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GB 5009.32-2016: Determination of propyl gallate in oils and fats
Status: Valid GB 5009.32: Evolution and historical versions
| Standard ID | Contents [version] | USD | STEP2 | [PDF] delivered in | Standard Title (Description) | Status | PDF |
| GB 5009.32-2016 | English | 459 |
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Determination of propyl gallate in oils and fats
| Valid |
GB 5009.32-2016
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| GB/T 5009.32-2003 | English | 199 |
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Determination of propylgallate in oils and fats
| Obsolete |
GB/T 5009.32-2003
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| GB/T 5009.32-1996 | English | 199 |
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Method for determination of propylgallate in oils and fats
| Obsolete |
GB/T 5009.32-1996
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| GB 5009.32-1985 | English | 199 |
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Method for determination of PG in oils and fats
| Obsolete |
GB 5009.32-1985
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PDF similar to GB 5009.32-2016
Basic data | Standard ID | GB 5009.32-2016 (GB5009.32-2016) | | Description (Translated English) | Determination of propyl gallate in oils and fats | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53 | | Word Count Estimation | 23,259 | | Date of Issue | 2016-12-23 | | Date of Implementation | 2017-06-23 | | Older Standard (superseded by this standard) | GB/T 23373-2009; GB/T 5009.32-2003 | | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 | | Issuing agency(ies) | National Health and Family Planning Commission of the People's Republic of China, State Food and Drug Administration | GB 5009.32-2016: Determination of propyl gallate in oils and fats---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Determination of propylgallate in oils and fats
National Standards of People's Republic of China
National Food Safety Standard
Determination of nine antioxidants
Published 2016-12-23
2017-06-23 implementation
National Health and Family Planning Commission People's Republic of China
China Food and Drug Administration issued
Foreword
Instead of the standard GB/T 5009.32-2003 "fat gallic acid propyl ester (PG) The measured" and GB/T 23373-2009 "Food
Determination of antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (of BHT) and tertiary butyl hydroquinone (TBHQ) of. "
As compared with the present standard GB/T 5009.32-2003, major changes are as follows.
--- standard name was changed to "determination of national food safety standards in food nine kinds of antioxidants";
--- increase the types of antioxidants;
--- increasing the scope of application of the method;
--- increased liquid chromatography, gas chromatography, liquid chromatography tandem mass spectrometry and gas chromatography mass spectrometry.
National Food Safety Standard
Determination of nine antioxidants
1 Scope
This standard specifies the food gallic acid propyl ester (PG), 2,4,5- trihydroxybenzene-butanone (THBP), tertiary butylhydroquinone
(TBHQ), nor-dihydro-nordihydroguaiaretic acid (NDGA), butylated hydroxy anisole (BHA), 2,6- di-tert-butyl-4-hydroxymethyl-phenol
(Ionox-100), octyl gallate (OG), 2,6- di-t-butyl-p-cresol (BHT), gallic acid antioxidant nine kinds of lauryl acrylate (DG)
Determination of 5 kinds agent --- HPLC liquid chromatography tandem mass spectrometry, gas chromatography mass spectrometry, gas chromatography and the ratio of
Colorimetric method.
This standard is applicable to liquid chromatography foods PG, THBP, TBHQ, NDGA, BHA, BHT, Ionox-100, OG, DG of
Determination; liquid chromatography-tandem mass spectrometry assay is suitable THBP, PG, OG, NDGA, DG in the diet; gas chromatography mass spectrometry is suitable for
Food BHA, BHT, TBHQ, measured Ionox-100; a gas chromatography is suitable for food, BHT measured BHA, TBHQ of;
PG is suitable for colorimetric assay grease.
The first HPLC method
Principle 2
Fat or oil sample is dissolved by an organic solvent using gel permeation chromatography (GPC) purge; solid food sample was dissolved in n-hexane, with ethyl
Nitriles extraction, solid-phase extraction cartridge. HPLC, external standard.
3 Reagents and materials
Unless otherwise indicated, all reagents used in the method of HPLC grade water to a water GB/T 6682 provisions.
3.1 Reagent
3.1.1 formic acid (HCOOH).
3.1.2 acetonitrile (CH3CN).
3.1.3 methanol (CH3OH).
3.1.4 hexane (C6H14). analytical grade, redistilled.
3.1.5 ethyl acetate (CH3COOCH2CH3).
3.1.6 cyclohexane (C6H12).
3.1.7 Sodium chloride (NaCl). AR.
3.1.8 anhydrous sodium sulfate (Na2SO4). analytically pure, burning 650 ℃ 4h, stored in a desiccator, cooled standby.
3.2 Reagent preparation
3.2.1 acetonitrile saturated n-hexane. n-hexane was added to the saturated acetonitrile.
3.2.2 hexane saturated with acetonitrile. n-hexane was added to the saturated acetonitrile.
3.2.3 a mixed solution of ethyl acetate and cyclohexane (1 1). Take 50mL ethyl acetate and 50mL of cyclohexane mix.
3.2.4 a mixed solution of acetonitrile and methanol (21). Take 50mL acetonitrile and 100mL methanol mixture.
3.2.5 saturated sodium chloride solution. water was added sodium chloride to saturation.
3.2.6 formic acid (0.1 99.9). Take 0.1mL acid into a 100mL volumetric flask, dilute to the mark.
3.3 Standard
3.3.1 butylated hydroxy anisole. purity ≥98%.
3.3.2 tert-butyl-p-cresol. purity ≥98%.
3.3.3 octyl gallate. purity ≥98%.
3.3.4 gallic acid lauryl acrylate. purity ≥98%.
3.3.5 gallic acid ester. purity ≥98%.
3.3.6 nor-dihydro nordihydroguaiaretic acid. purity ≥98%.
3.3.7 2,4,5-trihydroxybenzene-butanone. purity ≥98%.
Tertiary butylhydroquinone 3.3.8. purity ≥98%.
3.3.9 butyl-4-hydroxymethyl-phenol. purity ≥98%.
Note. The English name, molecular formula, and CAS numbers, see Appendix A.
3.4 Standard Preparation
3.4.1 antioxidant substances mixed standard stock solution. 0.1 g of accurately weighed (accurate to 0.1 mg) of solid antioxidant standard substance with acetonitrile
Brown soluble 100mL volumetric flask, dilute to volume, formulated at a concentration of 1000mg/L stock solution of a standard mixture, avoiding 0 ℃ ~ 4 ℃
Optical storage.
3.4.2 an antioxidant standard solution. Pipette an appropriate volume concentration of 1000mg/L antioxidant combined stock standard substance
Respectively, was diluted to a concentration of 20mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L of a mixed standard solution.
3.5 Material
3.5.1 C18 solid phase extraction column.2000mg/12mL.
3.5.2 Organic-based filter. pore size 0.22μm.
4 instruments and equipment
4.1 Centrifuge. speed ≥3000r/min.
4.2 rotary evaporator.
4.3 HPLC.
4.4 gel permeation chromatography.
4.5 Analytical balance. a sense of the amount of 0.01g and 0.1mg.
4.6 a vortex.
5 Step analysis
5.1 Sample Preparation
Solid or semi-solid sample is mixed pulverized, and then take the two quarters or 6/2 diagonals method, or a case of taking a representative sample according to
Sample, sealed; uniformly mixing liquid samples, take a representative sample, sealed and stored.
5.2 Determination of step
5.2.1 extract
5.2.1.1 Solid kinds of samples. Weigh 1g (accurate to 0.01g) sample (5.1) in 50mL centrifuge tubes, was added 5mL of acetonitrile saturated n
Hexane, 1min vortexing thoroughly mixed, soaking 10min. Was added 5mL saturated sodium chloride solution, 5mL of n-hexane saturated with acetonitrile
Solution was vortexed 2min, 3000r/min centrifugal 5min, acetonitrile layer was collected in a test tube, and then n-hexane was repeated using 5mL saturated solution in acetonitrile
Solution was extracted twice, and the combined extracts three times, plus 0.1% formic acid solution to adjust pH = 4, to be purified. At the same time a blank test.
5.2.1.2 oil. Weigh 1g (accurate to 0.01g) sample (5.1) in 50mL centrifuge tubes, 5mL of acetonitrile was added a saturated solution of n-hexane
Was dissolved sample, vortexed 1min, allowed to stand 10min, extracted with acetonitrile solution was vortexed for 2min 5mL hexane saturated, 3000r/min from
Heart 5min, acetonitrile layer was collected in a test tube, and then n-hexane was repeated using 5mL saturated acetonitrile solution was extracted twice, and the combined extracts three times,
To be purified. At the same time a blank test.
5.2.2 Purification
About 2g of anhydrous sodium sulfate was charged in a C18 solid phase extraction column, extraction column activated with 5mL methanol, then extracted with 5mL acetonitrile Ping Heng
Column, the effluent was discarded. All extracts (5.2.1) was poured into the column, the effluent was discarded, and then eluted with a mixed solution of acetonitrile and methanol 5mL,
All the collected eluate in a test tube, at 40 ℃ rotary evaporation to dryness, add acetonitrile to volume 2mL, through 0.22μm organic membrane for liquid chromatography
Spectroscopy.
5.2.3 Gel permeation chromatography (pure oil sample optional)
Weigh the sample (5.1) 10g (accurate to 0.01g) in 100mL volumetric flask, a mixed solution of ethyl acetate and cyclohexane to a volume moment
Degree, as a mother liquor; 5mL liquor taken in 10mL volumetric flask a mixed solution of ethyl acetate and cyclohexane to volume, to be purified.
Of 10mL test solution added to the gel permeation chromatography (GPC) in the sample line, purification using GPC (gel permeation chromatography purification conditions mitsuke
Recorded B), collecting the effluent, at 40 ℃ rotary evaporation to dryness, add acetonitrile to volume 2mL, through 0.22μm organic membrane, for measuring liquid chromatography
set. At the same time a blank test.
5.3 liquid chromatography conditions
a) Column. C18 column, column length 250mm, internal diameter 4.6mm, particle size 5 m, column, or equivalent;
b) Mobile phase A. 0.5% aqueous formic acid, mobile phase B. methanol;
c) elution gradient. 0 ~ 5min Mobile phase (A) 50%, 5min ~ 15min. Mobile phase (A) of from 50% to 20%, 15min ~
20min mobile phase (A) 20%, 20min ~ 25min. Mobile phase (A) of from 20% to 10%, 25min ~ 27min. flow
Phase (A) of from 10% to 50%, 27min ~ 30min. Mobile Phase (A) 50%;
d) Column temperature. 35 ℃;
e) Injection volume. 5μL;
f) Detection Wavelength. 280nm.
Standard curve prepared 5.4
Standard working solution concentration series were injected into the liquid chromatograph to measure the respective antioxidant, working standard solution at a concentration of a cross
Coordinates, in response to the value (such as. peak area, peak height, absorption value, etc.) for the vertical axis, the standard curve. 9 kinds of antioxidants FIG see enclosure liquid chromatography
C. record
5.5 Determination of the sample solution
The sample solution was injected into high performance liquid chromatography to give the corresponding peak response values of the standard curve obtained in the test solution Antioxidant
Concentration of the agent.
6 expression analysis results
Antioxidant content in the sample according to formula (1).
Xi = ρi ×
(1)
Where.
XI --- samples antioxidant content in milligrams per kilogram (mg/kg);
--- pi concentration of the antioxidant solution was obtained from the standard curve, in micrograms per milliliter (μg/mL);
V --- the final volume of the sample solution, in milliliters (mL);
m --- said mass of the sample taken, in grams (g).
The results rounded to three significant digits (or reserved to two decimal places).
7 precision
The absolute difference between two measurement results obtained under repeatable conditions not exceed 10% of the arithmetic mean.
8 Other
The detection limit of this method is not propyl gallate (PG). 2mg/kg, 2,4,5- trihydroxybenzene-butanone (THBP). 4mg/kg, tert-butyl
Hydroquinone (TBHQ). 10mg/kg, nor-dihydro-nordihydroguaiaretic acid (NDGA). 4mg/kg, butylated hydroxy anisole (BHA).
10mg/kg, 2,6- di-tert-butyl-4-hydroxymethyl-phenol (Ionox-100). 20mg/kg, octyl gallate (OG). 2mg/kg, 2,6- two
T-butyl-p-cresol (BHT). 4mg/kg, gallic acid lauryl acrylate (DG). 10mg/kg; the limit of quantitation were 20mg/kg.
The second method of liquid chromatography-tandem mass spectrometry
Principle 9
Fat or oil sample is dissolved by an organic solvent using gel permeation chromatography (GPC) purge; solid food sample was dissolved in n-hexane, with ethyl
Nitriles extraction, solid-phase extraction cartridge. Liquid chromatography tandem mass spectrometry assay, external standard.
10 Reagents and materials
Unless otherwise indicated, all reagents used in the method of HPLC grade water to a water GB/T 6682 provisions.
10.1 Reagents
With 3.1
10.2 reagent preparation
With 3.2
10.3 Standards
10.3.1 octyl gallate. purity ≥98%.
10.3.2 gallic acid lauryl acrylate. purity ≥98%.
10.3.3 gallic acid ester. purity ≥98%.
10.3.4 nor-dihydro nordihydroguaiaretic acid. purity ≥98%.
10.3.5 2,4,5-trihydroxybenzene-butanone. purity ≥98%.
Note. The English name, molecular formula, and CAS No. see Appendix A.
10.4 Standard Preparation
10.4.1 standard substance stock solution. 0.1 g of accurately weighed (accurate to 0.1 mg) of solid antioxidant standard substance, was dissolved in 100mL with acetonitrile
Brown volumetric flask, dilute to volume, to a concentration of 1000mg/L standard stock solution, 0 ℃ ~ 4 ℃ stored.
10.4.2 intermediate liquid standard substance. standard substance stock solution was pipetted 1.0mL in 100mL volumetric flask to volume with acetonitrile, the concentration of formulated
Of 10mg/L standard intermediate liquid mixture, 0 ℃ ~ 4 ℃ stored.
10.4.3 standard substance used was. Pipette appropriate volume of a standard substance solution were diluted to an intermediate concentration of 0.01mg/L, 0.02mg/L,
0.05mg/L, 0.1mg/L, 0.2mg/L, 0.5mg/L, 1mg/L, 2mg/L mixed standard solution.
The mobile phase (B) from 80% to 10%, 12.01min ~ 14min. Mobile Phase (B) 10%;
e) Column temperature. 35 ℃;
f) Injection volume. 2μL;
g) ionization mode. electrospray ionization;
h) the spray flow rate. 3L/min;
i) the drying gas flow rate. 15L/min;
j) Ion spray voltage. 3500V;
k) monitoring an ion pair, collision energy, dwell time and the retention time is given in Appendix D.
12.4 qualitative determination
When the test samples were measured under the same conditions, if the retention time of the standard sample detected peaks coincide, and deducting
Background of the sample spectrum, the selected ions are occurred, ion abundance ratio of the standard sample and the selected match (relative abundance
> 50%, ± 20% deviation allowed; relative abundance of > 20% to 50%, the allowable deviation of ± 25%; relative abundance of > 10% to 20%, the allowable
± 30% variation; relative abundance ≤10%, ± 50% deviation allowed), it may be determined that antioxidants present in the sample.
12.5 standard curve prepared
The series of standard working solution for liquid chromatography tandem mass spectrometry assay to quantify the area of the peaks corresponding to the standard ion concentration solution drawn subscript
Standard curve. 9 kinds of antioxidants multiple reaction monitoring (MRM) chromatograms in Appendix E.
12.6 Determination of the sample solution
The sample solution was subjected to liquid chromatography tandem mass spectrometer, a calibration curve obtained according to the concentration of the test solution of the antioxidant.
13 expression analysis results
Antioxidant content in the sample according to the formula (2).
Xi = ρi ×
(2)
Where.
XI --- samples antioxidant content in milligrams per kilogram (mg/kg);
--- pi concentration of the antioxidant solution was obtained from the standard curve, in micrograms per milliliter (μg/mL);
V --- the final volume of the sample solution, in milliliters (mL);
m --- said mass of the sample taken, in grams (g).
The results rounded to three significant digits (or reserved to two decimal places).
14. Precision
The absolute difference between two measurement results obtained under repeatable conditions not exceed 10% of the arithmetic mean.
Other 15
The detection limit of this method is not propyl gallate (PG). 0.05mg/kg, 2,4,5- trihydroxybenzene-butanone (THBP). 0.05mg/kg, to
A dihydro-nordihydroguaiaretic acid (NDGA). 0.05mg/kg, octyl gallate (OG). 0.005mg/kg, gallic acid lauryl acrylate (DG).
0.005mg/kg; limit of quantification was no propyl gallate (PG). 0.1mg/kg, 2,4,5- trihydroxybenzene-butanone (THBP). 0.1mg/kg, to
A dihydro-nordihydroguaiaretic acid (NDGA). 0.1mg/kg, octyl gallate (OG). 0.01mg/kg, gallic acid lauryl acrylate (DG).
0.01mg/kg.
The third gas chromatography - mass spectrometry
Principle 16
Fat or oil sample is dissolved by an organic solvent using gel permeation chromatography (GPC) purge; solid food sample was dissolved in n-hexane, with ethyl
Nitriles extraction, solid-phase extraction cartridge. Gas chromatography - mass spectrometry measurement, external standard.
17 Reagents and materials
Unless otherwise indicated, all reagents used in the method of HPLC grade water to a water GB/T 6682 provisions.
17.1 Reagents
With 3.1.
17.2 reagent preparation
With 3.2.
17.3 Standards
17.3.1 butylated hydroxy anisole. purity ≥98%.
Tertiary butylhydroquinone 17.3.2. purity ≥98%.
17.3.3 tert-butyl-p-cresol. purity ≥98%.
17.3.4 butyl-4-hydroxymethyl-phenol. purity ≥98%.
Note. The English name, molecular formula, and CAS numbers, see Appendix A.
17.4 Standard Preparation
17.4.1 standard substance stock solution. 0.1 g of accurately weighed (accurate to 0.1 mg) of solid antioxidant standard substance, was dissolved in 100mL with acetonitrile
Brown volumetric flask, dilute to volume, to a concentration of 1000mg/L standard stock solution, 0 ℃ ~ 4 ℃ stored.
17.4.2 mixed standard solution. Pipette an appropriate volume after concentration was 1000mg Antioxidant mixed standard stock solution/L, were diluted
Diluted to a concentration of 1mg/L, 2mg/L, 5mg/L, 10mg/L, 20mg/L, 50mg/L, 100mg/L, 200mg/L standard mixing
Use liquid.
17.5 Materials
With 3.5.
18 instruments and equipment
18.1 Centrifuge. speed ≥3000r/min.
18.2 rotary evaporator.
18.3 GC mass spectrometer.
18.4 gel permeation chromatography.
18.5 Analytical Balance. a sense of the amount of 0.01g and 0.1mg.
18.6 a vortex.
19 analysis steps
19.1 Preparation of samples
With 5.1.
19.2 Determination Step
With 5.2.
19.3 GC Mass Spectrometer Conditions
a) Column. 5% phenyl - methyl silicone capillary column, column length 30m, internal diameter 0.25mm, film thickness 0.25 m, or equivalent color
Column spectrum;
b) Column temperature program. 70 ℃ holding 1min, then warmed at 10 ℃/min to 200 ℃ program held 4min, then to
10 ℃/min was heated to 280 deg.] C held 4min;
c) Carrier gas. Helium, purity ≥99.999%, flow rate 1mL/min;
d) Inlet temperature. 230 ℃;
e) Injection volume. 1μL;
f) Injection mode. splitless, 1min after opening the valve;
g) Electron impact source. 70eV;
h) Ion source temperature. 230 ℃;
i) GC-MS Interface Temperature. 280 ℃;
j) Solvent delay 8min;
k) the selected ion monitoring. a quantitative each compound were selected ion, 2 ~ 3 qualitative ions. Each need to detect all
Ion peak according to the order, each sub-time detection. Retention time of each compound, ion quantitative and qualitative ion dwell time
Appendix F.
19.4 qualitative determination
When the test samples were measured under the same conditions, if the retention time of the standard sample detected peaks coincide, and deducting
Background of the sample spectrum, the selected ions are occurred, ion abundance ratio of the standard sample and the selected match (relative abundance
> 50%, ± 20% deviation allowed; relative abundance of > 20% to 50%, the allowable deviation of ± 25%; relative abundance of > 10% to 20%, the allowable
± 30% variation; relative abundance ≤10%, ± 50% deviation allowed), it may be determined that antioxidants present in the sample.
19.5 standard curve prepared
The series of standard working solution for mass spectrometry measured by gas chromatography to quantify the ion peak area corresponding to the standard concentration plotted standard
curve. 4 kinds of antioxidants selected ion monitoring GC-MS Appendix G. FIG.
19.6 Determination of the sample solution
The sample solution is injected into the gas chromatograph mass spectrometer to give the corresponding peak response value, the standard curve obtained in the test solution Antioxidant
Concentration of the agent.
20 expression analysis results
Antioxidant content in the sample (3) is calculated according to the formula.
Xi = ρi ×
(3)
Where.
XI --- samples antioxidant content in milligrams per kilogram (mg/kg);
--- pi concentration of the antioxidant solution was obtained from the standard curve, in micrograms per milliliter (μg/mL);
V --- the final volume of the sample solution, in milliliters (mL);
m --- said mass of the sample taken, in grams (g).
The results rounded to three significant digits (or reserved to two decimal places).
21 precision
The absolute difference between two measurement results obtained under repeatable conditions not exceed 10% of the arithmetic mean.
Other 22
The detection limit of this method is t-butyl hydroquinone (TBHQ). 0.5mg/kg, butylated hydroxy anisole (BHA). 1mg/kg,
2,6-di-t-butyl-4-hydroxymethyl-phenol (Ionox-100). 0.5mg/kg, 2,6- di-t-butyl-p-cresol (BHT). 0.5mg/kg; given
Limit both the amount of 1mg/kg.
The fourth gas chromatography method
Principle 23
Antioxidant sample extraction with an organic solvent, the gel permeation chromatography (GPC) purifying by gas chromatography flame ionization detector
Detecting, using qualitative retention time, external standard.
24 Reagents and materials
Unless otherwise indicated, all reagents used in the method of HPLC grade water to a water GB/T 6682 provisions.
24.1 Reagents
24.1.1 cyclohexane (C6H12).
24.1.2 ethyl acetate (CH3COOCH2CH3).
24.1.3 petroleum ether. boiling range 30 ℃ ~ 60 ℃ (redistilled).
24.1.4 acetonitrile (CH3CN).
24.1.5 acetone (CH3COCH3).
24.2 reagent preparation
A mixed solution of ethyl acetate and cyclohexane (1 1). Measure 50mL ethyl acetate and 50mL of cyclohexane mix.
24.3 Standards
24.3.1 BHA standards. purity ≥99.0%.
24.3.2 BHT standards. purity ≥99.3%.
24.3.3 TBHQ standards. purity ≥99.0%.
24.3.4 BHA, BHT, TBHQ standard stock solution. Weigh accurately BHA, BHT, TBHQ 50mg of each standard (the nearest
0.1 mg), a mixed solution of ethyl acetate and cyclohexane volume to 50mL, formulated as a stock solution 1mg/mL, and in the dark at 4 ℃
save.
24.3.5 BHA, BHT, TBHQ standard solution. draw stock standard solution 0.1mL, 0.5mL, 1.0mL, 2.0mL, 3.0mL,
4.0mL, 5.0mL set in a 10mL volumetric flask, a mixed solution of ethyl acetate and cyclohexane volume, concentration series of this standard
0.01mg/mL, 0.05mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, using now.
24.4 Materials
Organic filter. pore size 0.45μm.
25 instruments and equipment
25.1 Gas Chromatograph (GC). with a flame ionization detector (FID).
25.2 gel permeation chromatography (GPC), or equivalent separating means may be defatted.
25.3 Analytical Balance. a sense of the amount of 0.01g and 0.1mg.
25.4 rotary evaporator.
25.5 a vortex.
25.6 mill.
26 analysis steps
26.1 Preparation of samples
With 5.1.
26.2 Sample Processing
26.2.1 grease sample
Mixed oil sample, over 0.45μm filter, 0.5g accurately weighed (accurate to 0.1 mg), ethyl acetate and cyclohexane,
The exact volume to a mixed solution of 10.0mL, mixed to be purified.
26.2.2 moderate or high fat content of the samples (fat content more than 15% of the sample)
The actual amount of sample oil were weighed 5g mixed sample was...
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