Sucrose esters of fatty acids

Prepared at the 49th JECFA (1997)
superseding specifications prepared at the 44th JECFA (1995),
published in FNP 52 Addendum 3 (1995)

SYNONYMS

INS No. 473

DEFINITION

Mono-, di- and tri-esters of sucrose with food fatty acids, prepared from sucrose and methyl and ethyl esters of food fatty acids or by extraction from sucroglycerides. Only the following solvents may be used for the production: dimethyl formamide, dimethyl sulfoxide, ethyl acetate, isopropanol, propylene glycol, isobutanol and methyl ethyl ketone.

Assay

Not less than 80%

DESCRIPTION

Stiff gels, soft solids or white to slightly greyish white powders

FUNCTIONAL USES

Emulsifier

CHARACTERISTICS

IDENTIFICATION

Solubility

Sparingly soluble in water, soluble in ethanol

Test for fatty acids

Passes test
See description under TESTS

Test for sugar

Passes test
See description under TESTS

PURITY

Sulfated ash

Not more than 2%
Test 1 g of the sample as directed under the Test for Ash (Sulfated ash, Method I)

Acid value

Not more than 6

Free sucrose

Not more than 5%
See description under TESTS

Dimethyl formamide

Not more than 1 mg/kg
See description under TESTS

Dimethyl sulfoxide

Not more than 2 mg/kg
See description under TESTS

Ethyl acetate, isopropanol and propylene glycol

Not more than 350 mg/kg, singly or in combination
See description under TESTS

Isobutanol

Not more than 10 mg/kg
See description under TESTS

Methanol

Not more than 10 mg/kg
See description under TESTS

Methyl ethyl ketone

Not more than 10 mg/kg
See description under TESTS

Lead

Not more than 10 mg/kg
Prepare a sample solution as described for organic compounds in the Limit test, using 10 m g of lead ion (Pb) in the control

TESTS

IDENTIFICATION TESTS

Test for fatty acids

Add 1 ml of ethanol to 0.1 g of the sample, dissolve by warming, add 5 ml of dilute sulfuric acid TS, heat in a water bath for 30 min and cool. A yellowish white solid or oil is formed which is soluble in 3 ml of ether and has no odour of isobutyric acid.

Test for sugar

To 2 ml of the solution separated from the solid and oil in the test for fatty acids, add 1 ml of anthrone TS carefully down the inside of the test tube; the boundary surface of the two layers turns to blue or green.

PURITY TESTS

Free sucrose

Determine by gas liquid chromatography using the following conditions:
Reagents:
- Internal Standard: 5 mg/ml cholesterol in chloroform or 10 mg/ml tetracosane in chloroform
- Pyridine (dried over molecular sieve)
- N,O-Bis-(Trimethylsilyl)-acetamide (BSA)
- Trimethylchlorosilane (TMCS)
Procedure:
Weigh accurately 20-50 mg of the sample into a silylation vial, add 1 ml internal standard solution, 1 ml pyridine, and 0.5 ml each of BSA and TMCS. Seal vial, and heat at 70° for 30 min. Inject 1 m l into the gas liquid chromatograph.
Conditions:
Column:
- length: 0.3 m
- diameter: 4 mm (i.d.)
- material: glass
- packing: Dexil
Carrier gas: Nitrogen
Flow rate: 40 ml/min
Detector: FID
Temperature programme: Hold for 1 min at 160°, then 160-375° at 15°/min
Measure peak areas for sucrose and internal standard. The response factor (RF) is calculated from a number of gas liquid chromatography runs with standard solutions of sucrose containing internal standard.
Calculation:

and

Dimethyl formamide

Determine by hydrolysis to dimethylamine and analysis by gas liquid chromatography using the following conditions:
Reagents:
- Dimethyl formamide
- Dimethylamine hydrochloride
- Methanol
- Ethanol
- Hydrochloric acid
- Sodium hydroxide
Standard solutions:
Prepare 4.47 mg/ml (equivalent to 4.0 mg/ml of dimethyl formamide) stock solution of dimethylamine hydrochloride in ethanol, and prepare standard solutions equivalent to 4, 2 and 1 m g/ml of dimethyl formamide, respectively, by dilution of the stock solution with 0.1% sodium hydroxide solution in ethanol.
Sample preparation:
The apparatus for the hydrolysis is shown in the Appendix. Weigh accurately about 40 g of the sample into a 1000-ml round-bottomed flask. Add 500 ml of 5% methanolic solution of sodium hydroxide, and attach the flask to the apparatus. Set an Erlenmeyer flask containing 10 ml of 1% methanolic solution of hydrochloric acid to the apparatus. Heat the round-bottomed flask and let the content reflux for 1 hour, then distil to collect about 50 ml of the distillate while cooling water of the reflux condenser is stopped. Evaporate the distillate to almost dryness on a boiling water bath. Dissolve the residue with a small amount of ethanol, add 2.5 ml of 5% ethanolic solution of sodium hydroxide, and dilute to 25 ml with ethanol to prepare a sample solution.
Procedure:
Inject 2 m l of the sample solution into the gas liquid chromatograph under the conditions below.
Calibration curve:
Prepare a calibration curve by injecting each 2 m l of the standard solutions into the gas chromatograph.
Conditions:
Column:
- length: 2 m
- diameter: 2 mm (i.d.)
- material: Glass
- packing: 10% amine 220 and 10% KOH on 80/100 weak acid washed Chromosorb W
- conditioning: Heat to 130° overnight with 5 ml/min of nitrogen flow rate
Carrier gas: Nitrogen
Flow rate: 17 ml/min
Detector: FID
Temperatures
- injection port: 198±5°
- column: 60°
Calculation:

where
C_DFA = Concentration of dimethyl formamide
C = Concentration of dimethyl formamide detected
W = weight of sample taken

Dimethyl sulfoxide

Determine by gas liquid chromatography under the following conditions:
Reagents:
- Tetrahydrofuran
- Dimethyl sulfoxide
- Sucrose esters of fatty acids (dimethyl sulfoxide free, i.e. sucrose esters of fatty acids prepared without using dimethyl sulfoxide)
Standard solutions (prepared fresh monthly):
- Prepare a 0.25 mg/ml stock solution of dimethyl sulfoxide in tetrahydrofuran
- Dissolve 40 g of dimethyl sulfoxide free sucrose esters of fatty acids into tetrahydrofuran and dilute to 200 ml (Sucrose esters of fatty acids solution)
- Prepare a range of solutions containing 0.5, 1 and 5 m g/ml of dimethyl sulfoxide by dilution of the stock solution with the sucrose esters of fatty acids solution, respectively
Procedure:
Weigh accurately about 5 g of the sample, dissolve and dilute it with tetrahydrofuran to 25 ml to prepare a sample solution. Inject 3 m l of the sample solution into the gas chromatograph under the conditions below.
Calibration curve (prepared daily):
Prepare a calibration curve by injecting each 3 m l of the standard solutions into the gas chromatograph
Conditions:
Column:
- length: 2 m
- diameter: 3 mm (i.d.)
- material: Glass
- packing: 10% PEG 20M and 3% KOH on Gas Chrom Z
- conditioning: Raise the oven temperature to 180° at a rate of 10°/min and let stand for 24 to 48 h with 30 to 40 ml/min of nitrogen
Carrier gas: Nitrogen
Flow rate: 50 ml/min
Detector: Flame photometric (using 394 nm sulfur filter)
Temperatures
- injection port: 210°
- column: 160° (do not exceed 200°)
Calculation

where
C_DMSO = Concentration of dimethyl sulfoxide
C = Concentration of dimethyl sulfoxide detected
W = weight of sample taken

Propylene glycol

Determine by gas liquid chromatography using the following conditions:
Reagents:
- Propylene glycol
- 1,2-Butylene glycol
- Acetic anhydride
- Toluene
- Acetone
Standard solutions:
- Internal standard solution: Prepare 1000 mg/l solution of 1,2-butylene glycol in acetone. 200 m l of the solution contains 0.2 mg of 1,2-butylene glycol.
- Standard solution of propylene glycol: Prepare 1000 mg/l solution of propylene glycol in acetone.
Sample preparation:
Weigh accurately about 2 g of the sample in a 100-ml flat-bottomed flask, and add 10 ml of acetic anhydride and 200 m l of the internal standard solution. Attach a reflux condenser and heat the flask in a boiling water bath for 1 hour. Add 50 ml of water and heat the flask for another 10 min in a boiling water bath. After heating, let it cool to room temperature. Transfer the content to a 100-ml separating funnel and extract with 10 ml of toluene. After separation, discharge the lower layer (aqueous phase). Add 50 ml of water to the separating funnel and wash the extract. The upper layer (toluene phase) is used as a sample solution.
Procedure:
Inject 2 m l of the sample solution into the gas chromatograph under the following conditions. (Note: Since there would be a component of high boiling point, which may have a longer retention time, it is necessary at the end of each measurement to raise the oven temperature to 200° and to evacuate it from the column).
Calibration curve:
Follow the same procedure using 100, 200 and 500 m l of the standard solution of propylene glycol in place of the sample, and prepare a calibration curve.
Column:
- length: 2 m
- diameter: 3 mm (i.d.)
- material: Glass
- packing: 5% Alkyleneglycol phthalate on 80/100 Chromosorb W
- conditioning: Heat to 150° overnight with approximately 50 ml/min of nitrogen
Carrier gas: Nitrogen
Flow rate: 40 ml/min
Detector: Flame ionization
Temperatures:
- injection port: 230°
- column: 110°
Calculation:

where
C = Concentration of propylene glycol (mg/kg)
A_PG = Peak area of propylene glycol
A_IS = Peak area of internal standard
W_SPL = Weight of sample (g)
C_fPG = Sensitivity correction factor for propylene glycol (slope of the calibration curve)

Methanol, isopropanol, isobutanol, ethyl acetate and methyl ethyl ketone

Determine by gas chromatography with a head space sampler using the following conditions:
Reagents:
- Methanol
- Isopropanol
- Isobutanol
- Ethyl acetate
- Methyl ethyl ketone
Standard solutions:
Take each 1 g of methanol, isopropanol, isobutanol, methyl ethyl ketone and ethyl acetate in a volumetric flask and add water to total volume of 100 ml, and prepare 0.02-0.4 g/100 ml solutions by dilution of this solution.
If necessary, prepare standard solutions containing up to 7 g/100 ml of isopropanol and ethyl acetate.
Procedure:
Place 1 g (1.0 ± 0.1g) of powdered sample in a sample vial. Add 5 m l of water to the sample vial and seal it quickly with a septum. Set the sample vial in a pre-conditioned gas chromatograph and start the analysis under the below-mentioned conditions.
Calibration curve:
Take 1 g of powdered sucrose esters of fatty acids, solvent free or known residual solvent contents, in a sample vial, add 5 m l of the standard solution and seal it quickly with a septum. Set the sample vial in a pre-conditioned gas chromatograph and start the analysis under the following conditions and obtain calibration curves for each solvent.
Column:
- length: 30 m
- diameter: 0.53 mm (i.d.)
- material: Silica capillary
- film: 100% methyl polysiloxane
- conditioning: Heat to 60° for 2-3 h with approximately 10 ml/min of nitrogen
Carrier gas: Nitrogen
Flow rate: 5 ml/min
Detector: Flame ionization
Temperatures
- injection port: 110°
- column: 40°
- detector: 110°
Head space sampler:
- Sample volume: 1.0 g ± 0.1 g + 5 m l
- Sample heating temp.: 80°
- Sample heating time: 40 min
- Syringe temperature: 85° - Sample gas injection: 0.4 ml
Calculation:
C_i =A_i x Cf_i x 1000
where
C_i = Concentration of solvent i (mg/kg)
A_i = Peak area of solvent i (m v.sec.)
Cf_i = Conversion coefficient for solvent i (slope of the calibration curve) (m g/m v.sec)

METHOD OF ASSAY

Determine by high pressure liquid chromatography using the following conditions:
Sample preparation:
Add about 250 mg of the sample, accurately weighed to a 50 ml volumetric flask. Dilute to volume with tetrahydrofuran, and mix. Filter through a 0.5-m m membrane filter.
Procedure:
Inject 100 m l of the sample into the pre-stabilized high pressure liquid chromatograph.
Conditions:
Column: Styrene-divinylbenzene copolymer for gel permeation chromatography (TSK-GEL G2000 (Supelco) or equivalent)
Mobile phase: HPLC-grade degassed tetrahydrofuran
Flow rate: 0.7 ml/min
Detector: Refractive index detector
Temperatures:
Column: 38°
Detector: 38°
Record the chromatogram for about 90 min. Calculate the percentage of sucrose ester content in the sample taken by the formula:
100 A/T
where
A = the sum of peak areas for the three main components, the mono-, di-and triesters, eluting at about 65, 68 and 73 min, respectively
T = the sum of all peak areas eluting within 90 min

APPENDIX

SYNONYMS	INS No. 473
DEFINITION	Mono-, di- and tri-esters of sucrose with food fatty acids, prepared from sucrose and methyl and ethyl esters of food fatty acids or by extraction from sucroglycerides. Only the following solvents may be used for the production: dimethyl formamide, dimethyl sulfoxide, ethyl acetate, isopropanol, propylene glycol, isobutanol and methyl ethyl ketone.
Assay	Not less than 80%
DESCRIPTION	Stiff gels, soft solids or white to slightly greyish white powders
FUNCTIONAL USES	Emulsifier
CHARACTERISTICS
IDENTIFICATION
Solubility	Sparingly soluble in water, soluble in ethanol
Test for fatty acids	Passes test See description under TESTS
Test for sugar	Passes test See description under TESTS
PURITY
Sulfated ash	Not more than 2% Test 1 g of the sample as directed under the Test for Ash (Sulfated ash, Method I)
Acid value	Not more than 6
Free sucrose	Not more than 5% See description under TESTS
Dimethyl formamide	Not more than 1 mg/kg See description under TESTS
Dimethyl sulfoxide	Not more than 2 mg/kg See description under TESTS
Ethyl acetate, isopropanol and propylene glycol
	Not more than 350 mg/kg, singly or in combination See description under TESTS
Isobutanol	Not more than 10 mg/kg See description under TESTS
Methanol	Not more than 10 mg/kg See description under TESTS
Methyl ethyl ketone	Not more than 10 mg/kg See description under TESTS
Lead	Not more than 10 mg/kg Prepare a sample solution as described for organic compounds in the Limit test, using 10 m g of lead ion (Pb) in the control
TESTS
IDENTIFICATION TESTS
Test for fatty acids	Add 1 ml of ethanol to 0.1 g of the sample, dissolve by warming, add 5 ml of dilute sulfuric acid TS, heat in a water bath for 30 min and cool. A yellowish white solid or oil is formed which is soluble in 3 ml of ether and has no odour of isobutyric acid.
Test for sugar	To 2 ml of the solution separated from the solid and oil in the test for fatty acids, add 1 ml of anthrone TS carefully down the inside of the test tube; the boundary surface of the two layers turns to blue or green.
PURITY TESTS
Free sucrose	Determine by gas liquid chromatography using the following conditions: Reagents: - Internal Standard: 5 mg/ml cholesterol in chloroform or 10 mg/ml tetracosane in chloroform - Pyridine (dried over molecular sieve) - N,O-Bis-(Trimethylsilyl)-acetamide (BSA) - Trimethylchlorosilane (TMCS) Procedure: Weigh accurately 20-50 mg of the sample into a silylation vial, add 1 ml internal standard solution, 1 ml pyridine, and 0.5 ml each of BSA and TMCS. Seal vial, and heat at 70° for 30 min. Inject 1 m l into the gas liquid chromatograph. Conditions: Column: - length: 0.3 m - diameter: 4 mm (i.d.) - material: glass - packing: Dexil Carrier gas: Nitrogen Flow rate: 40 ml/min Detector: FID Temperature programme: Hold for 1 min at 160°, then 160-375° at 15°/min Measure peak areas for sucrose and internal standard. The response factor (RF) is calculated from a number of gas liquid chromatography runs with standard solutions of sucrose containing internal standard. Calculation: and
Dimethyl formamide	Determine by hydrolysis to dimethylamine and analysis by gas liquid chromatography using the following conditions: Reagents: - Dimethyl formamide - Dimethylamine hydrochloride - Methanol - Ethanol - Hydrochloric acid - Sodium hydroxide Standard solutions: Prepare 4.47 mg/ml (equivalent to 4.0 mg/ml of dimethyl formamide) stock solution of dimethylamine hydrochloride in ethanol, and prepare standard solutions equivalent to 4, 2 and 1 m g/ml of dimethyl formamide, respectively, by dilution of the stock solution with 0.1% sodium hydroxide solution in ethanol. Sample preparation: The apparatus for the hydrolysis is shown in the Appendix. Weigh accurately about 40 g of the sample into a 1000-ml round-bottomed flask. Add 500 ml of 5% methanolic solution of sodium hydroxide, and attach the flask to the apparatus. Set an Erlenmeyer flask containing 10 ml of 1% methanolic solution of hydrochloric acid to the apparatus. Heat the round-bottomed flask and let the content reflux for 1 hour, then distil to collect about 50 ml of the distillate while cooling water of the reflux condenser is stopped. Evaporate the distillate to almost dryness on a boiling water bath. Dissolve the residue with a small amount of ethanol, add 2.5 ml of 5% ethanolic solution of sodium hydroxide, and dilute to 25 ml with ethanol to prepare a sample solution. Procedure: Inject 2 m l of the sample solution into the gas liquid chromatograph under the conditions below. Calibration curve: Prepare a calibration curve by injecting each 2 m l of the standard solutions into the gas chromatograph. Conditions: Column: - length: 2 m - diameter: 2 mm (i.d.) - material: Glass - packing: 10% amine 220 and 10% KOH on 80/100 weak acid washed Chromosorb W - conditioning: Heat to 130° overnight with 5 ml/min of nitrogen flow rate Carrier gas: Nitrogen Flow rate: 17 ml/min Detector: FID Temperatures - injection port: 198±5° - column: 60° Calculation: where C_DFA = Concentration of dimethyl formamide C = Concentration of dimethyl formamide detected W = weight of sample taken
Dimethyl sulfoxide	Determine by gas liquid chromatography under the following conditions: Reagents: - Tetrahydrofuran - Dimethyl sulfoxide - Sucrose esters of fatty acids (dimethyl sulfoxide free, i.e. sucrose esters of fatty acids prepared without using dimethyl sulfoxide) Standard solutions (prepared fresh monthly): - Prepare a 0.25 mg/ml stock solution of dimethyl sulfoxide in tetrahydrofuran - Dissolve 40 g of dimethyl sulfoxide free sucrose esters of fatty acids into tetrahydrofuran and dilute to 200 ml (Sucrose esters of fatty acids solution) - Prepare a range of solutions containing 0.5, 1 and 5 m g/ml of dimethyl sulfoxide by dilution of the stock solution with the sucrose esters of fatty acids solution, respectively Procedure: Weigh accurately about 5 g of the sample, dissolve and dilute it with tetrahydrofuran to 25 ml to prepare a sample solution. Inject 3 m l of the sample solution into the gas chromatograph under the conditions below. Calibration curve (prepared daily): Prepare a calibration curve by injecting each 3 m l of the standard solutions into the gas chromatograph Conditions: Column: - length: 2 m - diameter: 3 mm (i.d.) - material: Glass - packing: 10% PEG 20M and 3% KOH on Gas Chrom Z - conditioning: Raise the oven temperature to 180° at a rate of 10°/min and let stand for 24 to 48 h with 30 to 40 ml/min of nitrogen Carrier gas: Nitrogen Flow rate: 50 ml/min Detector: Flame photometric (using 394 nm sulfur filter) Temperatures - injection port: 210° - column: 160° (do not exceed 200°) Calculation where C_DMSO = Concentration of dimethyl sulfoxide C = Concentration of dimethyl sulfoxide detected W = weight of sample taken
Propylene glycol	Determine by gas liquid chromatography using the following conditions: Reagents: - Propylene glycol - 1,2-Butylene glycol - Acetic anhydride - Toluene - Acetone Standard solutions: - Internal standard solution: Prepare 1000 mg/l solution of 1,2-butylene glycol in acetone. 200 m l of the solution contains 0.2 mg of 1,2-butylene glycol. - Standard solution of propylene glycol: Prepare 1000 mg/l solution of propylene glycol in acetone. Sample preparation: Weigh accurately about 2 g of the sample in a 100-ml flat-bottomed flask, and add 10 ml of acetic anhydride and 200 m l of the internal standard solution. Attach a reflux condenser and heat the flask in a boiling water bath for 1 hour. Add 50 ml of water and heat the flask for another 10 min in a boiling water bath. After heating, let it cool to room temperature. Transfer the content to a 100-ml separating funnel and extract with 10 ml of toluene. After separation, discharge the lower layer (aqueous phase). Add 50 ml of water to the separating funnel and wash the extract. The upper layer (toluene phase) is used as a sample solution. Procedure: Inject 2 m l of the sample solution into the gas chromatograph under the following conditions. (Note: Since there would be a component of high boiling point, which may have a longer retention time, it is necessary at the end of each measurement to raise the oven temperature to 200° and to evacuate it from the column). Calibration curve: Follow the same procedure using 100, 200 and 500 m l of the standard solution of propylene glycol in place of the sample, and prepare a calibration curve. Column: - length: 2 m - diameter: 3 mm (i.d.) - material: Glass - packing: 5% Alkyleneglycol phthalate on 80/100 Chromosorb W - conditioning: Heat to 150° overnight with approximately 50 ml/min of nitrogen Carrier gas: Nitrogen Flow rate: 40 ml/min Detector: Flame ionization Temperatures: - injection port: 230° - column: 110° Calculation: where C = Concentration of propylene glycol (mg/kg) A_PG = Peak area of propylene glycol A_IS = Peak area of internal standard W_SPL = Weight of sample (g) C_fPG = Sensitivity correction factor for propylene glycol (slope of the calibration curve)
Methanol, isopropanol, isobutanol, ethyl acetate and methyl ethyl ketone
	Determine by gas chromatography with a head space sampler using the following conditions: Reagents: - Methanol - Isopropanol - Isobutanol - Ethyl acetate - Methyl ethyl ketone Standard solutions: Take each 1 g of methanol, isopropanol, isobutanol, methyl ethyl ketone and ethyl acetate in a volumetric flask and add water to total volume of 100 ml, and prepare 0.02-0.4 g/100 ml solutions by dilution of this solution. If necessary, prepare standard solutions containing up to 7 g/100 ml of isopropanol and ethyl acetate. Procedure: Place 1 g (1.0 ± 0.1g) of powdered sample in a sample vial. Add 5 m l of water to the sample vial and seal it quickly with a septum. Set the sample vial in a pre-conditioned gas chromatograph and start the analysis under the below-mentioned conditions. Calibration curve: Take 1 g of powdered sucrose esters of fatty acids, solvent free or known residual solvent contents, in a sample vial, add 5 m l of the standard solution and seal it quickly with a septum. Set the sample vial in a pre-conditioned gas chromatograph and start the analysis under the following conditions and obtain calibration curves for each solvent. Column: - length: 30 m - diameter: 0.53 mm (i.d.) - material: Silica capillary - film: 100% methyl polysiloxane - conditioning: Heat to 60° for 2-3 h with approximately 10 ml/min of nitrogen Carrier gas: Nitrogen Flow rate: 5 ml/min Detector: Flame ionization Temperatures - injection port: 110° - column: 40° - detector: 110° Head space sampler: - Sample volume: 1.0 g ± 0.1 g + 5 m l - Sample heating temp.: 80° - Sample heating time: 40 min - Syringe temperature: 85° - Sample gas injection: 0.4 ml Calculation: C_i =A_i x Cf_i x 1000 where C_i = Concentration of solvent i (mg/kg) A_i = Peak area of solvent i (m v.sec.) Cf_i = Conversion coefficient for solvent i (slope of the calibration curve) (m g/m v.sec)
METHOD OF ASSAY	Determine by high pressure liquid chromatography using the following conditions: Sample preparation: Add about 250 mg of the sample, accurately weighed to a 50 ml volumetric flask. Dilute to volume with tetrahydrofuran, and mix. Filter through a 0.5-m m membrane filter. Procedure: Inject 100 m l of the sample into the pre-stabilized high pressure liquid chromatograph. Conditions: Column: Styrene-divinylbenzene copolymer for gel permeation chromatography (TSK-GEL G2000 (Supelco) or equivalent) Mobile phase: HPLC-grade degassed tetrahydrofuran Flow rate: 0.7 ml/min Detector: Refractive index detector Temperatures: Column: 38° Detector: 38° Record the chromatogram for about 90 min. Calculate the percentage of sucrose ester content in the sample taken by the formula: 100 A/T where A = the sum of peak areas for the three main components, the mono-, di-and triesters, eluting at about 65, 68 and 73 min, respectively T = the sum of all peak areas eluting within 90 min