Separation of Acetyleugenol from Cloves

Title: Separation of Acetyleugenol from Cloves

Aim: To isolate acetyleugenol from clove seed and characterize it using IR        

Introduction

Eugenol has wide-ranging applications, from dental materials to food additives. For example, the derivative of eugenol, acetyl eugenol itself play important role in food flavorings.1 It is therefore important to be able to produce eugenol and its acetyl derivative. Liquid-liquid extraction and column chromatography are common methods for the separation of natural product including eugenol and acetyleugenol. In column chromatography, the crude mixture is separated based on their partition difference between the mobile phase and a stationary phase.7 The aim of this experiment was to extract two natural products, eugenol and acetyleugenol from cloves and to separate individual compounds using Thin-layer Chromatography (TLC) and identify it using Infrared spectroscopy.3 TLC is a quick, inexpensive microscale technique that can be used to: determine the number of components in a mixture, verify a substances’ identity, monitor the progress of a reaction, determine appropriate conditions for column chromatography and analyse the fractions obtained from column chromatography. The infrared spectrum on the other hand provides a rich array of absorption bands which can provide a wealth of structural information about a molecule. It provides methods for studying materials in all three physical states i.e. gas, liquid or solid.4 IR analysis may simple involve the characterization of a material with respect to the presence or absence of a specific group frequency associated with one or more fundamental modes of vibration or by a complex pattern recognition or by a computer search-match algorithm when an unknown spectrum is compared to an existing spectral database. 5The spectral data is also used to measure one or more compounds in a simple or complex mixture.6

Experimental Procedure

The remainder of the dried extract from part 1 of the experiment was decanted into a separatory funnel and 20ml of 10% NaOH extract was added to a separatory funnel. The mixture was then shaken and vented three times to allow for separation of the mixture. A mass of 1 gram of drying agent Sodium Sulfate was added to a 50ml Erlenmeyer flask. The lower dichloromethane layer was drained out into a 50ml Erlenmeyer flask containing the drying agent. The upper water layer was drained out into a separate beaker and the dichloromethane extracts were dried for 5 minutes. The dried dichloromethane was the evaporated on a hot plate at a low setting under the ventilation hood. A volume of 50ml Erlenmeyer flask was then pre-weighed. The dried dichloromethane was decanted into the pre-weighed Erlenmeyer flask. The solid drying agent was then discarded.

B. SEPARATION OF EUGENOL FROM CLOVES

The aqueous NaOH extracts saved from the previous separation were used for the separation process. A 6-8ml volume of 6M HCl were added to the extracts adjusting the pH=1. The test was done using a pH paper and the color code (dark red color) or litmus paper (blue to red). The acidified extract from the first step above was then transferred to a 125ml separatory funnel and 20ml of dichloromethane was added to the separatory funnel and then shooked, vented and allowed to separate. A 1 gram pf NaSO4 drying agent was then added to a dry 50ml Erlenmeyer. The lower dichloromethane layer was allowed to drain into the 50ml Erlenmeyer flask. A 50ml Erlenmeyer flask was then pre-weighed and the mass recorded. After a 5 minutes drying time, the dried dichloromethane extract was decanted into the pre-weighed flask. The solid drying agent was the discarded. The dried extract was then evaporated under a ventilation hood on a low setting of a hot plate. The residue was then weighed and labelled a eugenol and saved for TLC analysis.

C: TLC OF EUGENOL, ACETYLEUGENOL, AND CLOVE OIL

A volume of 10ml of 2:1 ratio of Hexane/CH2Cl2 developing solvent was added to each of two separate 250ml beakers or TLC developing tanks. The developing solvent was then poured into the tank and covered a TLC strip 5×20cm was then taken and a pencil mark was drawn across the strip 1cm from the end. The clove oil and acetyleugenol were then spotted next to each other on the strip about 1cm from bottom of strip. The stopped TLC strip was then placed into the developing tank. The procedure was the repeated using a second TLC Strip using clove oil and eugenol. A duration of 20-30 minutes developing time was used. The strips were then taken out of the solvent and allowed to dry for 5 minutes. The strips were placed near a UV lamp or in beaker containing some crystals of iodine.

Results

Part A

Mass of actyleugenol=mass of acetyleugenol-mass of 50ml Erlenmeyer flask

                                  =73.226g-61.67g

                                  =11.592g

% Acetyleugenol= (mass of acetyleugenol/25) × 100

                           =11.592g/25 × 100

                           =46.37%

Part B

Mass of beaker=54.229g

Mass of beaker + eugenol=56.815g

Mass of eugenol=56.815g-54.299g

                          =2.516g

% eugenol = (mass of eugenol/25) × 100

                  =2.516g/25 × 100