2.8.3 Mechanical Expression Method
Mechanical expression involves the application of pressure using hydraulic or screw presses to force oil out of an oil-bearing material (Arisanu, 2013). Mechanical press methods are often used to extract vegetable oil from plant seeds having oil content higher than 20% (Sinha et al., 2015). There are two types of mechanical press methods namely, cold-press and hot-press methods. Cold-press or scarification method is carried out at low temperature (below 50oC). In cold-pressed oils, the purity and natural properties of seed oils are preserved (Azadmard- Damirchi et al., 2011; Bhatol, 2013), and this includes the retention of valuable nutraceuticals like phytosterols and tocopherols in the extracted oil (Kittiphoom et al., 2015). Because of these attractive qualities, there is growing global demand for cold-pressed oils. Cold-pressed oils are safer than hot-pressed seed oils because adverse effects caused by high temperatures are avoided. Some of the likely adverse effects of hot pressed oils are decreased oxidative stability, degradation of valuable oil components, and reduced oil keeping quality (Matthaus, and Brühl, 2003).
The hot-press method is carried out at elevated temperature and pressure and results into higher oil yield largely due to decreased oil viscosity at high temperatures. This enhances oil flow during extraction, thus high temperature increases the efficiency of the extraction process and oil yields of up to 80% possible (Patel et al., 2016).
Mechanical expression methods have the advantage of low operation cost and producing high quality light colored oil with low concentration of free fatty acids. However, it has a relatively low yield compared to solvent extraction, and is therefore comparatively inefficient, often with a large portion of oil left in the cake or meal after extraction (Bhuiya et al., 2015).
2.9 ANALYTICAL METHODS OF FATTY ACIDS AND HEAVY METALS
Worldwide, the most commonly employed techniques for analysis of fatty acid profiles of vegetable oils are gas chromatography (GC) and high performance liquid chromatograph (HPLC) (Botinestean et al., 2012).
HPLC is the most commonly used tool for the analysis of pharmaceutical products, but it is less successful in the quantification of fatty acids due to the absence of chromophores or fluorescent functional groups. Compared with the standard GC method, this method achieves acceptable separation and precision, but has poor sensitivity (Tsuyama et al., 1992).
GC is the most commonly used technology for the analysis of fatty acids which require derivatization to fatty acid methyl esters (FAME) due to the high boiling points of fatty acids, which are difficult to evaporate (Laakso et al., 2002).
2.9.1 Principles of Gas Chromatography
Chromatography is a separation technique based on the affinity difference between two phases, the stationary and mobile phases. A sample is injected into a column, either packed or coated with the stationary phase, and separated by the mobile phase based on the difference in interaction (distribution or adsorption) between compounds, and the stationary phase. Compounds with a low affinity for the stationary phase move faster through the column, and elute earlier. The compounds that elute from the end of the column are determined by a suitable detector. The mobile phase is always composed of an inert gas such as Nitrogen, Hydrogen or Helium which does not interact with the components in a mixture, but carries or transports them through the column, and the stationary phase is a “solid” which is either a packed column or a capillary column ( Al-Bukhaiti et al., 2017).
This technique enables the separation, identification, and purification of components of a mixture for qualitative and quantitative analysis. However, the use of Gas Chromatography is limited to volatile and thermally stable compounds or molecules that may undergo derivatization reactions to form volatile and thermally stable products (Lehotay and Hajslova, 2002).
During a Gas Chromatography separation process, the sample is vaporized and carried by the mobile gas phase (i.e., the carrier gas) through the column. The quality of separation (resolution) depends on how long the components to be separated stay in the stationary phase, and on how often they interact with this phase. The type of interaction between component and phase (selectivity) is determined by the functional groups. The polarity of the phase is a function of stationary phase substituents (Grob and Eugene, 2004).
To measure a sample with unknown concentration, a standard sample with a known concentration is injected into the Gas Chromatography. The standard sample peak retention time and area are compared to the test sample to calculate the concentration.
Major components of GC include; –
Gas supply – involves two types of gases, carrier gas to carry the sample through the column, and a detector support gas to support the flame in the flame ionization detector.
Introduction of sample – can be by hand or auto sampler. The most common type of sample introduction involves syringe injection of the sample into a heated injection port.
Column – separation of components of the sample takes place in the heated column, a long tube running from the sample injector to the detector.
Detector – detector function involves detection of sample components as they pass through the end of the column. The most common detector is a Flame Ionization Detector (FID), in which molecules of the sample are burned in the flame with produced ions detected.
A computer system – is used by most GC systems to collect and analyze the data. Signals from the detector are collected and converted into user friendly information. Generally, the system generates a chromatogram which is plotted using information on the amount of the component as peak area with elution time as retention time. Contents and concentration of various components of the sample are determined by comparing various chromatograms.
Chromatography/Flame Ionization Detector (GC/FID) is widely used analytical technique in quantitative analysis of fatty acids in vegetable oils, petrochemical, pharmaceuticals, and natural gas. The FID detector typically uses hydrogen/air flame into which the separated sample is oxidized and produce electrically charged particles (ions) which are collected and produce an electrical signal which is then measured. The GC/FID technique is robust, and of low cost compared to the Mass Spectrometry. This system has a challenge because the FID is extremely sensitive to hydrocarbon impurities from the hydrogen and air supply for the flame. The impurities can cause increased baseline noise and reduce the detector sensitivity.
Gas Chromatography/Mass Spectrometry (GC/MS) is a hyphenated technique developed from the coupling of GC and MS. The two instruments are highly compatible with each other, however, GC operates at high pressure (760 torrs) while the MS operates at a vacuum (5-l0torr). During the identification of compounds in the MS, the mass spectra acquired by this hyphenated technique offer more structure related information on the interpretation of fragments of the ions. The fragments ions with different relative abundances can be compared with the library spectra. Nowadays, GC/MS is integrated with various online MS databases for several reference compounds with search capabilities that could be useful for spectra match for the identification of separated components. Compounds that are adequately volatile, small, and thermostable in GC conditions can be easily analyzed by GC/MS. Sometimes polar compounds, with a number of hydroxyl groups, need to be derivatized prior to any analysis. The most common derivatization technique is the conversion of the analyte to its trimethylsilyl derivative. It is suitable for analysis of both volatile and nonpolar compounds.
GC/MS technique is limited in that, the compounds analyzed must be sufficiently volatile to allow transfer from liquid phase to mobile carrier gas and thus to elute from analytical column to the detector. Hence many compounds are too polar or too large to be analyzed with this technique. GC/MS has positive attributes in that it offers high efficiency separation with numerous columns, excellent limit of quantification, and also allows use of mass spectral library for identification of samples. During GC/MS analysis, vaporized sample is carried through the GC column with the help of heated carrier gas through the column where the components are separated. The separated components then enter MS through an interphase, and this is followed by ionization, mass analysis, and detection of mass-to-charge ratios of ions generated from each component by the mass spectrometer. The process of ionization not only ionizes the molecule, but also break the molecule into the positive or negative modes (Ruchira et al., 2012).
2.9.1.1 Selection of the Chromatographic Column
An appropriately selected column can produce a good chromatographic separation which provides an accurate and reliable analysis. An improperly used column can often generate confusion, inadequate, and poor separations which can lead to results that are invalid or complex to interpret. There are over 10,000 compounds that can be analyzed by Gas Chromatography, over 400 Gas Chromatography capillary columns (Jennings, 1990). The selection of proper capillary column for any application should be based on four significant factors which are stationary phase, column internal diameter, film thickness, and column length.
The differences in the chemical and physical properties of injected organic compounds, and their interactions with stationary phase are the basis of separation process. When strength of the analyte-phase interactions differs significantly for two compounds, one is retained longer than the other. How long they are retained in the column (retention time) is a measure of these analyte-phase interactions. Two compounds that co-elute (do not separate) on a particular stationary phase might separate on another phase of different physical and chemical property (Kupiec, 2004).
As a general rule, it is advisable to use similar polarities for phase and target compounds, for example, nonpolar molecules require nonpolar polysiloxane phases in the column. For FAME analysis, for example a capillary column with DB-23, 50% Cyanopropyl 50% methylpolysiloxane or HP-88, 88% Cyanopropyl 12% arylpolysiloxane can be used as a stationary phase because they are polar (Rahman et al., 2015).
2.9.1.2 Fatty Acids Methyl Ester Preparation (Derivatization)
Methyl esters are the favorite derivatives for Gas Chromatography analysis of fatty acids because they are more volatile than fatty acids (Christie, 1998). Derivatization is a process of converting fatty acids into fatty acid methyl esters, and this can be carried out through two possible approaches;
- By a two-steps reaction involving saponification followed by esterification.
- By a single step reaction known as transesterification.
The two main chemical reactions that occur during methylation are hydrolysis and esterification.
2.9.1.3 Hydrolysis of Fatty Acids
Hydrolysis results in a mixture of fatty acids and glycerol from triglycerides as indicated by the reaction in figure 5. Where R is a linear carbon chain.
Figure 5: Hydrolysis of fatty acids (Sigma, 2008)
2.9.1.4 Esterification (Methylation) of Fatty Acids
The esterification of fatty acids to fatty acid methyl esters is performed using an alkylation derivatization reagent. The esterification reaction involves the condensation of the carboxyl group of an acid, and the hydroxyl group of an alcohol in the presence of a catalyst. The catalyst protonates an oxygen atom of the carboxyl group making the acid much more reactive. An alcohol then combines with the protonated acid to yield an ester with the loss of water. The catalyst is removed with the water. Esterification is illustrated in reactions (2) and (3). The alcohol that is used determines the alkyl chain length of the resulting esters, for example, the use of methanol will result in the formation of methyl esters, whereas the use of ethanol will result in ethyl esters.
Figure 6: Methylation of fatty acids (Sigma, 2008)
2.9.1.5 Methylation Methods
2.9.1.5.1 Base Catalyzed and Acid Catalyzed (Two-Step) Method
In this procedure, a known amount of oil sample is placed into centrifuge tubes to which potassium hydroxide (10M) solution, and methanol are added. The reaction is performed at 55°C for 1.5h with mixing for 5s every 20min. After cooling to room temperature, 0.5mL sulphuric acid (10M) solution is added, and the reaction is continued at 55°C for 1.5h with mixing for 5s every 20min. After cooling to room temperature, n-hexane is added, and the mixture centrifuged for 5min. The hexane phase from the top layer of the solution is extracted, and transferred to screw cap for Gas Chromatography (GC) analysis (Christie, 1998).
2.9.1.5.2 Borontrifluoride/Methanol (BF3/CH3OH)
An aliquot of lipid extract is mixed with Borontrifluoride –methanol mixture and heated to a maximum temperature of 1000C for 2–90 min depending on the type of lipid. After cooling to ambient temperature, water and a non-polar solvent are added, vortexed, and the two phases separated by centrifugation. The upper organic phase containing the methyl esters is carefully removed to a new vial where it is removed under a stream of nitrogen gas (N2). The remaining residue containing fatty acid methyl esters is dissolved in n-hexane prior to GC analysis.
2.9.1.5.3 Sodium Methoxide/Methanol (NaOCH3/CH3OH)
Aliquot (20–40mg) is dissolved in dry toluene, 2ml 0.5M sodium methoxide (metallic sodium in anhydrous methanol) added and the samples heated at 60 oC for 20 min. This esterification step can also be carried out at room temperature (200C). A few drops of glacial acetic acid are added followed by 2ml saturated NaCl solution to eliminate excess methoxide. With 50ppm butylated hydroxy toluene (BHT) 2 ml n-hexane are added, and vortexed vigorously. The upper organic phase containing the extracted methyl esters is taken, dried over anhydrous sodium sulphate or 100 μl of a water scavenger such as 2, 2 dimethoxypropane added, and dried under a gentle stream of N2 at 40 0C. The fatty acid methyl esters are resuspended in 2 ml of n-hexane with 50ppm BHT and injected for GC analysis. Dilution of the sample in n-hexane may be necessary, depending on the GC response.
2.9.1.5.4 Potassium hydroxide/Methanol (KOH/CH3OH)
The oil sample is dissolved in n-hexane and KOH methanolic reagent added and the samples heated at 500C for 10–15min. A few drops of glacial acetic acid are added followed by water and n-hexane. The solution is vortexed vigorously and the upper organic phase taken and dried over anhydrous sodium sulphate prior to injection for GC analysis.
2.9.1.5.5 Sulphuric Acid/Methanol (H2SO4/CH3OH)
An aliquot is added to a 0.1M H2SO4 in methanol and the tubes heated to 100 oC for 30–60min. Sodium bicarbonate solution is added to neutralize the reagent and n-hexane to extract the FAMEs. The organic layer is carefully removed, then dried over anhydrous sodium sulphate. Successive n-hexane extracts are added and evaporated under a stream of N2. The residue is re-dissolved in n-hexane for GC analysis. (Wang et al., 2014).
2.10 ANALYTICAL INSTRUMENTS USED IN FATTY ACID ANALYSIS.
GC is extensively used in food analysis for routine qualitative and/or quantitative determination of components such as fatty acids, sterols, alcohols, oils, and low mass carbohydrates. Further applications in food analysis are devoted to the detection and quantification of food contaminants such as pesticides, environmental pollutants, natural toxins, veterinary drugs, and packaging materials. GC is reliable, efficient, and cost effective method of separation which requires small amount of sample, and also provides fast and highly accurate quantitative analysis.
2.11 THE PHYSICOCHEMICAL PROPERTIES OF VEGETABLE OILS
Physical and chemical properties of vegetable oil is determined in order to evaluate the nutritional quality and stability of the vegetable oils. These properties include viscosity, density, acid value, saponification value, iodine value, and peroxide value.
2.11.1 Viscosity
Oil viscosity is defined in two ways, either based on its absolute viscosity or its kinematic viscosity. The absolute viscosity is the resistance of oil to flow and shear due to internal friction, and it is measured in SI unit of Pa*s. Other units include (1poise = 100cP = 1 g*cm-1*s-1 = 0.1Pa*s).
The kinematic viscosity is the resistance of oil to flow and shear due to gravity, and it is measured in SI unit of m2/s. kinematic viscosity of oil can be obtained by dividing the oil absolute viscosity with its corresponding density (Singh and Heldman, 2001).
Viscosity of oil is used to determine the degree of unsaturation and molecular weight of the fatty acid. Viscosity increases with molecular weight of fatty acids and decreases with unsaturation (Kumar et al., 2010).
Viscosity is an important factor in selecting a good vegetable oil for food frying. The higher viscosity of frying oils the greater content of oil in the fried foods (Dana and Saguy, 2006), and this is because, high viscosity allows the oils to be accumulated more easily on the surface of fried foods, and enter inside the food during the cooling period (Maskan, 2003).
It has been well established that temperature has a strong influence on viscosity of vegetable oils, with viscosity generally decreasing with increase in temperature. The absolute viscosity of oil is an important parameter in determining quality of oils with regard to its fatty acid composition.
Yalcin et al. (2012) reported the viscosities of different vegetable oils from Turkey as olive (61.2mPa*s), hazelnut (59.7mPa*s), cottonseed (57.3mPa*s), canola (53.5 mPa*s), soybean (48.7mPa*s) and sunflower (48.2mPa*s) respectively.
The Principles of Viscosity Measurement is based on Ostwald’s viscometer, also known as U-tube viscometer or capillary viscometer, which is a device used to measure viscosity of a liquid with a known density. The method of determining viscosity with this instrument consist of measuring the time for a known volume of the liquid to flow through the capillary under influence of gravity. The instrument must first be calibrated with material of known density such as pure (deionized) water. Knowing the value of viscosity of one liquid, viscosity of other liquids can then be calculated.
=
η1 = is viscosity of liquid 1. η2 = is viscosity of liquid 2.
t1 = flowtime of liquid 1. 𝑡2 = 𝑓𝑙𝑜𝑤𝑡𝑖𝑚𝑒 𝑜𝑓 𝑙𝑖𝑞𝑢𝑖𝑑 2.
d1 = density of liquid 1. 𝑑2= 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑙𝑖𝑞𝑢𝑖𝑑 2.
Figure 7: Ostwald’s Viscometer (Generali, 2018)
2.11.2 Density
Density of oil is defined as mass per unit volume at specified pressure and temperature. The oil relative density or specific gravity of oil is defined as the ratio of the density of oil to that of water at the same specified pressure and temperature. Density of vegetable oils is temperature dependent, and decreases in value when temperature increases. The range is from 0.91 to 0.93g/cm3 between the temperature of 15oC and 25oC.
Ackman and Eaton (1977) indicated that a different proportion of fatty acids could be a major factor for the increase in density of vegetable oil. For example, the high density of soybean oil 0.9193g/cm3 reported was attributed to the higher content of linoleic acid (Ackman and Eaton, 1977).
Density is an important factor which influences oil absorption as it affects the drainage rate after frying, and also the mass transfer rate during the cooling stage of frying. The codex standard for density of vegetable oils range between 0.919 to 0.925gm/cm3 (Stahidi, 2005).
2.11.3 Acid Value (AV)
Acid value represents the mass of potassium hydroxide (KOH) in milligrams required to neutralize the free fatty acid in 1g of oil sample. Acid value (AV) is a good indicator of oil degradation caused by hydrolysis or enzymes (Othman and Ngassapa, 2010). Acid value increases with days of storage under ambient conditions. Acid value of oils determines the purity of oils.
The maximum recommended acid value for all vegetable oils is 4mgKOH/g of oil (FAO/WHO, 2019). A low acid value indicates the oil is stable and pure for human consumption (Orthoefer, 2007).
2.11.4 Saponification Value (SV)
Saponification value represents the number of milligrams of potassium hydroxide (KOH) required to saponify (hydrolyze) one (1g) of fat under the conditions specified. Saponification value is used as a measure of the average molecular weight (Chain Length) of all fatty acids present in the vegetable oil sample (Mohammed and Alli, 2015; Fazal et al., 2015).
The long chain fatty acids found in fats have a low saponification value, while the short chain fatty acids have a high saponification value (Musa et al., 2012). The recommended saponification values for different vegetable oils range between 187-196mgKOH/g for peanut, 185-193mgKOH/g for sesame, 188-194mgKOH/g for sunflower (FAO/WHO, 1999). Ezeagu et al. (1998) reported that a saponification value of 200mgKOH/g indicates a high proportion of short chain fatty acid of low molecular weight in a vegetable oil, and this property makes the vegetable oil potential for use in soap making, cosmetic industry, and sources of essential fatty acids in the body.
2.11.5 Peroxide Value (PV)
The peroxide value is defined as the weight of active oxygen contained in one gram of oil or fat (Horwitz, 1975), and It determines the degree of oxidation of oil which is an indication of the level of deterioration of oils and fats (Okechalu et al., 2011). A freshly refined oil should have nil peroxide value.
WHO/FAO (2019) recommended maximum peroxide value of 10meq/kg for all vegetable oils. A high peroxide value indicates high level of oxidative rancidity of the oil which may be caused by increase in storage time, temperature, and air contact with the oil (Kamau and Nanua, 2008). Oils exposed to both atmospheric oxygen and light may show a much larger increase in peroxide value during storage.
Low peroxidative value are indicative of low levels of oxidative rancidity of the oils and also suggest strong presence or high levels of antioxidant (Adelaja, 2006). Low peroxide value may also be used as a measure of shelf-life and freshness of the oil.
2.11.6 Iodine Value (IV)
Iodine value is the mass of iodine in grams that is required to react with 100g of fat or oil sample. The iodine value is used as a measurement of the total unsaturation of vegetable oils, and also as an indicator of their susceptibility to oxidation (Knothe, 2006). Vegetable oils can be divided into four major categories depending on their iodine values namely, saturated oils (iodine value between 5 and 50), mono-unsaturated oils (50 and 100), di-unsaturated oils (100 and 150), and tri-unsaturated oils (over150).
The higher the iodine value, the greater degree of unsaturation, and the more the oil becomes susceptible to oxidation (Onyeike, 2003). Vegetable oils with low iodine values have a high proportion of saturated fatty acids which are stable to oxidative degradation. The recommended iodine value for different vegetable oils range between 7.7 – 107 mg of I2/100 g for peanut oil, 105 – 120mg of I2/100 g for sesame oil, and 110 – 143mg of I2/100 g for sunflower oil respectively.
2.12 HEAVY METALS IN VEGETABLE OILS
Heavy metals are defined as metallic elements that have a relatively high density compared to water (Ferguson, 1990). Heavy metals can be classified as potentially toxic metals such as Arsenic (As), cadmium (Cd), and lead (Pb) which can be very harmful even at a low concentration when ingested over a long time period (Unak et al., 2007).
Micro essential elements such as iron (Fe), copper (Cu), and zinc (Zn) are required in small quantities for various biochemical and physiological functions. The essential elements can also produce toxic effects when the metal intake is excessively elevated (Gopalani et al., 2007).
2.12.1 Sources of Heavy Metals in Vegetable Oils
All heavy metals are naturally occurring elements that are found throughout the earth’s crust. Anthropogenic activities such as mining and smelting operations, industrial production and use, domestic and agricultural use of metals, and metal-containing compounds cause environmental contamination by heavy metals (He, 2005; Shallari et al., 1998). Environmental contamination can also occur through metal corrosion, atmospheric deposition, soil erosion of metal ions, and leaching of heavy metal ions, sediment re-suspension and metal evaporation from water resources to soil and ground water (Nriagu, 1989). Natural phenomena such as weathering and volcanic eruptions have also been reported to significantly contribute to heavy metal pollution (Ferguson, 1990; Bradl, 2002; He, 2005).
The presence of heavy metals and their chemical forms in vegetable oils depend on several factors. The metals might originate from the soil and fertilizers, and be absorbed by the plant roots. Heavy metals might be introduced during the production processes such as bleaching, hardening, refining, and deodorization. The metals may also be introduced into the vegetable oils by contamination through metal processing equipments (Leonardis et al., 2000).
For many years, it has been discovered that vegetable oil may contain heavy metals such as iron, nickel, lead, cadmium, copper, and Arsenic (Mendil et al, 2009). Various studies on vegetable oils consumed in different countries such as India, China, Nigeria, and others reported presence of heavy metals such as lead, cadmium, arsenic, nickel in vegetable oils sold in markets (Juszczak 2008).
Cadmium: This toxic element is easily transferred from sosil to plants, which are increasingly contaminated by cadmium from phosphate-based fertilizers. Cadmium can also be present in vegetable oils, as a result of contamination through refining process, the storage tank or the packing material such as a colorant or stabilizer in plastics (Mendil et al., 2009).
Copper: Copper is known to be both vital and toxic for many biological systems, and may enter the food materials from soil through mineralization by crops, food processing and environmental contamination such as application of copper based pesticides which are in common use in some countries (Onianwa et al., 2001; Koc et al., 2008).
Zinc: The presence of zinc in vegetable oil could be due to absorption by plants from soil or contamination of the vegetable oils during refining process, storage and packaging materials). According to WHO (2003) the Maximum Permissible Limit (MPL) of zinc in vegetable oils is 10000µg/g or mg/g.
Reported zinc levels in different vegetable oils from other parts of the world are in the ranges of 0.04-0.70 µg/g and 0.0484-0.2870 mg/kg (Garrido et al., 1994; Pehlivan et al., 2008).
Iron: Iron may enter vegetable oils from the soil through uptake of minerals by the roots, and also the reaction between the relatively high-unsaturated portion of the oil with the surface of iron containers used during transportation, storage, and processing of vegetable oils.
Lead: research study shows that the presence of lead in vegetable oil could be attributed to deposition or bioaccumulation from soil containing phosphate base fertilizer that were used in the plantation or may be due to water used for irrigation (Ansari et al., 2009). Other reports also reveal that contamination of vegetable oil with lead may also be due to industrial emission, combustion of fuel in refinery process, and from packaging material such as stabilizer and colorant in plastic (La Pera et al., 2004).
2.12.2 Effects of Heavy Metals on Human Health
Various studies around the world have confirmed the presence of heavy metals such as cadmium, lead, copper, iron, nickel, zinc, chromium, cobalt in vegetable oils, and reports indicate that toxic elements such as cadmium and lead can be very harmful even at low concentration when ingested over a long time period (Unak et al., 2007). The essential metals like zinc, copper, iron, and nickel can also produce toxic effects when the metal intake is excessively elevated (Gopalani et al., 2007).
Zinc: Zinc is an essential functional and structural element in most metabolic pathways in humans, and is regarded as cofactor for many enzymes. However, excessive levels of zinc in the body harms some physiological processes like breathing. Zinc deficiency can lead to growth retardation and immunological abnormalities (Tahsin and Yankov, 2007). According to WHO (2003) the Maximum Permissible Limit (MPL) of zinc in vegetable oils is 10000μg/g.
Cadmium: Cadmium is a highly toxic heavy metal with very low absorption levels in humans (3-5%) after exposure with contaminated foodstuffs. Cadmium accumulates in the human body and damages mainly the kidneys and liver. Cadmium is retained in the human body specifically in liver and kidney with a long biological half-life (10-30yrs). Joint expert committee on food additives of FAO and WHO offers the authorized index of provisional tolerable weekly intake for cadmium as 7μg/kg per body weight.
Iron: Iron deficiency is frequently associated with anemia, and thus reduces working capacity and impairs intellectual development (Schumann et al., 2007). It is known that adequate iron in diet is very important for decreasing the incidence of anemia. WHO (2003) established a Maximum Permissible Limit (MPL) for iron as 1.5mg/kg of body weight.
Lead: Lead is similar to cadmium because it has no beneficial role in human metabolism, and mainly producing progressive toxicity. The presence of lead in the body can lead to toxic effects, regardless of exposure pathway. Some researchers have suggested that lead continues to contribute significantly to socio-behavioral problems such as juvenile delinquency and violent crime (Needleman et al., 2002). WHO (1993) has established a provisional tolerable weekly intake (PTWI) for lead of 0.025mg/kg of body weight.
2.12.4 Determination of Heavy Metals in Vegetable Oils
2.12.4.1 Atomic Absorption Spectrometry (AAS)
Atomic absorption spectrometry (AAS), is an analytical technique that is used to determine the concentration of metals in samples. It is based on the principle that an atom in the ground state absorbs the light of wavelengths that are characteristic to each element when light is passed through the atoms in the vapour state, and since this absorption of light depends on the concentration of atoms in the vapour, the concentration of the target element in the sample is determined from the measured absorbance. The Beer-Lambert law describes the relationship between concentration and absorbance. Absorbance is directly proportional to concentration of the sample.
A = ɛbc,
Where A = absorbance (no unit),
ɛ = molar absorptivity constant (Lmol-1cm-1),
b = Path length of the sample (cm),
c = Concentration of sample in solution (molL-1).
Analyzing a sample to see if it contains a particular element means using light from that element. For example, with lead, a lamp containing lead emits light from excited lead atoms that produce the right mix of wavelengths to be absorbed by any lead atoms. Some of the radiation is absorbed by the lead atoms in the sample, and the greater the number of atoms in the vapour, the more radiation is absorbed.
The amount of light absorbed is proportional to the number of lead atoms. A calibration curve is constructed by running several samples of known lead concentration under the same conditions as the unknown.
The amount the standard absorbs is compared with the calibration curve and this enables the calculation of the lead concentration in the unknown sample. Consequently, an atomic absorption spectrometer needs the following three components: a light source; a sample cell to produce gaseous atoms; and a means of measuring the specific light absorbed.
2.12.4.1.1 Flame Atomic Absorption Spectrometry (FAAS)
In flame atomic absorption spectrometry, a sample is aspirated into a flame and atomized. A light beam from a hollow cathode lamp of the same element as the target metal is radiated through the flame, and the amount of absorbed light is measured by the detector. This method is much more sensitive than other methods, and free from spectral or radiation interference by co-existing elements. Pretreatment is either unnecessary or straightforward. However, it is not suitable for simultaneous analysis of many elements, because the light source is different for each target element.
