1 Scope
This standard specifies the determination method of carotene in foods.
Chromatographic condition I of this standard is applicable to the determination of α-carotene, β-carotene and total carotene in foods and chromatographic condition II is applicable to the determination of β-carotene in foods.
2 Principle
After subjecting to saponification, carotene in specimen is released as free form, extracted with petroleum ether, brought to volume with dichloromethane, separated by reversed-phase chromatography and quantitated by external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 α-amylase: enzyme activity ≥1.5U/mg.
3.1.2 Papain: enzyme activity ≥5U/mg.
3.1.3 Potassium hydroxide (KOH).
3.1.4 Anhydrous sodium sulfate (Na2SO4).
3.1.5 Ascorbic acid (C6H8O6).
3.1.6 Petroleum ether: boiling range of 30℃~60℃.
3.1.7 Methanol (CH4O): chromatographically pure.
3.1.8 Acetonitrile (C2H3N): chromatographically pure.
3.1.9 Trichloromethane (CHCl3): chromatographically pure.
3.1.10 Methyl tert-butyl ether [CH3OC(CH3)3]: chromatographically pure.
3.1.11 Dichloromethane (CH2Cl2): chromatographically pure.
3.1.12 Absolute ethanol (C2H6O): guaranteed reagent.
3.1.13 n-hexane (C6H14): chromatographically pure.
3.1.14 2,6-Di-tert-butyl-4-methylphenol (C15H24O, referred to as BHT).
3.2 Reagent preparation
Potassium hydroxide solution: weigh 500g of solid potassium hydroxide and dissolve it in 500mL water. Prepare this solution immediately before use.
3.3 Standards
3.3.1 α-carotene (C40H56, CAS No.: 7488-99-5): purity≥95% or the standard material approved and awarded with reference material certificate by the nation.
3.3.2 β-carotene (C40H56, CAS No.:7235-40-7): purity≥95% or the standard material approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solutions
3.4.1 α-carotene standard stock solution (500μg/mL): accurately weigh 50mg (accurate to 0.1mg) of α-carotene standard, add 0.25g of BHT, dissolve them with dichloromethane, then transfer the solution to a 100mL brown volumetric flask and bring the volume to the scale. Preserve the solution in dark place at below -20℃ for a validity period of three months at most. Prior to use, calibrate the standard stock solution by the specific operation detailed in Appendix A.
3.4.2 Standard intermediate solution of α-carotene (100μg/mL): accurately transfer 10.0mL of α-carotene standard stock solution into a 50mL brown volumetric flask and bring the volume to the scale with dichloromethane.
3.4.3 β-carotene standard stock solution (500μg/mL): accurately weigh 50mg (accurate to 0.1mg) of β-carotene standard, add 0.25g of BHT, dissolve them with dichloromethane, then transfer the solution to a 100mL brown volumetric flask and bring the volume to the scale. Preserve the solution in dark place at below -20℃ for a validity period of three months at most. Prior to use, calibrate the standard stock solution by the specific operation detailed in Appendix A.
Note: β-carotene standards are mainly all-trans (all-E) β-carotenes. During the process of storage, affected by factors as temperature and oxidation, some all-trans β-carotenes will be isomerized as cis-form-β-carotene, e.g. 9-cis-form (9Z)-β-carotene, 13-cis-form (13Z)-β-carotene and 15-cis-form (15Z)-β-carotene. If chromatographic condition I is adopted to determine β-carotene, the retention time of β-carotene isomer shall be confirmed according to Appendix B and the chromatographic purity of all-trans β-carotene standard solution is calculated.
3.4.4 Standard intermediate solution of β-carotene (100μg/mL): accurately transfer 10.0mL of β-carotene standard stock solution into a 50mL brown volumetric flask and bring the volume to the scale with dichloromethane.
3.4.5 Mixed standard working solutions of α-carotene and β-carotene (for chromatographic condition I): accurately transfer 0.50mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL and 10.00mL of the standard intermediate solution of α-carotene into six 100mL brown volumetric flasks and add 3.00mL of β-carotene intermediate solution into each flask, bring the volumes to the scale with dichloromethane to obtain the series mixed standard working solutions with α-carotene concentration of 0.5μg/mL, 1.0μg/mL, 2.0μg/mL, 3.0μg/mL, 4.0μg/mL and 10.00μg/mL respectively and all β-carotene concentration of 3.0μg/mL.
3.4.6 β-carotene standard working solutions (for chromatographic condition II): accurately transfer 0.50mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL and 10.00mL of the standard intermediate solution of β-carotene into six 100mL brown volumetric flasks. Bring the volumes to the scale with dichloromethane to obtain the series standard working solutions with concentration of 0.5μg/mL, 1.0μg/mL, 2.0μg/mL, 3.0μg/mL, 4.0μg/mL and 10.00μg/mL respectively.
4 Apparatuses
4.1 Refiner.
4.2 High speed disintegrator.
4.3 Thermostatic shaking water bath: with temperature control accuracy of ±1℃.
4.4 Rotatory evaporator.
4.5 Nitrogen blower.
4.6 Ultraviolet-visible spectrophotometer.
4.7 High-performance liquid chromatograph (HPLC): equipped with ultraviolet detector.
5 Analysis Procedures
Note: The whole experimental operation process shall be kept in a dark place.
5.1 Sample preparation
Cereal, bean and nut specimens, etc. shall be pulverized, grinded and sieved (the hole diameter of sieve plate is 0.3mm~0.5mm); vegetable, fruit, egg and alga specimens, etc. shall be mixed well with homogenizer; solid powder specimen and liquid specimen shall be shaken or stirred well prior to use. Specimens can be preserved for one week in 4℃ refrigerator.
5.2 Specimen treatment
5.2.1 Common food specimen
5.2.1.1 Pretreatment
Accurately weigh 1g~5g (accurate to 0.001g) of well-mixed specimen of common foods such as vegetables, fruits, homonemeae, cereals, beans and eggs, and 0.2g~2g (accurate to 0.001g) of oil specimen, transfer them into a 250mL conical flask, then add 1g of ascorbic acid and 75mL of absolute ethanol to oscillate for 30min in 60℃±1℃ water bath.
If the protein and starch contents in the specimen are high (>10%), add 1g of ascorbic acid, 15mL of 45℃~50℃ warm water, 0.5g of papain and 0.5g of α-amylase, cover the flask stopper and mix well, then put the flask into 55℃±1℃ thermostatic water bath for oscillation or ultrasonic treatment for 30min, add 75mL of absolute ethanol to oscillate for 30min in 60℃±1℃ water bath.
5.2.1.2 Saponification
Add into 25mL of potassium hydroxide solution and cover the flask stopper. Put the flask into preheated 53℃±2℃ thermostatic shaking water bath for saponification for 30min. Then take it out, keep it still and cool to room temperature.
5.2.2 Food specimen with β-carotene
5.2.2.1 Pretreatment
Solid specimen: accurately weigh 1g~5g (accurate to 0.001g) of specimen, put it into a 250mL conical flask, add 1g of ascorbic acid and 50mL of 45℃~50℃ warm water and mix well. Add 0.5g of papain and 0.5g of α-amylase (for starch-free specimen, α-amylase needs not to be added), cover the flask stopper, put the flask into 55℃±1℃ thermostatic water bath to subject to oscillation or ultrasonic treatment for 30min.
Liquid specimen: accurately weigh 5g~10g (accurate to 0.001g) of specimen, put it into a 250mL conical flask, and add 1g of ascorbic acid.
5.2.2.2 Saponification
Take pretreated specimen, add 75mL of absolute ethanol, shake well and add 25mL of potassium hydroxide solution, cover the flask stopper. Put the flask into preheated 53℃±2℃ thermostatic shaking water bath for saponification for 30min. Then take it out, keep it still and cool to room temperature.
Note: If the saponification is incomplete, the saponification time can be duly postponed to 1h.
5.3 Specimen extraction
Transfer the saponification solution into a 500mL separating funnel, add 100mL of petroleum ether and shake slightly, exhaust gas and cover the flask stopper to oscillate for 10min under room temperature, then keep it still for layering; after that, transfer water phase into another separating funnel and carry out extraction for the second time according to the above method. Combine organic phase and wash with water to near-neutral. Discard water phase, filter organic phase with anhydrous sodium sulfate for dehydration. Collect the filtrate into a 500mL evaporation flask, subject it to reduced pressure concentration at 40℃±2℃ on rotary evaporator to nearly dry. Blow dry with nitrogen, accurately add 5.0mL of dichloromethane with pipette, cover the flask stopper and dissolve the extract sufficiently. Filter with 0.45μm membrane, then collect the filtrate after discarding about 1mL of the initial filtrate into sample-injecting bottle for future use.
Note: If necessary, concentration or dilution may be carried out according to the content of carotene in to-be-determined sample solution to make the concentration of α-carotene and/or β-carotene in the to-be-determined sample solution within the range of 0.5μg/mL~10μg/mL.
5.4 Chromatographic determination
5.4.1 Chromatographic condition I (applicable to the determination of α-carotene, β-carotene and total carotene in foods)
5.4.1.1 Reference chromatographic conditions
Reference chromatographic conditions are as follows:
a) Chromatographic column: C30 column (150mm in column length, 4.6mm in inner diameter and 5μm in particle size) or equivalent;
b) Mobile phase: Phase A, methanol : acetonitrile : water = 73.5:24.5:2;
Phase B: methyl tert-butyl ether;
Table 1 Gradient Procedures
Time /min 0 15 18 19 20 22
A% 100 59 20 20 0 100
B% 0 41 80 80 100 0
c) Flow rate: 1.0mL/min;
d) Detection wavelength: 450nm;
e) Column temperature: 30℃±1℃;
f) Injection volume: 20μL.
5.4.1.2 Plotting of α-carotene standard curve and calculation of the response factor of all-trans β-carotene
Inject the mixed standard working solution of α-carotene and β-carotene into HPLC (see Figure C.1 for chromatogram), subject it to qualification by the retention time and determine the peak area of isomers of α-carotene and β-carotene.
For α-carotene, plot the standard curve according to the concentration and peak area of series standard working solutions by taking the concentration as horizontal coordinate and the peak area as longitudinal coordinate and calculate the regression equation.
For β-carotene, calculate the response factor of all-trans β-carotene according to the calibration concentration of standard working solution, the average value six determined peak areas of all-trans β-carotene and the chromatographic purity of all-trans β-carotene (CP, see Appendix B for calculation method) and according to Formula (1).
(1)
Where,
RF - the response factor of all-trans β-carotene, AU·mL/μg;
- the average value of chromatographic peak areas of all-trans β-carotene standard working solution, AU;
ρ - the calibration concentration of β-carotene standard working solution, μg/mL;
CP - the chromatographic purity of all-trans β-carotene, %.
5.4.1.3 Specimen determination
Inject the to-be-determined solution into liquid chromatograph under same chromatographic condition, subject it to qualification by retention time and quantitation by external standard method according to peak area. For α-carotene, calculate the concentration of α-carotene in to-be-determined solution according to the regression equation of standard curve; for β-carotene, make calculation according to the response factor of all-trans β-carotene.
5.4.2 Chromatographic condition II (applicable to the determination of β-carotene in foods)
5.4.2.1 Reference chromatographic conditions
Reference chromatographic conditions are as follows:
a) Chromatographic column: C18 column (250mm in column length, 4.6mm in inner diameter and 5μm in particle size) or equivalent;
b) Mobile phase, trichloromethane: acetonitrile: methanol = 3:12:85, containing 0.4g/L of ascorbic acid and subjecting to filter via 0.45μm membrane for future use;
c) Flow rate: 2.0mL/min;
d) Detection wavelength: 450nm;
e) Column temperature: 35℃±1℃;
f) Injection volume: 20μl.
Contents
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatuses
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Others
Appendix A Calibration Method for Concentration of Standard Solution
Appendix B Confirmation of Retention Time of β-carotene Isomer and Calculation of Chromatographic Purity of All-trans β-carotene
Appendix C Liquid Chromatogram of Carotene
Appendix D Percentage Absorption Coefficient of Carotenes
1 Scope
This standard specifies the determination method of carotene in foods.
Chromatographic condition I of this standard is applicable to the determination of α-carotene, β-carotene and total carotene in foods and chromatographic condition II is applicable to the determination of β-carotene in foods.
2 Principle
After subjecting to saponification, carotene in specimen is released as free form, extracted with petroleum ether, brought to volume with dichloromethane, separated by reversed-phase chromatography and quantitated by external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 α-amylase: enzyme activity ≥1.5U/mg.
3.1.2 Papain: enzyme activity ≥5U/mg.
3.1.3 Potassium hydroxide (KOH).
3.1.4 Anhydrous sodium sulfate (Na2SO4).
3.1.5 Ascorbic acid (C6H8O6).
3.1.6 Petroleum ether: boiling range of 30℃~60℃.
3.1.7 Methanol (CH4O): chromatographically pure.
3.1.8 Acetonitrile (C2H3N): chromatographically pure.
3.1.9 Trichloromethane (CHCl3): chromatographically pure.
3.1.10 Methyl tert-butyl ether [CH3OC(CH3)3]: chromatographically pure.
3.1.11 Dichloromethane (CH2Cl2): chromatographically pure.
3.1.12 Absolute ethanol (C2H6O): guaranteed reagent.
3.1.13 n-hexane (C6H14): chromatographically pure.
3.1.14 2,6-Di-tert-butyl-4-methylphenol (C15H24O, referred to as BHT).
3.2 Reagent preparation
Potassium hydroxide solution: weigh 500g of solid potassium hydroxide and dissolve it in 500mL water. Prepare this solution immediately before use.
3.3 Standards
3.3.1 α-carotene (C40H56, CAS No.: 7488-99-5): purity≥95% or the standard material approved and awarded with reference material certificate by the nation.
3.3.2 β-carotene (C40H56, CAS No.:7235-40-7): purity≥95% or the standard material approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solutions
3.4.1 α-carotene standard stock solution (500μg/mL): accurately weigh 50mg (accurate to 0.1mg) of α-carotene standard, add 0.25g of BHT, dissolve them with dichloromethane, then transfer the solution to a 100mL brown volumetric flask and bring the volume to the scale. Preserve the solution in dark place at below -20℃ for a validity period of three months at most. Prior to use, calibrate the standard stock solution by the specific operation detailed in Appendix A.
3.4.2 Standard intermediate solution of α-carotene (100μg/mL): accurately transfer 10.0mL of α-carotene standard stock solution into a 50mL brown volumetric flask and bring the volume to the scale with dichloromethane.
3.4.3 β-carotene standard stock solution (500μg/mL): accurately weigh 50mg (accurate to 0.1mg) of β-carotene standard, add 0.25g of BHT, dissolve them with dichloromethane, then transfer the solution to a 100mL brown volumetric flask and bring the volume to the scale. Preserve the solution in dark place at below -20℃ for a validity period of three months at most. Prior to use, calibrate the standard stock solution by the specific operation detailed in Appendix A.
Note: β-carotene standards are mainly all-trans (all-E) β-carotenes. During the process of storage, affected by factors as temperature and oxidation, some all-trans β-carotenes will be isomerized as cis-form-β-carotene, e.g. 9-cis-form (9Z)-β-carotene, 13-cis-form (13Z)-β-carotene and 15-cis-form (15Z)-β-carotene. If chromatographic condition I is adopted to determine β-carotene, the retention time of β-carotene isomer shall be confirmed according to Appendix B and the chromatographic purity of all-trans β-carotene standard solution is calculated.
3.4.4 Standard intermediate solution of β-carotene (100μg/mL): accurately transfer 10.0mL of β-carotene standard stock solution into a 50mL brown volumetric flask and bring the volume to the scale with dichloromethane.
3.4.5 Mixed standard working solutions of α-carotene and β-carotene (for chromatographic condition I): accurately transfer 0.50mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL and 10.00mL of the standard intermediate solution of α-carotene into six 100mL brown volumetric flasks and add 3.00mL of β-carotene intermediate solution into each flask, bring the volumes to the scale with dichloromethane to obtain the series mixed standard working solutions with α-carotene concentration of 0.5μg/mL, 1.0μg/mL, 2.0μg/mL, 3.0μg/mL, 4.0μg/mL and 10.00μg/mL respectively and all β-carotene concentration of 3.0μg/mL.
3.4.6 β-carotene standard working solutions (for chromatographic condition II): accurately transfer 0.50mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL and 10.00mL of the standard intermediate solution of β-carotene into six 100mL brown volumetric flasks. Bring the volumes to the scale with dichloromethane to obtain the series standard working solutions with concentration of 0.5μg/mL, 1.0μg/mL, 2.0μg/mL, 3.0μg/mL, 4.0μg/mL and 10.00μg/mL respectively.
4 Apparatuses
4.1 Refiner.
4.2 High speed disintegrator.
4.3 Thermostatic shaking water bath: with temperature control accuracy of ±1℃.
4.4 Rotatory evaporator.
4.5 Nitrogen blower.
4.6 Ultraviolet-visible spectrophotometer.
4.7 High-performance liquid chromatograph (HPLC): equipped with ultraviolet detector.
5 Analysis Procedures
Note: The whole experimental operation process shall be kept in a dark place.
5.1 Sample preparation
Cereal, bean and nut specimens, etc. shall be pulverized, grinded and sieved (the hole diameter of sieve plate is 0.3mm~0.5mm); vegetable, fruit, egg and alga specimens, etc. shall be mixed well with homogenizer; solid powder specimen and liquid specimen shall be shaken or stirred well prior to use. Specimens can be preserved for one week in 4℃ refrigerator.
5.2 Specimen treatment
5.2.1 Common food specimen
5.2.1.1 Pretreatment
Accurately weigh 1g~5g (accurate to 0.001g) of well-mixed specimen of common foods such as vegetables, fruits, homonemeae, cereals, beans and eggs, and 0.2g~2g (accurate to 0.001g) of oil specimen, transfer them into a 250mL conical flask, then add 1g of ascorbic acid and 75mL of absolute ethanol to oscillate for 30min in 60℃±1℃ water bath.
If the protein and starch contents in the specimen are high (>10%), add 1g of ascorbic acid, 15mL of 45℃~50℃ warm water, 0.5g of papain and 0.5g of α-amylase, cover the flask stopper and mix well, then put the flask into 55℃±1℃ thermostatic water bath for oscillation or ultrasonic treatment for 30min, add 75mL of absolute ethanol to oscillate for 30min in 60℃±1℃ water bath.
5.2.1.2 Saponification
Add into 25mL of potassium hydroxide solution and cover the flask stopper. Put the flask into preheated 53℃±2℃ thermostatic shaking water bath for saponification for 30min. Then take it out, keep it still and cool to room temperature.
5.2.2 Food specimen with β-carotene
5.2.2.1 Pretreatment
Solid specimen: accurately weigh 1g~5g (accurate to 0.001g) of specimen, put it into a 250mL conical flask, add 1g of ascorbic acid and 50mL of 45℃~50℃ warm water and mix well. Add 0.5g of papain and 0.5g of α-amylase (for starch-free specimen, α-amylase needs not to be added), cover the flask stopper, put the flask into 55℃±1℃ thermostatic water bath to subject to oscillation or ultrasonic treatment for 30min.
Liquid specimen: accurately weigh 5g~10g (accurate to 0.001g) of specimen, put it into a 250mL conical flask, and add 1g of ascorbic acid.
5.2.2.2 Saponification
Take pretreated specimen, add 75mL of absolute ethanol, shake well and add 25mL of potassium hydroxide solution, cover the flask stopper. Put the flask into preheated 53℃±2℃ thermostatic shaking water bath for saponification for 30min. Then take it out, keep it still and cool to room temperature.
Note: If the saponification is incomplete, the saponification time can be duly postponed to 1h.
5.3 Specimen extraction
Transfer the saponification solution into a 500mL separating funnel, add 100mL of petroleum ether and shake slightly, exhaust gas and cover the flask stopper to oscillate for 10min under room temperature, then keep it still for layering; after that, transfer water phase into another separating funnel and carry out extraction for the second time according to the above method. Combine organic phase and wash with water to near-neutral. Discard water phase, filter organic phase with anhydrous sodium sulfate for dehydration. Collect the filtrate into a 500mL evaporation flask, subject it to reduced pressure concentration at 40℃±2℃ on rotary evaporator to nearly dry. Blow dry with nitrogen, accurately add 5.0mL of dichloromethane with pipette, cover the flask stopper and dissolve the extract sufficiently. Filter with 0.45μm membrane, then collect the filtrate after discarding about 1mL of the initial filtrate into sample-injecting bottle for future use.
Note: If necessary, concentration or dilution may be carried out according to the content of carotene in to-be-determined sample solution to make the concentration of α-carotene and/or β-carotene in the to-be-determined sample solution within the range of 0.5μg/mL~10μg/mL.
5.4 Chromatographic determination
5.4.1 Chromatographic condition I (applicable to the determination of α-carotene, β-carotene and total carotene in foods)
5.4.1.1 Reference chromatographic conditions
Reference chromatographic conditions are as follows:
a) Chromatographic column: C30 column (150mm in column length, 4.6mm in inner diameter and 5μm in particle size) or equivalent;
b) Mobile phase: Phase A, methanol : acetonitrile : water = 73.5:24.5:2;
Phase B: methyl tert-butyl ether;
Table 1 Gradient Procedures
Time /min 0 15 18 19 20 22
A% 100 59 20 20 0 100
B% 0 41 80 80 100 0
c) Flow rate: 1.0mL/min;
d) Detection wavelength: 450nm;
e) Column temperature: 30℃±1℃;
f) Injection volume: 20μL.
5.4.1.2 Plotting of α-carotene standard curve and calculation of the response factor of all-trans β-carotene
Inject the mixed standard working solution of α-carotene and β-carotene into HPLC (see Figure C.1 for chromatogram), subject it to qualification by the retention time and determine the peak area of isomers of α-carotene and β-carotene.
For α-carotene, plot the standard curve according to the concentration and peak area of series standard working solutions by taking the concentration as horizontal coordinate and the peak area as longitudinal coordinate and calculate the regression equation.
For β-carotene, calculate the response factor of all-trans β-carotene according to the calibration concentration of standard working solution, the average value six determined peak areas of all-trans β-carotene and the chromatographic purity of all-trans β-carotene (CP, see Appendix B for calculation method) and according to Formula (1).
(1)
Where,
RF - the response factor of all-trans β-carotene, AU·mL/μg;
- the average value of chromatographic peak areas of all-trans β-carotene standard working solution, AU;
ρ - the calibration concentration of β-carotene standard working solution, μg/mL;
CP - the chromatographic purity of all-trans β-carotene, %.
5.4.1.3 Specimen determination
Inject the to-be-determined solution into liquid chromatograph under same chromatographic condition, subject it to qualification by retention time and quantitation by external standard method according to peak area. For α-carotene, calculate the concentration of α-carotene in to-be-determined solution according to the regression equation of standard curve; for β-carotene, make calculation according to the response factor of all-trans β-carotene.
5.4.2 Chromatographic condition II (applicable to the determination of β-carotene in foods)
5.4.2.1 Reference chromatographic conditions
Reference chromatographic conditions are as follows:
a) Chromatographic column: C18 column (250mm in column length, 4.6mm in inner diameter and 5μm in particle size) or equivalent;
b) Mobile phase, trichloromethane: acetonitrile: methanol = 3:12:85, containing 0.4g/L of ascorbic acid and subjecting to filter via 0.45μm membrane for future use;
c) Flow rate: 2.0mL/min;
d) Detection wavelength: 450nm;
e) Column temperature: 35℃±1℃;
f) Injection volume: 20μl.
Contents of GB 5009.83-2016
Contents
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatuses
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Others
Appendix A Calibration Method for Concentration of Standard Solution
Appendix B Confirmation of Retention Time of β-carotene Isomer and Calculation of Chromatographic Purity of All-trans β-carotene
Appendix C Liquid Chromatogram of Carotene
Appendix D Percentage Absorption Coefficient of Carotenes
