CHARACTERIZATION AND DETERMINATION OF FREE FATTY ACID IN DIFFERENT OILS



WRITTED BY


IDRIS ALHASSAN
ADM NO: - 1510202066




DEPARTMENT OF PURE AND APPLIED CHEMISTRY
INPARTIAL FULFILEMENT FOR THE REQUIREMENT OF THE AWARD (B.Sc Hons)
DEGREE IN APPLIED CHEMISTRY



FACULTY OF PHYSICAL SCIENCES, KEBBI STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY, ALIERO



SEPTEMBER, 2019

CERTIFICATIONS
This is to certify that this research project was conducted by Idris Alhassan with the admission umber 1510202066 of applied chemistry, faculty of physical science, Kebbi State University of Science and Technology, Aleiro.

__________________________               __________________
Dr Aliyu Muhammad     Date
          (Project Supervisor)


___________________________ ____________________
          Dr Hannatu Abubakar    Date
           (Project Coodinator)


___________________________              __________________
         Dr. Aliyu Muhammad               Date
        (Head of Department)





__________________________               __________________
            External Examiner               Date

DEDICATION
This project work is dedicated to Allah Almighty who game strength and enable me to accomplish this course of study successfully, and also goes to my beloved family for their love, support and encouragement.

DECLARATION
I Idris Alhassan do here by declare that this is an original work done by me in the department of Pure and Applied chemistry, Faculty of Physical Science, Kebbi State University of Science and Technology, Alieo from November 2015 to October 2019, under the supervision of Dr. Aliyu Muhammad.




Idris Alhassan
Sign:______________________
Date:______________________












ACKNOWLEDGEMENT
All glory and praised be to Allah almighty, the creator of the universe, Master of the Day of Judgment, to whom we all look forward to for sustenance. He alone possesses the knowledge of everything, and to him alone belongs the dominion of the earth.
I wish to express my profound gratitude and appreciation to my project supervisor, Dr. Aliyu Muhammad for his support, advise, and consistence without this dissertation wouldn’t have stand the test of time. He is among those people I will never forget in my life.
My special appreciation goes to my active family members for their care and prayers in my upbringing and supporting me morally.
 TABLE OF CONTENTS
Tittle page - -- - - - - - - - i
Certification - - - - - - - - - ii
Dedication - - - - - - - - - iii
Declaration - - - - - - - - - iv
Acknowledgment - - - - - - - - v
Table of contents - - - - - - - - vi-vii
Abstract - - - - - - - - - viii
CHAPTER ONE
1.0 Introduction - - - - - - - - 1-3
1.1 Palm oil - - - - - - - - 3-4
1.2 Free fatty acids - - - - - - - 4-5
1.3 Current method for determination of free fatty acids in palm oil - 5
1.4 Acid-base titration analysis - - - - - - 5-6
1.5 Literature review - - - - - - 6-7
1.6 Aim and Objectives - - - - - - - 7
CHAPTER TWO
2.0 Materials and Method - - - - - - 8
2.1 Preparation of reagent - - - - - - - 8-9
2.2 Determination of crude fiber - - - - - - 9-11
2.3 Determination of free fatty acid - - - - - 10-11
2.4. Iodine value - - - - - - - - 11-12
2.5 Methods - - - - - - - - 12
2.6 Current method for determination of free fatty acids in palm oil - - 12-13
2.7 Acid-base titration analysis: - - - - - - 13
2.7 Gas chromatography analysis - - - - - - 13-14
2.8 High performance liquid chromatography analysis - - - 15-16
2.9 Near-infrared reflectance spectroscopy (NIRS) analysis - - - 16-17
2.9 GC – MS Analysis of Fatty Acids - - - - - 18
2.10 Determination of Fatty Acid Composition - - - - 18
2.11 Determination of Free Fatty Acid Value - - - - 19
CHAPTER THREE
3.0 RESULTS AND DISCUSSION - - - - - 20
3.1 RESULTS - - - - - - - - 20
3.2.0 Discussion - - - - - - - - 21-22
CHAPTER FOUR
4.0 Conclusion and Recommendation - - - - - 23
4.1 Conclusion - - - - - -- - - 23 
4.2 Recommendation - - - - - - - 23
Appendix A - - - - - - - - - 24-25
Appendix B - - - - - - - - - 25-27

References - - - - - - - - - 28-29


LIST OF TABLES
Table 2.0 List of Reagents - - - - - - - 8
Table 3.1 Physical Properties of Palm Oil - - - - - 20
Table 3.2 Physical Properties of Shea butter Oil - - - - 20
Table 3.3 Fatty acids Composition of Oil - - - - - 22


ABSTRACT
The experiment was conducted outside the school area, which is held at National Research Institute of Chemical Technology (NRICT), Zaria, Nigeria to study the Determination of Free Fatty Acid in different oil samples and oil characterization.
The effect of light of different colors (wavelength) on the Free Fatty Acid (FFA) value of stored palm oil and Shea butter is hereby reported. Equal portions of the oil samples were stored in an environment of red, blue and green lights, respectively for a period of 21 days. Aliquots were taken from each of these samples at two days interval for analysis. And the FFA values obtained plotted against the number of days storage. Results obtained indicate that the FFA values of the oil samples obtained indicate that the FFA values of the oil samples increased with storage time. Also and more revealing is the fact that the FFA value of the samples did not follow any established order, especially as related to the spectrum of light.
Key words: Palm oil, Shea butter, Mesocarp, Fluorescent, Free Fatty acid, hydrolysis, and wavelength.
CHAPTER ONE
 1.0 INTRODUCTION
Fats and oils are compounds of glycerol (propan-1,2,3-triol)and fatty acids.The later formed from the hydrolysis of fats and oils with the help of the enzyme lipase in the presence of moisture, heat and catalyst(Njoku et al.,2010). The physical and chemical properties of fats and oils are essentially determined by the fatty acid composition of their tryclicerides.
The Sudanian Savanna of sub-Saharan Africa is the centre of origin of the shea tree (Vitellariapara), where it is also widely distributed. The species is known to occupy a 5000 km stretch of African savanna from senegal to ethiopia and Uganda (nikiema and Umali 2007). This is in addition to its reported occurrence in Dominica and honduras through human agency (Ahmad et al., 2000).The popularity of the species among indigenous peoples is predicated on its usefulness in innumerable ways. Apart from the fruit pulp which is edible, other plant parts like the leaves and roots are credited with various medicinal uses. Similarly, the sticky black residue left after butter clarification is used to fill cracks in walls (Ahmad et al,2000). Vitellariaparadoxa equally plays host to Cirinabutyrospermi whose protein-rich caterpillars are a cherished delicacy among some ethnic groups in Nigeria (Ande 2004, Ugese et al., 2005). The wood is heavy and invaluable in construction works and in the production of household and farm implements. it produces great heat and its fine charcoal is particularly valued by blacksmiths (2000).Author for correspondence however, outside the shores of Africa, specifically in europe and japan, the importance of the species is linked to the fat extract of its kernels.The estimated 10% (nikiema and Umali 2007) or 30% (FAO 2002) of annual shea nut collection that is exported, a major part is used in the food industry as cocoa butter substitute or improver. The unsaponifiable fraction is used for cosmetic manufacture which forms a small but important and high value end use (FAO 2002). Apart from allantoin, other constituents of the unsaponifiable fraction of shea fat like vitamin e (tocopherol) have been credited with skin hydrating and healing properties which has conferred on shea butter its excellent cosmetic effect on dry and damaged skin (nikiema and Umali 2007). it is also to be noted that even among African producer nations, shea fat is used in cooking, as illuminant as well as in soap and pomade preparations (Vickery and Vickery 1969, Awoleye 1995). interestingly, cosmetic industries in these countries are also finding it invaluable in their skin and hair cream formulations (Boffa et al. 1996). Generally, export of shea nuts is reported to have impacted favourably on the economies of the exporting countries (Boffa et al. 1996, Popoola and Tee 2001). In Nigeria, Vitellariaparadoxa occupies a wide expanse of savanna belt, occurring mainly across the guinean and sudanian savanna zones (Keay 1989). This wide distribution and high concentration of the trees presumably accounts for nigeria’s position as the leading producer of shea nuts in Africa (Umobong 2006, nikiema and Umali 2007). The work reported herein is an attempt at exploring this trait across the guinean and sudanian savanna zones of nigeria. such information could, among others, give a clue to identifying sources of shea nuts most suitable as either cocoa butter equivalent (CBe) or cocoa butter improver (CBi) in the food industry.
Oil palm (Elaeisguineensis) originates from Southeast Asia and Equatorial Africa. Palm oil is the leading vegetable oil in the world with the highest production of 38.5 million and dominating 25 % of total global oils and fats production in the year 2007. Malaysia's oil palm plantation area and crude palm oil production has been increasing gradually in the past five decades and Malaysia accounted for more than 40 % of the total world palm oil production. This might be due to the suitable climate and good management arising from R&D. In terms of production cost, palm oil is one of the cheapest oil in the market compared to other edible oils in the world such as soybean, rapeseed and sunflower oils(Nzikou et al;2007).
In order to achieve the country's Gross National Income(GNI) by the year 2020, improvements have to be done in palm oil industry especially in accelerating the production rate of high quality palm oil. High production rate of palm oil dependent on the methods used during laboratory works and this had been identified through eight entry point projects (EPPs) which some of it includes an accelerated replanting via a binding replanting policy, improving fresh fruit bunch yields, improving oil extraction rate, extraction of oil expediting growth of food and health-based segments of the industry and so forth. Meanwhile, the palm oil and crude palm oil quality are dependent on the oil contents itself such as free fatty acids, phosphatides, odoriferous matter, water and impurities which can be remove through several processes such as refining process. Free fatty acids content is one of the important parameters in palm oil industry. The free fatty acids content in palm oil indicates the level of deterioration of oil and it is responsible in dictating the price of the palm oil in industry. Furthermore, the nutritional status of palm oil is also determined by the types of fatty acids contained in palm oil. Therefore, in order to produce high quality of palm oil, it is crucial to determine the free fatty acids content in palm oil before it is marketed (MC Nought and Wilkinson, 1997).
1.1 Palm oil:
Palm oil is one of the most widely used edible oils in the world because of the lower price compared to other edible oils. The palm oil can be obtained from two distinct portions of palm fruit which are from the flesh of the fruit or known asmesocarp and also from seed of kernel of the fruit. High amount of oil can be obtained from the mesocarp of ripe fruits compared to the unripe one1. The extraction of crude palm oil (CPO) is carried out under high temperatures ranging from 90 to 140 °C. Crude palm oil appears as a semisolid, deep reddish orange-coloured viscous solution at ambient temperature. The reddish orange-coloured represents the existence of carotene in crude palm oil which also known as pro-vitamin A, however carotenes are discarded during the refinery process (Chong, 200; Sy am, et al;2009).
1.2 Free fatty acids
Free fatty acids in palm oil: The free fatty acids or also known as the acid value (AV) content of the palm oil determines the quality of the palm oil itself. Oils which are high in free fatty acids content have poor quality of oil and suffer significant losses during refining process. The maximum standard specifications set by the Palm Oil Refiners Association of Malaysia for the free fatty acids content (as palmitic acid) in crude palm oil (CPO) is 5 % and should be lower than 0.1 % in Refined Bleached Deodorized Oil (RBDO). Low free fatty acids content in crude palm oil produced good physico-chemical properties of crude palm oil products and could be useful for industrial applications.Basically, the production of free fatty acids in palm oil is performed through the hydrolysis of fatty acid during the palm oil processing. Apart from that, free fatty acids is also released naturally in crude palm oil and can be produced due to the action of enzyme in the palm fruits, by microbial lipases and by the reaction of oil with water during storage. Moreover, the damaged palm fruitsand lengthy storage of palm fruitsmay also increase the free fatty acids content, thus affect the quality of palm oil. According to Arzamendi and co-workers, fatty acids can be produced directly through transesterification and hydrolysis process of fats and oil. The transesterification process is very sensitive to free fatty acids, thus may lead to the undesirable saponification, low product yields and complication in the next separation processing steps. The free fatty acids also normally present in downstream by-products of edible oil processing.Based on previous study, it was found out there was an endogenous lipase which also known as triacylglycerol acylhydrolase found in oil palm fruits. The action of lipase may increase the free fatty acids level in crude palm oil. The contamination of fungi in palm oil may lead to the hydrolysis of glycerides and free fatty acids formation, thus affect the quality of palm oil. Short chain fatty acids in palm oil can be converted into a series of methyl ketones by certain xerophilic fungi and this phenomenon is known as ketonic rancidity (James, 200).
1.3 Current method for determination of free fatty acids in palm oil
Most of the methods for determination of free fatty acids in palm oil usually are the same with the determination of free fatty acids in other edible oils. free fatty acids determination in palm oil had been studied previously. However, most of the methods applied during the studies involved manual operation and time consuming. Basically the oil has to be extracted prior to the free fatty acids analysis by using chemical equipment. There are various ways of determining free fatty acids in palm oil which includes titration, Gas chromatography (GC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) analysis. The palm oil is extracted prior to the analytical separation either through supercritical fluid extraction, sox-hlet extraction, liquid-liquid extraction and solid-phase extraction methods.
1.4 Acid-base titration analysis:
The traditional way of determining free fatty acids in palm oil is through the acidbase titration method by titrating the sample against potassium hydroxide in hot 2-propanol solution, using phenolphthalein as an indicator and the result is expressed in mg KOH g-1 oil. Theoretically, the acids contain in the solution are neutralized during the titration and the value obtained represents the acid number of the sample which is proportional to free fatty acids content. Although this method is simple, it is sluggish, laborious and lack of accuracy. On top of that, during the neutralisation process, apart from free fatty acids, other substances which can react with KOH might be neutralized as well, thus leads to the inaccurate calculation of acid number value. Moreover, high amount of reagents and solvents are also consumed during the operation. Various researches has also been reported for determination of free fatty acids in various types of palm oil and other vegetable oils by using American Oil Chemists' Society (AOCS) standard titration
method (Chong,1995).
Determination of underivated fatty acids in palm oil and other vegetable oils by RP-HPLC has been carried out by Hein and Isengard (1990). This research intended to improve the previous method by skipping the derivatization process which used high amount of chemicals and also time consuming. Three different alternative RP-HPLC methods were introduced where different detectors and different mobile phases were performed. A good resolution relies on the pH value of mobile phase. It was observed that the optimum pH for the separation of fatty acid was in the range of 3.0-3.5. On top of that, based on this research it was proven that the vegetable oils can be differentiated based on the characteristic of fatty acid pattern in oils. By applying HPLC method for determination of fatty acids in oils, it was also possible to determine the amount of free fatty acids in the oil by calculating the acid number. The amount of free fatty acids content in oil was based on the concentration of free oleic acid determined from the vegetable oils.
1.7 LITERATURE REVIEW
Each year, about 120million MTof edible fats and oils are consumed by the world (Sirag, 2002). The leading oil that account for 30%  of world production of edible fat and oils is soya bean. In 2003 it is closely followed by palm oil, whereas rapeasedoil ranked third as only one third of the production tonnage of sybean oil palm oil with annual production 0f 760,000 MTin 2003, is the twelfth largest vegetable oil produced in the world, high in quality that olive oil and sour flour oil (FAO, 2003). The production of palm oil increased 20% in the recent ten years, it was 632,000 MT in 1992. China has almost double the production of palm oil (from 142,000 to 210,000 MT) whereasindia has decreased the production by 44$ from (from 236,000 to 131,000MT0 in the above period.
1.8 Aim and Objectives
The aim of this research work is to compare and analyze the fatty acid composition of the palm oil and Shea butter oils.
1.9    Objectives
1) To determine the free fatty acid composition of the oil using GC-MS analysis.
2) To compare the physico-chemical and chemical properties, acid values, saponification value and iodine value of the oil.







CHAPTER TWO
2.0 MATERIALS AND METHOD
Table 2.0: List of Reagents
Reagents
Types
Purity
Manufacture

N-Hexane (C6H14)
450ml
99.5%
BDH England

Hydrochloric acid (HCL)
100ml
99%
Phillip Harris

Chloroform (CCl4)
250ml
99.66%
BDH England

Sulphuric acid (H2S04)
100ml
99.6%
BDH England

Potassium hydroxide (KOH)
250ml
99%
BDH England

Sodium hydroxide (NaOH)
250ml
99.7%
Avishkar

Methanol (CH4O)
250ml
99.6%
Phillip Harris


2.1 Preparation of reagent
0.01m Hydrochloric acid: this was prepared by preparing 0.6cm3 of concentrate hydrochloric acid (36% SG1.18) to 1dm3 volumetric flask containing 40cm3 of distilled water. The flask was then made up to the mark with distilled water.
2% Boric acid: 2.0g of pure boric acid was put in to 100cm3 volumetric flask followed by 20cm3 of methanol and 50cm3 of distilled water and well shaken thoroughly, few drop of 0.01m HCL were added to adjust the pH (to paint red) and the mixture was then diluted to the mark with distilled water.
40% Sodium hydroxide (W/V): 40% NaOH was prepared by dissolving 40g of NaOH pellet in 20cm3 of distilled water in a beaker. The solution then transferred and washed in to a 100cm3 volumetric flask and diluted to the mark of the flask with distilled water.
10% of Tetraoxosulphate (iv) acid (W/V): this was prepared by taking 10cm3 of concentrated tetraoxosuphate (iv) acid and diluting to 100cm3 volumetric flask with distilled water.
2.2 Determination of crude fiber
Procedure
2.0g of sample was mixed with Kjeldashl Catalyst in Kjeldah digestion tube 20cm3 of concentrated sulphuric acid was placed in a fume cupboard heated until it become clear. This indicate that, organic nitrogen and carbon where concentrate to ammonium sulphate and Carbon (iv) oxide respectively.
After the digestion, the tube was remove form the digestion and diluted to 50cm3 with distilled water. 10cm3 of the distilled digested sample was pipette in to the distilled flask containing 20cm3 of 40% NaOH solution. The content was diluted with 40cm3 distilled water and distilled using micro Kjeldha distillation apparatus. The distillate was retrieved in to a receiving flask containing 25cm3 of boric acid indictor solution (i.e distillate + boric acid)  was then titrated against 0.01m HCL to the end point (green – purple). The crude protein was calculated using the formula below:
   

2.3 Determination of free fatty acid
About 0.5g of oil was boiled with 5cm3 of ethanol and allowed to cool and 2 drops of phenolphthalein indicator was added, then titrated with 0.1N NaOH until color disappear (AOAC, 1998). Free fatty acid was calculated using the expression below:

Where: V = Titer value
Na = Normality of acid
F = Equivalent weight of free fatty acid
Ws = Weight of sample.
Preparation of reagents
5cm3 of Ethanol
5% Ethanol; 5cm3 of concentrated ethanol was dissolved in distilled water and made up to 100cm3.
Phenolphthalein indicator
1% phenolphthalein indicator, 1g of phenolphthalein was dissolved in 100cm3 of ethanol and distilled water.
0.1N NaOH
1N NaOH; 40g of sodium hydroxide pellet was weighed and dissolved in 1 litter of distilled water.
2.4. Iodine value
Iodine value is the measure of the degree of unsaturation in oil samples, it is the weight of iodine absorbed by 100 parts by weight of the sample. This reaction only occurs in unsaturated liquids (oils). Thus, the higher the iodine value, the higher the degree of unsaturation.
Procedure
Weight 0.5ml or any required amount into a 500ml glass-stopper flask and add 10ml of chloroform.
Pipette 10ml of hannus solution into the flask and allow it to stand in a dark place for 30 minutes with occasional shaking.
After incubation in the dark, add 10ml of potassium iodide solution and shake thoroughly.
Also add 100ml of freshly boiled and cooled water, washing down any free iodine on the stopper.
Add few drops of starch indicator and titrate against 0.1M sodium thiosulphate shaking it vigoriously so that any iodine remaining in the chloroform can be taken up by the KI solution.
Blank should be carried out in the same way but without a sample.
Record the volume of titrant used.

Calculations

2.5 METHODS
Fallen fruits of the Shea tree were collected from nine locations across the guinean and Sudaniansavanna zones in August, 2019. The collection was in respect to a more elaborate study of the species in Nigerivolving determinations of metric and chemical traits of fruits and nuts and seedling growth evaluation. The specific locations were: Lokoja, Makurdi, Akwanga, Minna(Southern Guinean Savanna), Kachia, Jalingo(Northern Guinean Savanna), Yolaand Kano (sudanian savanna). number of trees sampled in each location varied from 15 to 25, with each sampled tree occurring at least 100 m from each other (Schmidt 2000). From each location, forty-five (45) nuts, obtained from depulped fruits, were randomly selected. The 45 nuts were contributed equally by each sampled tree where possible, otherwise at least a nut from each tree. As such the depulped and sun dried nuts of the shea tree from the eight locations were decorticated, chopped into pieces and finely milled. The 45 nuts from each location were mixed together before performing the analysis.
2.6 Current method for determination of free fatty acids in palm oil:
Most of the methods for determination of free fatty acids in palm oil usually are the same with the determination of free fatty acids in other edible oils. free fatty acids determination in palm oil had been studied previously. However, most of the methods applied during the studies involved manual operation and time consuming. Basically the oil has to be extracted prior to the free fatty acids analysis by using chemical equipment. There are various ways of determining free fatty acids in palm oil which includes titration, Gas chromatography (GC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) analysis. The palm oil is extracted prior to the analytical separation either through supercritical fluid extraction, soxhlet extraction, liquid-liquid extraction and solid-phase extraction methods.
2.7 Acid-base titration analysis:
The traditional way of determining free fatty acids in palm oil is through the acidbase titration method by titrating the sample against potassium hydroxide in hot 2-propanol solution, using phenolphthalein as an indicator and the result is expressed in mg KOH g-1 oil. Theoretically, the acids contain in the solution are neutralized during the titration and the value obtained represents the acid number of the sample which is proportional to free fatty acids content. Although this method is simple, it is sluggish, laborious and lack of accuracy. On top of that, during the neutralisation process, apart from free fatty acids, other substances which can react with KOH might be neutralized as well, thus leads to the inaccurate calculation of acid number value. Moreover, high amount of reagents and solvents are also consumed during the operation. Various researches has also been reported for determination of free fatty acids in various types of palm oil and other vegetable oils by using American Oil Chemists' Society (AOCS) standard titration
Method.
2.8 Gas chromatography analysis
Gas liquid chromatography has been widely used and among the popular choice for half of century. The advantages of using gas chromatography are because it offers rapid analysis, high sensitivity and provide good reproducible analysis. It also relatively low cost in terms of analysis and convenience. Principally, fatty acids are separated by the interaction between stationary phase and mobile phase along the column before being detected by the detector. Flame ionization detector (FID) is the most common and widely used detector in chromatographic analysis for free fatty acids detection. On top of that, FID detector also able to detect various compounds as small as pictogram level concentration. Normally, gas chromatography chromatogram shows sharp and symmetric peaks. Nonetheless, the interpreted resolution of gas chromatography is highly dependent on the column length and polarity of stationary phase used basically, the choices of capillary column types, stationary phase polarity and column length are crucial in order to obtain good resolution and separation in gas chromatography analysis.
The problem with gas chromatography analysis is it might face difficulty while dealing with non-volatile samples as compared to the volatile one. In order to improve this limitation, free fatty acids has to be esterified before being injected into the gas chromatography column. In GC/FID, comparison of retention time between standard and sample is used to identify free fatty acid. However, complexity of fatty acid composition and limitation of fatty acid standard is clearly a major difficulty in order to identify some peaks with conventional FID detector. Identification by using GC/FID may also reflect by the presence of contaminants or coeluting compounds. Besides, using GC/FID requires adequate standards and standards are not available for some fatty acids especially for the complicated fatty acid compounds like polyunsaturated fatty acids. Therefore, fatty acid analysis can be done by converting free fatty acids into fatty acid methyl ester (FAME) before injecting into the gas chromatography column. The combination of GC/FID and GC/Mass spectrometry (GC/MS) can be applied in order to ensure the accurate identification of a peak. Several works had been reported in literature for determination of free fatty acids in various kinds of palm oils and vegetable oils.

2.9 High performance liquid chromatography analysis
High performance liquid chromatography is one of the methods that is widely used for determination of free fatty acids either in palm oil or other vegetable oils. HPLC method has been widely chosen for determination of free fatty acids nowadays due to the fact that it has excellent selectivity and it ables to quantify high diversity of analytes. The main difference between HPLC and gas chromatography method lies on types of the mobile phase used where liquid mobile phase is used in HPLC while gas mobile phase is used in gas chromatography. The free fatty acids is widely analyzed by using RP-HPLC. According to Christie, the separation of free fatty acids is based on the chain length and degree of unsaturation. Octadecylsilyl (ODS) is normally used as stationary phase and acetonitrile or methanol in water is used as mobile phase for free fatty acids analysis. The free fatty acids is detected by the UV detector between the wavelength of 205 and 210 nm.
Determination of underivated fatty acids in palm oil and other vegetable oils by RP-HPLC has been carried out by Hein and Isengard (1988). This research intended to improve the previous method by skipping the derivatization process which used high amount of chemicals and also time consuming. Three different alternative RP-HPLC methods were introduced where different detectors and different mobile phases were performed. A good resolution relies on the pH value of mobile phase. It was observed that the optimum pH for the separation of fatty acid was in the range of 3.0-3.5. On top of that, based on this research it was proven that the vegetable oils can be differentiated based on the characteristic of fatty acid pattern in oils. By applying HPLC method for determination of fatty acids in oils, it was also possible to determine the amount of free fatty acids in the oil by calculating the acid number. The amount of free fatty acids content in oil was based on the concentration of free oleic acid determined from the vegetable oils (Nelkon and Parker,1988).
Determination of free fatty acids, partial acylglycerols and tocols in palm oil products using HPLC with evaporative light scattering detector (ELSD) was performed by Moh and coworkers. Different mobile phases were used which are heptanes and 2-propanol with the flow rate of 1 mL min-1. The prepared samples were dissolved in dichloromethane (DCM) before being injected into the HPLC column. It was found out that the total free fatty acids content in crude palm oil and crude palm olein is high (3.80-5.30 %) but low in the refinedbleached-deodorized palm olein (< 0.1 %). The results obtained from HPLC analysis were compared with the results obtained from GC/FID analysis for validation. Although the steps of derivatization was eliminated in HPLC analysis, however high consumption of solvents are still being used as mobile phase and HPLC analysis also possess long time of analysis.
2.10 Near-infrared reflectance spectroscopy (NIRS) analysis
Several studies had also been conducted for determination of free fatty acids by using near-infrared reflectance spectroscopy (NIRS). Che Man and Moh developed an near-infrared reflectance spectroscopy calibration for determination of free fatty acids in crude palm oil, refined-bleached-deodorized palm olein and refined-bleached-deodorized palm oil using the NIR reflectance approach in order to replace the current wet chemical methods which is using a lot of hazardous solvents48. The palm oil sample was prepared by spiking a 400 g of sample with 0.15 % w/w enzyme and was incubated at 60 °C and 200 rpm. The sample was then analyzed by using NIR spectroscopic instrument and was performed in a Dutch cup. The determination of free fatty acids in palm oil by NIR was based on the C=O stretching bands in the region of 1850-2050 nm. The absorption obtained from the NIR was correlated with standard AOCS titration method to validate the results.
Multiple linear regression (MLR) was carried out due to the chemical interactions of the absorbing species and molecules. This interaction will interfere with the linearity of the relationship between the absorbance and concentration. Good calibration is shown in MLR analysis and the R2 for crude palm oil, refined-bleached-deodorized palm olein and refined-bleached-deodorized palm oil are 0.994, 0.961 and 0.971 respectively. The total analysis time is short as compared to the conventional wet chemical method which is less than 5 min per sample. On top of that, NIR analysis also able to test hundred samples daily without using much solvents.
Houmøller and co-workers had conducted an experiment to determine the solid fat content (SFC) and Free fatty acids in blends of palm stearin, coconut oil and rapeseed oil with the presence of Thermomyceslanuginosalipase at 70°C. The edible oils were then analyzed by using near infrared spectroscopy (NIRS). According to Houmølleret al., nearinfrared reflectance spectroscopy analysis is rapid and does not requires any sample pre-treatment. They used the partial least squares regression (PLSR) method to calibrate the quantitative determination of solid fat content and free fatty acids at various temperatures ranging from 10 to 40 °C. From the data obtained, it was proven that the near-infrared reflectance spectroscopy could be used to replace the traditional methods for determining free fatty acids and solid fat content in vegetable oils. This method can also be applied in quality control, process control and optimization purposes. The activity of the immobilized enzyme for esterification of margarine oils can also be monitored through the prediction of equivalent reaction time in a batch reactor from NIR spectra. Determination of free fatty acids in other edible oils by using FT-NIR spectroscopy had also been reported previously.
2.11 GC – MS Analysis of Fatty Acids
The analysis of the fatty acids in the oil sample was done at National Research Institute of Chemical Technology (NARICT), Zaria, Nigeria, a Shimadzu Qp210 plus series gas chromatography coupled with Shimadzu Qp2010 plus mass spectroscopy doctor (GC – MS) system was used, the temperature programmed was set up from 700C to 2800C the carrier gas used was helium. The injection volume was 2UL with injection temperature of 2800C and a column flow of 1.80 millimeter per minute for the gas chromatography. For the mass spectroscopy, ACQ mode canner with scan range of 30 – 700 Atomic mass per unit at the speed of 2478 was used.
2.12 Determination of Fatty Acid Composition
Chemical analysis: the fatty acid composition was determine by conversion of oil samples to fatty acid methyl esters prepared by of n-hexane 50ml of oil followed by 50ml of sodium methoxide using the method of Cocks et al; (1966). The mixture was vortex for 5sec ad allowed to settle for 5minutes. The top layer was injected into a gas chromatograph (model GC – 14A, Shimadzu Corporation, Kyoto, Japan) equipped with a flame – ionization detector and a polar capillary column (Bpx 70 0.25), 0.32mm internal diameter, 60m length and 0.25m film thickness (SGE Incorporated, USA) to obtain individual peaks of fatty acid methyl esters. The detector temperature was 2400C and column temperature was 1100C held for one minute and increased at the rates of 80C. Min G1 to 2200C and held for one minute. The run time was 32 minutes. The fatty acid methyl esters peaks were identified by comparing their retention time with those of standards. Percent relative fatty acid was calculated base on the peak area of a fatty acid species to the total peak area of all fatty acids in the samples.
2.13 Determination of Free Fatty Acid Value
The FFA value of the oil sample was determined in duplicates using standard analytical methods for fats and oils by the American oil Chemists Society ADCS (1990) at 45% confidence limit. The percentage FFA value was calculated form the equation below: 

Where: w = Weight (in grams of samples),
V = Volume (in milliliters) of sodium hydroxide solution used,
m = Molarity of sodium hydroxide solution used and
M = Molecular weight of the FFA









CHAPTER THREE
3.0 RESULTS AND DISCUSSION
3.1 RESULTS
Table 3.1 Physical Properties of Palm Oil

Qualitative Parameters
Composition/characterization

-
Percentage yield oil
54%

-
Color
Light Yellow

-
Nature
At room temperature

-
Saonification value
55.90 ± 0.01 (MgKOH/g)

-
Iodin value
9.50 ± 0.10 (1g/100g)

-
Acid value
1.40 ± 0.10 (1g/100g)

-
Peroxide value
0.70 ± 0.10 (MgKOH/g)

-
Free falty acid
1.80 ± 0.10%


Result presented in mean ± standard deviation triplicate data
Table 3.2 Physical Properties of Shea butter Oil

Qualitative Parameters
Composition/characterization

-
Percentage yield oil
42.2 ± 0.2/100g

-
Color
Yellow

-
Nature
Liquid at room temperature

-
Crude fiber
1.7 ± 30%

Result: Mean ± standard deviation Triplicate Data

3.2.0 Discussion
3.2.1
Table 3.1 displays the physiochemical properties of Free Falty Acid (FFA) value of palm oil sample kept under different color, the color is quite different from normal white, as reported by Ana (1993). The values in the table are obtained at 54% confidence level, this value is closely related to the volume (58.3%) reported by Ana (2015). This implies that the percent of the oil is very high making it a good source of oil. The low saponification value (56.28 ± 0.25) suggested that the oil can be used for candle and soap production and as chemical feed stock for lubrication, this value signifies a maximum purity and made it suitable for soap production (Shina et al., 1986). The free falty acid content of the oil if is 0.30 this is low when compared with 0.61 recorded by (Usman et al., 2009). The low free falty acid value (1.80 ± 0.10) suggested that oil has potential edible oil after detoxification. The iodine value (9.50 ± 0.10) indicate that the oil is semi drying oil, it also suggested the potential of the oil in the production of alkyl  resin, shoe, polish and varnishes among others, the peroxide value of (0.7 ± 0.10) was obtained which is relatively lower than the 30Meg/kg reported by (Dundo et al., 2013). High peroxide value (0.7 ± 0.10) is associated with high rancidity rate thus, with this fact, the peroxide value obtained from the Oil is simply a indication that oil is less liable to rancidity at room temperature (Abdullahi et al., 2013). The acid value for palm oil was found to be 1.40. Based on the acidity, it can be edible since the value fall below maximum acceptable value of 4.0MgKOH/g of oil as recommended by Codex Alimentarus Commission by only when it is detoxified (Abeye et al., 1998). GC – MS as recorded in table 3.2 are clearly indicate that, the oil may contain the following fatty acids: in table 3.3
Table 3.3 Fatty acids Composition of Oil

Name of fatty Acid
Molecular Formula (MF)
Molecular Weight (Mw) (g)
Similarity Index (SI) (%)

-
3-cyclohexyl-1-propanol
C7H18O
256
82

-
Methyl ester oleic acid
C19H36O2
284
90

-
Palmitic Acid
C16H32O2
282
94

-
Oleic Acid
C18H34O2
280
92

-
Stearic Acid
C18H36O2
294
90

-
Palmitoeic
C16H30O2
338
85

-
CIS – CIS linoleic Acid
C18H32O2
262
90



CHAPTER FOUR
4.0 Conclusion and Recommendation
4.1 Conclusion 
Shea butter and palm oil see is a good source of oil due to its high percentage oil yield obtained in the present study. The both oil contain glycoside that make it toxic and therefore it would be detoxified before used. The oil has a crude and free fatty acid value. In addition, saponification value revealed that the oil may be used industrially for soap making.
Characterization of the oil also indicated that high oil quality which suggested that the sweetablity of the oil for various industrial uses such as in cosmetics, paints and lubricants because of there plants grows widely, it can easily be harnessed and ploited for it oil to be used as industrial raw materials. The study revealed that the both oils of oleic proved as a potential local source of oleic and linoteic acid for exploration.
4.2 Recommendation
The storage of palm oil and Shea butter oil under different colors of light has some obvious implication on the Free Fatty Acid (FFA) value of the oil. Generally, the effect of light on storing these oils is that of increasing not only the rate of oxidation but also that hydrolysis as well, since light is a source of energy.
It is therefore suggested that palm oil and Shea butter oil samples for storage should be kept and also in more opaque containers to inhibit the effects of light generally, measure should also be taken to prevent over heating in storage environment.


Appendix A
Acid value
Biuret Reading
1st
2nd
3rd

Final reading
1.50
1.40
1.30

Initial reading
0.00
0.00
0.00

Total
1.50
1.40
1.30




Average mea for Acid vale = 1.40m/s

       
       
Acid value  =  0.79g/ml
By using standard deviation formula,

Xi
Xi – X
(Xi – X)2

1.50
0.01
0.01

1.40
0
0

1.30
-0.01
0.01

Total
-0.01
0.02


APPENDIX B

IDRIS ALHASSAN (SAMPLE A.)

FIGURE 1.0 GC-MS CHROMTOGRAM OF PALM OIL


FIGURE 1.1 CHART FOR PEAK LINE (1) OF THE CRUDE PALM OIL








FIGURE 1.2 CHART FOR PEAK LINE (2) OF THE PALM OIL.

IDRIS ALHASSAN (SAMPLE  B)
FIGURE 2.0 GC-MS CHROTOMOGRAM OF SHEA BUTTER OIL PEAK LINE
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