In recent years a challenging task is to find trace amount of adulterated components in oils and fats at even very low concentration per parts per million to parts per trillion is concern for health. Today, mixing of different substances to formulate completely miscible by using low price raw materials is an increasing in the market (adulteration).1 Sometimes contamination may also occur unknowingly either by using refined bleached deodorized palm olefin (RBDPOo), partly hydrolyzed oils (PHOs) or deodorization processing.2 According to AOAC Official method and World Health Organisation (WHO) recommendations existence of more quantity of saturated fatty acids increases level of low density lipoprotein (LDL) cholesterol in the blood and trans fatty acid isomer (>1%) deleterious cause cardiovascular and cerebrovascular diseases.3 As molecular structure point of view triacylglycerols (TAG) which comprises three hydroxy (OH-) functional groups connected to -carboxylic acid (COOH) of different fatty acid (FA) through ester bond.4 However, majority of oils are extracted from different plants or seeds may contain larger number of fatty acid chain length with different degree of unsaturatation (mono or poly) and also may vary position double bonds in hydrocarbon chain. Predominantly, the characteristic features of oils like physicochemical absorption, unsaponifiable and nutritional values are highly depended on the purity of oil substances.58 At present there are many titration methods as well as spectroscopic techniques are available to identify adulterated materials for quantification in food industry. Among them, gas chromatography mass spectrometry (GC-MS/MS), high-performance liquid chromatography (HPLC), high-performance liquid chromatography mass spectrometry (LC-MS/ MS), High-performance thin layer chromatography (HPTLC), supercritical fluid chromatography etc.9 However, in all these equipments are quite labour-intensive, time-consuming, requiring extensive experience in sample preparation, involves series of chemical manipulating steps, required high purity of mobile phase, further difficult to analyse the data interpretation. In addition to, many of the chemicals were used in chromatography are caused hazardous to the analyst as well as the environment.10 Conversely, in the present study non-destructive FTIR and 1H-NMR are easy and fast techniques to get information based on distinct peak shift, intensity, peak area. Recent times nuclear magnetic resonance (NMR) has become one of the most promising technique to find atomic level structure information (topology) in complex matrices both in solid as well as liquid state, mainly in the area of food processing sector or quality control.1113 As complete structural assignment of individual linking segments provides an advantage to correlate the quantitatively minimum level of the product. Hence, in the present study focused on FTIR and 1H-NMR spectroscopic methods to assign structural connectivity and purity of the oils.14,15
MATERIALS AND METHODS
Three different oils namely Sunflower, Peanut, and Gingelly oils were chosen for the study. The FTIR experiment was performed on Perki-nElmer instrument. Initially dry nitrogen was purged in samples to keep away from interference with atmospheric carbon dioxide along with water vapors. Approximately, 10μL of oil sample was placed on a potassium bromide polished plate. Immediately a uniform thin film was formed on the surface of the potassium bromide plate.11 Consequently a high resolution spectra was obtained, hence this method was considered as a neat spectrum because no solvent was used. The sample was scanned at room temperature and collected absorbance frequency data from 600 to 4000 cm-1
For 1H-NMR analysis, spectrum was obtained from NMReady-60 bench top spectrometer with the resonance frequency of 60 MHz in permanent magnet. The sample was prepared in a confirmed within the enclosure of standard 5 mm NMR tubes (NORELL) and dissolved in 500 uL of deuterated chloroform (CDCl3) and solvent peak was noticed at 7.25 ppm. Collected the data in the range 0-15 ppm as spectral width and number of scans given for each sample is 128 scans typically 0.06 sec per scan at room temperature. Tetra methyl silane (TMS) used as internal standard reference peak at 0.0 ppm. Raw data were exported from the NMReady to be a phase, baseline corrected was done using mestRenova software. All the lines in NMR spectrum was measured in δ (ppm) with reference chemical shift of standard TMS.16
RESULTS AND DISCUSSION
In this study, the molecular functional group of triacylglyceride linkage (TAGs) was confirmed by using FTIR and 1H NMR spectroscopy.
FTIR spectrum analysis
In Figure 1-3 and Table 1 represents FTIR spectrum of three oils namely Peanut, Sunflower, Gingelly oils respectively. All the three oils spectras were appeared with similar features. Among the peaks, the high intense vibration frequency band was noticed at 1744.07, 1744.28, 1744.24 cm-1 is corresponding to ester carbonyl functional group of triacylglyceride of Peanut, Sunflower, Gingelly oils. Association of three carbonyl functional groups is responsible for higher stretching frequency further evident consist of three carbonyl (O-C=O) connectivity.17 The important observation (Figure 1-3) is stronger absorption for C=O, whereas for double bond olefin has shown quite low absorbance band typically at 1652.50, 1647.41, 1655.30 cm-1 result of double bond C=C frequency is not interfere with C=O stretching frequency. Unsaturated long chain cis olefinic C-H stretching frequency appeared at ~3008 cm-1 while saturated carbon-carbon symmetrical and asymmetrical stretching observed at 2923, 2922, 2853 cm-1. However, methylene (CH2) symmetrical bending frequency seen in the range 1464-1377 cm-1. Ester bond of C-O stretching was noted around 1160.23, 1118.91, 1097.39 cm-1. The appearance of broad band in the range 721.92, 721.73, 722.00 cm-1 is a loop of cis di-substituted is less symmetry than trans conformation. The spectrum also shows overtone of a weak band at 3481.5, 3476.0 cm-1 associated with the glyceride ester carbonyl absorption. The FTIR studies significantly implies variation in the position as well as absorbance of the bands owing to variation in composition of triglyceride fatty acid structures.18
1H NMR structural analysis
In proton NMR study, three oils were subjected to investigate the structural assignment and as well as confirmation of purity. Figure 4-6 shows the proton NMR spectra of three oils recorded in deuterated chloroform at room temperature. Inspection of spectra shows well resolved triplet in deshielded region at 5.39 ppm (Table 2) associated with olefinic protons of double bond signify presence of unsaturated linkages. Very low intense unresolved peak was noticed at 5.03 ppm represents central methine signal of tertiary triglyceride position (>CHOCOR) owing to presence of three bulky substitutes creates more steric effect leads to more shielded shift compare to double bond protons.19 The chemical shift values in the range 4.2-3.8 ppm assigned methylene protons of ester connected triglyceride molecule. Bisallyic (=CH-CH2-CH=) and adjacent to carbonyl protons (O-C=O-CH2-) were seen typically at 2.8-2.1 ppm (allyl, bisallylic, trisallylic). The remaining more number of methylene protons of adjacent double bond appeared at 1.38-1.26 as multiplet (m) peak. Terminal methyl shows approximately at 0.8 ppm as triplet.20
By integration of each individual peak in spectrum further may utilise to quantify ratios of saturated and unsaturated fatty acids in TAGs profile. The appearance of triplet peak at 5.3 ppm (Z-vinyl CH) further confirms absence of epoxide formation through free radical mechanism.21 It is known that olefinic protons are more susceptible site to undergo oxidation conversion C=C in presence of oxygen under high temperature. Further, peak appeared at 5.3 ppm as district triplet due to arrangement of double bonds as cis (Z) conformation in hydrocarbon chain.22 Comparison of NMR data of all the three oils (Table 2) a slight variation in chemical shift as well as intensity. Remarkably bis-allylic position chemical shift values in sunflower oil is low intense than peanut and gingelly oils. It may be due to slightly lower in saturated fatty acid content.23 The spectrum (Figure 4-6) clearly support presence of different chemical environment. In all the cases, lines appeared in lower chemical shift region (aliphatic region) 5.3 to 0.8 ppm support protons of oleic, linoleic, linolenic and saturated fatty acids respectively.24
In this work, we attempted to analyse TAGs profile of fatty acids in three different oils by using FTIR and 1H NMR. The analysis confirms the presence of functional groups in the Peanut, Sunflower and Gingelly oils all absorptions in the region 722-3000 cm-1 corresponding to C-H, H-C-H -CH3, C-O and O-C=O, C=C with well resolved stretching frequencies was noticed. In proton NMR spectra of olefin protons clearly separated from glyceride methylene protons depicted at 5.3 ppm whereas bis-allylic as triplet seen at 2.82-2.73 ppm. The absence of doublet of doublet or multiplet peaks and appearance in shielded region in the span of 5.30-0.8 ppm reveals the double bonds both in mono and poly unsaturated fatty acids retains in Z or cis conformation. Further NMR spectrum clearly evident disappearance of signals for epoxide ring, aldehyde, mono and diacylglycerol protons as a result of more oxidative stability of all the three oils. The slight variation of peak values both in FTIR and proton NMR result of variation of content between saturated and unsaturated fatty acids. The used methodology is an interesting and quite valuable for the authentication of edible oils both in food industry, quality control as well as human health. Its main advantage is simplicity, speed, less time consume high-sensitivity and no require for sample pre-treatment.