WORKING DRAFT
ISO/WD 9832
Animal and vegetable fats and oils — Determination of residual technical hexane
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Scope
This International Standard specifies two methods for the determination of the residual technical hexane content of animal and vegetable fats and oils (referred to as fats hereinafter), from 0,5 mg/kg to 1 500 mg/kg. The method A is suitable for the determination of hexane contents between 10 mg and 1 500 mg per kilogram of fat. The method B is suitable for the determination of hexane contents between 0,5 mg and 10 mg per kilogram of fat.
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Normative reference
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 661, Animal and vegetable fats and oils — Preparation of test sample
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Term and definition
For the purposes of this International Standard, the following term and definition applies. 3.1 residual technical hexane content content of volatile hydrocarbons such as those remaining in fats following processing involving the use of hydrocarbon solvents, when determined by the method specified in this International Standard NOTE
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The content is expressed as milligrams of hexane per kilogram of sample.
Principle
Desorption of volatile hydrocarbons by heating at 80 °C in a closed vessel after addition of an internal standard. Determination of the particular volatile hydrocarbons content of the headspace by gas chromatography using packed or capillary columns. NOTE This analysis is very sensible and the manipulation should be performed in a room where presence of hydrocarbon solvents is excluded.
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ISO/WD 9832
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Reagents
WARNING — Attention is drawn to the regulations which specify the handling of dangerous substances. Technical, organizational and personal safety measures shall be followed. Use only reagents of recognized analytical grade. 5.1
n-Octane, free from traces of hexane and heptane
5.2 Technical hexane, with a composition similar to that of hexane used in industrial processing or, if this is not available, n-hexane. It is recommended that technical hexane be used for the calibration. This reagent usually has an n-hexane content of 50 % (mass fraction) and consists mainly of C6 isomers but may include C5 and C7 hydrocarbons. 5.3 Solution of technical hexane in n-octane, mass concentration 3,35 mg/ml Transfer exactly 1 ml of technical hexane (5.2) in a volumetric flask of 200 ml capacity (6.1) and dilute to the mark with n-octane (5.1). 5.4
Internal standard, n-heptane.
5.5 Solution of n-heptane (internal standard) in n-octane, mass concentration 6,84 mg/ml Transfer exactly 1 ml of n-heptane (5.4) in a volumetric flask of 100 ml capacity (6.1) and dilute to the mark with n-octane (5.1). 5.6 Carrier gas, e.g. hydrogen, nitrogen or helium, etc., thoroughly dried and with an oxygen content of less than 10 mg/kg. 5.7 Auxiliary gases, hydrogen (99,9 % pure, free from organic impurities) and air (free from organic impurities). 5.8 Calibration fat, freshly refined and deodorized vegetable fat, the technical hexane content of which is negligible. This material should be free from peroxides or other components likely to decompose with the formation of volatile material which could be confused with hydrocarbons during the test.
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Apparatus
Usual laboratory equipment and, in particular, the following. 6.1
Volumetric flasks, of 100 ml capacity.
6.2
Septum vials, of 20 ml capacity.
6.3 Septa, inert to fats and solvents, made of a material such as butyl rubber or red rubber free from hydrocarbon solvent residues and of a suitable quality that they will not swell under the conditions of use, aluminium caps suitable for use with the vials (6.1) and crimping pliers. 6.4
Tongs, suitable for holding the vials (6.1).
6.5 Syringes, of 10 µl capacity, used only for the analysis of residual technical hexane. They shall not be cleaned with hydrocarbon solvent.
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ISO/WD 9832
6.6 Syringes, of 1 µl capacity, used only for the analysis of residual technical hexane. They shall not be cleaned with hydrocarbon solvent. 6.7 Syringes, of 1 000 µl capacity, gas-tight, used only for the analysis of residual technical hexane. They shall not be cleaned with hydrocarbon solvent. 6.8 Syringes, of 5 000 µl capacity, gas-tight, used only for the analysis of residual technical hexane. They shall not be cleaned with hydrocarbon solvent. 6.9 Gas chromatograph, with a flame ionization detector (FID) or mass spectrometer (MS), equipped with either a)
a packed glass column, 2 m to 4 m long and of internal diameter 3,2 mm approximately, packed with an acid-washed and silanized diatomaceous earth support of particle size 150 µm to 180 µm (Chromosorb P NAW 60 mesh to 80 mesh1) is suitable), and coated with 10 % squalane or any other phase permitting the chromatographic separation required, or
b)
a glass capillary column, approximately 30 m long and of 0,32 mm internal diameter, coated with methylpolysiloxane of film thickness between 0,2 µm and 3 µm.
c)
a fused silica capillary column, approximately 30 m long and of 0,53 mm internal diameter, coated with methylpolysiloxane of film thickness 5 µm;
NOTE For analyses in series, a headspace gas chromatograph with automatic sample injection and tempering oven has been shown to be satisfactory. In this case, manual injection is not necessary.
6.10 Heating bath, equipped with clamps for holding septum vials, regulated thermostatically at 80 °C 2 °C. NOTE
For continuous operation, glycerol is recommended as the heating medium.
6.11 Shaking machine (or vortex) with horizontal movement preventing projection of oil onto the septa.
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Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 5555. It is essential that the sample be protected from gain or loss of solvent residues.
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Preparation of test sample
Prepare the test sample in accordance with ISO 661, taking care to prevent gain or loss of solvent. Therefore samples should not be heated or filtered before the analysis in order to prevent loss of solvent.
1) Chromosorb P NAW 60 mesh to 80 mesh is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.
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ISO/WD 9832
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Procedure A: technical hexane content between 10 mg/kg and 1 500 mg/kg
9.1
Chromatographic conditions
The injector and detector temperature shall be set at 100 °C and the oven temperature at 50 °C. If a capillary column [see 6.9 b)] is used, the apparatus shall have a 1/100 split injection system.
9.2
Calibration Weigh 5 g 0,1 g of calibration fat (5.8) into each of four vials (6.2).
9.2.1
To three of the four vials (6.2) add using a syringe (6.5 or 6.6), 10 µl of technical hexane (5.2) and 5 µl of internal standard (5.4), to obtain a mass concentration of 1 340 mg/kg in technical hexane (or 1 320 mg/kg in n-hexane). Close rapidly each vial with a septum and a cap (6.3). Do not add technical hexane to the fourth vial but by means of a syringe (6.5) add 5 µl of internal standard (5.4). Shake vigorously the four vials, in the shaking machine (6.11) at room temperature. Allow to stand for 1 h. Verify that the fat does not touch the septum during shaking. If this happens, reject the vial and start again with a further portion of calibration fat. CAUTION - If there is fat on the septum it will contaminate the needle when the headspace gas is sampled and the contaminant may be transferred to the column; it is particularly important that such contamination be avoided when using capillary columns. 9.2.2 At intervals of about 15 min (i.e. the retention time of the internal standard), place one vial at a time up to its neck in the heating bath (6.10) set at 80 °C to allow equilibrium between the fat and the gaseous phase to be reached. 9.2.3 From each vial which has been in the heating bath for 60 min 1 min, take (without removing it from the bath) 1 000 µl of the gaseous phase by means of a syringe (6.7) warmed to 60 °C. Immediately inject into the gas chromatograph the gaseous phase so removed. 9.2.4 From the chromatogram corresponding to the vial to which no technical hexane was added, calculate the hexane content Ac, expressed as a percentage of the total peak areas. 9.2.5 From each of the chromatograms corresponding to each of the vials to which solvent was added, calculate the calibration factor F using the formula F
w h Ais At Ac Ais wis
where Ac
is the hexane content calculated in 9.2.4
Ais is the content of the internal standard in the calibration fat (5.8) with added technical hexane, expressed as a percentage of the total peak areas At
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is the total hydrocarbons content, including the internal standard, in the calibration fat (5.8) with added solvent, expressed as a percentage of the total peak areas
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ISO/WD 9832
wh is the content of technical hexane (5.2) in the calibration fat (5.8) with added technical hexane, expressed in milligrams per kilogram, i.e. 1 340 mg/kg wis is the content of the internal standard in the calibration fat (5.8) with added technical hexane, expressed in milligrams per kilogram, i.e. 680 mg/kg. Express the results to the third decimal place. The calibration factors of the three calibration samples should be approximately equal. Calculate the arithmetic mean value F , which should be about 0,45 with a relative standard deviation not higher than 15 %.
9.3
Determination
9.3.1 Weigh a test portion of 5 g 0,1 g of the test sample (clause 8) into a vial (6.2) as quickly as possible. By means of a syringe (6.5), inject 5 µl of the internal standard (5.4) and close immediately with a septum and a cap (6.3). 9.3.2 Mix the contents vigorously by hand for about 1 min by moving the vial with a circular motion in a horizontal plane in such a way that the fat does not touch the septum. If this happens, reject the vial and start again with a further test portion. (See CAUTION in 9.2.1.) Place the vial up to its neck in the heating bath (6.10) set at 80 °C for 60 min 1 min. 9.3.3 Then take 1 000 µl from the gaseous phase by means of a syringe (6.7) warmed to 60 °C, without removing the vial from the heating bath. Immediately inject into the gas chromatograph the gaseous phase so removed. NOTE
In case of pressure controlled systems a similar injection volume shall be guaranteed.
9.3.4 Determine the residual technical hexane content of the sample from the chromatogram (see the example given in Figure 1), measuring those peaks identified as being from hexane and not from decomposition products.
9.4
Number of determinations
Carry out the determination on two test portions from the same test sample in rapid succession.
10 Procedure B: technical hexane content between 0,5 mg/kg and 10 mg/kg 10.1 Chromatographic conditions Chromatographic conditions may be chosen taking into account the characteristics of the capillary column and the nature of the carrier gas, in order to get a good resolution between the internal standard and the isomers of technical hexane. As an example, the following oven temperature program is suitable for the determination of traces of residual technical hexane: Initial oven temperature: Temperature increase: Final temperature:
50 °C for 8 min 10 °C/min, from 80 °C to 120 °C 120 °C for 5 min.
The injector temperature may be set at 200 °C and the detector temperature at 150 °C. The carrier gas pressure should be adjusted in order to elute the n-hexane in 3 minutes and to keep a flow rate in the column around 13 ml / min. The injector split should be reduced to a flow rate about 12 ml/min. The sensitivity of the detector should be adjusted to its higher set.
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ISO/WD 9832
10.2 Calibration 10.2.1 Weigh 5 g 0,1 g of calibration fat (5.8) into each of six vials (6.2). To three of the four vials (6.2) add using a syringe (6.5), 2 µl of the solution of technical hexane (5.3) and 5 µl of the solution of internal standard (5.5), to obtain a mass concentration of 1,34 mg/kg in technical hexane and 6,84 mg/kg of n-heptane (internal standard). Do not add technical hexane to the fourth vial but by means of a syringe (6.5) add 5 µl of the solution of internal standard (5.5). Close each vial with a septum and a cap (6.3). Shake vigorously the four vials, in the shaking machine (6.11) at room temperature. Verify that the fat does not touch the septum during shaking. If this happens, reject the vial and start again with a further portion of calibration fat. CAUTION - If there is fat on the septum it will contaminate the needle when the headspace gas is sampled and the contaminant may be transferred to the column; it is particularly important that such contamination be avoided when using capillary columns. 10.2.2 At intervals of about 20 min, place one vial at a time up to its neck in the heating bath (6.10) set at 80 °C to allow equilibrium between the fat and the gaseous phase to be reached. 10.2.3 From each vial which has been in the heating bath for 60 min 1 min, take (without removing it from the bath) 2 500 µl of the gaseous phase by means of a syringe (6.8) warmed at 60 °C. Immediately inject into the gas chromatograph the gaseous phase so removed. 10.2.4 From the chromatogram corresponding to the vial to which no technical hexane was added, calculate the technical hexane content Ac, expressed as a percentage of the total peak areas. In the condition of the analysis given in clause 10.1, the different components of technical hexane are the followings (retention times in brackets): 2-methylpentane (2,56 min), 3-methylpentane (2,84 min), n-hexane (3,18 min), methylcyclopentane (3.92 min), cyclohexane (5,02 min) as presented in figure 2. 10.2.5 From each of the chromatograms corresponding to each of the vials to which solvent was added, calculate the calibration factor F using the formula F
w h Ais At Ac Ais wis
where Ac
is the hexane content calculated in 10.2.4
Ais is the content of the internal standard in the calibration fat (5.8) with added technical hexane, expressed as a percentage of the total peak areas At
is the total hydrocarbons content, including the internal standard, in the calibration fat (5.8) with added technical hexane, expressed as a percentage of the total peak areas
wh is the content of the technical hexane in the calibration fat (5.8) with added technical hexane, expressed in milligrams per kilogram, i.e. 1,34 mg/kg. wis is the content of the internal standard in the calibration fat (5.8) with added technical hexane, expressed in milligrams per kilogram, i.e. 6,84 mg/kg. Express the results to the third decimal place.
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ISO/WD 9832
The calibration factors of the three calibration samples should be approximately equal. Calculate the arithmetic mean value F , which should be about 0,45 with a relative standard deviation lower or equal to 15 %. NOTE Residual peak of technical hexane in the blank sample should represent less than 0,5 mg/kg otherwise reagents and procedure should be reconsidered.
10.3 Determination 10.3.1 Weigh a test portion of 5 g 0,1 g of the test sample (clause 8) into a vial (6.2) as quickly as possible. By means of a syringe (6.5), inject 5 µl of the internal standard solution (5.5) and close immediately with a septum and a cap (6.3). 10.3.2 Mix the contents vigorously by hand for about 1 min by moving the vial with a circular motion in a horizontal plane in such a way that the fat does not touch the septum. If this happens, reject the vial and start again with a further test portion. (See CAUTION in 10.2.1.) Place the vial up to its neck in the heating bath (6.10) set at 80 °C for 60 min 1 min. 10.3.3 Then take 2 500 µl from the gaseous phase by means of a syringe (6.7) warmed at 60 °C, without removing the vial from the heating bath. Immediately inject into the gas chromatograph the gaseous phase so removed. NOTE
In case of pressure controlled systems a similar injection volume shall be guaranteed.
10.3.4 Determine the residual technical hexane content of the sample from the chromatogram (see the example given in Figure 2), measuring those peaks identified as being from technical hexane and not from decomposition products.
10.4 Number of determinations Carry out the determination on two test portions from the same test sample in rapid succession.
11 Expression of results The residual technical hexane content of the sample, w, in milligrams per kilogram, is given by the formula w
At Ais F wis Ais
where Ais
is the content of the internal standard in the sample, expressed as a percentage of the total peak areas;
At
is the total hydrocarbons content, including the internal standard, of the sample, expressed as a percentage of the total peak areas;
F
is the mean calibration factor determined in 9.2.5 or 10.2.5;
wis
is the content of the internal standard in the sample, expressed in milligrams per kilogram, i.e. 680 for n-heptane for procedure A, and 6,84 for n-heptane for procedure B.
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ISO/WD 9832
Take as the final result the arithmetic mean of the two determinations provided that the repeatability requirement (12.2) is met. If the repeatability requirement is not met, disregard the results and carry out two new determinations on test portions taken from the same test sample.
Figure 1 — Example of a gas chromatogram of hexane hydrocarbons for procedure A
Figure 2 — Example of a gas chromatogram of hexane hydrocarbons for procedure B
Peak identification 1 2-Methylpentane 2 3-Methylpentane 3 n-Hexane 4 Methylcyclopentane 5 Cyclohexane 6 n-Heptane (internal standard)
12 Precision 12.1 Interlaboratory test Details of an interlaboratory test on the precision of the method are summarized in Annex B. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given.
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ISO/WD 9832
12.2 Repeatability The absolute difference between two independent single test results, obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases exceed the values of r given in the tables in Annex A.
12.3 Reproducibility The absolute difference between two single test results, obtained with the same method on identical test material in different laboratories by different operators using different equipment, will in not more than 5 % of cases exceed the values of R given in the tables in Annex A.
13 Test report The test report shall specify: all information necessary for the complete identification of the sample; the sampling method used, if known; the test method used, with reference to this International Standard; all operating details not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the results; the test results obtained, or, if the repeatability has been checked, the final quoted result obtained.
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ISO/WD 9832
Annex A (informative) Results of interlaboratory tests
An international collaborative test involving __ laboratories in __ countries was carried out on the following samples: A: B: C: D: E: The test was organized by the _________ Member Body in 200_ and the results obtained were subjected to statistical analysis in accordance with ISO 5725-1 [2] and ISO 5725-2 [3] to give the precision data shown in Table A.1 for the visual method and in table A.2 for the potentiometric method.
Table A.1 – Sample
A
B
C
D
E
Number of laboratories participating Number of laboratories after eliminating outliers Number of test results from remaining Laboratories Mean value, meq/kg Repeatability standard deviation, sr, meq/kg Repeatability relative standard deviation, % Repeatability limit r (2,8 sr), meq/kg Reproducibility standard deviation, sR, meq/kg Reproducibility relative standard deviation, % Reproducibility limit R (2,8 sR), meq/kg
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ISO/WD 9832
Bibliography
[1]
ISO 5555, Animal and vegetable fats and oils — Sampling
[2]
ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results — Part 1: General principles and definitions
[3]
ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method
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