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GAS CHROMATOGRAPHIC (GC) ASSAY OF FLAVOUR CHEMICALS

This procedure applies both to the assay of flavour chemicals and to the quantitation of minor components in flavour chemicals. Analysts following this procedure and performing the test, should obtain sufficient resolution of major and even trace components of a mixture to calculate accurately the concentration of the desired component. They should be familiar with the general principles, usual techniques, and instrumental variables normally met in gas chromatographic analysis. They should pay particular attention to the following:

1. Stability of baseline, return to baseline before and after each peak of interest, and minimum use of recorder attenuation.

2. Any incompatibility between a sensitive sample component and column support, liquid substrate, or construction material.

3. The response to different components of the same or different detectors. Since sizeable errors may be encountered in correlating area percent directly to weight percent, the methods for calculating response factors should be known.

4. Where limits for minor components are specified in the column entitled Other Requirements in the above tabular specifications for flavour chemicals, analysts should use authentic materials to confirm the retention times of minor components. Determine the quantity of components following the instructions below under Calculations and Methods.

GC CONDITIONS FOR ANALYSIS

Column: open tubular capillary column of fused silica or deactivated glass 30 m long x 0.25 to 0.53 mm id

Stationary phase:

1. For a non-polar column: methyl silicone gum, or equivalent (preferably a bonded and cross-linked dimethyl polysiloxane)

2. For a polar column: polyethylene glycol, or equivalent (preferably a bonded and cross-linked polyethylene glycol)

3. The stationary phase coating should have a thickness of 1 to 3 µm

Carrier gas: helium flowing at a linear velocity of 20 to 40 ml/s

Sample size: 0. 1 to 1.0 µl

Split ratio: (for 0.25 mm to 0.35 mm id columns only) 50:1 to 200:1, typically. Make sure that no individual component exceeds the capacity of the column

Inlet temperature: 225 to 275º

Detector temperature: 250 to 300º

Detectors: use either a thermal conductivity or flame ionization detector operating both as recommended by the manufacturer

Oven program: 50 to 240º, increasing the temperature by 5º/min, hold at 240º for 5 min

Analysts can also use any GC conditions providing separations equal to (or better than) those obtained with the above method, but in the case of a dispute, the above method must stand.

CALCULATIONS AND METHODS

A. Peak area integration with total area detected normalized to 100%, using electronic integrators: Use an electronic peak integrator in accordance with the manufacture's recommendations. Ensure that the integration parameters permit proper integration of the peaks of a variety of shapes and magnitudes and do not interpret baseline shifts and noise spikes as area contributed by the sample. Use internal or external standards as needed to confirm that the total GC peak area corresponds to 100% of the components present in the sample.

B. Results obtained as described above are based on the assumption that the entire sample has eluted and the peaks of all of the components have been included in the calculation. They will be incorrect if any part of the sample does not elute or if all the peaks are not measured. In such cases, and in all methods described above, the internal standard method may be used to determine percentages based on the total sample. For this method, measurements are required of the peaks of the component(s) being assayed and of the internal standard.

An accurately weighed mixture of the internal standard and the sample is prepared and chromatographed, the area ratio(s) of the component(s) to the standard is computed, and the percentage(s) of the component(s) is calculated.

If this calculation is to be applied, the substance used as the standard should be one that meets the following criteria:

a. Its detector response is similar to that of the component(s) to be determined. In general, the more nearly the chemical structure of the component resembles that of the standard, the closer the response will be.

b. Its retention time is close to, but not identical with, that of the component(s).

c. Its elution time is different from that of any other component in the sample so that its peak does not superimpose on any other.

The weight ratio of the internal standard to the sample should be such that the internal standard and the component sought produce approximately equal peaks. This is not possible, of course, if several components of interest are at different levels of concentration.

If the internal standard method is applied properly, it may be assumed that the ratio of the weight of component to the weight of internal standard is exactly proportional to the peak area ratio, and under these conditions no correction factor is needed. The sample is first run by itself to determine whether the internal standard would mask any component by peak superimposition. If there is no interference, a mixture is prepared of the sample and of the internal standard in the specified weight ratio, and the percentages of the internal standard and of the sample in the mixture are calculated. The mixture is chromatographed, and the areas of the component peak and the internal standard peak are calculated by one of the methods described above.

The calculations are as follows:

1.1. % Component in Mixture/ %Internal Standard in Mixture = Component Area/Internal Standard Area, or

1.2. % Component in Mixture = % Internal Standard in Mixture x (Component Area/ Internal Standard Area)

2. % Component in Sample = (%Component in Mixture x 100) / %Sample in Mixture

Should calibration be necessary, mixtures should be prepared of internal standard and component, of either 100% or of known purity. The number of mixtures and the weight ratios to be used depend on the component being analyzed. Usually, three mixtures will be required. The weight ratio of one is chosen so that the heights of component and standard are equal. The ratios of the other two may be two-thirds and four-thirds of their value. Each mixture should be chromatographed at least three times, and areas calculated. The factor for each chromatograph should be calculated as specified below, and the averages taken for each mixture. An overall average factor is calculated from them. The calibration should be performed periodically.

1. Factor = [(Weight of Component x % Purity) / (Weight of Internal Standard x % Purity)] x [(Internal Standard Area) / Component Area]

2. % Component in Sample Mixture = (Component Area x Factor x % Internal Standard in Sample Mixture) / Internal Standard Area

3. % Component in Sample = (% Component in Sample Mixture x 100) / Sample in Sample Mixture

GC SYSTEM SUITABILITY TEST SAMPLE

The GC system suitability test sample consists of an equal-weight mixture of food-quality acetophenone, benzyl alcohol, benzyl acetate, linalool, and hydroxycitronellal.

Using the test sample described below, periodically test the performance of and resolution provided by the gas chromatograph employed. The test sample must display results comparable in quantitative composition, peak shape, and elution order to those specified below. The quantitative composition should not deviate from the results listed below by more than 10%. Analyze the GC test sample using the GC Conditions for Analysis given above.

Component in

Test Sample

Order of Elution

Non-polar   Polar

Normalized%   Area (FID)

Non-polar           Polar

Benzyl alcohol

1

4

22.0

21.3

Acetophenone

2

2

21.1

21.4

Linalool

3

1

20.8

21.0

Benzyl acetate

4

3

18.6

19.1

Hydroxycitronellal

5

5

16.7

16.7

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