CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 DESCRIPTION OF STUDY AREA
Dry clean seeds of peanut, sesame, and sunflower were purchased from farmers from four different locations Nebbi, Arua, Yumbe, and Zombo in West Nile sub region, Northern Uganda between the period of September to December 2017. The seeds (5kg) of peanut, sesame, and sunflower were packed in black polythene bags, and were transported to the Department of Chemistry Kyambogo University for analysis.
3.2 SAMPLE PREPARATION
Clean undamaged seeds were selected, and sundried for 24 hrs to get rid of moisture. Sunflower and peanut were de-hulled and de-shelled respectively before grinding of the seeds. Seeds were then ground mechanically into a fine homogeneous powder using electric grinder machine (Brooks Crompton series 2000, UK). The powders were then packed in plastic bags, and stored in a cool dry cupboard at 25oC for two days before oil extraction.
3.3 OIL EXTRACTION USING SOXHLET APPARATUS
Each sample (50g) was placed into a thimble, and extracted using n-hexane (bp 68oC) in a 5L soxhlet extractor for 8hrs using adapted method of Pena et al. (1992). The oil was then recovered by evaporating the solvent using a rotary evaporator, and residual solvent was removed by drying in an oven at 60oC for 1hr, and flushing with 99.9% nitrogen gas. The oil obtained after the extraction was transferred into a measuring cylinder which was placed over water bath for 30min at 70oC to ensure complete evaporation of solvent, and volume of the oil was recorded and expressed as oil content (%).
The oil content was calculated as follows;
Oil content
The extracted oil was stored in a freezer at -20C for immediate determination of physicochemical parameters, fatty acid profile, and heavy metal contents.
3.4 DETERMINATION OF PHYSICOCHEMICAL PROPERTIES
3.4.1 Density
The densities of different vegetable oils were determined by the pycnometer method using standard method described in AOCS (2009).
The pycnometer was cleaned, dried, and weighed to determine its mass. Dry pycnometer was filled with oil sample at 27oC and the weight recorded. The pycnometer was then filled with water at 27oC and weighed. The sample weight was then compared with the weight of water to determine its density (AOAC, 1998);
Density (ρ) .
3.4.2 Viscosity
Absolute viscosity (dynamic viscosity) is the product of kinematic viscosity and density of the oil. The kinematic viscosity of the oil sample was determined with a viscometer at 27oC. Oil sample (25mL) was placed in a viscometer placed in a temperature controlled vessel equipped with a thermostat which maintained a temperature with an accuracy of ±0.1oC. Kinematic viscosities (ν) expressed in centistokes were calculated from the measured flow time (t), and instrument constant (c) by means of the equation below;
ν = ct.
Density was measured using a 25mL pycnometer immersed in a temperature-controlled circulating water bath. Dynamic viscosities (ƞ) expressed in centipoise (cP), were calculated from the kinematic viscosities (ν), and the densities (ρ) by using equation below;
ƞ = ρν.
3.4.3 Peroxide Value (PV)
The oil sample (3g) was accurately weighed into a conical flask, and chloroform (10 mL) was added to dissolve the oil by swirling. Glacial acetic acid (15 mL) and freshly prepared saturated aqueous potassium iodide solution were also added, and the flask was stoppered and shaken for 1 minute and placed in a water bath at 40oC. Water (75mL) was added, and the mixture was titrated with standard sodium thiosulphate solution (0.002M) using soluble starch solution (1%) as an indicator. Titration was also performed for the blank. The peroxide value was calculated using the formula given below;
Peroxide Value (PV) = (S-B) xWxN,
Where B = the volume of sodium thiosulphate used for the blank,
W = the weight of the sample,
S = the volume of sodium thiosulphate consumed by the sample oil,
N = the normality of standard sodium thiosulphate.
3.4.4 Saponification Value (SV)
The oil sample (2g) was placed in a conical flask to which 25mL alcoholic KOH (0.5M) was added. The mixture was heated in a reserved condenser and cooled. After cooling the mixture, phenolphthalein (1mL) was added and titrated with HCl (0.2M) until a pink end point was reached. A blank titration was performed under the same time conditions.
Where B= Volume (mL) of HCl required by blank,
T= Volume (mL) of HCl required by oil sample,
N= Normality of HCl,
W=Weight of oil in gm.
3.4.5 Iodine Value (IV)
Carbon tetrachloride (200mL) was added to oil sample (0.4g), and 25mL of Wijs solution (8g) of iodine trichloride in 200mL of glacial acetic acid was also added to the mixture in the flask using a safety pipette in a fumed chamber. The flask was stoppered, and the content of the flask was vigorously swirled. The flask was placed in the dark for 2hr 30min, at the end of this period, 20mL of potassium iodide solution (10%) and water (120mL) was added to the flask. The content of the flask was titrated with sodium thiosulphate solution (0.1M) using starch indicator until end point. A blank titration was performed for other samples. The iodine value (IV) was calculated by the formula;
Iodine Value
Where C = concentration of sodium thiosulphate,
V1= volume of sodium thiosulphate used for blank,
V2= volume of sodium thiosulphate used for oil sample,
M= mass of the oil sample.
3.4.6 Acid Value (AV)
An oil sample (5g) was weighed into a conical flask and then neutral ethyl alcohol (25mL) was added to it and then the mixture was boiled on water bath. Phenolphthalein indicator solution (1-2 drops) was added to the mixture while hot and was titrated against standard potassium hydroxide solution with shaking until end point when the first pink colour persisted for 30 seconds. The acid value was calculated by the formulae below;
Acid value
Where V= volume of standard KOH solution in mL,
N= normality of standard KOH solution,
W= weight of oil sample in grams.
3.5 FATTY ACIDS ANALYSIS
The fatty acid profile was determined using Gas Chromatography/Mass Spectrometry after the oil samples were esterified into Fatty Acid Methyl Esters (FAMEs) suitable for analysis.
3.5.1 Preparation of FAME Standards
The fatty acid methyl ester standards (FAMEs): Palmitic (C16:0), Palmitoleic (C16:1), Stearic (C18:0), Oleic (C18:1), Linoleic (C18:2), Linolenic (C18:3) and Erucic (C22:0) acid were purchased from Sigma (Sigma-Aldrich, Germany), and were used to prepare the stock solution. Individual FAMEs standards were used for preparation of stock standard mixture (50 mg/mL). Identification and contents of the fatty acids were carried out by comparing sample FAME peak retention times with those obtained for FAME mix standard and by mass spectrometry.
3.5.2 Preparation of Fatty Acid Methyl Esters (FAMEs)
Derivatization was performed according to standard reference method (AOAC, 2000). Oil sample (50mg) was saponified (esterified) for 5 min at 95oC with 3.4mL of KOH (0.5M) in dry methanol. The mixture was then neutralized by using HCl (0.7M). 3mL of boron trifluoride (14%) in methanol was added. The mixture was heated for 5min at 90oC to achieve complete methylation process. The fatty acid methyl esters were thrice solvent extracted from the mixture with redistilled n-hexane and pooled together.
3.5.3 Gas Chromatography/Mass spectrometry (GC/MS) Analysis
The fatty acid methyl esters (FAMEs) were analyzed in gas chromatography/mass spectrometry (GC/MS) using an Agilent Technologies gas chromatograph (GC-5975T, Little Fall, NY, USA) equipped with an Agilent auto sampler 7683-B injector and Mass selective (MS-5975) detector. The pooled extract was concentrated to 1mL for GCMS analysis and 1μL was injected on Agilent J&W GC capillary column, HP-88 containing 88% cyanopropyl arylpolysiloxane as stationary phase (30m, 0.25mm i.d., 0.25μm film thickness). The injector and detector temperatures were 240oC and 260oC, respectively. The initial temperature of 140 oC was maintained for 2 min, raised to 230 oC at the rate of 4 oC/min, and kept at 230 oC for 5 min. The split ratio was 1:50, and helium was used as a carrier gas with a flow rate of 0.8mL/min.
The mass spectrometer was operated in the electron impact (EI) mode at 70eV; with an ion source temp: (230oC), a quadruple temp: (150oC), and a translating line temperature of 270oC. The mass scan was found in the range between 50 and 550m/z with an em voltage, 1035V. Peak identifications for the FAs in oil samples were performed by the comparison with MS spectra and retention times (Rt) of the standards.
3.6 DETERMINATION OF HEAVY METAL CONTENTS IN THE OIL SAMPLES
3.6.1 Sample Preparation
Samples of vegetable oils were weighed, and subsequently digested using a microwave unit. After digestion with a mixture of nitric acid and hydrogen peroxide clear solutions were obtained, and the analytes were determined using FAAS. In the procedure, each sample of oil (1g) was weighed into the digestion vessels. The digestions were performed by adding 3.5mL of HNO3 (68%) conc. and 1.0mL H2O2 (30%) to the sample. The microwave oven heating programme was performed in five steps using 35 Bar of pressure, as depicted in Table 1. The fifth step was a cooling down procedure of the system through forced ventilation over 20 min. After cooling all the digests were transferred into 10mL volumetric flasks, and diluted to volume with HNO3 (1% v/v). The digestion procedure was done in triplicate for each sample and reagent blanks were prepared similar to the samples.
The different oil samples were digested before analysis using the procedures described by Anwar et al. (2004) below;
Oil samples (1g) were weighed into separate digestion flasks. 5mL of concentrated nitric acid (65%) was added, and the contents heated at 70-80oC for 2-3 hours on a hot plate. Heating was continued at about 150oC for 3hours, 3-5mL of concentrated sulphuric acid (98%) and hydrogen peroxide (30%) each was added, and the mixture heated to completely decompose the organic matter. All contents of the flasks were evaporated until a semi-dried mass was obtained, and this mass was dissolved in a small amount of deionized water (approx. 5mL), filtered through Whatman filter paper No 42 and made up to a final volume of 25mL in volumetric flasks with 2M nitric acid.
3.6.2 Preparation of Standard Calibration Curves
Preparation of standard calibration curve working standards of lead, iron, zinc, and cadmium metals were prepared from the certified standard solutions in freshly prepared 2M nitric acid. A series of standard solutions for lead, iron, zinc, and cadmium of each metal ion in the range of absorbance noted for unknown samples were simultaneously run on FAAS model AA-6300 Shimadzu under the same set of analytical conditions. Standard calibration curves were obtained for concentrations verses absorbance data that was statistically analyzed using fitting of straight line by least square method.
3.6.3 Method Validation and Quality Control
In order to validate the analytical method, the following method validation parameters such as instrumental detection limit, limit of detection, limit of quantification, precision and accuracy studies were carried out.
3.6.4 Instrumental Detection Limit
Instrumental detection limit (IDL) is the smallest signal above background noise that an instrument can detect reliably. The IDL is calculated to be the concentration equal to three times the standard deviation of the blank signal. In this study, IDL for each metal was determined from analysis of seven replicates of calibration blank, and the concentration was calculated as;
IDL = 3xSbl
3.6.5 Limit of Detection
Limit of detection (LOD) is the minimum concentration of analyte that can be detected, but not necessarily quantified with an acceptable uncertainty. LOD for each metal was determined from analysis of seven replicates of method blanks which were digested in the same digestion procedure as the actual samples. LOD was calculated as;
LOD = 3xSbl
Where Sbl = Is the standard deviation of the method blank
3.6.6 Limit of Quantification
The limit of quantification (LOQ) is the lowest concentration of an analyte in a sample which can be quantitatively determined with acceptable uncertainty. LOQ was obtained from triplicate analysis of seven method blanks which were digested in the same digestion procedure as the actual samples. The LOQ was calculated as;
LOQ = 3xSbl
where Sbl = Is the standard deviation of the method blank
3.6.7 Precision and Accuracy
Precision and accuracy of the results were assessed by determining recovery and repeatability of the analysis of matrix spike, matrix spike duplicate, and laboratory control samples. For doing so, each sample was spiked in replicates of five at near mid-range calibration concentration. The spiked sample were digested and analyzed following the same analytical procedure as the oil samples. Precision was expressed as relative standard deviation (RSD) of replicate results. The relative standard deviations of the sample were obtained as;
%RSD = 100
The percentage recoveries of the analyte were calculated to evaluate the accuracy of the analytical procedure. Recovery was then calculated as;
%R = .
3.6.8 Analysis of Matrix Spike and Matrix Spike Duplicate Sample
Both matrix spikes (MS) and matrix spikes (MSD) were prepared by spiking 0.5g of each oil sample with 2mL of a mixture of spiking standards so that the spike level was 4 mg/L of Fe and 2 mg/L of Zn, and 1 mg/L of Cd and Pb. They were all carried through the same digestion and analysis steps as the unspiked sample. The relative percent differences (RPD) between the MS and MSD results were calculated as;
RPD = .
3.6.9 Analysis of Laboratory Control Sample
Five replicates of 0.5g lithium carbonate spiked with 2mL of a mixture of spiking standards so that the spike level were 4 mg/L of Fe, 2 mg/L of Zn and 1 mg/L of Cd and Pb. These were undergoing the same digestion procedure described for the oil sample but with no added sample. The % LCS recoveries for each metal were calculated as;
where % R = Percent recovery,
LCS = Laboratory Control Sample Results,
S = Amount of spike added.
3.6.10 Sample Analysis
The absorbance of the clear supernatant was measured using FAAS model AA-6300 Shimadzu under the following operating conditions; Deuterium arc background correction equipped with a hollow cathode lamp was used for the determination of Cd, Pb, Fe, and Zn, and an air-acetylene burner was used, the wavelengths and (spectral band pass) were for Cd: 228.8nm (0.5nm), Pb: 217.0nm (0.5nm), Fe:238.2 (0.5nm) and Zn: 213.9nm (0.5nm). The nebulizer flowrate was 5.0mLmin-1.
Final concentrations of the metals in the vegetable oil samples were calculated using the following formula;
Concentration (mg/kg)
Where C= Concentration of metal ions (mg/L),
V = Final volume (50mL) of solution,
W= Initial weight (0.5g) of sample measured.
3.6.11 Statistical Analyses
In this study, all measurements were carried out in triplicate and reported as means±standard deviation. Statistical analysis were performed using minitab Versin 13.3, statistical package (Minitab Inc. state college, P.A. USA). All variables were subjected to a one way ANOVA to test statistical significant difference among the different categories. A probability value at P<0.05, was considered to denote the statistically significant difference.
3.6.12 Limitations of the study
The findings of this study have been seen in light of some limitation , the purchase of oil seeds were done based on random sampling which is subject to biases and sampling error. This may influence overall analysis towards null hypothesis.
The study was limited to only three heavy metals and only oil seeds crops.
