Thông tư 27/2010/TT-BYT Quy chuẩn kỹ thuật quốc gia phụ gia thực phẩm

• Số hiệu: 27/2010/TT-BYT
• Loại văn bản: Thông tư
• Nơi ban hành: Bộ Y tế
• Người ký: Trịnh Quân Huấn
• Ngày ban hành: 20/05/2010

BỘ Y TẾ -------	CỘNG HÒA XÃ HỘI CHỦ NGHĨA VIỆT NAM Độc lập – Tự do – Hạnh phúc ---------
Số: 27/2010/TT-BYT	Hà Nội, ngày 20 tháng 5 năm 2010

THÔNG TƯ

BAN HÀNH QUY CHUẨN KỸ THUẬT QUỐC GIA VỀ PHỤ GIA THỰC PHẨM - PHẨM MÀU

BỘ TRƯỞNG BỘ Y TẾ

Căn cứ Luật Tiêu chuẩn và Quy chuẩn kỹ thuật ngày 29 tháng 6 năm 2006 và Nghị định số 127/2007/NĐ-CP ngày 01 tháng 8 năm 2007 của Chính phủ quy định chi tiết thi hành một số điều của Luật Tiêu chuẩn và Quy chuẩn kỹ thuật;
Căn cứ Pháp lệnh Vệ sinh an toàn thực phẩm ngày 07 tháng 8 năm 2003 và Nghị định số 163/2004/NĐ-CP ngày 07 tháng 9 năm 2004 của Chính phủ quy định chi tiết thi hành một số điều của Pháp lệnh Vệ sinh an toàn thực phẩm;
Căn cứ Nghị định số 188/2007/NĐ-CP ngày 27 tháng 12 năm 2007 của Chính phủ quy định chức năng, nhiệm vụ, quyền hạn và cơ cấu tổ chức của Bộ Y tế;
Theo đề nghị của Cục trưởng Cục An toàn vệ sinh thực phẩm, Vụ trưởng Vụ Khoa học và Đào tạo, Vụ trưởng Vụ Pháp chế,

QUY ĐỊNH:

Điều 1. Ban hành kèm theo Thông tư này:

QCVN 4-10:2010/BYT - Quy chuẩn kỹ thuật quốc gia về phụ gia thực phẩm - Phẩm màu.

Điều 2. Thông tư này có hiệu lực từ ngày 01 tháng 01 năm 2011.

Điều 3. Cục trưởng Cục An toàn vệ sinh thực phẩm, Thủ trưởng các đơn vị thuộc Bộ Y tế, các đơn vị trực thuộc Bộ Y tế; Giám đốc Sở Y tế các tỉnh, thành phố trực thuộc trung ương và các tổ chức, cá nhân có liên quan chịu trách nhiệm thi hành Thông tư này. /.

Nơi nhận: - VPCP (Văn xã, Công báo, Cổng TTĐT Chính phủ); - Các Bộ, cơ quan ngang Bộ, cơ quan thuộc CP; - Bộ Tư pháp (Cục Kiểm tra VBQPPL); - Bộ trưởng Nguyễn Quốc Triệu (để báo cáo); - UBND các tỉnh, thành phố trực thuộc TW; - Viện Kiểm sát nhân dân tối cao; - Toà án nhân dân tối cao; - Sở Y tế các tỉnh, thành phố trực thuộc TW; - Chi cục ATTP các tỉnh, thành phố trực thuộc TW; - TTYTDP các tỉnh, thành phố trực thuộc TW; - Các cơ quan KTNN về thực phẩm nhập khẩu; - Tổng Cục Tiêu chuẩn - Đo lường - Chất lượng; - Website Bộ Y tế; - Lưu: VT, K2ĐT, PC, ATTP.

KT. BỘ TRƯỞNG THỨ TRƯỞNG THỨ TRƯỞNG Trịnh Quân Huấn

FILE ĐƯỢC ĐÍNH KÈM THEO VĂN BẢN

Tiếng Anh (English)

THE MINISTRY OF HEALTH -------	THE SOCIALIST REPUBLIC OF VIETNAM Independence - Freedom - Happiness ---------
No. 27/2010/TT-BYT	Hanoi, May 20, 2010

= Khối lượng cặn x 107,4 CIRCULAR PROMULGATING NATIONAL TECHNICAL REGULATION ON FOOD ADDITIVES - COLOURS THE MINISTER OF HEALTH Pursuant to the Law on Standards and Technical Regulations dated June 29, 2006 and Government’s Decree No. 127/2007/ND-CP dated August 01, 2007 elaborating some Articles of the Law Standards and Technical Regulations; Pursuant to the Ordinance on Food Safety dated August 07, 2003 and the Government’s Decree No. 163/2004/ND-CP dated September 07, 2004 elaborating some Articles of the Ordinance on Food Safety; Pursuant to the Government’s Decree No. 188/2007/ND-CP dated December 27, 2007 defining functions, tasks, powers and organizational structure of the Ministry of Health; At the request of the Director General of the Vietnam Food Administration, the Director General of the Science and Education Department, the Director General of the Department of Legislation, HEREBY DECIDES: Article 1. Promulgated together with this Circular is: QCVN 4-10:2010/BYT - National technical regulation on food additives - Colours. ... ... ... Article 3. Director General of Vietnam Food Administration, heads of units affiliated to the Ministry of Health, units affiliated to the Ministry of Health; Directors of Departments of Health of provinces and central-affiliated cities, organizations and individuals concerned are responsible for the implementation of this Circular./. PP. TH MINISTER THE DEPUTY MINISTER Trinh Quan Huan NATIONAL TECHNICAL REGULATION QCVN 4-10:2010/BYT ON FOOD ADDITIVES - COLOURS ... ... ... Foreword QCVN 4-10:2010/BYT is developed by the Drafting Board for National technical regulation on food additives and food processing aids, submitted by the Vietnam Food Administration for approval and promulgated together with the Circular No. 27/2010/TT-BYT dated May 20, 2010 of the Minister of Health. NATIONAL TECHNICAL REGULATION ON FOOD ADDITIVES - COLOURS I. GENERAL 1. Scope This National technical regulation (hereinafter referred to as “the Regulation”) provides for specifications and regulatory requirements for quality, hygiene and safety of colours used as food additives. 2. Regulated entities This Regulation applies to: ... ... ... 2.2. Relevant regulatory bodies. 3. Interpretation of terms and acronyms: 3.1. “colour” means a food additive used for the purpose of adding or restoring color in a food product. 3.2. JECFA monograph 1 - Vol. 4 (JECFA monographs 1 - Combined compendium of food additive specifications; Joint FAO/WHO expert committee on food additives; Volume 4 - Analytical methods, test procedures and laboratory solutions used by and referenced in the food additive specifications; FAO, 2006) is developed by JECFA and was published by FAO in 2006. 3.3. “C.A.S number” (Chemical Abstracts Service) refers to registry numbers of chemical substances identified by the American Chemical Society. 3.4. “TS” stands for test solution. 3.5. “ADI” stands for Acceptable daily intake. 3.6. “INS” stands for international numbering system. II. SPECIFICATIONS, TESTS AND SAMPLING ... ... ... 1.1. Annex 1: Specifications and tests for curcumin 1.2. Annex 2: Specifications and tests for riboflavin 1.3. Annex 3: Specifications and tests for tatrazine 1.4. Annex 4: Specifications and tests for quinoline 1.5. Annex 5: Specifications and tests for sunset yellow FCF 1.6. Annex 6: Specifications and tests for carmines 1.7. Annex 7: Specifications and tests for carmoisine 1.8. Annex 8: Specifications and tests for amaranth 1.9. Annex 9: Specifications and tests for ponceau 4R ... ... ... 1.11. Annex 11: Specifications and tests for red 2G 1.12. Annex 12: Specifications and tests for allura red AC 1.13. Annex 13: Specifications and tests for indigotine 1.14. Annex 14: Specifications and tests for brilliant blue FCF 1.15. Annex 11: Specifications and tests for chlorophylls 1.16. Annex 16: Specifications and tests for chlorophyllin copper complexes 1.17. Annex 17: Specifications and tests for chlorophyllins, copper complexes sodium and potassium salts 1.18. Annex 18: Specifications and tests for green S 1.19. Annex 19: Specifications and tests for caramel ... ... ... 1.21. Annex 21: Specifications and tests for brown HT 1.22. Annex 22: Specifications and tests for synthetic β-carotene 1.23. Annex 23: Specifications and tests for carotenes, vegetable 1.24. Annex 24: Specifications and tests for annatto extracts 1.25. Annex 25: Specifications and tests for β-Apo-carotenal 1.26. Annex 26: Specifications and tests for β-Apo-8'-Carotenoic acid ethyl ester 1.27. Annex 27: Specifications and tests for grape skin extract 1.28. Annex 28: Specifications and tests for titanium dioxide 1.29. Annex 29: Specifications and tests for iron oxides ... ... ... 2. Specifications specified in this Regulation are tested under JECFA monograph 1 - Vol. 4, except for some specific tests described in the annexes. The tests provided in this Regulation are optional. Other equivalent tests may be used. 3. Sampling adheres to the guidelines in the Circular No. 16/2009/TT-BKHCN dated June 02, 2009 by the Ministry of Science and Technology and relevant regulations of law. III. REGULATORY REQUIREMENTS 1. Declaration of conformity 1.1. Conformity of colours shall be declared in accordance with the regulations set out in this Regulation. 1.2. Methods and procedures for declaration of conformity shall comply with the Regulation on certification and declaration of conformity with regulations and standards under the Decision No. 24/2007/QĐ-BKHCN dated September 28, 2007 of the Minister of Science and Technology and regulations of laws. 2. Inspection of colours The quality, hygiene and safety of colours must be inspected in accordance with the regulations of law. IV. RESPONSIBILITIES OF ENTITIES ... ... ... 2. Entities are entitled to import, export, produce, sell and use colours only after their completion of registration of declarations of conformity and their compliance with regulations of law on quality, hygiene, safety and labeling. V. IMPLEMENTATION 1. The Vietnam Food Administration shall preside over and cooperate with competent authorities concerned to provide guidance on and organize the implementation of this Regulation. 2. The Vietnam Food Administration shall, according to its managerial duties, suggest amendments to this Regulation to the Ministry of Health. 3. In the cases where any of the international guidelines for tests and regulations of law referred to in this Regulation is amended or replaced, the newest one shall apply. ANNEX 1 SPECIFICATIONS AND TESTS FOR CURCUMIN 1. Synonyms ... ... ... INS: 100i ADI = 0 – 3mg/kg bw. 2. Definition Curcumin is obtained by solvent extraction of turmeric i.e., the ground rhizomes of Curcuma longa L. (Curcuma domestica Valeton). In order to obtain a concentrated curcumin powder, the extract is purified by crystallization. The product consists essentially of curcumins; i.e. the colouring principle 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene3,5-dione (synonyms: Curcumin, Diferuloylmethane, CI Natural Yellow 3, CI (1975) 75300) and its desmethoxy- and bis-desmethoxy-derivatives in varying proportions. Minor amounts of oils and resins naturally occurring in turmeric may be present. Only the following solvents may be used in the extraction and purification: acetone, methanol, ethanol, isopropanol, hexane and ethyl acetate. Supercritical carbon dioxide may also be used in the extraction. Chemical names Principal colouring components: I. 1,7-Bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione II. ... ... ... III. 1,7-Bis-(4-hydroxyphenyl)-hepta-1,6-diene-3,5-dione C.A.S. number I. 458-37-7 II. 33171-16-3 III. ... ... ... Chemical formula I. C21H20O6 II. C20H18O5 III. C19H16O4 ... ... ... I. R1= R2=OCH3 II. R1= OCH3; R2=H III. R1= R2=H Formula weight ... ... ... 368.39 II. 338.39 III. 308.39 3. Description Orange-yellow crystalline powder. 4. Functional uses ... ... ... 5. Specifications 5.1. Identification Solubility Insoluble in water and in diethyl ether; soluble in ethanol and in glacial acetic acid. Colour reactions A solution of the sample in ethanol is characterized by a pure yellow colour and a light green fluorescence. Addition of this ethanol extract to concentrated sulfuric acid produces a deep crimson colour. Thin Layer Chromatography Passes test (described in “Tests”). ... ... ... Residual solvents Acetone: Not more than 30 mg/kg Hexane: Not more than 25 mg/kg Methanol:   Ethanol: ... ... ... Ethyl acetate: Lead Not more than 2 mg/kg 5.3. Content Not less than 90 % total colouring matters. 6. Tests 6.1. Identification ... ... ... Treat an aqueous or dilute ethanolic solution of the sample with hydrochloric acid until a slightly orange colour begins to appear. Divide the mixture into 2 parts and add some boric acid powder or crystals to one portion. A marked reddening will be quickly apparent, best seen by comparison with the portion to which the boric acid has not been added. The test may also be made by dipping pieces of filter paper in an ethanolic solution of colouring matter, drying at 100º, and then moistening with a weak solution of boric acid to which a few drops of hydrochloric acid have been added. On drying, a cherry-red colour will develop. Thin Layer Chromatography Spot 5 µl of test solution (0.01 g of sample in 1 ml of 95% ethanol) on a TLC (Microcrystalline cellulose, 0.1 mm) plate. Develop the plate in a chamber containing a mixture of 3-methyl-1-butanol/ethanol/ water/ammonia (4:4:2:1) as solvent and allow the solvent front to ascend 10-15 cm. Examine under daylight and under UV-light and observe: Two or three yellow spots with Rf between 0.2 and 0.4 under daylight and UV-light. Spots with Rf about 0.6 and 0.8 under UV-light All spots show distinct yellow fluorescence under UV-light. 6.2 Purity Lead ... ... ... 6.3. Method of assay Accurately weigh about 0.08 g of the sample in a 200-ml volumetric flask and dissolve by shaking with ethanol. Make up to volume with ethanol and mix. Pipette 1.0 ml of solution into a 100-ml volumetric flask and make up to volume with ethanol. Determine the absorbance (A) at 425 nm in a 1-cm cell. Calculate the total colouring matters content of the sample using the following equation: Where: A = absorbance of sample W = weight of sample ... ... ... The determination must be performed without delay, because the colour fades. ANNEX 2 SPECIFICATIONS AND TESTS FOR RIBOFLAVIN 1. Synonyms Vitamin B2, Lactoflavin INS: 101i  ADI = 0 – 0.5 mg/kg bw. 2. Definition ... ... ... Chemical names Riboflavin; 3,10-dihydro-7,8-dimethyl-10-[(2S,3S,4R)-2,3,4,5- tetrahydroxypentyl]benzo-[g]pteridine-2,4-dione; 7,8-dimethyl-10-(1'-Dribityl)isoalloxazine. C.A.S. number 83-88-5 Chemical formula C17H20N4O6 Structural formula Formula weight ... ... ... 3. Description Yellow to orange-yellow crystalline powder, with slight odour. 4. Functional uses Colour. 5. Specifications 5.1. Identification Solubility Very slightly soluble in water; practically insoluble in alcohol, chloroform, acetone and ether; very soluble in dilute alkali solutions. ... ... ... Using the aqueous solution from the Assay, determine the absorbance (A) at 267 nm, 375 nm and 444 nm. The ratio A375/A267 is between 0.31 and 0.33. The ratio A444/A267 is between 0.36 and 0.39. Specific rotation [a]20, D: Between -115^o and -140^o. Colour reaction Passes test (described in “Tests”). 5.2. Purity ... ... ... Not more than 1.5%. Sulfated ash Not more than 0.1%. Subsidiary colouring matters Passes test (described in “Tests”). Primary aromatic amines Not more than 100 mg/kg calculated as aniline. Lead  Not more than 2 mg/kg. ... ... ... Not less than 98%. 6. Tests 6.1. Identification Specific rotation Dry the sample at 100^o for 4 h. Dissolve 50.0 mg in 0.05 N sodium hydroxide free from carbonate and dilute to 10.0 ml with the same solvent. Measure the optical rotation within 30 min of dissolution. Colour reaction Dissolve about 1 mg of sample in 100 ml of water. The solution has a pale greenish-yellow colour by transmitted light, and by reflected light has an intense yellowish-green fluorescence which disappears on the addition of mineral acids and alkalis. 6.2. Purity ... ... ... Loss on drying At 105^o, 4 h Sulfated ash - Tested as directed under JECFA monograph 1 - Vol. 4. - Test 2 g of the sample Subsidiary colouring matters Prepare the standard for this test for the absence of lumiflavin by diluting 3 ml of 0.1 N potassium dichromate with water to 1000 ml. Pour some chloroform through an alumina column to remove any ethanol. To 10 ml of this chloroform add 35 mg of the sample, shake for 5 min and filter. The colour of the filtrate should be no more intense than that of 10 ml of the standard when viewed in identical containers. Lead Tested as directed under JECFA monograph 1 - Vol. 4. Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in Annex 1: General tests, “Instrumental Methods.” ... ... ... Carry out the assay in subdued light. In a brown glass 500 ml volumetric flask, suspend 65.0 mg of the sample in 5 ml of water, ensuring that it is completely wetted, and dissolve in 5 ml of 2 N sodium hydroxide solution. As soon as dissolution is complete, add 100 ml of water and 2.5 ml of glacial acetic acid and dilute to 500.0 ml with water. Place 20.0 ml of this solution in a brown glass 200 ml volumetric flask, add 3.5 ml of a 1.4% w/v solution of sodium acetate and dilute to 200.0 ml with water. Measure the absorbance (A) at the maximum at 444 nm. % Riboflavin = [(A×5000) / (328×W)] × 1,367 Where: A = absorbance of the sample solution at 444 nm W = weight of sample in g. ANNEX 3 ... ... ... 1. Synonyms CI Food Yellow 4, FD&C Yellow No. 5; CI (1975) No. 19140 INS: 102 ADI = 0 - 7.5 mg/kg bw. 2. Definition Consists of trisodium 4,5-dihydro-5-oxo-1-(4-sulfophenyl)-4-[(4- sulfophenyl)azo]-1H-pyrazole-3-carboxylate and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Trisodium4,5-dihydro-5-oxo-1-(4-sulfophenyl)-4-[(4-sulfophenyl)azo]- 1H-pyrazole-3-carboxylate ... ... ... 1934-21-0 Chemical formula C16H9N4Na3O9S2 Structural formula Formula weight 534.37 3. Description Light orange powder or granules. ... ... ... Colour. 5. Specifications 5.1. Identification Solubility Soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test. 5.2. Purity ... ... ... Loss on drying at 135 °C Not more than 15% together with chloride and sulfate calculated as sodium salts. Water insoluble matter Not more than 0.2%. Lead  Not more than 2 mg/kg. Subsidiary colouring matters  Not more than 1%. Organic compounds other than colouring matters ... ... ... Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. Ether extractable matter Not more than 0.2%. 5.3. Content Not less than 85% total colouring matters 6. Tests 6.1. Purity ... ... ... Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph, Volume 4, “Instrumental Methods.” Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 12 cm. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use HPLC under the following conditions: HPLC elution gradient: 2 to 100% at 2% per min. ... ... ... Proceed as directed under Total Content by Titration with Titanous Chloride (in Annex 1: General tests), using the following: Weight of sample: 0.6 - 0.7 g; Buffer: 15 g sodium hydrogen tartrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 13.56 mg. ANNEX 4 SPECIFICATIONS AND TESTS FOR QUINOLINE ... ... ... CI Food Yellow 13, CI (1975) No. 47005, INS: 104 ADI = 0 – 10 mg/kg bw. 2. Definition Prepared by sulfonating 2-(2-quinolyl)-1,3-indandione or a mixture containing about two-thirds 2-(2-quinolyl)-1,3-indandione and one-third 2- [2-(6-methyl-quinolyl)]1,3-indandione; consists essentially of sodium salts of a mixture of disulfonates (principally), monosulfonates and trisulfonates of the above compounds and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters apply. Chemical names Disodium 2-(1,3-dioxo-2-indanyl)-6,8-quinolinesulfates; disodium 2-(2- quinolyl)-indan-1,3-dionedisulfonates (principal component) C.A.S. number ... ... ... Chemical formula C18H9NNa2O8S2 (principal component) Structural formula Principal component 6 salt: R1 = SO3Na, R2 = H 8 salt: R1, R2 = SO3H Formula weight 477.38 (Principal components) ... ... ... Yellow powder or granules. 4. Functional uses Colour. 5. Specifications 5.1. Identification Solubility Soluble in water; sparingly soluble in ethanol Identification of colouring matters ... ... ... 5.2. Purity Loss on drying at 135°C Not more than 30% together with chloride and sulfate calculated as sodium salts. Water insoluble matter Not more than 0.2%. Lead Not more than 2 mg/kg. Zinc ... ... ... Subsidiary colouring matters Not more than 4 mg/kg: of 2-(2-quinolyl)-1,3-indandione and 2-[2-(6- methylquinolyl)]-1,3-indandione Organic compounds other than colouring matters Not more than 0.5%, sum of 2-methylquinoline, 2-methylquinolinesulfonic acid, phthalic acid, 2,6-dimethylquinoline, 2,6-dimethylquinolinesulfonic acid Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. Ether extractable matter Not more than 0.2%. 5.3. Content ... ... ... - not less than 80% shall be disodium 2-(2-quinolyl)-indan1,3-dionedisulfonates; - not more than 15% shall be sodium 2-(2-quinolyl)-indan-1,3- dionemonosulfonates; - not more than 7% shall be trisodium 2-(2-quinolyl)-indan-1,3- dionetrisulfonate; 6. Tests 6.1. Purity Lead Tested as directed under JECFA monograph 1-Vol. 4. Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph, Volume 4, “Instrumental Methods.” Subsidiary colouring matters ... ... ... Use the apparatus and ether quality described in Ether Extractable Matter and carry out an extraction following the details given there under. Wash the ether extract with two 25-ml portions of water. Evaporate the ether extract to about 5 ml and then transfer it to an oven at 105^o to remove the remaining ether. Dissolve the residue in chloroform, and dilute the solution to exactly 10 ml. Determine the absorbance at the wavelength of maximum absorption (approximately 420 nm) using chloroform as the reference solution. The absorbance corresponding to the limit figure of 4 mg/kg 2-(2-quinolyl-1,3-indandione is 0.27. Any 2-[2-(6-methylquinolyl)]- 1,3-indandione is assessed as 2-(2-quinolyl)-1,3-indandione. Organic compounds other than colouring matters Organic compounds other than colouring matters in Quinoline prepared from 2-(2-quinolyl)-1,3-indandione (only). Determine by liquid chromatography, using the following conditions: Instrument: High performance liquid chromatograph fitted with a gradient elution accessory. Detector: A UV HPLC detector recording absorbances at 254 nm. Column: 250 x 4 nm, Nucleosil C18, 7 µm. Solvent system: A: Acetate Buffer pH 4.6: water (1:10) (Acetate Buffer is 1M NaOH:1 M acetic acid:water (5:10:35). ... ... ... Sample concentration: 1% weight/volume in Solvent A. Gradient: Min %A %B 0 100 0 15 ... ... ... 35 20 50 50 25 0 100 36 0 ... ... ... 42 100 0 Flow rate: 1 ml/min 6.2. Method of assay Proceed as directed under Total Content by Spectrophotometry (JECFA monograph 1 - Vol. 4). Solvent: pH 7 phosphate buffer ... ... ... Absorptivity (a): 86.35 Approximate wavelength of maximum absorption: 415 nm Determination of the percentages of di-, mono- and trisulfonates in Quinoline prepared from 2-(2-quinolyl)-1,3-indandione (only): Use the HPLC conditions prescribed in the Determination of Organic Compounds other than Colouring Matters with a sample solution of concentration 0.05% in HPLC Solvent A in place of the sample solution of concentration 1%. Express the results as percentages of the Total colouring matters present. ANNEX 5 SPECIFICATIONS AND TESTS FOR SUNSET YELLOW FCF 1. Synonyms CI Food Yellow 3, FD&C Yellow No. 6; CI (1975) No. 15985 ... ... ... ADI = 0 - 2.5 mg/kg bw. 2. Definition Consists essentially of disodium 6-hydroxy-5-(4-sulfonatophenylazo)-2- naphthalene-6-sulfonate and subsidiary colouring matters together with NaCl and/or Na2SO4 as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium 6-hydroxy-5-(4-sulfonatophenylazo)-2-naphthalene-sulfonate C.A.S. number 2783-94-0 Chemical formula ... ... ... Structural formula Formula weight 452.38 3. Description Orange-red powder or granules. 4. Functional uses Colour. 5. Specifications ... ... ... Solubility Soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135°C Not more than 15% together with chloride and sulfate calculated as sodium salts. ... ... ... Not more than 0.2%. Lead Not more than 2 mg/kg. Subsidiary colouring matters Not more than 5%. Not more than 2% shall be colours other than trisodium 2-hydroxy-1-(4- sulfonatophenylazo)naphthalene-3,6-disulfonate. Organic compounds other than colouring matters Not more than 0.5%, sum of 4-amino-1-benzenesulfonic acid; 3-hydroxy2,7-naphthalenedisulfonic acid; ... ... ... 7- hydroxy-1,3-naphthalenedisulfonic acid; 4,4'- diazoaminodibenzenesulfonic acid; 6,6'-oxydi-2-naphthalenesulfonic acid. Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. Ether extractable matter Not more than 0.2%. 5.3. Content Not less than 85% total colouring matters ... ... ... 6.1. Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm. Organic compounds other than colouring matters ... ... ... - Proceed as directed under Determination by High Performance Liquid Chromatography using an elution gradient of 2 to 100% at 4% per min followed by elution at 100%. 6.2. Method of assay Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following: Weight of sample: 0.5 - 0.6 g; Buffer: 10 g sodium citrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 11.31 mg. ... ... ... SPECIFICATIONS AND TESTS FOR CARMINES 1. Synonyms Cochineal carmin, CI Natural Red 4, CI (1975) No. 75470, INS 120 A group ADI of 0-5 mg/kg bw for carmines, as ammonium carmine or the equivalent of Ca, K and Na salts. 2. Definition Obtained by aqueous extraction of cochineal, which consists of the dried bodies of the female insect Dactylopius coccus Costa; the colouring principle is a hydrated aluminium chelate of carminic acid in which aluminium and carminic acid are thought to be present in the molar ratio 1:2. In commercial products the colouring principle is present in association with ammonium, calcium, potassium or sodium cations, singly or in combination, and these cations may also be present in excess. Products may also contain proteinaceous material derived from the source insect, and may also contain free carminate or a small excess of aluminium cations. Chemical names ... ... ... C.A.S. number 1390-65-4 (carmine) 1260-17-9 (carminic acid) Chemical formula Carminic acid: C22H20O13 Structural formula Carminic acid ... ... ... Aluminium complex of carminic acid Formula weight Carminic acid: 492.39 3. Description Red to dark red, crumbly solid or powder 4. Functional uses Colour ... ... ... 5.1. Identification Solubility The solubility of carmine preparations varies depending on the nature of the cations present. Products where the major cation is ammonium (ammonium carminate) are freely soluble in water at pH 3.0 and pH 8.5. Products where the major cation is calcium (calcium carminate) are very slightly soluble in water at pH 3.0 but freely soluble at pH 8.5. Colour reaction Passes test (described in “Tests”). 5.2. Purity Loss on drying ... ... ... Total ash Not more than 12%. Protein Not more than 25%. Matter insoluble in dilute ammonia Not more than 1%. Lead Not more than 2 mg/kg. Microbiological criteria ... ... ... 5.3. C22H20O13 content Not less than 50% of C22H20O13 on the dry basis. 6. Tests 6.1 Identification Colour reactions - Make a solution of the sample slightly alkaline by adding 1 drop of 10% sodium hydroxide or potassium hydroxide solution. A violet colour is produced. - Add a small sodium dithionite (Na2S2O4) crystal to acid, neutral or alkaline solutions of the sample. The solutions are not decolourized. ... ... ... - Acidify a dispersion of the sample in water with 1/3 volume of hydrochloric acid TS and shake it with amyl alcohol. Wash the amyl alcohol solution 2-4 times with an equal volume of water to remove hydrochloric acid. Dilute the amyl alcohol solution with 1-2 volumes of petroleum ether (40-60^o) and shake with a few small portions of water to remove colour. Add, dropwise, 5% uranium acetate, shaking thoroughly after each addition. A characteristic emerald-green colour is produced. 6.2 Purity Loss on drying - Tested as directed under JECFA monograph 1-Vol. 4. - At 135^oC, 3 h. Total ash - Tested as directed under JECFA monograph 1-Vol. 4. - Test 1 g of the sample as directed in the test for Ash. ... ... ... - Tested as directed under JECFA monograph 1-Vol. 4. - Proceed as directed under Nitrogen Determination (non-ammonia N x 6.25) Matter insoluble in dilute ammonia Dissolve about 0.25 g of the sample, previously dried and accurately weighed, in 2.5 ml of dilute ammonia solution (160 ml of strong ammonia TS, made up to 500 ml) and dilute to 100 ml with water: the solution is clear. Filter through a sintered glass filter. Wash with a 0.1% ammonia solution and dry to constant weight at 105º. Lead Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” 6.3. Method of assay ... ... ...  (100 × A × 100) / (1,39 × W) where: A = absorbance of the sample solution; W = weight, in mg, of the sample taken; and 1.39 = absorbance of a solution of carminic acid having a concentration of 100 mg per 1000 ml; If the measured absorbance of the solution is not within the range 0.650 to 0.750, prepare another sample and adjust the weight accordingly. ANNEX 7 SPECIFICATIONS AND TESTS FOR CARMOISINE ... ... ... CI Food Red 3, Azorubine; CI (1975): 14720 INS: 122 ADI = 0 - 4 mg/kg bw. 2. Definition Consists essentially of disodium 4-hydroxy-3-(4-sulfonato-1-naphthylazo)- 1-naphthalenesulfonate and subsidiary colouring matters together with NaCl and/or Na2SO4 as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium 4-hydroxy-3-(4-sulfonato-1-naphthylazo)-1-naphthalenesulfonate. C.A.S. number ... ... ... Chemical formula C20H12N2Na2O7S2 Structural formula Formula weight 502.44 3. Description Red powder or granules. 4. Functional uses ... ... ... 5. Specifications 5.1. Identification Solubility Soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test. 5.2. Purity ... ... ... Not more than 15% together with chloride and sulfate calculated as sodium salts. Water insoluble matter Not more than 0.2%. Lead Not more than 2 mg/kg. Subsidiary colouring matters Not more than 1%. Organic compounds other than colouring matters Not more than 0.5% of 4-Amino-1-naphthalenesulfonic acid and 4-Hydroxy-1-naphthalenesulfonic acid together. ... ... ... Not more than 0.01% calculated as aniline. Ether extractable matter Not more than 0.2%. 5.3. Content Not less than 85% total colouring matters. 6. Tests 6.1 Purity Lead ... ... ... - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use HPLC under the following conditions: HPLC elution gradient: 1 to 100% at 2.0% per min. 6.3. Method of assay ... ... ... Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following: Weight of sample: 0.5 - 0.6 g; Buffer: 15 g sodium hydrogen tartrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 12.56 mg ANNEX 8 SPECIFICATIONS AND TESTS FOR AMARANTH 1. Synonyms CI Food Red 9, Naphtol Rot S.; CI (1975): 16185 ... ... ... ADI = 0 - 0.5 mg/kg bw. 2. Definition Consists essentially of trisodium 3-hydroxy-4-(4-sulfonato-1-naphthylazo)- 2,7-naphthalenedisulfonate and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Trisodium 3-hydroxy-4-(4-sulfonato-1-naphthylazo)-2,7-naphthalene disulfonate C.A.S. number 915-67-3 Chemical formula ... ... ... Structural formula Formula weight 604.48 3. Description Reddish brown to dark reddish brown powder or granules. 4. Functional uses Colour. 5. Specifications ... ... ... Solubility Soluble in water; sparingly soluble in ethanol Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135 °C Not more than 15% together with chloride and sulfate calculated as sodium salts. ... ... ... Not more than 0.2%. Lead Not more than 2 mg/kg. Subsidiary colouring matters Not more than 3%. Organic compounds other than colouring matters Not more than 0.5%, sum of 4-amino-1-benzenesulfonic acid; 3- hydroxy-2,7-naphthalenedisulfonic acid, 6-hydroxy-2-naphthalenesulfonic acid, 7-hydroxy-1,3-naphthalenedisulfonic acid and 7-hydroxy-1,3,6- naphthalene-trisulfonic acid. Unsulfonated primary aromatic amines ... ... ... Ether extractable matter Not more than 0.2%. 5.3. Content Not less than 85% total colouring matters. 6. Tests 6.1 Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm, then 1 h further development. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use HPLC with elution gradient: 2 to 100% at 4.0% per min. 6.2. Method of assay ... ... ... Weight of sample: 0.7 - 0.8 g; Buffer: 10 g sodium citrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 15.11 mg. ANNEX 9 SPECIFICATIONS AND TESTS FOR PONCEAU 4R 1. Synonyms CI Food Red 7, Cochineal Red A ; New Cochineal; CI (1975): 16255 ... ... ... ADI = 0 - 4 mg/kg bw. 2. Definition Consists essentially of trisodium d-2-hydroxy-1-(4-sulfonato-1- naphthylazo)-6,8-naphthalenedisulfonate, and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Trisodium-2-hydroxy-1-(4-sulfonato-1-naphthylazo)-6,8- naphthalenedisulfonate C.A.S. number 2611-82-7 Chemical formula ... ... ... Structural formula Formula weight 631.51 3. Description Red powder or granules. 4. Functional uses Colour. 5. Specifications ... ... ... Solubility Soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135 °C Not more than 20% together with chloride and sulfate calculated as sodium salts. ... ... ... Not more than 0.2%. Lead Not more than 2 mg/kg. Subsidiary colouring matters Not more than 1%. Organic compounds other than colouring matters Not more than 0.5% of sum of: 4-amino-1-naphthalenesulfonic acid, 7-hydroxy-1,3- naphthalenedisulfonic acid, 3-hydroxy-2,7-naphthalenesulfonic acid, 6- hydroxy-2-naphthalenesulfonic acid, 7-hydroxy-1,3,6- naphthalenetrisulfonic acid. Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. ... ... ... Not more than 0.2%. 5.3. Content Not less than 85% total colouring matters 6. Tests 6.1. Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” ... ... ... - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 3; Height of ascent of solvent front: approximately 17 cm, then 1 h further development Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use HPLC under the following conditions: HPLC elution gradient: 2 to 100% at 2% per min. 6.2. Method of assay Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following: ... ... ... Buffer: 10 g sodium citrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 15.78 mg. ANNEX 10 SPECIFICATIONS AND TESTS FOR ERYTHROSINE 1. Synonyms CI Food Red 14, FD&C Red No. 3; CI (1975): 45430 INS 127 ADI = 0 - 0,1 mg/kg bw ... ... ... Consists essentially of disodium salt of 9-(o-carboxyphenyl)-6-hydroxy2,4,5,7-tetraiodo-3-isoxanthone monohydrate and subsidiary colouring matters together with water, sodium chloride and/or sodium sulfate as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium salt of 9-(o-carboxyphenyl)-6-hydroxy-2,4,5,7-tetraiodo-3- isoxanthone monohydrate C.A.S. number 16423-68-0 Chemical formula C20H6I4Na2O5.H2O Structural formula ... ... ... Formula weight 897.88 3. Description Red powder or granules. 4. Functional uses Colour. 5. Specifications 5.1. Identification ... ... ... Soluble in water, slightly soluble in ethanol Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135 °C Not more than 13% together with chloride and sulfate calculated as sodium salts. Inorganic iodides Not more than 0.1% calculated as sodium iodide. ... ... ... Not more than 0.2%. Zinc Not more than 50 mg/kg. Lead Not more than 2 mg/kg. Subsidiary colouring matters Not more than 4% (except fluorescein). Fluorescein Not more than 20 mg/kg. ... ... ... Tri-iodoresorcinol: Not more than 0.2% 2-(2,4-dihydroxy-3,5-di-iodobenzoyl) benzoic acid: Not more than 0.2% Ether extractable matter From a solution of pH not less than 7, not more than 0.2%. 5.3. Content Not less than 87% total colouring matters. 6. Tests 6.1. Purity ... ... ... Weigh 1.0 g of the sample into a 100-ml beaker. Add 75 ml distilled water and the magnetic follower. Stir to dissolve. Immerse a iodide specific electrode and a reference electrode in the solution and set a suitable millivoltmeter to read the potential of the system in millivolts. Add 0.001 M silver nitrate solution from a burette initially in 0.5 ml aliquots, reducing these to 0.1 ml as the end-point approaches as indicated by an increasing change in potential for each addition. After allowing time for the reading to stabilize, record the millivolt readings after each addition. Continue the titration until further additions make little change in the potential. Plot the millivolt readings against the volume of silver nitrate solution added. The equivalent point is the volume corresponding to the maximum slope of the curve. The percentage of sodium iodide in sample is: Titre x 0.015%. Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 5; Height of ascent of solvent front: approximately 17 cm. Note: Take special care not to allow the chromatograms to be exposed to direct sunlight. ... ... ... - Tested as directed under JECFA monograph 1-Vol. 4. - Proceed as directed under Column chromatography, using, for example, the following absorptivities: Tri-iodoresorcinol: 0.079 mg L^-1cm^-1 at 233 nm (acidic). 2(2,4-dihydroxy-3,5-di-iodobenzoyl)benzoic acid: 0.047 mg L^-1cm^-1 at 348 nm (alkaline). Fluorescein Principle: The fluorescein is separated from the sample by TLC and compared with a standard chromatogram prepared from fluorescein at the concentration corresponding to the limit figure. Solvent: Methanol+water+ammonia (s.g. 0.890) (500 ml+400 ml+100 ml). ... ... ... Weigh 1.0 g of the sample, dissolve in about 50 ml solvent and dilute to 100 ml in a volumetric flask. Standard: Weigh an amount of fluorescein, previously purified by recrystallisation from ethanol, equal to 1 g x the colouring matter content of the sample as determined under Assay. Dissolve in water (or in water with 10 ml ammonia s.g. 0.890 if fluorescein-free acid is being used) and dilute to 100 ml. Make further sequential dilutions as follows: 1 ml to 100 ml with water. 1 ml to 100 ml with water. 20 ml to 100 ml with solvent. Chromatography solvent: n-Butanol+water+ammonia (s.g. 0.890)+ethanol (100 ml+44 ml+1 ml+ 22.5 ml). Procedure: ... ... ... Note: Take special care not to allow the chromatograms to be exposed to direct sunlight. 6.2. Method of assay Dissolve about 1 g of the sample, accurately weighed, in 250 ml of water, transfer to a clean 500-ml beaker, add 8.0 ml of 1.5 N nitric acid and stir well. Filter through a sintered glass crucible (porosity 3, diameter 5 cm) which has been weighed containing a small glass stirring rod. Wash thoroughly with 0.5% nitric acid until the filtrate gives no turbidity with silver nitrate TS, and then wash with 30 ml water. Dry to constant weight at 135±5º, carefully breaking up the precipitate by means of the glass rod. Cool in a desiccator and weigh Determination of Hydrochloric Acid-insoluble Matter in Erythrosine Lake ... ... ... Concentrated hydrochloric acid Hydrochloric acid 0.5% v/v Dilute ammonia solution (dilute 10 ml ammonia, s.g. 0.890 to 100 ml with water). Procedure: Accurately weigh approximately 5 g of the lake into a 500-ml beaker. Add 250 ml water and 60 ml concentrated hydrochloric acid. Boil to dissolve the alumina while the Erythrosine converts to its "free acid" form, which is insoluble in acid. Filter through a tared No. 4 sintered glass crucible. Wash the crucible with a small amount of hot 0.5% hydrochloric acid and then with some hot distilled water. Remove the acid filtrate from the filter flask, replace the crucible and wash with hot dilute ammonia solution until the washings are colourless. Dry the crucible to constant weight at 135º. Express the residue as a percentage of the weight taken. ANNEX 11 SPECIFICATIONS AND TESTS FOR RED 2G 1. Synonyms ... ... ... INS: 128 ADI = 0 - 1 mg/kg bw. 2. Definition Consists essentially of disodium 8-acetamido-1-hydroxy-2-phenylazo3,6-naphthalenedisulfonate and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium 8-acetamido-1-hydroxy-2-phenylazo-3,6-naphthalenedisulfonate C.A.S. number 3734-67-6 ... ... ... C18H13N3Na2O8S2 Structural formula Formula weight 509.43 3. Description Red powder or granules. 4. Functional uses Colour. ... ... ... 5.1. Identification Solubility Freely soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135 °C ... ... ... Water insoluble matter Not more than 0.2%. Lead Not more than 2 mg/kg. Subsidiary colouring matters Not more than 2%. Organic compounds other than colouring matters Not more than 0.3% of sum of 5-acetamido-4-hydroxy-2,7-naphthalene2,7-disulfonic acid and 5-Amino-4-hydroxy-2,7-naphthalene-2,7- disulfonic acid. Unsulfonated primary aromatic amines ... ... ... Ether extractable matter Not more than 0.2%. 5.3. Content Not less than 80% total colouring matter. 6. Tests 6.1. Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use HPLC under the following conditions: HPLC elution gradient: 2 to 100% at 2% per min. 6.2. Method of assay ... ... ... Weight of sample: 0.6 - 0.7 g; Buffer: 15 g sodium hydrogen tartrate; Weight: (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 12,74 mg ANNEX 12 SPECIFICATIONS AND TESTS FOR ALLURA RED AC 1. Synonyms CI Food Red 17, FD&C Red No. 40; CI (1975): 16035 INS: 129 ... ... ... 2. Definition Consists of disodium 6-hydroxy-5-(2-methoxy-5-methyl-4- sulfonatophenylazo)-2-naphthalenesulfonate and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium 6-hydroxy-5-(2-methoxy-5-methyl-4-sulfonatophenylazo)-2- naphthalenesulfonate C.A.S. number 25956-17-6 Chemical formula C18H14N2Na2O8S2 ... ... ... Formula weight 496.43 3. Description Dark red powder or granules. 4. Functional uses Colour. 5. Specifications 5.1. Identification ... ... ... Solubility Soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135 °C Not more than 15% together with chloride and sulfate calculated as sodium salts. Water insoluble matter ... ... ... Lead Not more than 2 mg/kg. Subsidiary colouring matters Not more than 3%. Organic compounds other than colouring matters Not more than 0.3% of sodium 6-hydroxy-2-naphtalene sulfonate; Not more than 0.2% of 4-amino-5-methoxy-2-methyl-benzene-sulfonic acid; Not more than 1.0% of disodium 6,6'-oxybis(2-naphthalene-sulfonate); Unsulfonated primary aromatic amines ... ... ... Ether extractable matter Not more than 0.2%. 5.3. Content Not less than 85% total colouring matters 6. Tests 6.1 Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use liquid chromatography under the following conditions: HPLC elution gradient: O to 18% at 1% per min (linear) then 18% to 62% at 7% per min (linear) followed by elution at 100%. Flow rate: 0.6 ml per min 6.2. Method of assay ... ... ... Weight of sample: 0.5 - 0.6 g; Buffer: 15 g sodium hydrogen tartrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 12.41 mg. ANNEX 13 SPECIFICATIONS AND TESTS FOR INDIGOTINE 1. Synonyms Indigocarmin; CI Food Blue 1, FD&C Blue No. 2; CI (1975): 73015 INS: 132 ... ... ... 2. Definition Consists essentially of a mixture of disodium 3,3' -dioxo-[delta^2,2'- biindoline]-5,5'-disulfonate (principal component) and disodium 3,3'- dioxo-[delta^2,2'-biindoline]-5,7'-disulfonate (isomer) and subsidiary colouring matters together with NaCl and/or Na2SO4 as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium 3,3'-dioxo-[delta^2,2'-biindoline]-5,5'-disulfonate (principal component) C.A.S. number 860-22-0 (5,5' isomer) Chemical formula C16H8N2Na2O8S2 ... ... ... Formula weight 466.36 3. Description Blue powder or granules. 4. Functional uses Colour. 5. Specifications 5.1. Identification ... ... ... Solubility Soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135 °C Not more than 15% together with chloride and sulfate calculated as sodium salts. Water insoluble matter ... ... ... Lead Not more than 2 mg/kg. Subsidiary colouring matters Not more than 1% (except disodium 3,3'-dioxo-[delta^2,2'-biindoline]- 5,7'- disulfonate). Organic compounds other than colouring matters Not more than 0.5% of sum of isatin-5-sulfonic acid, 5-sulfoanthranilic acid and anthranilic acid Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. Ether extractable matter ... ... ... 5.3. Content Not less than 85% total colouring matters. Not more than 18% of disodium 3,3'-dioxo-[delta^2,2'-biindoline]-5,7'- disulfonate 6. Tests 6.1. Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” ... ... ... - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 3; Height of ascent of solvent front: approximately 17 cm. Note 1. The 5,7' isomer is separated as a wide blue zone just in front of the main blue band. Do not include this zone in the subsidiary colouring matter zones which are cut out and measured. Note 2. The 15 ml sodium hydrogen carbonate solution used in the general procedure is replaced by 15 ml 0.05 N hydrochloric acid in order to avoid the decomposition which the sulfonated indigo undergoes in alkaline solution. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use liquid chromatography under the following conditions: HPLC elution gradient: 2 to 100% gradient followed by elution at 100%. Ether extractable matter - Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... 6.2. Method of assay Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following: Weight of sample: 1.0 - 1.1 g; Buffer: 15 g sodium hydrogen tartrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 23.32 mg. Isomer content by paper chromatography: Refer to the conditions for the determination of subsidiary colouring matters. Cut the isomer band from the chromatogram in the manner detailed for the subsidiary bands, extract into solvent and measure the absorbance at its Smax. Measure the absorbance of the corresponding blank at the same wavelength. As a standard use 0.1 ml of an 0.20% solution of the sample applied to the 18 cm x 0.7 cm rectangle. ... ... ... % = [A / As] x 20% x [D / 100] where: A is the net absorbances (of the corresponding blank) of the isomer. As is the net absorbances (of the corresponding blank) of the standard. D is the total colouring matters content of the sample. Isomer content by HPLC: The 5,7' isomer separates under the HPLC conditions detailed above for the separation of subsidiary colouring matters, and the amount present can be quantified. ANNEX 14 ... ... ... 1. Synonyms CI Food Blue 2, FD&C Blue No. 1; CI (1975): 42900 INS: 133 ADI = 0 - 1 mg/kg bw. 2. Definition Consists essentially of Disodium 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3- sulfonatobenzyl)-amino] phenyl] (2-sulfonatophenyl)methylene]-2,5- cyclohexadiene-1-ylidene] ammoniomethyl] benzenesulfonate and its isomers and subsidiary colouring matters together with NaCl and/or Na2SO4 as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3-sulfonatobenzyl)- amino]phenyl](2-sulfonatophenyl)methylene]-2,5-cyclohexa-diene-1- ylidene]ammoniomethyl]-benzenesulfonate. ... ... ... C.A.S. number 3844-45-9 Chemical formula C37H34N2Na2O9S3 Structural formula Formula weight 792.86 3. Description ... ... ... 4. Functional uses Colour. 5. Specifications 5.1. Identification Solubility Soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test. ... ... ... Loss on drying at 135 °C Not more than 15% together with chloride and sulfate calculated as sodium salts. Water insoluble matter Not more than 0.2%. Lead Not more than 2 mg/kg. Chromium Not more than 50 mg/kg. ... ... ... Not more than 6%. Organic compounds other than colouring matters Not more than 1.5%, sum of 2-, 3- and 4-formylbenzenesulfonic acids. Not more tan 0.3% 3-[[N-ethyl-N-(4-sulfophenyl) amino] methyl] benzenesulfonic acid. Leuco base Not more than 5%. Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. Ether extractable matter ... ... ... 5.3. Content Not less than 85% total colouring matter. 6. Tests 6.1. Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” Subsidiary colouring matters ... ... ... - Use the following conditions: Developing solvent: No. 4; Develop chromatogram for approximately 20 hours. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Proceed as directed under Column chromatography, using, for example, the following absorptivities: 3-formylbenzenesulfonic acid: 0.0495 mgL^-1/cm^-1 at 246 nm in dilute HCl. 3-[[N-ethyl-N-(4-sulfophenyl)amino] methyl] benzenesulfonic acid: 0.078 mgL^-1/cm^-1 at 277 nm in dilute ammonia. Leuco base Tested as directed under JECFA monograph 1-Vol. 4. Weigh accurately 120±5 mg sample and proceed as directed under Leuco Base in Sulfonated Triarylmethane Colours (Vol. 4) Absorptivity (a) = 0.164 mgL^-1cm^-1at 629 nm Ratio = 0.9706. 6.2. Method of assay ... ... ... Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following: Weight of sample: 1.8 - 1.9 g; Buffer: 15 g sodium hydrogen tartrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 39.65 mg. ANNEX 15 SPECIFICATIONS AND TESTS FOR CHLOROPHYLLS 1. Synonyms ... ... ... Clorophyllin; C.I. Natural Green 3; C.I. (1975): 75810; INS 140 ADI “Not limited”. 2. Definition Obtained by solvent extraction of grass, lucerne, nettle and other plant material; during the subsequent removal of solvent, the naturally present coordinated magnesium may be wholly or partly removed from the chlorophylls to give the corresponding phaeophytins; the principal colouring matters are the phaeophytins and magnesium chlorophylls. The extracted product, from which the solvent has been removed, contains other pigments such as carotenoids as well as oils, fats and waxes derived from the source material. Only the following solvents may be used for the extraction: acetone, dichloromethane, methanol, ethanol, propan-2-ol and hexane. Chemical names The major colouring principles are: Phytyl (13^2R,17S,18S)-3-(8-ethyl-13^2-methoxycarbonyl-2,7,12,18-tetramethyl13'-oxo-3-vinyl-13^1 -13^2 -17,18-tetrahydrocyclopenta [at]-prophylrin-17- yl)propionate, (Pheophytin a), or as the magnesium complex (Chlorophyll a). Phytyl (13^2R,17S,18S)-3-(8-ethyl-7-formyl-132 -methoxycarbonyl-2,12,18- trimethyl-13'-oxo-3-vinyl-13^1 -13^2-17,18-tetrahydro-cyclopenta [at]-prophylrin17-yl)propionate, (Pheophytin b), or as the magnesium complex (Chlorophyll b). ... ... ... Phaeophytin a, Magnesium complex: 479-61-8 Phaeophytin b, Magnesium complex: 519-62-0 Chemical formula Phaeophytin a Magnesium complex (Chlorophyll a): C55H72MgN4O5 Phaeophytin a: C55H74N4O5 Phaeophytin b Magnesium complex (Chlorophyll b): C55H70MgN4O6 Phaeophytin b: C55H72N4O6 Structural formula ... ... ... X = CH3 for the "a" series X = CHO for the "b" series. Loss of Mg converts chlorophyll into the corresponding phaeophytin Formula weight Phaeophytin a magnesium complex (Chlorophyll a): 893.51 Phaeophytin a: 871.22 Phaeophytin b magnesium complex (Chlorophyll b): 907.49 Phaeophytin: 885.20 3. Description ... ... ... 4. Functional uses Colour. 5. Specifications 5.1. Identification Solubility Insoluble in water; soluble in ethanol, diethyl ether, chloroalkanes, hydrocarbons and fixed oils. Thin-layer chromatography Passes test (described in “Tests”). ... ... ... Residual solvents Acetone, methanol, ethanol, propan-2-ol, hexane: Not more than 50 mg/kg, singly or in combination. Dichloromethane: Not more than 10 mg/kg Arsenic Not more than 3 mg/kg.  (Tested as directed under JECFA monograph 1-Vol. 4 - Method II). Lead ... ... ...  (Tested as directed under JECFA monograph 1-Vol. 4. Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” 5.3. Content Content of total combined phaeophytins and their magnesium complexes is not less than 10%. 6. Tests 6.1. Identification Thin-layer chromatography Apply a 1 in 20 solution of the sample in chloroform as a band of the length of 2 cm to a Silica 60 TLC plate. After drying, develop the plate by a mixture of 50% hexane, 45% chloroform and 5% ethanol (General purpose reagent grade chloroform is supplied with 2% of added ethanol as a stabilizer. ... ... ... Phaeophytin a: 0.77; grey/brown Phaeophytin b: 0.75; yellow/brown Chlorophyll a: 0.50; blue/green Chlorophyll b: 0.63; yellow/green In addition spots may be visible for b-carotene at Rf 0.81 and xanthophyll at Rf 0.47 and 0.23. 6.2. Purity Residual solvents Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... Arsenic - Tested as directed under JECFA monograph 1-Vol. 4. - Method II. Lead  - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” 6.1. Method of assay Accurately weigh about 100 mg of the sample and dissolve in diethyl ether, making the volume to 100 ml. Dilute 2 ml of this solution to 25 ml with diethyl ether. The concentration of the sample should not give an absorbance at 660.4 nm that is in excess of the working range for Absorbance measurements, i.e., not in excess of 0.7. Measure the absorbances of the solution in a 1 cm cell against a diethyl ether blank at 660.4 nm, 642.0 nm, 667.2 nm and 654.4 nm. (These being the absorbance maxima in diethyl ether for chlorophyll a, chlorophyll b, phaeophytin a, and phaeophytin b, respectively). In addition measure at 649.8 nm and 628.2 nm. To the remaining diluted solution add one crystal of oxalic acid and after dissolution and mixing, remeasure the absorbances at the same wavelengths.  "dA" is the difference between the absorbances between the absorbance at the respective wavelengths, before and after addition of oxalic acid. ... ... ... Chlorophyll a = 17.7 d A (660.4 nm) + 7.15 d A (642.0 nm) Chlorophyll b = 19.4 d A (642.0 nm) - 2.92 d A (660.4 nm) Phaeophytin a = -4.89 A (649.8 nm) + 0.0549 A (628.2 nm) +18.7 A (667.2 nm) + 0.0575 A (654.4 nm) - chlorophyll a Phaeophytin b = -71.0 A (649.8 nm) + 2.51 A (628.2 nm) - 13.5 A (667.2 nm) + 84.3 A (654.4 nm) - chlorophyll b Convert the figures in mmol/L to percentages using the following equations: ... ... ... ANNEX 16 SPECIFICATIONS AND TESTS FOR CHLOROPHYLLS, COPPER COMPLEXES 1. Synonyms Copper chlorophyllin, copper phaeophytin C.I. Natural Green 3; C.I. (1975) No. 75810; INS: 141i ... ... ... 2. Definition Obtained by addition of an organic salt of copper to the substance obtained by solvent extraction of grass, lucerne, nettle and other plant material; the product, from which the solvent has been removed, contains other pigments such as carotenoids as well as fats and waxes derived from the source material. The principal colouring matters are the copper phaeophytins. Only the following solvents may be used for the extraction: acetone, dichloromethane, methanol, ethanol, propan-2-ol and hexane. Chemical names The major colouring principles are: [Phytyl (13^2R,17S,18S)-3-(8-ethyl-13^2 -methoxycarbonyl-2,7,12,18- tetramethyl-131 -oxo-3-vinyl-13^1 ,13^2 ,17,18-tetra-hydrocyclopenta [at]- prophyrin-17-yl)propionate] copper (II) (Copper chlorophyll a) [Phytyl (13^2R,17S,18S)-3-(8-ethyl-7-formyl-132 -methoxycarbonyl-2,12,18- trimethyl-131 -oxo-3-vinyl-13^1,13^2,17,18-tetrahydro-cyclopenta [at]prophyrin17-yl)propionate] copper (II) (Copper chlorophyll b). C.A.S. number 65963-40-8 Chemical formula ... ... ... Copper phaeophytin b: C55H70CuN4O6 Structural formula where: X = CH3 for the "a" compound X = CHO for the "b" compound Formula weight Copper phaeophytin a: 932.75 Copper phaeophytin b: 946.73 ... ... ... Waxy solid ranging in colour from blue green to dark green depending on the source material. 4. Functional uses Colour. 5. Characteristics 5.1. Identification Solubility Insoluble in water; soluble in ethanol, diethyl ether, chloroalkanes, hydrocarbons and fixed oils. Spectrophotometry ... ... ... Thin-layer chromatography Passes test (described in “Tests”). 5.2. Purity Residual solvents Acetone, methanol, ethanol, propan-2-ol, hexane: Not more than 50 mg/kg, singly or in combination. Dichloromethane: Not more than 10 mg/kg. Free ionizable copper Not more than 200 mg/kg. ... ... ... Not more than 8% of the total copper phaeophytins. Arsenic Not more than 3 mg/kg. Lead Not more than 5 mg/kg. 5.3. Content Not less than 10% of total copper phaeophytins. 6. Tests 5.1. Identification ... ... ... Thin-layer chromatography Apply a 1 in 20 solution of the sample in chloroform as a band of the length of 2 cm to a Silica 60 TLC plate. After drying, develop the plate by a mixture of 50% hexane, 45% chloroform and 5% ethanol (General purpose reagent grade chloroform is supplied with 2% of added ethanol as a stabilizer. The 5% ethanol in the solvent mixture is in addition to this), until the solvent ascends to a point 15 cm above the initial spots. Allow the solvent to evaporate, then visually examine the separated spots and identify the components of interests by their Rf values and colours. Approximate Rf values and colour of the spots are as follows: Copper phaeophytin a: 0.5; green Copper phaeophytin b: 0.73; yellow/green In addition spots may be visible for b-carotene at Rf 0.81 and xanthophyll at Rf 0.47 and 0.23. 5.2. Purity Residual solvents - Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... Free ionizable copper Accurately weigh about 1 g of the sample and dissolve in 20 ml of arachid oil, with the aid of gentle heat. Add exactly 200 ml of water, stir mechanically, and adjust to pH 3.0 by careful addition of 0.5 N hydrochloric acid (avoid overshooting). Allow the mixture to stand for 10 min. If necessary readjust to pH 3.0 by careful addition of 0.5 N hydrochloric acid. Transfer to a separating funnel and allow to stand for about 20 min.  Filter the aqueous phase through a No. 50 Whatman filter paper, rejecting the first 10 ml.Subject this solution to analysis for copper by atomic absorption spectrometry - JECFA monograph 1-Vol. 4. Total copper Ignite about 0.1 g, accurately weighed, of the sample contained in a silica dish, at a temperature not exceeding 500º, until all carbon is removed; moisten with one or two drops of concentrated sulphuric acid and re-ash. Dissolve the ash by boiling with 3 portions (each of 5 ml) of 10% (w/w) hydrochloric acid, filtering each addition through the same small filter paper into a 100 ml volumetric flask. Cool, and make up to volume with purified water. Subject this solution to analysis for copper by atomic absorption spectrometry - See JECFA monograph 1-Vol. 4. Arsenic - Tested as directed under JECFA monograph 1. Vol.4. - Method II. Lead - Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... 6.3. Method of assay Accurately weigh about 100 mg of the sample and dissolve in diethyl ether, making the volume to 100 ml. Dilute 2 ml of this solution to 25 ml with diethyl ether. The concentration of the sample should not give an absorbance at 660.4 nm that is in excess of the working range for Absorbance measurements, i.e., not in excess of 0.7. Measure the absorbances (A) of the solution in a 1 cm cell against a diethyl ether blank at 667.2 nm, 654.4 nm, 649.8 nm and 628.2 nm. (The latter two wavelengths being the absorbance maxima in diethyl ether for copper phaeophytin a and copper phaeophytin b respectively).  Calculate the concentration of the individual compounds in (mol/L from the following equations: Copper phaeophytin a = 45.6 A (649.8nm) - 2.75 A (628.2nm) + 3.10 A (667.2nm) - 35.4 A (654.4nm) Copper phaeophytin b = -8.46 A (649.8nm) + 20.7 A (628.2nm) - 1.69 A (667.2nm) + 5.13 A (654.4nm) Convert the figures in mmol/L to percentages using the following equations: ... ... ... ANNEX 17 SPECIFICATIONS AND TESTS FOR CHLOROPHYLLINS, COPPER COMPLEXES SODIUM AND POTASSIUM SALTS 1. Synonyms Sodium copper chlorophyllin; potassium copper chlorophyllin Chlorophyllins, C.I. (1975) No. 75810; INS 141ii ... ... ... 2. Definition The alkali salts of Copper Chlorophyllins are obtained by the addition copper to the product obtained by the saponification of a solvent extraction of grass, lucerne, nettle and other plant material; the saponification removes the methyl and cyclophytol ester groups and may partially cleave the pentenyl ring; after addition of copper to the purified chlorophyllins, the acid groups are neutralised to form the salts of potassium and/or sodium; the commercial products may be presented as aqueous solutions or dried powders. Only the following solvents may be used for the extraction: acetone, dichloromethane, methanol, ethanol, propan-2-ol and hexane. Chemical names The major colouring principles are: 3-(10-Carboxylato-4-ethyl-1,3,5,8-tetramethyl-9-oxo-2-vinylphorbin-7- yl)propionate, copper complex (Copper chlorophyllin a) 3-(10-carboxylato-4-ethyl-3-formyl-1,5,8-trimethyl-9-oxo-2-vinylphorbin-7- yl)propionate, copper complex (Copper chlorophyllin b) Depending on the degree of hydrolysis the cyclopentenyl ring may be cleaved with the resultant production of a third carboxyl function. Chemical formula Copper phaeophytin a: C34H32CuN4O5 ... ... ... Structural formula where: X = CH3 for the "a" series X = CHO for the "b" compound. M = Potassium and/or sodium Formula weight Copper chlorophyllin a: 640.20 Copper chlorophyllin b: 654.18 ... ... ... 3. Description Dark green to blue/black powder or dark green solution. 4. Functional uses Colour. 5. Specifications 5.1. Identification Solubility Soluble in water; very slightly soluble in lower alcohols and ketones and diethyl ether; insoluble in chloroalkanes, hydrocarbons and fixed oils. ... ... ... Passes test (described in “Tests”). Test for copper Passes test (described in “Tests”). Test for sodium Passes test. Test for potassium Passes test. 5.2. Purity ... ... ... Passes test (described in “Tests”). Residual solvents Acetone, methanol, ethanol, propan-2-ol, hexane: Not more than 50 mg/kg, singly or in combination. Dichloromethane: Not more than 10 mg/kg Free ionizable copper Not more than 200 mg/kg. Total copper Not more than 8% of the total copper phaeophytins. Arsenic ... ... ... Lead Not more than 5 mg/kg. 5.3. Content Not less than 95% total copper chlorophyllins after drying (100º, 1 h). 6. Tests 6.1. Identification Spectrophotometry A (1%, 1 cm) of the sample, dried at 100º for 1 h, at 405 nm in pH 7.5 phosphate buffer is not less than 540. ... ... ... Dissolve the sulfated ash of the sample (using 1 g of sample, Method I) in 10 ml of dilute hydrochloric acid TS by heating on a water bath. Filter if the solution is not clear, and dilute to 10 ml with water. Use this solution as the test preparation for the following tests: To 5 ml of the test preparation add ammonia TS to make the solution alkaline. Blue colour should appear. To 5 ml of the above test preparation add 0.5 ml of a 1 in 1000 sodium diethyldithiocarbamate solution. A brown precipitate should be formed. Test for sodium Tested as directed under JECFA monograph 1-Vol. 4 - Test the solution described under Tests, identification tests Test for copper. Test for potassium Tested as directed under JECFA monograph 1-Vol. 4 - Test the solution described under Tests, identification tests Test for copper. 6.2 Purity ... ... ... To 5 ml of a 0.5% aqueous solution of the sample in a test- tube add 1 ml of 1 N hydrochloric acid and add 5 ml of diethyl ether. Mix thoroughly and allow to separate. The ether layer should be no darker than pale green. Residual solvents Determine by gas chromatographically using either the method of entrainment distillation (Determination of Residual Solvents) or headspace analysis (Limit Test for Solvent Residues). (Tested as directed under JECFA monograph 1-Vol. 4) Free ionizable copper Accurately weigh about 1 g of the sample and mix in 20 ml of arachid oil, with the aid of gentle heat. Add exactly 200 ml of water, stir mechanically, and adjust to pH 3.0 by careful addition of 0.5 N hydrochloric acid (avoid overshooting). Allow the mixture to stand for 10 min. If necessary readjust to pH 3.0 by careful addition of 0.5 N hydrochloric acid. Transfer to a separating funnel and allow to stand for about 20 min. Filter the aqueous phase through a No. 50 Whatman filter paper, rejecting the first 10 ml. Subject this solution to analysis for copper by atomic absorption spectrometry (see JECFA monograph 1-Vol. 4). Total copper Ignite about 0.1 g, accurately weighed, of the sample contained in a silica dish, at a temperature not exceeding 500º, until all carbon is removed; moisten with one or two drops of concentrated sulphuric acid and re-ash. Dissolve the ash by boiling with 3 portions (each of 5 ml) of 10% (w/w) hydrochloric acid, filtering each addition through the same small filter paper into a 100 ml volumetric flask. Cool, and make up to volume with purified water. Subject this solution to analysis for copper by atomic absorption spectrometry (see JECFA monograph 1-Vol. 4). Arsenic Tested as directed under JECFA monograph 1-Vol. 4- Method II ... ... ... Tested as directed under JECFA monograph 1-Vol. 4. Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” 6.3. Method of assay Weigh accurately about 1 g of the sample, dried previously at 100º for 1 h, then dissolve in 20 ml Phosphate Buffer Solution (pH 7.5) and dilute to 1000 ml with distilled water. Dilute 10 ml of this solution to 100 ml with Phosphate Buffer Solution (pH 7.5). Measure the optical density of the final solution (0.001% w/v) in a suitable spectrophotometer, using a 1 cm cell and slit width of 0.10 mm at 403-406 nm, recording the maximum within this range. The percentage of sodium copper chlorophyllin is given by the expression: Optical density ´ 10^4 -------------------------------------- 565 ´ weight of sample (g) ... ... ... ANNEX 18 SPECIFICATIONS AND TESTS FOR GREEN S 1. Synonyms CI food green 4, food green S; CI (1975): 44090 INS: 142 ADI “Not specified”. 2. Definition Consists essentially of sodium N-[4-[[4-(dimethylamino)phenyl](2- hydroxy-3,6-disulfo-1-naphthalenyl)-methylene]-2,5-cyclohexadien-1- ylidene]-N-methylmethanaminium and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured compounds. ... ... ... Chemical names Sodium N-[4-[[4-(dimethylamino)phenyl](2-hydroxy-3,6-disulfo-1- naphtalenyl)-methylene]-2,5-cyclohexadien-1-ylidene]-N-methylmethanaminium; or Sodium 5-[4-dimethylamino-I-(4-dimethyliminiocyclohexa-2,5- dienylidene) benzyl]-6-hydroxy-7-sulfonato-naphthalene-2-sulfonate. C.A.S. number 860-22-0 Chemical formula C27H25N2NaO7S2 Công thức cấu tạo ... ... ... 576.63 3. Description Dark green powder or granules. 4. Functional uses Colour. 5. Specifications 5.1. Identification Solubility ... ... ... Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135 °C Not more than 20% together with chloride and sulfate calculated as sodium salts. Water insoluble matter Not more than 0.2%. Lead ... ... ... Chromium  Not more than 50 mg/kg. Subsidiary colouring matters  Not more than 1%. Organic compounds other than colouring matters Not more than 0.1% of 4,4'-Bis (dimethylamino) benzhydryl alcohol. Not more than 0.1% of 4,4'-Bis (dimethylamino) benzophenone. Not more than 0.2% of 3-Hydroxynaphthalene-2,7-disulfonic acid. Unsulfonated primary aromatic amines ... ... ... Leuco base Not more than 5.0%. Ether extractable matter Not more than 0.2%. 5.3. Content Not less than 80% total colouring matter. 6. Tests 6.1. Purity ... ... ... - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: Developing solvent: No. 2; Height of ascent of solvent front: approximately 17 cm. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use liquid chromatography under the following conditions: HPLC elution gradient: 2 to 100% at 2% per min. Leuco base ... ... ... - Weigh accurately 110±5 mg of sample and proceed as directed under Leuco Base in Sulfonated Triarylmethane Colours. Absorptivity: (a) = 0.1725 mg L^-1 cm^-1 at approximately 634 nm. Ratio = 0.9600 6.2. Method of assay Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following: Weight of sample: 1.4 - 1.5 g; Buffer: 15 g sodium hydrogen tartrate; Weight (D) of colouring matters equivalent to 1.00 ml 0.1 N TiCl3: 28.83 mg. ... ... ... SPECIFICATIONS AND TESTS FOR CARAMEL 1. Synonyms Caramel colours are divided into four classes. The synonyms for each class are: Class I: Plain caramel, caustic caramel; INS No. 150a Class III: Ammonia caramel; INS No. 150c Class IV: Sulfite ammonia caramel , INS No. 150d ADI for class I “Not specified”; ADI for class III = 0-160 mg/kg bw ADI for class IV = 0-200 mg/kg bw (0-150 mg/kg on solids basis) ... ... ... Complex mixtures of compounds, some of which are in the form of colloidal aggregates, manufactured by heating carbohydrates either alone or in the presence of food-grade acids, alkalis or salts; classified according to the reactants used in their manufacture as follows: Class I: Prepared by heating carbohydrates with or without acids or alkalis; no ammonium or sulfite compounds are used. Class III: Prepared by heating carbohydrates with or without acids or alkalis in the presence of ammonium compounds; no sulfite compounds are used. Class IV: Prepared by heating carbohydrates with or without acids or alkalis in the presence of both sulfite and ammonium compounds. In all cases the carbohydrate raw materials are commercially available foodgrade nutritive sweeteners consisting of glucose, fructose and/or polymers thereof. The acids and alkalis are food-grade sulfuric or citric acids and sodium, potassium or calcium hydroxides or mixtures thereof. Where ammonium compounds are used they are one or any of the following: ammonium hydroxide, ammonium carbonate and ammonium hydrogen carbonate, ammonium phosphate, ammonium sulfate, ammonium sulfite and ammonium hydrogen sulfite. Where sulfite compounds are used they are one or any of the following: sulfurous acid, potassium, sodium and ammonium sulfites and hydrogen sulfites. Food-grade anti-foaming agents may be used as processing aids during manufacture. 3. Description ... ... ... 4. Functional uses Colour 5. Specifications 5.1. Identification Solubility Miscible with water Identification of colouring matters Passes test. ... ... ... Class I: Not more than 50% of the colour is bound by DEAE Cellulose and not more than 50% of the colour is bound by Phosphoryl Cellulose. Class III: Not more than 50% of the colour is bound by DEAE Cellulose and more than 50% of the colour is bound by Phosphoryl Cellulose. Class IV: More than 50% of the colour is bound by DEAE Cellulose and it exhibits an Absorbance Ratio of not more than 50. See description under TESTS. 5.2. Purity Note: Arsenic and lead metals limits apply to all classes of caramel and are expressed on the basis of the product as is: Other limits and ranges apply to the individual classes as indicated and, unless otherwise stated, are expressed on a solids basis. Solid content Class I: 62-77% Class III: 53-83% ... ... ... Colour intensity Class I: 0.01-0.12 Class III: 0.08-0.36 Class IV: 0.10-0.60 Total nitrogen Class I: max 0.1% Class III: 1.3 -6.8% Class IV: 0.5-7.5% Total sulfur ... ... ... Class III: max 0.3% Class IV: 1.4-10.0% Sulfur dioxide Class I: - Class III: - Class IV: max 0.5% Ammoniacal nitrogen ... ... ... Class III: max 0.4% Class IV: max 2.8% 4-Methylimidazole (MEI) Class I: - Class III: max 300 mg/kg & max 200 mg/kg on an equivalent colour basis Class IV: max 1000 mg/kg & max 250 mg/kg on an equivalent colour basis 2-Acetyl-4-tetrahydroxybutylimidazole (THI) ... ... ... Class III: max 40 mg/kg & max 25 mg/kg on an equivalent colour basis. Class IV: - Arsenic Not more than 1 mg/kg Lead Not more than 2 mg/kg 6. Tests 6.1. Identification ... ... ... Classification/Colour bound by DEAE Cellulose For the purposes of this specification, colour bound by DEAE cellulose is defined as the percentage of decrease in absorbance of a caramel colour solution at 560 nm after treatment with DEAE Cellulose. Special reagent: DEAE (diethylaminoethyl) Cellulose of 0.7 meq/gram capacity, e.g. Cellex D from Bio-Rad or equivalent DEAE Celluloses of higher or lower capacities in proportionately higher or lower quantities. Procedure: Prepare a caramel colour solution of approximately 0.5 absorbance unit at 560 nm by transferring an appropriate amount of caramel colour into a 100- ml volumetric flask with the aid of 0.025 N hydrochloric acid. Dilute to volume with 0.025 N hydrochloric acid and centrifuge or filter, if solution is cloudy. Take a 20 ml aliquot of the caramel colour solution, add 200 mg of DEAE Cellulose, mix thoroughly for several min, centrifuge or filter, and collect the clear supernatant. Determine the absorbance of the caramel colour solution and the supernatant in a 1-cm cell at 560 nm, with a suitable spectrophotometer previously standardized using 0.025 N hydrochloric acid as reference. Calculate the percentage of Colour Bound by DEAE Cellulose by the formula: where: ... ... ... A2 is the absorbance of the supernatant after DEAE Cellulose treatment at 560 nm. Classification/Colour bound by phosphoryl cellulose For the purposes of this specification colour bound phosphoryl cellulose is defined as the percentage of decrease in absorbance of a caramel colour solution at 560 nm after treatment with Phosphoryl Cellulose. Special reagent: Phosphoryl Cellulose of 0.85 meq/gram capacity, e.g. Cellex P from Bio-Rad or equivalent Phosphoryl Celluloses of higher or lower capacities in proportionately higher or lower quantities. Procedure: Transfer 200-300 mg of caramel colour into a 100-ml volumetric flask, dilute to volume with 0.025 N hydrochloric acid, and centrifuge or filter, if solution is cloudy. Take a 40 ml aliquot of the caramel colour solution, add 2.0 g Phosphoryl Cellulose and mix thoroughly for several min. Centrifuge or filter, and collect the clear supernatant. Determine the absorbance of the caramel colour solution and the supernatant in a 1-cm cell at 560 nm, with a suitable spectrophotometer previously standardized using 0.025 N hydrochloric acid as reference. Calculate the percentage of Colour Bound by Phosphoryl Cellulose by the formula: ... ... ... A1 is the absorbance of the caramel colour solution at 560 nm A2 is the absorbance of the supernatant after Phosphoryl Cellulose treatment at 560 nm. Classification/ Absorbance ratio For the purposes of this specification, Absorbance Ratio is defined as the absorbance of caramel colour at 280 nm divided by the absorbance of caramel colour at 560 nm. Procedure: Transfer 100 mg of caramel colour into a 100-ml volumetric flask with the aid of water, dilute to volume, mix and centrifuge if solution is cloudy. Pipet a 5.0 ml portion of the clear solution into a 100-ml volumetric flask, dilute to volume with water, and mix. Determine the absorbance of the 0.1% solution in a 1-cm cell at 560 nm and that of the 1:20 diluted solution at 280 nm with a suitable spectrophotometer previously standardized using water as reference. (A suitable spectrophotometer is one equipped with a monochromator to provide a band width of 2 nm or less and of such quality that the stray-light characteristic is 0.5% or less.) Calculate the Absorbance Ratio of the caramel colour by dividing the absorbance units at 280 nm multiplied by 20 (dilution factor) by the absorbance units at 560 nm. 6.2. Purity ... ... ... Solid content The solids content of Caramel Colour is determined by drying a sample upon a carrier composed of pure quartz sand that passes a No. 40 but not a No. 60 sieve and has been prepared by digestion with hydrochloric acid, washed acid-free, dried and ignited. Mix 30.0 g of prepared sand accurately weighed with 1.5-2.0 g Caramel Colour accurately weighed and dry to constant weight at 60o under reduced pressure 50 mm/Hg (6.7 kPa). Record the final weight of the sand plus caramel. Calculate the % solids as follows: % solids = where: m3 = final weight of sand plus caramel (g) m2 = weight of sand (g) m1 = weight of caramel initially added (g) Calculation on a solids basis ... ... ... Ci ´ 100 % solids Cs = Colour intensity For the purpose of this specification, Colour Intensity is defined as the absorbance of a 0.1% (w/v) solution of Caramel Colour solids in water in a 1 cm cell at 610 nm. Procedure: ... ... ... A610 ´ 100 % solids Colour intensity = Determine % solids as described under Solids content. Calculation on an equivalent colour basis: Where additional limits for 4-MEI and THI are expressed on an equivalent colour basis the concentrations are first calculated on a solids basis as directed under "Calculations on a solids basis", and then expressed on an equivalent colour basis according to the formula: CS ´ 0,1 Colour intensity Equivalent colour basis = ... ... ... where: CS = concentration on a solids basis. This gives content expressed in terms of a product having a Colour Intensity of 0.1 absorbance units. Total nitrogen Tested as directed under JECFA monograph 1 - Vol. 4- Determine as directed under Nitrogen Determination (Kjeldahl Method) using Method II Total sulfur In the largest available casserole that fits in an electric muffle furnace, place 1-3 g MgO or equivalent quantity of Mg(NO3)2·6H2O (6.4 - 19.2 g), 1 g powdered sucrose, and 50 ml HNO3. Add 5-10 g caramel colour. Place same quantities of reagents in another casserole for blank. Evaporate on steam bath to paste. Place casserole in cold electric muffle (25º) and gradually heat until all NO2 fumes are driven off. Cool, dissolve and neutralize with HCl (1+2.5), adding excess of 5 ml. Filter, heat to boiling, and add 5 ml 10% BaCl2·2H2O solution dropwise. Evaporate to 100 ml, let stand overnight, filter, wash, ignite, and weigh the BaSO4. Correct result for BaSO4 obtained in blank and report as mg S/100 g. Commercial instruments that analyse for total sulfur such as, the Leco-Combustion/Titration procedure can also be used and are recommended for sample amounts of about 200 mg. ... ... ... Apparatus Use a modified Monier-Williams apparatus (available from 5GA Scientific, Inc., Bloomfield, N.J., USA) for the determination of sulfurous acid, or construct the apparatus as shown in the figure. The assembly consists of a 1000-ml three-neck round-bottom distillation flask having 24/40 standardtaper ground-glass joints. A 30-cm Allihn condenser is attached in the reflux position to an outer neck of the flask, and the other end of the condenser is connected with 1/4-inch. Tygon or silicon tubing (preboiled with 1 in 20 hydrochloric acid solution and rinsed with water) to the absorption tube assembly (having 35/20 ball joints or the equivalent). Connect the centre neck of the flask with a 125-ml cylindrical separator, and attach a piece of tubing to a short U-tube inserted through a rubber stopper in the neck of the separator. Attach a curved glass inlet tube, reaching nearly to the bottom of the flask, to the other outer neck of the flask, and connect the inlet tube to a 250-ml gas-washing bottle with a piece of the tubing. The gas-washing bottle, in turn, is connected by tubing to a nitrogen cylinder. Grind 4.5 g of pyrogallol (pyrogallic acid) with 5 ml of water in a small mortar, and transfer the slurry to the gas-washing bottle. Grind the residue again, and transfer it quantitatively to the bottle with two 5-ml portions of water. Pass nitrogen from the cylinder to the bottle to flush out air, and then add to the bottle, through a long-stem funnel, a cooled solution of 65 g of potassium hydroxide in about 85 ml of water. Place the head of the bottle in position, and bubble nitrogen through it to remove air from the headspace. Clamp off the tubing on both sides of the bottle, and connect it to the glass inlet tube of the distillation flask. The gas-washing bottle must be prepared with fresh pyrogallol solution as described on the day of use. To each U-tube of the absorption tube assembly add the following: two pieces of 8-mm glass rod about 25 mm in length, 10 ml of 3-mm glass beads at the exit side, 10.0 ml of 3% hydrogen peroxide solution, and 1 drop of methyl red TS. Assemble all pieces of the apparatus, and check for leaks by blowing gently into the tubing attached to the neck of the separator. While blowing, close the stopcock of the separator. Let stand for a few min; if the liquid levels in the U-tubes equalize, reseal all connections and test again. If the system is airtight proceed as directed below. Procedure: ... ... ... When the joint at the top of the condenser cools, remove the connecting assembly and rinse it into the second U-tube, leaving the crossover tube attached to the exit joint of the first U-tube but disconnected from the entrance of the second U-tube. Rotate the crossover tube until the free end almost touches the entrance of the first U-tube. Add 1 drop of methyl red TS to the first U-tube, and titrate with 0.1 N sodium hydroxide just to a clear yellow colour, mixing with a gentle rocking motion. After titrating the first Utube, remove the crossover tube, attach it to the second U-tube exit, and titrate similarly. Record the sum of the two titers as Vt, in ml. Perform a blank determination, and record the volume of 0.1 N sodium hydroxide required as Vo. Calculate the percentage of sulfur dioxide in the sample by the formula Where: m is the weight of the sample taken, in g. Ammoniacal nitrogen Add 25 ml of 0.1 N sulfuric acid to a 500-ml receiving flask, and connect it to a distillation apparatus consisting of a Kjeldahl connecting bulb and a condenser such that the condenser delivery tube is immersed beneath the surface of the acid solution in the receiving flask. Transfer about 2 g of caramel colour, accurately weighed, into an 800-ml long-neck Kjeldahl digestion flask, and to the flask add 2 g of magnesium oxide (carbonatefree), 200 ml of water, and several boiling chips. Swirl the digestion flask to mix the contents, and quickly connect it to the distillation apparatus. Heat the digestion flask to boiling, and collect about 100 ml of distillate in the receiving flask. Wash the tip of the delivery tube with a few ml of water, collecting the washings in the receiving flask, then add 4 or 5 drops of methyl red indicator (500 mg of methyl red in 100 ml of alcohol), and titrate with 0.1 N sodium hydroxide, recording the volume, in ml, required as Vt. Conduct a blank determination, and record the volume, in ml, of 0.1 N sodium hydroxide required to neutralize as Vo. Calculate the percentage of ammoniacal nitrogen in the sample by the formula: Ammoniacal nitrogen ... ... ... m is the weight of the sample taken, in g. 4-Methylimidazole Note: Information on an improved method is sought. The following materials and reagents are required (the reagents should be ACS grade or equivalent where applicable). Materials Pyrex glasswool, 22 x 300 mm chromatography column with PTFE stopcock (e.g. Kimax 17800); 150 ml polypropylene beaker (e.g. Nalge 1201); 250 ml round-bottom flask (e.g., Pyrex 4320); 75 mm powder funnel; 5 cm spatula; rotary vacuum evaporator; hot plate; pan for water bath; disposable Pasteur pipets; 5 ml volumetric flask. Reagents  Acetone; Celite 545; methylene chloride; sodium hydroxide; and tetrahydrofuran. ... ... ... After thoroughly mixing the caramel colour sample by shaking or stirring, weigh a 10.00 g aliquot into a 150 ml polypropylene beaker. Polypropylene is considered superior to glass because of its hydrophobic surface which facilitates quantitative sample transfer. A 5.0 g portion of 3.0 N NaOH is added and thoroughly mixed to ensure that the pH of the entire sample exceeds 12. A 20 g portion of Celite 545 is added to the beaker, and the contents are mixed until a semi-dry mixture is obtained. This normally requires approximately 2 to 3 min. With samples of unusually high water content, the resultant caramel colour-Celite 545 mixture may be overly wet. In such cases, a 5.00 g aliquot of caramel colour may be mixed with 2.5 g of 3.0 N NaOH and 15 g of Celite 545 and carried through the remainder of the analysis. A plug of Pyrex glasswool is placed in the bottom of a 22 x 300 mm chromatographic column with PTFE stopcock. The caramel colour-Celite 545 mixture is placed in the column with the aid of a 75 mm powder funnel. The column contents are settled by repeatedly allowing the column to fall vertically about 10 cm to a padded surface. When properly settled, the caramel colour-Celite 545 mixture should occupy approximately the lower 250 mm of the column. Care should be exercised at this point to avoid a column bed which is either too loosely or too tightly packed. Loose packing will allow too rapid elution of the methylene chloride and possibly incomplete extraction. A too tightly packed column, e.g., the result of tamping down the column contents, can result in regions of the bed which are relatively inaccessible to the extraction solvent. This can also result in incomplete extraction. With the stopcock open, the column is filled with methylene chloride poured from the sample beaker. When the solvent reaches the glasswool plug, the stopcock is closed and the solvent is allowed to stand in contact with the bed for 5 min. The stopcock is then opened and the column is further eluted with methylene chloride until 200 ml have been collected in a 250 ml roundbottom flask. A 1.00 ml aliquot of 2 MEI internal standard solution (50.0 mg of 2 MEI/50.0 ml of methylene chloride) is added to the collected eluate. The 2 MEI is well separated from the 4 MEI under the GLC conditions employed and has not been found in caramel colour. The bulk of the solvent is then removed from the eluate on a rotary vacuum evaporator operated at 45-50 kPa and with the round-bottom flask maintained at 35^o in a water bath. The extracted residue is transferred quantitatively to a 5 ml volumetric flask with a disposable Pasteur pipet, by rinsing the round-bottom flask several times with small (ca. 0.75 ml) portions of either tetrahydrofuran or acetone. Both solvents have been used with equal success. After mixing the contents thoroughly by several inversions of the flask, the extract is ready for GLC analysis. The extracts should be analysed as soon as possible after their preparation, because stability problems have occasionally been encountered with extracts more than 1 day old. The GLC analysis is carried out using a gas chromatograph equipped with a hydrogen flame detector. The column is glass, 1 mm x 6 mm o.d. x 4 mm i.d., filled with 7.5% Carbowax 20M + 2% K0H on 90/100 mesh Anakrom ABS. The GLC parameters are as follows: carrier, nitrogen, 50 ml/min; hydrogen, 50 ml/min; oxygen, 80 ml/min; injection port, 200^o ; column isothermal, 180^o ; detector, 250^o ; sample size, 5 µl. All quantitation is done by using the internal standard technique. 2-Acetyl-4-tetrahydroxybutylimidazole (THI) Note: Information on an improved method is sought. THI is converted into its 2,4-dinitrophenylhydrazone (THI-DNPH). This derivative is separated from excess reagent and carbonylic contaminants by HPLC on RP-8, then determined by its absorbance at 385 nm. ... ... ... Caramel colour (200-250 mg) is weighed accurately, then dissolved in water (3 ml). The solution is transferred quantitatively to the upper part of a Combination Column. Elution with water is started, and a total of about 100 ml of water is passed through the columns. The upper column is then disconnected. The lower column is eluted with 0.5 N HCl. The first 10.0 ml of eluate are discarded, then a volume of 35 ml is collected. The solution is concentrated to dryness at 40^o and 15 torr. The syrup residue is dissolved in carbonyl-free methanol (250 µl) and the 2,4- dinitrophenylhydrazine reagent (250 µl) is added. The reaction mixture is transferred to a septum-capped vial and stored for 5 h at room temperature. A volume of 5 µl (but also from 1 to 25 µl) is injected onto a LiChrosorb RP-8 (10 µm) HPLC column. The mobile phase is MeOH/0.1 M H3PO4 50/50 (v/v). Adjustments in mobile phase composition may be needed as column characteristics vary, depending upon the manufacturer. (Use of LiChrosorb RP-8, 10 µm, 250 x 4 mm "Vertex" column manufactured by Knauer, Bad Homburg, F.R.G. is strongly recommended). At a mobile phase flow rate of 2 ml/min and column dimensions of 250 x 4.6 mm, THI-DNPH is eluted at about 6.3±0.1 min. It is detected at 385 nm and the peak height is measured. The amount is calculated from a calibration curve prepared with THI-DNPH in methanol. Materials: - 2,4,-Dinitrophenylhydrazine hydrochloride reagent: Commercial 2,4- dinitrophenylhydrazine (5 g) is added to concentrated hydrochloric acid (10 ml) in a 100-ml Erlenmeyer flask, and the latter is gently shaken until the free base (red) is converted to the hydrochloride (yellow). Ethanol (100 ml) is added and the mixture is heated on a steam bath until all the solid has dissolved. After crystallization at room temperature, the hydrochloride is filtered off, washed with ether, dried at room temperature and stored in a desiccator. On storage the hydrochloride is slowly converted to the free base. The latter can be removed by washing with dimethoxyethane. The reagent is prepared by mixing 0.5 g of 2,4-dinitrophenylhydrazine hydrochloride in 15 ml of 5% methanol in dimethoxyethane for 30 min. It should be stored in the refrigerator and be checked periodically by HPLC. - Cation-exchange resin (strong): Dowex 50 AG x 8, H+ , 100-200 mesh. - Cation-exchange resin (weak): Amberlite CG AG 50 I, H+ , (100-200 mesh). (Sediment two or three times prior to use). - Methanol, carbonyl-free: Methanol is prepared after Y. Peleg and C.H. Mannheim, J. Agr. Fd. Chem, 18 (1970) 176, by treatment with Girard P reagent. - Dimethoxyethane: If impure, dimethoxyethane is purified by distillation from 2,4-dinitrophenylhydrazine in the presence of acid and redistilled from sodium hydroxide. Immediately prior to use it is passed through a column of neutral aluminium to remove peroxides. ... ... ... Combination Columns: Similar to the set-up described in J. Agr. Fd. Chem. 22 (1974) 110. The upper column (150 x 12.5 mm, filling height max. 9 cm, or 200 x 10 mm, filling height max. 14 cm, with capillary outlet of 1 mm i.d.) is filled with weakly acidic cation-exchanger; bed height, approx. 50-60, or 80-90 mm, respectively. The lower column (total length 175 mm, i.d. 10 mm, with capillary outlet and Teflon stopcock) is filled with strongly acidic cationexchanger to a bed-height of 60 mm. As a solvent reservoir, a dropping funnel (100 ml) with Teflon stopcock is used. All parts are connected by standard ground-glass joints (14.5 mm). HPLC: With the column specified above and an ultraviolet detector capable of reading at 385 nm. Calibration: THI-DNPH is dissolved in absolute, carbonyl-free methanol (about 100 mg/l; calculated concentration of THI: 47.58 ng/µl). A portion of this solution is diluted tenfold with methanol (4.7 ng THI/µl). THI-DNPH standard solutions are stable for at least twenty weeks when stored in the refrigerator. Arsenic - Tested as directed under JECFA monograph 1-Vol. 4. - Method II. Lead ... ... ... - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” ANNEX 20 SPECIFICATIONS AND TESTS FOR SUNSET BRILLIANT BLUE FCF 1. Synonyms CI food Black 1, Black PN; CI (1975): 28440 INS: 151 ADI = 0 - 1 mg/kg bw. 2. Definition ... ... ... May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Tetrasodium 4-acetamido-5-hydroxy-6-[7-sulfonato-4-(4-sulfonatophenylazo)- 1-naphthylazo]-1,7-naphthalene-disulfonate C.A.S. number 2519-30-4 Chemical formula C28H17N5Na4O14S4 Structural formula ... ... ... 867.69 3. Description Black powder or granules 4. Functional uses Colour. 5. Specifications 5.1. Identification Solubility ... ... ... Identification of colouring matters Passes test. 5.2. Purity Loss on drying at 135 °C Not more than 20% together with chloride and sulfate calculated as sodium salts. Water insoluble matter Not more than 0.2%. Lead ... ... ... Subsidiary colouring matters  Not more than 4%. Organic compounds other than colouring matters Not more than 0.8%, sum of 4-Acetamido-5-hydroxy-1,7-naphthalenedisulfonic acid, 4-Amino-5-hydroxy-1,7-naphthalenedisulfonic acid, 8-Amino-2-naphthalenesulfonic acid, Sulfanilic acid, 4,4'-Diazoaminodi(benzenesulfuric acid) Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. Ether extractable matter Not more than 0.2%. 5.3. Content ... ... ... 6. Tests 6.1. Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” Subsidiary colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use the following conditions: ... ... ... Chromatogram (ii): No. 4. Height of ascent of solvent front: (i): approximately 17 cm (ii): approximately 17 cm Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use liquid chromatography under the following conditions: HPLC elution gradient: 2 to 100% at 2% per min (linear). 6.2. Method of assay ... ... ... Weight of sample: 0.6 - 0.7 g; Buffer: 15 g sodium hydrogen tartrate; Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 10.86 mg. ANNEX 21 SPECIFICATIONS AND TESTS FOR BROWN HT 1. Synonyms CI Food Brown 3, Chocolate brown HT, CI (1975) No. 20285 INS: 155 ... ... ... 2. Definition Consists essentially of disodium 4,4'- (2,4-dihydroxy-5-hydroxymethyl-1,3- phenylene-bisazo) di-1-naphthalene-sulfonate and subsidiary colouring matters together with NaCl and/or Na2SO4 as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium 4,4'-(2,4-dihydroxy-5-hydroxymethyl-1,3-phenylene-bisazo di-1- naphthalenesulfonate. C.A.S. number 4553-89-3 Chemical formula C27H18N4Na2O9S2 ... ... ... Formula weight 652.57 3. Description Brown powder or granules. 4. Functional uses Colour. 5. Specifications 5.1. Identification ... ... ... Solubility Soluble in water; insoluble in ethanol. Identification of colouring matters Passes test.   5.2. Purity Loss on drying at 135 °C Not more than 30% together with chloride and sulfate calculated as sodium salts. Water insoluble matter ... ... ... Lead  Not more than 2 mg/kg. Subsidiary colouring matters  Not more than 10%. Organic compounds other than colouring matters Not more than 0.7% of 4-aminonaphthalene-1-sulfonic acid. Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. Ether extractable matter ... ... ... 5.3. Content Not less than 70% total colouring matter. 6. Tests 6.1 Purity Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” Subsidiary colouring matters ... ... ... - Use the following conditions: Prepare the standard in the following manner: Dilute 1.0 ml of the 1% dye solution to 100 ml with water and mix well. Transfer 0.10 ml of this solution to a test tube; add 5.0 ml of water: acetone (1:1 by vol.) and then 14.9 ml of 0.05 N sodium hydrogen carbonate solution and shake the tube to ensure mixing. Determine the net absorbance (AS) of the standard. Developing solvent: No.6. Develop chromatogram for approximately 14 h. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. - Use liquid chromatography under the following conditions: HPLC elution gradient: 2 to 100% at 2.0% per min. 6.2. Method of assay ... ... ... Proceed as directed under Total Content by Spectrophotometry (in JECFA monograph 1-Vol. 4), using the following conditions: Solvent: pH 7 phosphate buffer; Dilution of solution A: 10 ml to 250 ml. Absorptivity (a): 40.3 Approximate wavelength of maximum absorption: 460 nm. ANNEX 22 SPECIFICATIONS AND TESTS FOR β-CAROTENE, SYNTHETIC 1. Synonyms ... ... ... INS: 160ai ADI = 0 - 5 mg/kg bw. 2. Definition These specifications apply to predominantly all trans (Z) isomer of b-carotene together with minor amounts of other carotenoids; diluted and stabilized forms are prepared from b-carotene meeting these specifications and include solutions or suspensions of b-carotene in edible fats or oils, emulsions and water dispersible powders; these preparations may have different cis/trans isomer ratios; the analytical methods described for the parent colour are not necessarily suitable for the assay of or determination of impurities in the stabilized forms (appropriate methods should be available from the manufacturer). Chemical names b-Carotene, b,b-carotene C.A.S. number 7235-40-7 Chemical formula ... ... ... Structural formula Formula weight 536.88 3. Description Red to brownish-red crystals or crystalline powder; sensitive to oxygen and light and should therefore be kept in a light-resistant container under inert gas. 4. Functional uses Colour. 5. Specifications ... ... ... Solubility Insoluble in water; practically insoluble in ethanol; slightly soluble in vegetable oils; soluble in chloroform. Spectrophotometry Determine the absorbance of the sample solution C (See Method of Assay) at 455 nm and 483 nm. The ratio A455/A483 is between 1.14 and 1.19. Determine the absorbance of the sample solution C at 455 nm and that of sample Solution B (See Method of Assay) at 340 nm. The ratio A455/A340 is not lower than 15. Test for carotenoid ... ... ... Carr-Price reaction A solution of the sample in chloroform turns blue on addition of an excess of Carr-Price reagent TS. 5.2. Purity Sulfated ash Not more than 0.1%. Subsidiary colouring matters Carotenoids other than b-carotene: Not more than 3% of total colouring matters. Lead ... ... ... 5.3. Content Not less than 96% total colouring matters, expressed as b-carotene. 6. Tests 6.1. Identification Test for carotenoid The colour of a solution of the sample in acetone disappears after successive additions of a 5% solution of sodium nitrite and 1 N sulfuric acid. 6.2. Purity ... ... ... - Tested as directed under JECFA monograph 1-Vol. 4. - Test 2 g of the sample (Method I) Subsidiary colouring matters Carotenoids other than b-carotene Dissolve about 80 mg of sample in 100 ml chloroform. Apply 400 µl of this solution as a streak 2 cm from the bottom of a TLC-plate (Silicagel 0.25 mm). Immediately develop the chromatogram with a solvent mixture of 95 parts dichloromethane and 5 parts diethyl ether in a saturated chamber, suitably protected from light, until the solvent front has moved 15 cm above the initial streak. Remove the plate, allow the main part of the solvent to evaporate at room temperature and mark the principal band as well as the bands corresponding to other carotenoids. Remove the silicagel adsorbent that contains the principal band, transfer it to a glass-stoppered 100 ml centrifuge tube and add 40.0 ml chloroform (solution 1). Remove the silicagel adsorbent that contains the combined bands corresponding to the other carotenoids, transfer it to a glass-stoppered, 50 ml centrifuge tube and add 20.0 ml chloroform (solution 2). Shake the centrifuge tubes by mechanical means for 10 min and centrifuge for 5 min. Dilute 10.0 ml of Solution 1 to 50.0 ml with chloroform (solution 3). Determine, with a suitable spectrophotometer, the absorbances of Solutions 2 and 3 in 1-cm cells at the wavelength maximum about 464 nm, using chloroform as blank. Calculation: ... ... ... where: A2 = absorbance of Solution 2 A3 = absorbance of Solution 3 Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” 6.2. Method of assay ... ... ... Proceed as directed under Total Content by Spectrophotometry in JECFA monograph 1-Vol. 4 using the following conditions: W = 0.08 g V1 = V2 = V3 = 100 ml v1 = v2 = 5 ml A^1%1cm = 2500 lmax ~ 456 nm. ANNEX 23 SPECIFICATIONS AND TESTS FOR CAROTENES, VEGETABLE ... ... ... Natural b-carotene, carotenes-natural; CI Food Orange 5, mixed carotenes; CI (1975) No. 75130; CI (1975) No. 40800 (b-Carotene). INS 160aii ADI "acceptable", provided the level of use does not exceed the level normally found in vegetables. 2. Definition Carotenes (vegetable) are obtained by solvent extraction of carrots (Daucus carota), oil of palm fruit (Elaeis guinensis), sweet potato (Ipomoea batatas) and other edible plants with subsequent purification. The main colouring principles are a- and b-carotenes of which b-carotenes account for the major part. Minor amounts of g- and d-carotenes and other pigments may be present. Besides the colour pigments, this substance may contain oils, fats and waxes naturally occurring in the source material. The only solvents used for the extraction are acetone, methanol, ethanol, propan-2-ol, hexane, carbon dioxide and vegetable oils. The main articles of commerce are solutions or suspensions in food grade vegetable/plant oil. This is for ease of use and to improve stability as carotenes easily oxidise. Class Carotenoid C.A.S. number ... ... ... Chemical formula C40H56 (-β carotene) Structural formula All-trans-b-carotene Formula weight 536.88 (b-Carotene) 3. Description Red-brown to brown or orange to dark orange solid or liquid. ... ... ... Colour. 5. Specifications 5.1. Identification Solubility Insoluble in water. Spectrophotometry A cyclohexane solution of the sample (1 in 200,000) shows maximum absorptions at 440-457 and 470-486 nm. Colour reaction ... ... ... 5.2. Purity Residual solvents Not more than 50 mg/kg, singly or in combination, of acetone, hexane, methanol, ethanol and propan-2-ol. Lead Not more than 2 mg/kg. 5.3. Content Content of carotenes (calculated as b-carotene) is not less than declared. 6. Tests ... ... ... Colour reaction A spot of a solution of the sample in toluene (about 400 µg /ml of ß-carotene) on a filter paper turns blue 2-3 min after application of a spray or drop of 20% solution of antimony trichloride solution in toluene. 6.2 Purity Lead Tested as directed under JECFA monograph 1-Vol. 4. Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” 6.3. Method of assay ... ... ... Proceed as directed under Total Content by Spectrophotometry in JECFA monograph 1-Vol. 4 using the following conditions: W = amount of the sample to obtain adequate absorbance (g) V1 = V2 = V3 = 100 ml v1 = v2 = 5 ml A^1%1cm = 2500 lmax = 440 - 457 nm. ANNEX 24 SPECIFICATIONS AND TESTS FOR CARAMEL EXTRACTS ... ... ... Annatto F; Orlean; Terre orellana; R. Orange; CI (1975) No. 75120 (Natural Orange 4). INS 160bii ADI = 0 - 12 mg/kg bw (for bixin); ADI = 0 - 0.6 mg/kg bw (for norbixin and its salts) The colouring matters bixin and norbixin derived from annatto extracts (solvent-extracted bixin; solvent-extracted norbixin; aqueous-processed bixin; alkali-processed norbixin, acid-precipitated; and alkali-processed norbixin, not acid-precipitated) are included in the ADIs for bixin and norbixin. 2. Definition Alkali-processed norbixin (acid-precipitated) is prepared by removal of the outer coating of the seeds of the annatto tree (Bixa orellana L) with aqueous alkali. The bixin is hydrolysed to norbixin in hot alkaline solution and is acidified to precipitate the norbixin. The precipitate is filtered, dried and milled to give a granular powder. Alkali-processed norbixin contains several coloured components; the major colouring principle is cis-norbixin, a minor colouring principle is trans-norbixin; thermal degradation products of norbixin may also be present as a result of processing. Products supplied to the food industry may be formulated with appropriate carriers of food grade quality. ... ... ... cis-Norbixin: 6,6'-Diapo-Ψ,Ψ-carotenedioic acid cis-Norbixin dipotassium salt: Dipotassium 6,6'-diapo-Ψ,Ψcarotenedioate cis-Norbixin disodium salt: Disodium 6,6'-diapo-Ψ,Ψ-carotenedioate C.A.S. number cis-Norbixin: 542-40-5 cis-Norbixin dipotassium salt: 33261-80-2 cis-Norbixin disodium salt: 33261-81-3 Chemical formula C24H28O4; C24H26K2O4; C24H26Na2O4 ... ... ... Formula weight 380.5 (acid), 456.7 (dipotassium salt), 424.5 (disodium salt) 3. Description Dark red-brown to red-purple powder. 4. Functional uses Colour. 5. Specifications 5.1. Identification ... ... ... Solubility Soluble in alkaline water, slightly soluble in ethanol. UV/VIS absorption The sample in 0.5% potassium hydroxide solution shows absorbance maxima at about 453 nm and 482 nm. Thin-layer chromatography Passes test (described in “Tests”). 5.2. Purity Arsenic ... ... ... Lead Not more than 2 mg/kg. Mercury Not more than 1 mg/kg. 5.3. Content Not less than 35% colouring matter (expressed as norbixin) 6. Tests 6.1. Identification ... ... ... Activate a TLC plate (e.g. LK6D SILICA GEL 60 A (layer thickness: 250 μm, size: 5 x 20 cm)) for 1 h at 110˚. Prepare a 5% solution of the sample in 95% ethanol and apply 10 µl to the plate. Allow to dry and develop using a mixture of n-butanol, methyl ethyl ketone and 10% aqueous ammonia (3:2:2 by volume) until the solvent front has ascended about 10 cm. Allow to dry. Bixin and norbixin appear as yellow spots with Rf values of about 0.50 to 0.45, respectively. Spray with 5% sodium nitrite solution and then with 0.5 mol/l sulfuric acid and the spots immediately decolourise. 6.2 Purity Arsenic - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an ICP-AES/AAS-Hydride technique. Alternatively, determine arsenic using Method II of the Arsenic Limit Test. The selection of sample size and method of sample preparation may be based on the principles of the methods described in JECFA monograph 1-Vol. 4. Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an AAS ICP-AES technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the methods described in JECFA monograph 1-Vol. 4. ... ... ... - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using cold vapour atomic absorption technique. Select sample size appropriate to the specified level. 6.3. Method of assay Proceed as directed in Food Colours, Colouring Matters Content by Spectrophotometry in JECFA monograph 1-Vol. 4 - procedure 1, using the following conditions: Solvent: 0.5 % potassium hydroxide Absorbance at lmax: about 482 nm. Specific absorbance A^1%1cm = 2870. ... ... ... ANNEX 25 SPECIFICATIONS AND TESTS FOR β-APO-CAROTENAL 1. Synonyms CI food Orange 6; CI (1975) No. 40820 INS 160e ADI = 0 - 5 mg/kg bw. 2. Definition Specifications apply to predominantly all trans (Z) isomer of b-apo-8'-carotenal together with minor amounts of other carotenoids; diluted and stabilized forms are prepared from b-apo-8'-carotenal meeting these specifications and include solutions or suspensions of b-apo-8'-carotenal in edible fats or oils, emulsions and water dispersible powders; these preparations may have different cis/trans isomer ratios; the analytical methods described for the parent colour are not necessarily suitable for the assay of or determination of impurities in the stabilized forms (appropriate methods should be available from the manufacturer). Chemical names ... ... ... C.A.S. number 1107-26-2 Chemical formula C30H40O Structural formula Formula weight 416.65 3. Description ... ... ... 4. Functional uses Colour. 5. Specifications 5.1. Identification Solubility Insoluble in water, slightly soluble in ethanol, sparingly soluble in vegetable oils, soluble in chloroform. Spectrophotometry Determine the absorbance of the sample solution (See Method of Assay) at 461 nm and 488 nm. ... ... ... Carotenoid Passes test (described in “Tests”). Carr-Price reaction A solution of the sample in chloroform turns blue on addition of an excess of Carr-Price reagent TS. 5.2. Purity Sulfated ash Not more than 0.1%. Subsidiary colouring matters ... ... ... See description under TESTS. Lead  Not more than 2 mg/kg. 5.3. Content Not less than 96% of total colouring matters. 6. Tests 5.1. Identification Carotenoid ... ... ... 6.2. Purity Sulfated ash - Tested as directed under JECFA monograph 1. Vol.4. - Test 2 g of the sample (Method I) Subsidiary colouring matters Dissolve about 80 mg of sample in 100 ml chloroform. Apply 400 µl of this solution as a streak 2 cm from the bottom of a TLC-plate (Silicagel 0.25 mm). Pretreat the thin-layer plate by soaking in a tank with 3% KOH in methanol so that it is completely wetted. Then dry the plate for 5 min in the air and activate for 1 h at 110^o in an oven. Let cool over CaCl2 and keep in a desiccator over CaCl2. Immediately after applying the carotenoid solution to the plate, develop the chromatogram with n-hexane/chloroform/ethylacetate (70+20+10) in a saturated chamber suitably protected from light, until the solvent front has moved 10 cm above the initial streak. Remove the plate, allow the main part of the solvent to evaporate at room temperature and mark the principal band as well as the bands corresponding to other carotenoids Remove the silicagel adsorbent that contains the principal band, transfer it to a glass-stoppered 100 ml centrifuge tube and add 40.0 ml chloroform (solution 1). ... ... ... Shake the centrifuge tubes by mechanical means for 10 min and centrifuge for 5 min. Dilute 10.0 ml of Solution 1 to 50.0 ml with chloroform (solution 3). Determine, with a suitable spectrophotometer, the absorbances of Solutions 2 and 3 in 1-cm cells at the wavelength maximum in chloroform at about 474 nm, using chloroform as a blank. Calculation: Carotenoids other than b-apo-8'-carotenal: where: A2 = absorbance of Solution 2 A3 = absorbance of Solution 3 Lead - Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... 6.3. Method of assay Weigh accurately about 80 mg of the sample and proceed as directed under Total Content by Spectrophotometry in JECFA monograph 1-Vol. 4 with the following conditions. Absorptivity (a) = 2640 lmax ~ 461 nm. ANNEX 26 SPECIFICATIONS AND TESTS FOR β-APO-8'-CAROTENOIC ACID ETHYL ESTER ... ... ... CI food Orange 7; CI (1975) No. 40825 INS 160f ADI = 0 - 5 mg/kg bw. 2. Definition Specifications apply to predominantly all trans (Z) isomer of ß-apo-8'- carotenoic acid ethyl ester together with minor amounts of other carotenoids; diluted and stabilized forms are prepared from ß-apo-8'-carotenoic acid ethyl ester meeting these specifications and include solutions or suspensions of ßapo-8'-carotenoic acid ethyl ester in edible fats or oils, emulsions and water dispersible powders; these preparations may have different cis/trans isomer ratios. The analytical methods described for the parent colour are not necessarily suitable for the assay of or determination of impurities in the stabilized forms (appropriate methods should be available from the manufacturer). Chemical names b-Apo-8'-carotenoic acid ethyl ester, ethyl 8'-apo-b-caroten-8'-oate. C.A.S. number 1109-11-1 ... ... ... C32H44O2 Structural formula Formula weight 460.70 3. Description Red to violet-red crystals or crystalline powder; sensitive to oxygen and light and should therefore be kept in a light-resistant container under inert gas. 4. Functional uses Colour. ... ... ... 5.1. Identification Solubility Insoluble in water, very slightly soluble in ethanol, slightly soluble in vegetable oils, soluble in chloroform. Spectrophotometry Determine the absorbance of the sample solution (See Method of Assay) at 449 nm and 475 nm. The ratio A^475/A^449 is between 0.82 and 0.86. Carotenoid Passes test (described in “Tests”). ... ... ... A solution of the sample in chloroform turns blue on addition of an excess of Carr-Price reagent TS. 5.2. Purity Sulfated ash Not more than 0.1%. Subsidiary colouring matters Not more than 3% of total colouring matters. Lead  Not more than 2 mg/kg. ... ... ... Not less than 96% of total colouring matters. 6. Tests 5.1. Identification Carotenoid The colour of a solution of the sample in acetone disappears after successive additions of a 5% solution of sodium nitrite and 1 N sulfuric acid. 6.2. Purity Sulfated ash ... ... ... Subsidiary colouring matters Carotenoids other than ß-apo-8'-carotenoic acid ethyl ester: Dissolve about 80 mg of sample in 100 ml chloroform. Apply 400 µl of this solution as a streak 2 cm from the bottom of a TLC-plate (Silicagel 0.25 mm). Pretreat the thin-layer plate by soaking in a tank with 3% KOH in methanol so that it is completely wetted. Then dry the plate for 5 min in the air and activate for 1 h at 110^o in an oven. Let cool over CaCl2 and keep in a desiccator over CaCl2. Immediately after applying the carotenoid solution to the plate, develop the chromatogram with n-hexane/chloroform/ethylacetate (70+20+10) in a saturated chamber suitably protected from light, until the solvent front has moved 10 cm above the initial streak. Remove the plate, allow the main part of the solvent to evaporate at room temperature and mark the principal band as well as the bands corresponding to other carotenoids. Remove the silicagel adsorbent that contains the principal band, transfer it to a glass-stoppered 100 ml centrifuge tube and add 40.0 ml chloroform (solution 1). Remove the silicagel adsorbent that contains the combined bands corresponding to the other carotenoids, transfer it to a glass-stoppered, 50 ml centrifuge tube and add 20.0 ml chloroform (solution 2). Shake the centrifuge tubes by mechanical means for 10 min and centrifuge for 5 min. Dilute 10.0 ml of Solution 1 to 50.0 ml with chloroform (solution 3). Determine, with a suitable spectrophotometer, the absorbances of Solutions 2 and 3 in 1-cm cells at the wavelength maximum in chloroform at about 455 nm, using chloroform as a blank. Calculation: Carotenoids other than b-apo-8'-carotenoic acid ethyl ester (%): ... ... ... A2 = absorbance of Solution 2 A3 = absorbance of Solution 3 Lead - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” 6.2. Method of assay Weigh accurately about 80 mg of the sample and proceed as directed under Total Content by Spectrophotometry in JECFA monograph 1-Vol. 4 with the following conditions: ... ... ... lmax ~ 449 nm. ANNEX 27 SPECIFICATIONS AND TESTS FOR GRAPE SKIN EXTRACT 1. Synonyms Enociania, Eno, Grape skin extract. INS 163 (ii) ADI = 0 - 2.5 mg/kg bw. 2. Definition ... ... ... Chemical names The principal colouring matters are anthocyanins, glucosides of anthocyanidins (2-phenylbenzopyrylium salts) such as peonidin, malvidin, delphinidin, and petunidin. Chemical formula Peonidin : C16H13O6 X Malvidin : C17H15O7 X Delphinidin : C15H11O7 X Petunidin : C16H13O7 X X: acid moiety Structural formula ... ... ... Peonidin : R = OCH3 ; R’= H Malvidin : R, R’= OCH3 Delphinidin : R, R’= OH Petunidin : R = OCH3 ; R’= OH X: acid moiety 3. Description Purplish-red liquid, lump, powder or paste, having a slight characteristic odour. 4. Functional uses Colour ... ... ... 5.1. Identification Solubility Soluble in water Spectrophotometry At pH 3 the absorbance maximum is about 525 nm Colour reaction Passes test (described in “Tests”). 5.2. Purity ... ... ... Sulfur dioxide Not more than 0.005% per 1 colour value. Basic colouring matters Passes test (described in “Tests”). Other acidic colouring matters Passes test (described in “Tests”). Lead Not more than 2 mg/kg 5.3. Content ... ... ... 6. Tests 6.1. Identification Colour reaction Add 0.1 g of the sample to 50 ml of water and shake thoroughly. Filter if necessary. The solution shows red to purplish-red colour and it turns to blue or dark green on the addition of sodium hydroxide TS. 6.2. Purity Sulfur dioxide Distil 1 g of the sample with 100 ml of water and 25 ml of phosphoric acid solution (2 in 7) in a distilling flask with the Wagner tube (Figure 1). In an absorption flask, place 25 ml of lead acetate solution (1 in 50) previously prepared. Insert the lower end of condenser into lead acetate solution in the absorption flask. Distil until the liquid in the absorption flask reaches about 100 ml and rinse the end of the condenser with a little amount of water. To the distilled solution add 5 ml of hydrochloric acid and 1 ml of starch TS, and titrate with 0.01 N iodine. ... ... ... Figure 1 : Wagner Tube Basic colouring matters Add 1 g of the sample to 100 ml sodium hydroxide solution (1 in 100) and shake well. Take 30 ml of this solution and extract with 15 ml of ether. Extract this ether extract twice with each 5 ml of dilute acetic acid TS. The acetic acid extract is colourless. Other acidic colouring matters Add 1 ml of ammonia TS and 10 ml of water to 1 g of the sample and following the directions Chromatography place 0.002 ml of the solution on the chromatographic sheet and dry it. Use a mixture of pyridine and ammonia TS (2:1 by volume) as developing solvent and stop the development when the solvent front reaches about 15 cm height from the point where the sample solution was placed. No spot is observed at the solvent front after drying under daylight. If any spot is observed, it should be decolourized when sprayed with a solution of stannous chloride in hydrochloric acid (2 in 5). Lead ... ... ... 6.3. Method of assay In the absence of an assay method, a measurement of colour intensity by the following method may be used. Prepare approximately 200 ml of pH 3.0 citric acid - dibasic sodium phosphate buffer solution: Mix 159 volumes of 2.1% citric acid solution and 41 volumes of 0.16% dibasic sodium phosphate solution, and adjust the pH to 3.0, using the citric acid solution or Na2HPO4 solution. Weigh accurately an adequate amount of the sample so that the measured absorbance is between 0.2 and 0.7, and add pH 3.0 citric acid - dibasic sodium phosphate buffer solution to make up a 100-ml solution. Measure the absorbance A of this solution in a 1 cm cell at the wavelength of maximum absorption around 525 nm, using pH 3.0 citric acid - dibasic sodium phosphate buffer solution as the blank.  Colour value = m = weight of sample (g) ... ... ... SPECIFICATIONS AND TESTS FOR TITANIUM DIOXIDE 1. Synonyms Titania, CI pigment white 6, CI (1975) No. 77891 Titanium dioxide INS 171 ADI “Not limited”. 2. Definition Titanium dioxide is produced by either the sulfate or the chloride process. Processing conditions determine the form (anatase or rutile structure) of the final product. In the sulfate process, sulfuric acid is used to digest ilmenite (FeTiO3) or ilmenite and titanium slag. After a series of purification steps, the isolated titanium dioxide is finally washed with water, calcined, and micronized. ... ... ... Commercial titanium dioxide may be coated with small amounts of alumina and/or silica to improve the technological properties of the product. C.A.S. number 13463-67-7 Chemical formula TiO2 Formula weight 79.88 3. Description White to slightly coloured powder ... ... ... Colour 5. Specifications 5.1. Identification Solubility Insoluble in water, hydrochloric acid, dilute sulfuric acid, and organic solvents. Dissolves slowly in hydrofluoric acid and hot concentrated sulfuric acid. Colour reaction Passes test (described in “Tests”). ... ... ... Loss on drying Not more than 0,5%. At 105°, 3h. Loss on ignition Not more than 1.0% on the dried basis. At 800°. Aluminium oxide and/or silicon dioxide Not more than 2%, either singly or combined Acid-soluble substances Not more than 0.5%; Not more than 1.5% for products containing alumina or silica. ... ... ... Not more than 0,5%. Impurities soluble in 0.5 N hydrochloric acid Antimony Not more than 2 mg/kg Arsenic Not more than 1 mg/kg Cadmium Not more than 1 mg/kg ... ... ... Not more than 10 mg/kg Mercury Not more than 1 mg/kg 5.3. Method of assay Not less than 99.0% on the dried basis (on an aluminium oxide and silicon dioxide-free basis) 6. Tests 6.1. Identification ... ... ... Add 5 ml sulfuric acid to 0.5 g of the sample, heat gently until fumes of sulfuric acid appear, then cool. Cautiously dilute to about 100 ml with water and filter.  To 5 ml of this clear filtrate, add a few drops of hydrogen peroxide; an orange-red colour appears immediately. 6.2. Purity Acid-soluble substances Suspend 5 g of the sample in 100 ml 0.5 N hydrochloric acid and on steam bath for 30 min with occasional stirring. Filter through a Gooch crucible fitted with a glass fibre filter paper. Wash with three 10-ml portions of 0.5 N hydrochloric acid, evaporate the combined filtrate and washings to dryness and ignite at a dull red heat to constant weight. Water-soluble matter ... ... ... Impurities soluble in 0.5 N hydrochloric acid Antimony, arsenic, cadmium and lead Tested as directed under JECFA monograph 1-Vol. 4: General tests. Transfer 10.0 g of sample into a 250-mL beaker, add 50 ml of 0.5 N hydrochloric acid, cover with a watch glass, and heat to boiling on a hot plate. Boil gently for 15 min, then pour the slurry into a 100 to 150-mL centrifuge bottle, and centrifuge for 10 to 15 min, or until undissolved material settles. Decant the supernatant extract through a Whatman No. 4 filter paper, or equivalent, collecting the filtrate in a 100-mL volumetric flask and retaining as much as possible of the undissolved material in the centrifuge bottle. Add 10 ml of hot water to the original beaker, washing off the watch glass with the water, and pour the slurry into the centrifuge bottle. Form a slurry, using a glass stirring rod, and centrifuge. Decant through the same filter paper, and collect the washings in the volumetric flask containing the initial extract. Repeat the entire washing process two more times. Finally, wash the filter paper with 10 to 15 ml of hot water. Cool the contents of the flask to room temperature, dilute to volume with water, and mix. Determine antimony, cadmium, and lead using an AAS/ICP-AES technique appropriate to the specified level. Determine arsenic using the ICP-AES/AAS-hydride technique. Alternatively, determine arsenic using Method II of the Arsenic Limit Test, taking 3 g of the sample rather than 1 g. The selection of sample size and method of sample preparation may be based on the principles of the methods described in JECFA monograph 1-Vol. 4: general tests. Mercury ... ... ... Aluminium oxide Reagents and sample solutions 0.01 N Zinc Sulfate Dissolve 2.9 g of zinc sulfate (ZnSO4·7H2O) in sufficient water to make 1000 ml. Standardize the solution as follows: Dissolve 500 mg of high-purity (99.9%) aluminium wire, accurately weighed, in 20 ml of concentrated hydrochloric acid, heating gently to effect solution, then transfer the solution into a 1000-ml volumetric flask, dilute to volume with water, and mix. Transfer a 10 ml aliquot of this solution into a 500 ml Erlenmeyer flask containing 90 ml of water and 3 ml of concentrated hydrochloric acid, add 1 drop of methyl orange TS and 25 ml of 0.02 M disodium ethylenediaminetetraacetate (EDTA) Add, dropwise, ammonia solution (1 in 5) until the colour is just completely changed from red to orange-yellow. Then, add: (a): 10 ml of ammonium acetate buffer solution (77 g of ammonium acetate plus 10 ml of glacial acetic acid, dilute to 1000 ml with water) and (b): 10 ml of diammonium hydrogen phosphate solution (150 g of diammonium hydrogen phosphate in 700 ml of water, adjusted to pH 5.5 with a 1 in 2 solution of hydrochloric acid, then dilute to 1000 ml with water). Boil the solution for 5 min, cool it quickly to room temperature in a stream of running water, add 3 drops of xylenol orange TS, and mix. Using the zinc sulfate solution as titrant, titrate the solution to the first yellow-brown or pink end-point colour that persists for 5-10 sec. (Note: This titration should be performed quickly near the end-point by adding rapidly 0.2 ml increments of the titrant until the first colour change occurs; although the colour will fade in 5-10 sec, it is the true end-point. Failure to observe the first colour change will result in an incorrect titration. The fading end-point does not occur at the second end-point.) Add 2 g of sodium fluoride, boil the mixture for 2-5 min, and cool in a stream of running water. Titrate this solution, using the zinc sulfate solution as titrant, to the same fugitive yellow-brown or pink end-point as described above. ... ... ... T = 18,896 m / V where: T is the mass (mg) of Al2O3 per ml of zinc sulfate solution m is the mass (g) of aluminium wire V is the ml of the zinc sulfate solution consumed in the second titration 18.896 = (R × 1000 mg/g × 10 ml/2)/1000 ml and R is the ratio of the formula weight of aluminium oxide to that of elemental aluminium. Sample Solution A Accurately weigh 1 g of the sample and transfer to a 250-ml high-silica glass Erlenmeyer flask. Add 10 g of sodium bisulfate (NaHSO4·H2O). (Note: Do not use more sodium bisulfate than specified, as an excess concentration of salt will interfere with the EDTA titration later on in the procedure.) Begin heating the flask at low heat on a hot plate, and then gradually raise the temperature until full heat is reached. ... ... ... Sample Solution B Prepare 200 ml of an approximately 6.25 M solution of sodium hydroxide. Add 65 ml of this solution to Sample Solution A, while stirring with the magnetic stirrer; pour the remaining 135 ml of the alkali solution into a 500-ml volumetric flask. Slowly, with constant stirring, add the sample mixture to the alkali solution in the 500-ml volumetric flask; dilute to volume with water, and mix. (Note: If the procedure is delayed at this point for more than 2 hours, store the contents of the volumetric flask in a polyethylene bottle.) Allow most of the precipitate to settle (or centrifuge for 5 min), then filter the supernatant liquid through a very fine filter paper. Label the filtrate Sample Solution B. Sample Solution C Transfer 100 ml of the Sample Solution B into a 500-ml Erlenmeyer flask, add 1 drop of methyl orange TS, acidify with hydrochloric acid solution (1 in 2), and then add about 3 ml in excess. Add 25 ml of 0.02 M disodium EDTA, and mix. [Note: If the approximate Al2O3 content is known, calculate the optimum volume of EDTA solution to be added by the formula:  (4 ´ % Al2O3) + 5]. Add, dropwise, ammonia solution (1 in 5) until the colour is just completely changed from red to orange-yellow. Then add10 ml each of Solutions 1 and 2 (see above) and boil for 5 min. Cool quickly to room temperature in a stream of running water, add 3 drops of xylenol orange TS, and mix. If the solution is purple, yellow-brown, or pink, bring the pH to 5.3 - 5.7 by the addition of acetic acid. At the desired pH, a pink colour indicates that not enough of the EDTA solution has been added, in which case, discard the solution and repeat this procedure with another 100 ml of Sample Solution B, using 50 ml, rather than 25 ml, of 0.02 M disodium EDTA. Procedure Using the standardized zinc sulfate solution as titrant, titrate Sample Solution C to the first yellow-brown or pink end-point that persists for 5-10 sec. (Important: See Note under “0.01 Zinc sulfate”.) This first titration should require more than 8 ml of titrant, but for more accurate work a titration of 10-15 ml is desirable. ... ... ... Calculation: Calculate the percentage of aluminium oxide (Al2O3) in the sample taken by the formula: % Al2O3 = 100 × (0,005VT)/m where: V is the number of ml of 0.01 N zinc sulfate consumed in the second titration, T is the titre of the zinc sulfate solution, m is the mass (g) of the sample taken, and 0.005 = 500 ml / (1000 mg/g × 100 ml). Silicon dioxide ... ... ... Accurately weigh 1 g of the sample and transfer to a 250-ml high-silica glass Erlenmeyer flask. Add 10 g of sodium bisulfate (NaHSO4 · H2O). Heat gently over a Meeker burner, while swirling the flask, until decomposition and fusion are complete and the melt is clear, except for the silica content, and then cool. (Caution: Do not overheat the contents of the flask at the beginning, and heat cautiously during fusion to avoid spattering.) To the cooled melt add 25 ml of sulfuric acid solution (1 in 2) and heat carefully and slowly until the melt is dissolved. Cool, and carefully add 150 ml of water by pouring very small portions down the sides of the flask, with frequent swirling to avoid over-heating and spattering. Allow the contents of the flask to cool, and filter through fine ashless filter paper, using a 60 degree gravity funnel. Rinse out all the silica from the flask onto the filter paper with sulfuric acid solution (1 in 10). Transfer the filter paper and its contents into a platinum crucible, dry in an oven at 120°, and heat the partly covered crucible over a Bunsen burner. To prevent flaming of the filter paper, first heat the cover from above, and then the crucible from below.  When the filter paper is consumed, transfer the crucible to a muffle furnace and ignite at 1000^o for 30 min. Cool in a desiccator, and weigh. Add 2 drops of sulfuric acid (1 in 2) and 5 ml of concentrated hydrofluoric acid (sp.gr. 1.15), and carefully evaporate to dryness, first on a low-heat hot plate (to remove the HF) and then over a Bunsen burner (to remove the H2SO4). Take precautions to avoid spattering, especially after removal of the HF. Ignite at 1000^o for 10 min, cool in a desiccator, and weigh again. Record the difference between the two weights as the content of SiO2 in the sample. 6.3. Method of assay Accurately weigh about 150 mg of the sample, previously dried at 105^o for 3 hours, and transfer into a 500-ml conical flask. Add 5 ml of water and shake until a homogeneous, milky suspension is obtained. Add 30 ml of sulfuric acid and 12 g of ammonium sulfate, and mix. Initially heat gently, then heat strongly until a clear solution is obtained. Cool, then cautiously dilute with 120 ml of water and 40 ml of hydrochloric acid, and stir. Add 3 g of aluminium metal, and immediately insert a rubber stopper fitted with a U-shaped glass tube while immersing the other end of the U-tube into a saturated solution of sodium bicarbonate contained in a 500-ml wide-mouth bottle, and generate hydrogen. Allow to stand for a few minutes after the aluminium metal has dissolved completely to produce a transparent purple solution. Cool to below 50^o in running water, and remove the rubber stopper carrying the U-tube. Add 3 ml of a saturated potassium thiocyanate solution as an indicator, and immediately titrate with 0.2 N ferric ammonium sulfate until a faint brown colour that persists for 30 seconds is obtained. Perform a blank determination and make any necessary correction. Each ml of 0.2 N ferric ammonium sulfate is equivalent to 7.990 mg of TiO2. ... ... ... SPECIFICATIONS AND TESTS FOR IRON OXIDES 1. Synonyms Iron oxides; Iron Oxide yellow: CI Pigment Yellow 42 and 43; CI(1975) No. 77492; INS No. 172(iii) Iron Oxide Red: CI Pigment Red 101 and 102; CI (1975) No. 77491; INS No. 172(ii) Iron Oxide Black: CI Pigment Black 11; CI (1975) No. 77499; INS No. 172(i) ADI = 0 - 0.5 mg/kg bw. 2. Definition Iron oxides are produced from ferrous sulfate by heat soaking, removal of water, decomposition, washing, filtration, drying and grinding. ... ... ... Chemical names Iron Oxide Yellow: Hydrated ferric oxide, hydrated iron (III) oxide Iron Oxide Red: Iron sesquioxide, anhydrous iron (III) oxide Iron Oxide Black: Ferroso ferric oxide, iron (II,III) oxide C.A.S. number Iron Oxide Yellow: 51274-00-1 Iron Oxide Red: 1309-37-1 Iron Oxide Black: 1317-61-9 Chemical formula ... ... ... Iron Oxide Red: Fe2O3 Iron Oxide Black: FeO · Fe2O3 Formula weight 88.85 FeO(OH) 159.70 Fe2O3 231.55 FeO . Fe2O3 3. Description Yellow, red, brown or black powder 4. Functional uses ... ... ... 5. Specifications 5.1. Identification Solubility Insoluble in water and organic solvents; soluble in concentrated mineral acids. Water-soluble matter Not more than 1,0%. 5.2. Purity ... ... ... Iron Oxide Red : Not more than 1.0% (105°, 4 h) Arsenic Not more than 3 mg/kg. Cadmium Not more than 1 mg/kg. Lead Not more than 10 mg/kg Mercury Not more than 1 mg/kg ... ... ... Not less than 60% of iron. 6. Tests 6.1. Purity Arsenic, cadmium and lead Weigh 5 g of the sample and transfer to a beaker. Add 50 ml concentrated hydrochloric acid and heat on a hot plate until totally dissolved. Dilute with water to 100 ml in a volumetric flask. Determine cadmium and lead using an atomic absorption technique appropriate to the specified level and arsenic using Method II of JECFA monograph 1-Vol. 4. Mercury - Tested as directed under JECFA monograph 1-Vol. 4. - Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in “Instrumental Methods.” ... ... ... Weigh accurately about 0.2 g of the sample, add 10 ml of 5 N hydrochloric acid and heat cautiously to boiling in a 200-ml conical flask until the sample has dissolved. Allow to cool, add 6 to 7 drops of 30% hydrogen peroxide solution and again heat cautiously to boiling until all the excess hydrogen peroxide has decomposed (about 2-3 min). Allow to cool, add 30 ml of water and about 2 g of potassium iodide and allow to stand for 5 min. Add 30 ml of water and titrate with 0.1 N sodium thiosulfate adding starch TS as the indicator towards the end of the titration. Each ml of 0.1 N sodium thiosulfate is equivalent to 5.585 mg of Fe (III). ANNEX 30 SPECIFICATIONS AND TESTS FOR FAST GREEN FCF 1. Synonyms CI food green 3,CI (1975): 42053 INSL: 143 ... ... ... 2. Definition Consists essentially of disodium 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3- sulfonatobenzyl)amino]phenyl](4-hydroxy-2-sulfonatophenyl)methylene]-2,5-cyclohexadien-1-ylidene]ammoniomethyl]benzenesulfonate and isomers and subsidiary colouring matters together with water, NaCl and/or Na2SO4 as the principal uncoloured components. May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies. Chemical names Disodium 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3-sulfonatobenzyl) amino] phenyl](4-hydroxy-2-sulfonatophenyl)methylene]-2,5-cyclohexadien-1- ylidene] ammoniomethyl]-benzenesulfonate; Inner disodium salt of N-ethyl-N-[4[[4-ethyl[(3- sulfophenyl)methyl]amino]phenyl](4-hydroxy-2-sulfophenyl) methylene]- 2,5-cyclohexadien-1-ylidene]-3-sulfobenzene-methanaminium hydroxide. C.A.S. number 2353-43-9 Chemical formula ... ... ... Structural formula Formula weight 808.86 3. Description Red to brown-violet powder or crystals. 4. Functional uses Colour. 5. Specifications ... ... ... Solubility Soluble in water; sparingly soluble in ethanol. Identification of colouring matters Passes test.   5.2. Purity Loss on drying at 135°C Not more than 15% together with chloride and sulfate calculated as sodium salts. ... ... ... Not more than 0.2%. Lead  Not more than 2 mg/kg. Chromium  Not more than 50 mg/kg. Subsidiary colouring matters  Not more than 6%. Organic compounds other than colouring matters Not more than 0.5%, sum of 2-, 3-, and 4-Formylbenzenesulfonic acids, sodium salts. ... ... ... Not more than 0.5% of 2-Formyl-5-hydroxybenzenesulfonic acid, sodium salt. Unsulfonated primary aromatic amines Not more than 0.01% calculated as aniline. Leuco base Not more than 5.0%. Ether extractable matter Not more than 0.4%. 5.3. Content Not less than 85% total colouring matter. ... ... ... 6.1. Purity Lead Tested as directed under JECFA monograph 1-Vol. 4. Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1-Vol. 4, “Instrumental Methods.” Subsidiary colouring matters Prepare a 1% solution of the colour. By means of a syringe spot as a band 1 mg of the colour on an 20 x 20 cm silica gel thin layer chromatographic plate. Allow the plate to dry about 20 min in the dark before placing it in a chromatographic tank containing the following developing solution: acetonitrile/isoamyl alcohol/methyl ethyl ketone/water/ammonia (50/50/15/10/5 by volume). Develop the plate until the solvent front is near the top of the plate. Remove the plate and allow it to dry. The subsidiary colours will appear in the following positions: the two top bands are the lower sulfonated subsidiary colours followed by the isometric and the main band or fast Green FCF near the bottom of the plate. Scrape off all the bands above the isometric colour. Extract the colour from the silica gel with ethyl alcohol. Filter through a sintered glass funnel and examine spectrophotometrically. The Standard absorptivity of the lower subsidiary colour is 0.126 mgL^-1cm^-1. Note: In the calculation of the subsidiary colour, it is assumed that its absorptivity at its peak is the same as that of the parent compound at its peak. Standard solutions must be prepared and their absorptivities measured within 1 h. Spectrophotometric measurements of the extracted subsidiary colours must be made as promptly as possible. Organic compounds other than colouring matters - Tested as directed under JECFA monograph 1-Vol. 4. ... ... ... 3-formylbenzenesulfonic acid: 0.495 mg L^-1 cm^-1 at 246 nm in dilute HCl 3-[(ethyl)(4-sulfophenyl)amino]methylbenzenesulfonic acid: 0.078 mg L^-1 cm^-1 at 277 nm in dilute ammonia 2-formyl-5-hydroxybenzenesulfonic acid: 0.080 mg L^-1 cm^-1 at 335 nm in dilute ammonia Leuco base - Tested as directed under JECFA monograph 1-Vol. 4. - Weigh accurately 110 ± 5 mg sample and proceed as directed under Leuco Base in Sulfonated Triarylmethane Colours. Absorptivity (a) = 0.156 mg L^-1 cm^-1 at approx. 625 nm. Ratio = 0.971. 6.2. Method of assay Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following: ... ... ... Buffer: 15 g sodium hydrogen tartrate; Weight (D) of colouring matters equivalent to 1.00 ml 0.1 N TiCl3: 40.45 mg. Bạn Chưa Đăng Nhập Thành Viên! Vì chưa Đăng Nhập nên Bạn chỉ xem được Thuộc tính của văn bản. Bạn chưa xem được Hiệu lực của Văn bản, Văn bản liên quan, Văn bản thay thế, Văn bản gốc, Văn bản tiếng Anh,... Nếu chưa là Thành Viên, mời Bạn Đăng ký Thành viên tại đây Bạn Chưa Đăng Nhập Thành Viên! Vì chưa Đăng Nhập nên Bạn chỉ xem được Thuộc tính của văn bản. Bạn chưa xem được Hiệu lực của Văn bản, Văn bản liên quan, Văn bản thay thế, Văn bản gốc, Văn bản tiếng Anh,... Nếu chưa là Thành Viên, mời Bạn Đăng ký Thành viên tại đây Bạn Chưa Đăng Nhập Thành Viên! Vì chưa Đăng Nhập nên Bạn chỉ xem được Thuộc tính của văn bản. Bạn chưa xem được Hiệu lực của Văn bản, Văn bản liên quan, Văn bản thay thế, Văn bản gốc, Văn bản tiếng Anh,... Nếu chưa là Thành Viên, mời Bạn Đăng ký Thành viên tại đây Bạn Chưa Đăng Nhập Thành Viên! Vì chưa Đăng Nhập nên Bạn chỉ xem được Thuộc tính của văn bản. Bạn chưa xem được Hiệu lực của Văn bản, Văn bản liên quan, Văn bản thay thế, Văn bản gốc, Văn bản tiếng Anh,... Nếu chưa là Thành Viên, mời Bạn Đăng ký Thành viên tại đây Circular No. 27/2010/TT-BYT dated May 20, 2010 on promulgating National technical regulation on food additives - Colours Tải Văn bản tiếng Việt Tải Văn bản tiếng Việt (docx) Tải Văn bản tiếng Anh (Download English translation) Bạn Chưa Đăng Nhập Thành Viên! Vì chưa Đăng Nhập nên Bạn chỉ xem được Thuộc tính của văn bản. Bạn chưa xem được Hiệu lực của Văn bản, Văn bản liên quan, Văn bản thay thế, Văn bản gốc, Văn bản tiếng Anh,... Nếu chưa là Thành Viên, mời Bạn Đăng ký Thành viên tại đây Văn bản liên quan 1 National Technical Regulation QCVN 4-10:2010/BYT for Food additives - Colours | Lược đồ | Liên quan hiệu lực | Tải về Ban hành: 20/05/2010 Tình trạng: Đã biết Cập nhật: 14/03/2025 2 Decree No. 188/2007/ND-CP of December 27, 2007, defining the functions, tasks, powers and organizational structure of the Ministry of Health. Tiếng Anh | Lược đồ | Liên quan hiệu lực | Tải về Ban hành: 27/12/2007 Hiệu lực: Đã biết Tình trạng: Đã biết Cập nhật: 13/03/2009 3 Decree No. 127/2007/ND-CP of August 01, 2007, detailing the implementation of a number of articles of the Law on Standards and Technical Regulations Tiếng Anh | Lược đồ | Liên quan hiệu lực | Tải về Ban hành: 01/08/2007 Hiệu lực: Đã biết Tình trạng: Đã biết Cập nhật: 19/03/2012 4 Law No. 68/2006/QH11 of June 29, 2006 on standards and technical regulations Tiếng Anh | Lược đồ | Liên quan hiệu lực | Tải về Ban hành: 29/06/2006 Hiệu lực: Đã biết Tình trạng: Đã biết Cập nhật: 01/02/2009 5 Decree of Government No.163/2004/ND-CP of September 7, 2004 detailing the implementation of a number of articles of the ordinance on food hygiene and safety Tiếng Anh | Lược đồ | Liên quan hiệu lực | Tải về Ban hành: 07/09/2004 Hiệu lực: Đã biết Tình trạng: Đã biết Cập nhật: 01/02/2009 6 Ordinance No. 12/2003/PL-UBTVQH11 of July 26, 2003, on food hygiene and safety Tiếng Anh | Lược đồ | Liên quan hiệu lực | Tải về Ban hành: 26/07/2003 Hiệu lực: Đã biết Tình trạng: Đã biết Cập nhật: 01/02/2009 Thư mục cha: Chọn thư mục cha Tạo thư mục 238 Bạn Chưa Đăng Nhập Thành Viên! Vì chưa Đăng Nhập nên Bạn chỉ xem được Thuộc tính của văn bản. Bạn chưa xem được Hiệu lực của Văn bản, Văn bản liên quan, Văn bản thay thế, Văn bản gốc, Văn bản tiếng Anh,... Nếu chưa là Thành Viên, mời Bạn Đăng ký Thành viên tại đây Bạn Đang Đăng Nhập Thành Viên Free! Vì Đăng Nhập Thành Viên Free nên Bạn chỉ xem được Thuộc tính và Nội dung của văn bản. Bạn chưa xem được Nội dung toàn văn, Hiệu lực của Văn bản, Văn bản liên quan, Văn bản thay thế, Văn bản gốc, Văn bản tiếng Anh,... Nếu muốn làm Thành Viên Basic / Pro, mời Bạn Chuyển Đổi loại Thành Viên tại đây. Bạn Đang Đăng Nhập Thành Viên Basic! Vì Đăng Nhập Thành Viên Basic nên Bạn chỉ xem được Thuộc tính, Nội dung của văn bản, Nội dung toàn văn, Hiệu lực của Văn bản, Văn bản liên quan, Văn bản thay thế, Văn bản gốc. Bạn chưa xem được Văn bản tiếng Anh, ... Nếu muốn làm Thành Viên Pro, mời Bạn Chuyển Đổi loại Thành Viên tại đây. Circular 27/2010/TT-BYT National technical regulation on food ... 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