experinment 1 Quality Control of Eucalyptus Leaf Extract INTRODUCTION The major

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experinment 1 Quality Control of Eucalyptus Leaf Extract

INTRODUCTION
The major component in most eucalyptus oils is 1,8-cineole; C10H18O or 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane.

The ability to quantitatively analyse extracts of Eucalyptus leaves is essential for many reasons since the oil has a variety of uses. In particular, there are stringent specifications when it is used for food and medical purposes. Gas phase chromatography is the only efficient method to analyse such a complex mixture of organic compounds. Such analyses have been useful in identifying species and cultivars that produce relatively high concentrations of 1,8-cineole.
The extraction procedure developed for cineole is designed to provide maximum convenient ‘through-put’ of samples while retaining accuracy in the analysis. The procedure is simply to place a known weight of Eucalyptus leaves (3 g) in a known volume of ethanol (50 mL) and allow the mixture to stand for a minimum of two weeks in order to achieve maximum extraction of the oil.
In this experiment you are going to optimise the operating parameters for the GC and then quantitatively analyse an extract of Eucalyptus oil.
To optimise the analysis you will run a standard mixture at a number of different column temperatures and assess the efficiency of the column by calculating resolution, the number of theoretical plates, and the plate height. These column efficiency parameters will then be used to work out which temperature is best for the analysis. Using these conditions, you will utilise nonanol as an internal standard to quantify the amount of cineole present in the unknown sample.

Lastly, you are going to look at the water content in the cineole extract. This gives us some idea of how pure the extract is. To do this we will use the Karl Fischer analysis which is a conductometric titration method. The Karl Fischer reagent is prepared by the action of sulfur dioxide upon iodine dissolved in pyridine (C5H5N) and methanol.

The reaction of this reagent with small amounts of water is complex but is believed to be as follows:
2C5H5N + I2 + H2O + SO2 2C5H5N+SO3- (1)
C5H5NSO3 + CH3OH C5H5NHOSO2OCH3 (2)

Reaction (1) only takes place in the presence of an oxygenated molecule which leads to the intermediate compound.
Reaction (2) prevents the pyridine complex from reacting with another molecule of water or other active hydrogen compounds. Hence one molecule of water is equivalent to one molecule of iodine.
So the aims of the experiment can be summarised as:
(a) Optimise the gas chromatography conditions for the analysis of cineole in Eucalyptus extracts;
(b) Use the internal standard method to determine the amount of 1,8-cineole in the extract; and
(c) Determine the amount of water in the leaves of the Eucalyptus species used to make the extract using an electrochemical method.

BACKGROUND ON THE INSTRUMENTS
The Karl Fischer analyser in this exercise is the Metrohm KF Titrino 718. This is basically an autotitrator. The end-point in the titration is detected electrometrically.
The procedure involves the "dead-stop" technique. If a small voltage is applied across two platinum electrodes immersed in the reaction mixture, a small current will flow as long as free iodine is present. When the iodine is removed the current decreases to zero, this being the end-point of the titration and the titre is recorded.
Substances which interfere with the titration are:
1. oxidising agents, eg, CrO42-, Cr2O72-, Cu2+, Fe3+, higher oxides, and peroxides;
2. reducing agents, eg, S2O32-, Sn2+ and S2-;
3. compounds which can be regarded as forming water with the components of the reagent, eg, basic oxides such as ZnO, salts of weak oxy-acids such as NaHCO3, borates H3BO3.

EXPERIMENTAL
There are 4 main parts to this experiment.
1. Determine the optimum oven temperature for the analysis
2. Identify which of the peaks is your internal standard and which is cineole
3. Do a quantitative analysis of cineole
4. Determine the water content of the Eucalyptus extract.
Determination of GC conditions for the Eucalyptus oil analysis.
In this section you will determine the optimum GC oven temperature for the separation of the components of the standard solution, containing cineole and nonanol. You will investigate the separation of the components in the standard solution at 4 different temperatures by an incremental increase of 30C.
The initial conditions set on the instrument initially are your starting point for the first injection of the standard solution and your demonstrator will give you a brief outline of how to change those. Details of how to use the instrument software and the correct order for conducting the experiment will be provided in the lab.
What is below is a quick summary of that procedure:

Inject 1 L of the standard solution, containing cineole and nonanol (made up in ethanol) and start the analysis.
Raise the column oven temperature 30°C and re-run the analysis. Repeat using temperature increments of 30°C until the components no longer resolve.
While the chromatograms are running calculate the number of theoretical plates, the height equivalent to a theoretical plate, and the resolution factor for your report. The formulae for these calculations can be found in Chapter 26 of your textbook.
From your data select the optimum analysis temperature; discuss this with your demonstrator.

Internal standard calibration
Now you will use the GC conditions established in the previous section in conjunction with the software to analyse the cineole content of eucalyptus oil using nonanol as the internal standard.
Set up the GC analysis as detailed in the instruction handout in the lab. Inject the Internal Standard Solution containing only nonanol in ethanol and compare the retention times with retention times with your previously collected data at the same temperature to determine the identity of each component.
Follow the handout instructions to conduct the calibration.
When the calibration is finished re-inject your standard solution. If it reports the correct values you can continue to analyse the unknown. If not you may need to re-calibrate or you may have some other problem such as a faulty syringe. In this case see your demonstrator or a technician.

Determination of cineole in unknown
The unknown contains no internal standard. In order to produce a sample that can be quantitatively analysed use a micropipette and deliver equal volumes of the unknown cineole solution and the internal standard solution into a screw cap sample vial; this is the sample that is injected into the GC. Before you inject, your sample mixture will probably have a different internal standard concentration therefore you must edit the sample table. See the instruction handout for how to do this.

Karl Fischer water analysis
In summary the technique involves:
1. Titrating any residual water that has diffused into the titration vessel since last use and then refilling and resetting the burette, i.e. zeroing the instrument.
2. Injecting a known volume of water, 25 L, then titrating to determine (tw).
3. Injecting a known volume of the unknown sample, 250 L, and then titrating to determine (te).
4. Injecting a known volume of pure solvent, 250 L, then titrating to determine (ts). We will use the Internal Standard solution as our solvent (i.e. contains mostly ethanol).
The Karl Fischer instrument will calculate the volume of reagent added to dry your sample. That can then be substituted into the calculations below. In order to produce good results you must follow the instructions provided next to the instrument precisely.

Please answer following questions

1.The ‘unknown’ cineole sample in this experiment isn’t really derived from a Eucalypt extract. If it was, you might expect there to be a number of other compounds extracted into the ethanol. How would you change your GC analysis in order to cope with this? What conditions have other researchers used for similar analyses?

2.In this experiment you measured the water content via a coulometric titration. What is another method for measuring the water content of the leaf extracts? You need to take into account that the cineole you are measuring is volatile.

Explanation / Answer

There are various studies took place for identification of chemical constituents in the leaves of Eucalyptus. GC-MS (Gas chromatography-Mass spectrometry) were done to identify different components in test sample.

The conditions used under this method are as follows:

Column: Poly Ethylene Glycol (PEG), 60m X 0.32 mm, ID X 1.0mm film,

GC: Inject 0.2 mL, Split 80:1, He flow 0.5 mL/min, Column 45 oC, 8 min hold ramp to 230oC @ 8oC/min-hold 10 min.

MS: 220oC

Interface temperature: 200oC scan

Range EI: 30 - 300

Results:

2. Karl Fischer titration method is the best method to determine wate content in leaf extracts. There are other Gas production methods that would provide similar resutls.

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