain the sterilization or quality of food. The principal role of food packaging is to protect food products from outside influences and damage in three major aspects: chemical, biological, and physical. Chemical protection minimizes compositional changes triggered by environmental factors such as exposure to air, moisture, or light. Biological protection shields the food from microorganisms, insects, and rodents. Physical protection secures food from mechanical damage, such as shock and vibration experienced during transportation and distribution (Marsh & Bugusu, 2007). Presently, laminates are commonly used as food packaging materials owing to their improved performance over single-layer polymers. Polyesters and polyolefins are the most widely used polymers in food packaging, both of which possess several beneficial properties, including improved flexibility, strength, stability, chemical resistance, and ease of processability (Marsh & Bugusu, 2007). Polyamides, well known as Nylon, offer good chemical resistance, toughness, Received August 17, 2014; accepted March 20, 2015 *Corresponding author. [email protected]

and prevent gas diffusion (Armstrong et al., 2012). Typically, when two layers are chemically incompatible because of different polarities, adhesive layers are required to construct multilayered structures. The manufacturing process for these laminates has been highly engineered, either by addition polymerization or condensation polymerization, followed by the operation of laying-up into a “sandwich” form (Yablon, 2014). The inspection for structural integrity and functional performance during the manufacturing process is vital to the quality control of the laminate products. There are numerous variables to monitor in the course of the polymer laminate manufacturing process. Effective quality control methods for these composite systems must provide structural information about the thickness and consistency of each layer, as well as molecular information regarding each composite’s identity. In particular, by examining the polymer laminate cross-sections, quality control of laminate layers can be routinely carried out to verify the microscopic structure and to identify defects and/or contaminants. Nevertheless, there is a lack of an appropriate technique that can provide molecular composition information at the high spatial resolution required for analysis of these structures. The focus of this investigation is to develop a novel approach—an attenuated total internal reflectionFourier transform infrared (ATR-FTIR) imaging technique to analyze the cross-sections of the polymer laminate. Specific emphasis will be placed on simplifying the sample preparation process and extracting accurate molecular and spatial information from individual polymeric layers.

Advantages of an ATR Infrared Microspectroscopic Imaging Technique

P REVIOUS STUDY FOR POLYMER L AMINATES In order to monitor the manufacturing process and characterize the complex laminate structures, current techniques for polymer laminate analysis include a range of optical microscopy (Joyce et al., 1997), infrared and Raman spectroscopy (Jawhari & Pastor, 1992; Sarac & Springer, 2002; Xiao et al., 2004), thermal microprobe analysis (Price et al., 1999; Morikawa et al., 2006), X-ray photoelectron spectroscopy (Gilbert et al., 2013), nuclear magnetic resonance imaging (Papon et al., 2011), scanning electron microscopy (Gupper et al., 2002; Kotera et al., 2012), transmission electron microscopy (Chou et al., 2010), and atomic force microscopy (Drake et al., 1988). In most cases, when one is analyzing a multilayer polymer laminate, the first step is to microtome a thin section perpendicular to the layer thickness to expose individual layers and avoid smearing (contamination) from one layer to the next. Optical microscopy is one of the oldest and least expensive techniques used to study the structure of materials such as phase separation, compatibility, size, and size distribution. Conventional optical microscopy is solely based on refractive index differences between layers. The specimens are analyzed in the form of thin films, usually between 1 and 50 μm thick (de Carvalho & Bretas, 1995; Bretas, 2003). The preparation of thin sections in this range usually requires expertise in microtomy. Even then, the layers can separate or curl, requiring further sample preparation and treatment. Vibrational spectroscopy provides contrast based on molecular composition or more specifically, functional groups contained within the molecular structure of the material. Both infrared and Raman spectroscopy have been extensively employed to examine microscopic areas in polymers for more than three decades (Davies et al., 1985). With the coupling of an infrared interferometer to a microscope equipped with specialized detectors, FTIR spectroscopy is a well-established technique to examine and visualize multicomponents in the field of view. Obtaining FTIR spectra from polymer samples is relatively fast, straightforward, and routine. Both Raman and infrared spectroscopy are often used to monitor the evolution of complex reactions like copolymerization and cross-linking reactions during the polymer manufacturing process, or to quantitatively measure the number or fraction of interacting groups in certain hydrogen-bonded polymer blend systems (Coleman & Painter, 1995; Skrifvars et al., 2004). Raman spectroscopy is a suitable method for monitoring the conversion of C = C bonds from methacrylic, vinylic, and unsaturated polyesters because they exhibit relatively intense Raman signals (Nelson & Scrantont, 1996). In 2004, Xiao et al. utilized confocal Raman microscopy to image a laminated polymer (Paramount film) and further evaluated the thickness variations with this technique (Xiao et al., 2004). Layers of polyethylene terephthalate and low-density polyethylene were observed. The center-to-center separation was measured to be 22.5 μm with an error of ~11–16%. However, confocal Raman

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has been shown to be subject to positional uncertainties; the Raman signal was weak and easily interfered by fluorescence (Everall, 2000a, 2000b). Transmission infrared spectroscopy is dependent on infrared radiation passing through a thin sample with photons of specific wavelengths being absorbed by specific functional groups. It has been employed as the primary technique for polymer characterization for more than six decades. A general rule of thumb is that the desired sample thickness for most common materials should be on the order of 6 μm. For thicker samples, the absorptions can saturate which diminishes the qualitative and quantitative capacity of the method. Like optical microscopy the requirement of a thin film can be problematic. Nishioka et al. (1992) have reported the application of FTIR microspectroscopy to analyze the interaction between polyurethane coating layer and an ethylene acrylic acid copolymer layer. They found that a mixed phase was formed along the interface and observed an interaction between the chemistry associated with each layer. Chalmers et al. (2002) investigated a three-layer film laminate comprising an ethylene/vinyl acetate (EVA) copolymer layer sandwiched between layers of polyethylene (PE) and polyester using transmission measurements. By highlighting different wavenumbers, layer discrimination and improved contrast were observed. Lavalle et al. (2004) further modified transmission infrared microspectroscopy by immersing a 5-μm-thick laminate sample between two ZnSe hemispheres, which improved the spatial resolution of the system. However, the ideal sample for transmission mode has to be sufficiently thin to allow for radiation to travel through the sample without total absorption. In preparing such a sample, laminates tend to curl and layers are prone to separate when they are prepared as thin sections. Hence, thin sample sectioning of a polymer laminate requires experience and expertise in addition to the time and effort demanded to obtain optimal thickness and integrity. A solution for many of these problems is the use of ATR infrared spectroscopy and microspectroscopy. This method continues to increase in popularity owing to the facts that little sample preparation is required, the method’s inherent surface sensitivity allows highly absorbing samples to be studied and, most importantly, the optical path length is typically independent of sample thickness. ATR microspectroscopy utilizes an internal reflection element (IRE) to reflect infrared radiation off the IRE/sample interface. At the point of reflection an evanescent wave is created that penetrates into the sample ~1–2 μm. ATR microspectroscopy is an immersion method by its nature, where the sample is immersed in a medium with high refractive index. Typical materials used as the IRE include, but are not limited to, germanium, zinc selenide, silicon, and diamond. Consequently, the spatial resolution and photometric accuracy are improved. These benefits will be discussed in more detail later in this paper. Through the use of ATR microspectroscopy, intractable samples analyzed by transmission or reflection modes become routine. The only requirement for ATR is that the

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sample be in intimate contact with the IRE. Several methods of ATR imaging using a hemisphere are routinely utilized including point-by-point mapping, focal plane array (FPA) imaging, and off-axis imaging.

H ISTORICAL DEVELOPMENT OF ATR I MAGING As mentioned earlier, by its very nature, ATR is an immersion method. The concept of immersion has been known and employed for a long time to collect more of the available radiation and focus light into smaller spatial domains. The first specialized infrared ATR microscope using a germanium IRE was constructed by Nakano & Kawata (1992). The microscope incorporated a confocal aperture for both the source and primary image of the sample to isolate the sample area of interest. The sample was attached to the Ge IRE and then focused beneath the microscope. The IRE/ sample composite was aligned with radiation entering the hemisphere normal to its surface, called the on-axis position. By moving the hemisphere off-axis, the radiation entered the hemisphere at a slight incident angle and through refraction came to focus at an off-axis position; thus, different sampling sites could be interrogated (Nakano & Kawata, 1994). This off-axis ATR-FTIR mapping technique was adapted and further developed by Lewis & Sommer (1999, 2000). The hemisphere/sample combination was initially centered at the microscope’s focus and then mapping was conducted by moving the composite off-axis using a motorized stage (Spragg et al., 2004). This movement continued using small adjustments in the x and/or y-direction until the full map was collected. Through this approach they were able to obtain maps of soft materials using a 1.5-mm radius Ge hemisphere as the IRE. Owing to the small radius of the hemisphere the area mapped was limited to 100 × 100 μm2. Further, because the microscope employed an aperture to isolate the region of interest on the sample, the theoretical improvement in spatial resolution was not realized. Sommer et al. (2001) and Tisinger (2002) imaged various samples using an infrared microscope equipped with an FPA detector. A germanium hemisphere was employed as the IRE. Their results demonstrated the feasibility of rapidly obtaining molecular-specific images with an improvement in spatial resolution. They reported a method to measure the spatial resolution by generating a step function in the absorption profile and their spatial resolution was determined to be close to the diffraction limit of the microscope (~8 μm for light of 6.3 μm wavelength). Based on the investigations done by Lewis & Sommer (2001), Perkin Elmer introduced an ATR accessory for use on the Spotlight 300 infrared imaging microscope in 2007 (Perkin Elmer, Inc., Waltham, MA). This microscope employed a 16-element linear array detector and a conventional rapid scan interferometer. This combination made routine analysis using the ATR imaging technique feasible. Later, Patterson & Havrilla (2006) conducted ATR imaging using a large-radius (12.5 mm) Ge IRE. With this accessory

an area of 2,500 × 2,500 μm2 ATR imaging was realized, and a more constant penetration depth across the image area was achieved. Based on these investigations, Perkin Elmer introduced a large area ATR accessory for the Spotlight microscope in 2012.

ATR-FTIR IMAGING G OALS The primary goal of the investigation is to demonstrate the capabilities and benefits of an off-axis ATR microspectroscopy imaging approach over transmission imaging methods. A second goal is to determine the ultimate spatial resolution of the ATR-FTIR imaging approach. A future goal is to extend this type of analysis to small particle studies and determine the detection limit in terms of the smallest particle that can be detected in a matrix.

MATERIALS

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METHODS

Sample Preparation As discussed earlier, when FTIR imaging of polymer laminates is performed in transmission mode, typically the total sample thickness should be

The Advantages of an Attenuated Total Internal Reflection Infrared Microspectroscopic Imaging Technique for the Analysis of Polymer Laminates.

Until recently, the analysis of polymer laminates using infrared microspectroscopy involved the painstaking separation of individual layers by dissect...
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