**1. Introduction**

Agricultural biomass residue is a naturally occurring polymeric compound comprised of cellulose, hemicellulose, lignin, and extractives [1]. Biomass has the potential to be used as raw material for the biofuel industry. Cellulose occurs as crystalline fibers with random amorphous portions. The crystallinity of cellulose fibers is a key obstacle for effective biomass enzymatic hydrolysis [2]. Hemicellulose functions as a matrix for the cellulose and acts as a linkage between the crystalline cellulose and amorphous lignin [1-2]. Lignin is a polyphenolic compound that acts as a cementing material for biomass [1]. Lignin binds the hemicellulose and cellulose within the matrix. It provides rigidity and mechanical strength, holding it together [3]. The complex structure of lignocellulosic biomass presents resistance and 'recal‐ citrance' to biological and chemical degradation during enzymatic hydrolysis/saccharification and the subsequent fermentation process. This leads to a very low conversion rate, which makes the process less economically feasible [4-7]. Pretreatment strategies are employed to overcome the aforementioned challenges by deconstructing/disrupting the lignocellulosic matrix [8], while making cellulose and hemicellulose accessible for the production of bioetha‐ nol.

Radio frequency (RF)-alkaline and steam explosion pretreatment of biomass barley straw have shown that pretreatment enhanced the modification of the natural binding characteristics of lignocellulosic biomass [8-9]. Subsequently, this improved the physical properties of the produced pellets from the pretreated biomass [10]. RF heating has been considered as an energy efficient heating method that is an alternative to conventional methods (such as hot air). In RF heating, there is a volumetric heat generation inside the product, which is a result

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of the interaction between the RF waves and the ions/molecules of the product [11]. Therefore, heat flows from inside the product to the outside, unlike conventional heating methods in which heat is transferred from the heating medium to the product via conduction or convection [8]. RF heating has the following advantages: 1) uniform electric field strength inside the application chambers, therefore preventing uncontrolled heating, overheating, local hot spots, and product degradation; 2) large penetration depth (10-30 m); and 3) higher energy efficiency [11-13]. RF has been successfully applied to leather drying [13], quantification of hydroxycin‐ namic acids and lignin in perennial forage and energy grasses [14], thermal therapy [15], and in other research fields such as food processing (blanching, tempering, pasteurisation, sterilisation) and medicine [11]. Recently, Izadifar et al. (2009) [12] demonstrated that RF can be used for the extraction of podophyllotoxin from rhizomes of *Podophyllum peltatum*.

Steam explosion is operated by introducing the feedstock into the reactor and heating under steam pressure (2000-5000 kPa; 200-260°C) for a few minutes [16]. Steam explosion induces chemical effects because "water itself acts as an acid at high temperatures" [17]. The sudden thermal expansion involved in the termination of the reaction causes the particulate structure of the biomass to open up [17]. Lam et al. 2011 [18] investigated the steam explosion of Douglas Fir (*Pseudotsuga menziesii*) at a reaction temperature of 200-220o C and a retention time of 5-10 min. Excoffier et al. (1991) [19] and Ferro et al. [20] (2004) have applied steam explosion on lignocellulosic biomass. These authors reported that steam explosion is effective in the pretreatment of biomass.

The traditional methods of determining the chemical compositions of biomass involve gravimetric and analytical procedures. These procedures are time-consuming, laborious and expensive to perform, with low sample throughput and often results in a corresponding degradation of natural polymers [1, 21-22]. In contrast, procedures involving infrared (IR) spectroscopy are useful tools in rapidly extracting information about the structure of biomass constituents and the associated chemical changes resulting from various biomass treatments [1]. Infrared spectroscopy offers researchers an alternative method that is easier, robust and rapid. Fourier transform infrared (FTIR) spectroscopy has been successfully applied in a variety of species in wood surface characterization, for estimating the carbohydrate and lignin components [1, 22]. The majority of the carbon based molecules in plants and animals alike are highly active in the IR. FTIR is associated with superior spectral resolution and provides information on the fundamental molecular vibrations [22]. This permits better discrimination of structural and compositional differences, and often better structural interpretation [22]. Furthermore, FTIR analysis requires only small amounts of biomass plant material, which helps when screening samples available in limited quantities [22]. Applications of FTIR have also been found in biomedical weld and medicine research such as cancer and bone [23-24].

Mid-infrared spectroscopy and near infrared reflectance spectroscopy (NIRS) are the two types of IR spectroscopy that have found application for the measurement of chemical composition in lignocellulosic biomass. NIRS has been used in the prediction of the chemical composition in bulk plant samples. While mid-IR spectroscopy in contrast to NIRS predicts the fundamental molecular

vibrations, as such it gives a better information and insight into the molecular bonds present in the sample [25]. FTIR has been successfully applied for the comparison of Fourier transform infrared (FTIR) spectra of individual cells acquired using synchrotron and conventional sources [23]. The authors reported that the main beneficiaries of this rapid development (FTIR) on the instrumental front are the applications-based sciences, such as biomedical and cellbiological fields, for which results have been produced that are unavailable by standard methodologies. Stewart et al. (1995) [26] used FTIR spectroscopy to investigate the changes in composition and structure of oak wood and barley straw that has been subjected to chemical and biochemical treatments. The samples were also analyzed gravimetrically for residual neutral sugar composition, lignin and uronic acid contents. The spectroscopic techniques provided complementary information to the traditional approach. The FTIR fingerprint of wheat endosperm arabinoxylan (AX) has been investigated using a set of polysaccharides exhibiting variation of their degree of substitution and xylo-oligosaccharides comprising xylose units mono-or disubstituted by arabinose residues [27]. Yu (2005) [28] applied syn‐ chrotron FTIR microspectroscopy for the investigation of the molecular chemistry of various feed tissues. These images revealed spatial intensity and distribution of chemical functional groups in various feeds tissues within cellular dimensions. The researcher reported that such information can be used for plant breeding program for selecting superior variety of plant for targeted feed purposes and for prediction of feed quality and nutritive value. This proved that synchrotron FTIR microspectroscopy can be used for biological structure study.

Changes in plant cell wall composition following pretreatment largely influence the end use performance. The ability to easily monitor these compositional changes is, therefore, impor‐ tant. Hence, the objective of this investigation is to study and rapidly estimate the relative chemical composition of lignin, cellulosic and hemicellulosic polymers in pretreated and nontreated lignocellulosic barley straw using FTIR-PAS. This work focuses on the effects of radio frequency-alkaline and steam explosion pretreatments on the cell compositions of barley straw.
