Figure 1 Separation of Ginger oil by (a) Cold Maceration (b) Hot Extraction (c) Liquid- Liquid Extraction -

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1 Effect of Methods of Separation on Ginger Oil Method of extractions affects how many components of a sample is successfully extracted from the sample. Figure one shows the chromatogram obtained for ginger oil using three separation methods. The GC/EIMS chromatogram of the cold macerations method shows thirty peaks (30), hot extraction, fifty four (54 peaks) and the liquid-liquid method, thirty peaks (30) respectively. The hot extraction method, the soxhlet method, extracted the oil the most out of the three separation methods of this sample, ginger oil. Table 1 shows components extracted by each method. The components comprises of different classes of compounds as indicated in table. Figure 1 Separation of Ginger oil by (a) Cold Maceration (b) Hot Extraction (c) Liquid- Liquid Extraction -

2 Table 1 s of Giger oil Extracted by Three Different Methods Hexanoic (C7H14O) Hexanoic methyl ester (C7H14O) 3.0 (C7H15O) ,,3- Trimethyl 3 * 4.10 (S)-1-piperideine-6- carboxylate (C6HNO) isopropyltoulene (C9H1) ,,4- Trimethyl (C9H1) ethyl-- methyl (C9H1) (CH15O) ,3,7- Octatriene (C10H16) (C9H13) ,4-diethyl Decanoic, (C11HO) Methyl-3- propyl 9.1 -Butyl--octenal (C1HO) Carene-- ol (C10H16O) β cubebene Ethyl-1,3- dimethyl Hexanoic (C7H14O) 3.31 Ethyl-- pentanal (C7H0O) 3 3. (C7H3O) 4 * 4.1 (S)-1- piperideine-6- carboxylate (C6HNO) Isopropyl toluene (C9H19O) Borrneol (C10H1O) 6.76 Octenol (CH16O) Isobutyl-,5- dimethyl benzene (C1H1) α copaene (1-Methyl-1- propenyl) δ-elemene (Cubebene, C15H4) (C10H1) Borneol (C10H1O) (CH15) 11.4 Caprylate (CH15O - )

3 Dodecanoic Methyl ester (C13H6O) Farnesyl group (C15H5) Bisabolol oxide Pentadecanoic (C15H30O) Hexadecanoic -methyl ester (C17H34O) ,- Hexadodecanediol (C16H34O) Palmitic (C16H3O) ,1- Octadecadienoic, methyl ester (C19H34O) Methyl Oleate (C19H36O) Octadecanoic,methyl ester (C19H3O) 17.6 Linoleic (C1H3O) Octadecanoic (C1H36O) (N-Benzoyl- aminomethyi)-,3-dihydro-- methyllh- Isoindole (C10H1N) Phenyl-4- pentenyl-1- alcohol (C11H14O) (C10H11) 15.1 (C10H7) (C10H19O) Octenal, - butyl (C1HO) (C9H1) Elemene Farnesene Germacrene 10.6 Farnesol (C15H6O) γ- Muurolene Decanoate (C10H19O) δ-elemene (Cubebene, C15H4) (C9H19O) Bisabolol oxide Hexadecanoic, (C17H34O) Hexadecanoic, (C16H3O) ,1Octadecadienoic, (C19H34O) Methyl Oleate (C19H36O) Octadecanoic (C19H3O) Oleic (C1H34O) 17.1 Octadecanoic (C1H36O) Stearidonic

4 Pea k Pea k Pea k (min ) Stearidonic Oleic anion Eicosadienoic (C0H36O) (min ) α- Muurolene Methyl laurate (C13H6O) Methyl -9- tetradecenoat e (C15HO) (min ) Stearidonic Eicosadienoic (C0H36O) (C1H33O) Oleic anion 13,16- Docosadienoic methyl ester (C3H4O) (C3H40O) (C3H37) (-Hydroxy- -propanyl)- 1,4a-dimethyl decahydro-1- naphthalenol (C15HO) α-bisabolol Oxide Pentadecanoi c (C15H30O) (1S,4aS,7R,S ) -7-(- Hydroxy-- propanyl)- 1,4a-dimethyl decahydro-1- naphthalenol (C15HO) (C1H5O) Methyl Tetra decanoate (C15H30O) β-bisabolol Oxide ,16- Docosadienoic methyl ester (C3H4O) 13Z, 16Z - Docosadienoic (CH40O) 9.3 (Oleic fragment) 30.5 (C1H34O) Adrenic / 7Z,10Z,13Z,16Z - Docosatetraenoi c (CH36O)

5 Geranyl isopentanoate Palmitoleic (C16H30O) (C10H1) ,-Hexa decanediol (C16H34O) Palmitic, (C17H34O) Oleic (C1H34O) Methyl Linoleate (C19H34O) Methyl Linolenate (C19H3O) Methyl Stearate (C19H3O) (C16H33) Oleic anion Stearidonic EICOSADIE NOIC ACID (C0H36O)

6 (C15H7O) (C1H3) (C3H41O) ,16- Docosadienoi c (CH40O) Adrenic (CH36O) (C4HO) = fragments of high mass components * needs further clarification Table Classes of Compounds in Ginger Oil Class of Compound Cold Maceration Extraction Method Hot Extraction Method Liquid Liquid Extraction Method Fatty s C7-C0 C7- C C7-C Oxygenated C7- C3 C10 - C4 C- C1 Hydrocarbons Hydrocarbons C10, C15 C9, C15 C15 Heterocycles C6 C10 C6 Unsaturated C1 C10 C7 Hydrocarbons Aromatic Hydrocarbons None C9,C10 C10,C1 Isomers C15 C15, C1, C19 Table shows compounds of higher molecular weight compounds than reported in an earlier paper for ginger oil (Indu Sasidharan et al, 010). We can conclude that the hot extraction method is the best method that extracted the most components and all classes of compounds but

7 one may need to use more than one extraction method to completely extract the components of a sample. Table also shows that the cold maceration method did not extract aromatic hydrocarbons. The hot method did not extract lower molecular weight oxygenated hydrocarbons or may be the lower molecular oxygenated hydrocarbons are very volatile and has evaporated when the oil is cooled down before analysis. Most of the oxygenated hydrocarbons, hydrocarbons, heterocycles and unsaturated hydrocarbons are terpene or sesquiterpene compounds. Reference 1. Indu Sasidharan, A. Niramala Menon, (010) Comparative Chemical Composition And Anti microbial Activity Fresh & Dry Ginger Oils (ZINGIBER OFFICINALE ROSCOE). International Journal of CURRENT PHARMACEUTICAL RESEARCH Vol, Issue 4, 40-43

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