T.C.
TARIM ve KÖYİŞLERİ BAKANLIĞI
Koruma ve Kontrol Genel Müdürlüğü
The Analysis Methods for Purity Criteria of Food Additives
Authorization Law: Turkish Food Codex
The Official Gazette: 17.01.2001-24643
Communication No : 2001/46
Objective
Article 1 – The objective of this Communiqué is to determine the analysis methods for verifying that additives used in the foodstuffs satisfy the purity criteria.
Scope
Article 2 - This Communiqué covers the analysis methods for purity criteria of antioxidants, preservatives and colouring agents.
Legal basis
Article 3 - This Communiqué has been prepared in accordance with the “Turkish Food Codex Regulation” published in the Official Gazette, dated 16/11/1997, reiterated no 23172.
Analysis methods
Article 4 – The analysis methods should be applied according to the General Principles Annex-I as follows:
a) Substances extractable with diethylether in water soluable sulphanated organic colouring matters used in foodstuffs are determined according to the method 1 in ANNEX-II.
b) Acetic acid (E260), potassium acetate (E261), sodium diacetate (E262) and formic acid in calcium acetate (E263), formates and other oxidizable substance (2) are determined according to the method 2 in Annex-II.
c) Non-volatile substances in propionic acid (E280) are determined according to the method 3 in Annex-II.
d) The mass loss that comes about after drying in sodium nitrate (E250) can be determined according to the method 4 in Annex-II.
e) Salicylic acid in; ethyl p-hydroxy benzoate (E214), ethyl p-hydroxy benzoate sodium salt (E215), n-propyl p-hydroxy benzoate (E216), n-propyl p-hydroxy benzoate sodium salt (E217), metyl p-hydroxy benzoate (E218) and methyl p-hydroxy benzoate sodium salt (E219) can be determined according to the method 5 in Annex-II.
f) Free acetic acid in sodium diacetate (E262) is determined according to the method 6 in Annex-II.
g) Sodium acetate in sodium diacetate (E262) is determined according to the method 7 in Annex-II.
h) The limit tests to determine aldehydes in sorbic acid (E200), sodium, potassium and calcium sorbates (E201, E202, E203) and propionic acid (E280) are made according to the method 8 in Annex II.
i) Peroxide limit number in lecithin (E322) is determined according to the method 9 in Annex-II.
j) In case of lecithin (E322), substances that do not dissolve in toluene are determined according to the method 10.
k) The limit tests of reducing substances in sodium, potassium and calcium lactates (E325, E326 and E327) are made according to the method 11 in Annex-II.
l) Volatile acids in ortho-phosphoric acid (E338) are determined according to the method 12 in Annex-II.
m) The limit test to determine the nitrate in ortho-phosphoric acid (E338) is made according to the method 13 in Annex-II.
n) Water insoluble substances in mono-di, tri-sodium orthophosphate and mono-, di- and tri-potassium ortho-phosphate [E339 (i), E339 (ii), E339 (iii), E340 (i), E340 (ii), E340 (iii)] are determined according to the method 14 in Annex-II.
o) pH in food additives is determined according to the method 15 in Annex-I.
Compliance with European Union
Article 5 – This Communiqué has been prepared within the frame compliance with European Union considering the Commission Directive 81/712/EEC for “Analysis Methods for Purity Criteria of Food Additives”.
This Communiqué does not prevent the laboratories from using the methods experienced according to their own regulations and having scientific validity. This Communiqué is applied compulsorily to the relations with European Union.
Provisional Article 1 – Presently active laboratories using the analysis methods included in this Communiqué are obliged to apply the provisions of this Communiqué within 1 year.
Enforcement
Article 6 – This Communiqué enters into force on the date of publication.
Execution
Article 7 – The provisions of this Communiqué are executed by the Minister of Agriculture and Rural Affairs and the Minister of Health.
ANNEX-I
Basic Principles
1. Preparation of the Sample for Analysis
1.1. General
50 grams of sample is taken from the laboratory sample to be used in analysis. This amount can, however, only be increased under special circumstances.
1.2. Preparation of the sample
The sample should be made homogeneous before the analysis.
1.3. Preservation
The sample prepared should always be kept in an air-tight and moisture-tight container to prevent its deterioration.
2. Reagents
2.1. Water
2.1.1. In the Communiqué, “water” is; water used for, solution, dilution or for washing, distilled water or demineralised water at the equal level of purity.
2.1.2. Solution and dilution address, unless otherwise specified, to the “aqueous solution”.
2.2. Chemicals
All reagents should be analytically pure. Otherwise, their properties shall be mentioned.
3. Equipment and Materials
3.1. The list of equipment covers the technical characteristics and details of the equipment.
3.2. The analytical balance should at least be have a sensitivity of 0.1 mg..
4. Expression of the Results
4.1. Results
The result mentioned in the official analysis report should be the mean value of an analysis with double parallel meanings.
4.2. Calculation of the percentage
Unless otherwise indicated, the results should be provided as a percentage of the original sample as a mass.
4.3. The number of significant figures after the comma should be mentioned according to the precision of the method.
ANNEX-II
Analysis Methods
Method 1: The determination of Substances that can be extracted by diethyl ether in water-soluble Sulphonated Organic colouring materials Used in Foodstuffs.
1. Scope and field of Application
This method determines the substances that can be extracted by diethyl ether in water-soluble sulphonated organic colouring materials and which are not mixed with other substances.
2. Definition
Substances that can be extracted by diethyl ether: The determination of substances that can be extracted by diethyl ether through the method defined.
3. Principle
The diethyl ether extract in the colouring substance is determined after extraction with diethyl ether and after weighing the residue following vaporisation of ether in the extract.
4. Reagents
4.1. Diethyl ether, anhydrous and peroxide-free (dried with the help of calcium chloride)
5. Equipment
5.1. Soxelate apparatus
5.2. Dessicator, containing newly activated silica gel or an equivalent dehydrating substance or moisture determining indicator.
5.3. Analytical balance
5.4. Oven, with thermostatic control and that can be adjusted to 85±2oC.
6. Procedure
10 grams of sample is weighed with ±10 mg sensitivity on filter paper. The paper is folded, put into the cartridge and closed with fat free cotton wool. The soxhlet flask is weighed empty and placed. In the soxhlet (5.1), extraction is performed for 6 hours with diethyl ether (4.1). The temperature is adjusted to the lowest possible level. The soxhlet flask that contains extraction residue is kept in oven (5.4) at 85±2oC for 20 minutes to dry. The flask is taken into dessicator (5.2) and left to cool. The cold flask is weighed.
The procedures of drying and weighing are repeated until two consecutive weightings are less than 0.5 milligrams. If any increase in the mass is observed the lowest reading is used in calculation.
7. Expression of the Results
7.1. Formula and calculation method:
The content of the substance that can be extracted with ether, can be calculated as the percentage of the sample with the method mentioned below:
m1 x 100
-----------
m0
m1= The mass of the residue expressed in grams after vaporization,
mo= The amount of the sample, g.
7.2. Repeatability
The difference between the results of the two analyses carried out under the same conditions, by the same analyst, at the same time or rapidly, one after the other for the same sample, must not be more than 0.02 grams for each 100 grams of the same sample.
Method 2: The definition of Acetic Acid (E260), Potassium Acetate (E261), Sodium Diacetate (E262) and Formic acid in Calcium Acetate (E263), Formats and Other Additives than can be Oxidized.
1. Scope and Field of Application
This method determines the formic acid, formates and other substances that can be oxidized in acetic acid (E260), potassium acetate (E261), sodium diacetate (E262), and calcium acetate (E263) in terms of formic acid.
2. Definition:
Formic acid, format and other substances that can be oxidized: the determination of formic acid, format and other substances that can be oxidized by the defined method.
3. Principle:
The sample solution is subjected to reaction with excess of standard potassium permanganate in alkali conditions with the aim of producing manganese dioxide. The excess of manganese dioxide and potassium permanganate is determined under acid conditions iodometrically and the concentrations of substances that can be oxidized is calculated and determined in terms of formic acid .
4. Reagents:
4.1. Potassium iodide
4.2. Potassium permanganate solution, 0.02 M
4.3. Anhydrous sodium carbonate
4.4. Sodium thiosulfate solution, 0.1 M.
4.5. Starch solution, 1% weight/volume
4.6. Diluted sulfuric acid solution; 90 ml its density 1.84 g/ml sulfuric acid, is added into the water and completed to 1 liter.
5. Equipment and Materials:
5.1. Water bath, with a property to boil.
5.2. Analytical scale
6. Procedure:
In free acid samples, 10 grams of sample is weighed with 10 milligrams sensitivity. It is diluted with 70 milliliters of water. A solution that contains 10 grams of anhydrous sodium carbonate in 30 milliliters of water is added into it. In salt samples, 10 grams of sample is weighed with 10 milligrams sensitivity; is diluted with 100 milliliters of water. One gram of anhydrous sodium carbonate is added; needs to be shake for dissolution. 20 milliliters of 0.02 M potassium permanganate is added and is heated in boiling water bath for 15 minutes. The mixture is cooled. 50millilitres of dissolved sulfuric acid and 0.5 grams of potassium iodide are added. The precipitated manganese dioxide is shaken until dissolved. The solution is titrated with 0.1 M sodium thiosulphate until it turns light yellow. A few drops of starch solution is added and titration is continued until the solution turns colourless.
7. Expression of the Results:
7.1. Formula and calculation method:
Formic acid, formate and other additives that can be oxidized are calculated by the formula given below in terms of formic acid.
2.3x b 100 x a
--------- X ( ----------- - V)
m0 b
a = Potassium permanganate molarity
b = Sodium thiosulphate molarity
m0 = Sample quantity, grams
V = volume of 0.1 M Sodium thiosulphate, spent during titration, in milliliters.
7.2. Repeatability
The difference between the results of the two analysis made under the same conditions, by the same analyst, at the same time or rapidly and one after the other and for the same sample, must not be more than 5 milligrams for each 100 grams of the sample.
8. Notes:
8.1. The volume of 11.3 millilitres 0.1 M sodium thiosulphate is equal to 0.2 % formic acid in 10 grammes of sample.
8.2. If there is no formate, the volume needed for potassium permanganate is 20 millilitres; if there is more formic acid than a moss of 0.27 % (mass/mass), the excess of potassium permanganate will be sufficient and a minimum stable volume of 8 millilitres will be obtained. In this case the measurement shall be repeated using a sample with less weight.
Method 3: Determination of Non-Volatile Substances in Propionic Acid (E280)
1. Scope and Field of Application
This method is used to determine non-volatile substances in propionic acid (E280)
2. Definition
The amount of non-volatile substance in propionic acid is the determination of non-volatile substance in propionic acid with the defined method.
3. Principle
The sample is vaporized and dried at 103±2oC and the residue is determined as gravimetrically.
4. Equipment and Materials:
4.1. Evaporation vessel made of silica or platinum, with a capacity of 100 grams.
4.2. Oven that can be kept at 103±2oC thermostatically.
4.3. Analytical scale
4.4. Water bath.
4.5. Dessicator, containing newly activated silica gel or an equivalent dehydrating substance or moisture determining indicator.
5. Procedure:
100 g propionic acid sample is weighed in a previously dried and weighed container, with 0.1 g sensitivity. All the propinic acid is evaporated over the water bath boiling in fume cupboard. Kept at the autoclave at 103±2oC for 1 hour. Cooled in the dessicator and weighed. The heating, cooling and weighing procedures are repeated until the differences between consecutive measurements is less than 0.5 milligrams. If any increase is observed in the mass, the lowest value that can be read is used in the calculation.
6. Expression of the Results:
6.1. Formula and calculation method:
The content of the non-volatile substance is calculated with the following formula as a sample percentage:
m0 - m1
--------------- X 100
m0
m1= Residue after vaporisation, in grammes
mo= Sample quantity, grammes.
6.2. Repeatability:
The difference between the two analyses made under the same conditions by the same analyst, at the same time or rapidly, one after the other for the same sample, must not be more than 5 milligrammes for every 100 grammes of the sample.
Method 4: Determination of Loss in Drying in Sodium Nitrite (E250)
1. Scope and Field of Application
This method, is used to determine the loss in drying in sodium nitrite (E250)
2. Definition
This moisture content of sodium nitrite: the determination of the loss produced as a result of drying, by the defined method
3. Principle
The loss of mass, produced as a result of drying is determined by the calculation of loss of heating, weighing and mass in the oven at 103±2oC.
4. Equipment and Materials
4.1. Oven that can be kept at 103±2oC thermostatically.
4.2. Petry dish, with flat bottom for weighing with a diameter of 60-80 millimetres, 25 millimetres deep, with a lid.
4.3. Dessicator, newly activated silica gel or an equivalent dehydrating substance and an moisture determining indicator.
5. Procedure:
Petry dish and its lid is brought to constant weight in 1 hour in the oven at 103±2oC. At the end of this period, is taken into the dessicator and cooled to room temperature. Petri dish and the lid is weighed with 10 mg sensitivity. The petry dish is kept with the lid open at 103±2oC for 1 hour. Then, it is taken into the dessicator with the lid closed and cooled to room temperature. It is weighed with 10 milligrammes sensitivity. The petry (4.2) is closed with its cover and placed into a dessicator (4.3) to cool down to a room temperature. The heating, cooling and weighing procedures are repeated until the difference between consecutive measurements is less than 10 mg. If any increase in the mass is observed, the lowest value read is used in the calculation.
6. Expression of the Results:
6.1. Formula and calculation method:
The content of mass loss with drying is calculated as sample percentage with the following formula:
100 x (m2 – m3)
--------------------------
(m2 – m3)
m1= empty mass of the petri dish in grammes
m2= The mass of petri container and the sample, before drying, in grammes.
m3= The mass of petri container and sample after drying, in grammes.
6.2. Repeatability:
The difference between the two analyses made under the same circumstances, by the same analyst, at the same time or rapidly, one after the other for the same sample, must not be more than 100 milligrammes for each 100 grammes of the sample.
Method 5: The Determination of Salicylic Acid in Ethyl p-hydroxy benzoate (E214), Ethyl p-hydroxy benzoate sodium salt (E215), n-Propyl p-hydroxy benzoate (E216), n-Propyl p-hydroxy benzoate sodium salt (E217), Methyl p-hydroxy benzoate (E218), Methyl p-hydroxy benzoate and Methyl p-hydroxy benzoate sodium salt (E219).
1. Scope and Field of Application
This method is used in the determination of salicylic acid in ethyl p-hydroxy benzoate (E214), n-propyl p-hydroxy benzoate (E216), methyl p-hydroxy benzoate (E218) and in sodium salts (E215, E217 and E219).
2. Definition
Salicylic acid concentration: the determination of salicylic acid with the defined method.
3. Principle
The formation of colour purple is observed as a result of the reaction between ammonium iron-III sulfate and sample solution.
4. Solutions
4.1. Ammonium iron-III sulfate solution, 0.2 % mass/volume, 0-2 grammes ammonium iron-III sulfate dodecahydrate is dissolved in 50 millilitres water 10 millilitres 10% mass/volume nitric acid is added and is completed to 100 millilitres by water.
4.2. Ethanol 95% volume/volume.
4.3. Salicylic acid solution, 0-1 gramme/liter.
4.4. Sulfuric acid solution, 1 M
5. Equipment and Materials
5.1. Nessler tubes, 50 milliliters
6. Precedure
6.1. For ethyl, n-propyl and methyl p-hydroxy benzoate samples;
6.1.1. 0.1 gramme sample is weighed with 1 milligram sensitivity and dissolved in 10 milliliters 95% (volume/volume) ethanol. Solution is placed in a Nessler cylinder and completed to 50 milliliters with water. It is stirred and while being stirred, 1 milliliter iron-III solution is added. It is left to wait for 1 minute.
6.1.2. At the same time the solution for comparison is prepared by repeating 6.1.1; but 1 milliliter salicylic acid solution is used instead of 0-1 gramme sample.
6.1.3. The colour formation in the sample solution is compared with the solution prepared for comparison.
6.2. For ethyl, n-propyl and methyl p-hydroxyl benzoate sodium salt samples.
6.2.1. The procedure in 6.1.1. is repeated before adding up to 50 milliliters by using 1 M sulphuric acid to adjust its pH to 5.
6.2.2. is repeated.
6.2.3. is repeated.
7. Expression of the Results
7.1. Interpretation of the results
If the formation of red-purple colour in the sample solution is denser than in the solution for comparison, the experiment is positive and the sample contains more than 0.1 % salicylic acid.
7.2. Sensitivity
The experiment limit is 30 milligrammes of salicylic acid for every 100 grammes.
7.3. Repeatability
The results of two analyses, made under the same circumstances by the same analyst, at the same time or rapidly one after the other for the same sample must be the same.
Method 6: The Determination of Free Acetic Acid in Sodium Diacetate (E262)
1. Area and Purpose of Application
This method is used in the determination of the acid in sodium diacetate (E262)
2. Definition
Acetic acid content: is the determination of the acetic acid amount through the method defined.
3. Principle
The titration of acetic acid in the sample is determined by using standard sodium hydroxide solution and phenolphthalein.
4. Reagents
4.1. Phenolphthalein solution, in ethanol, 1% (mass/volume)
4.2. Sodium hydroxide solution, 1 M.
5. Equipment and Materials
5.1. Analytical balance
6. Procedure
3 grammes of sample is weighed with 1 milligramme sensitivity and dissolved in 50 milliliters of water. 2 or 3 drops of phenolphthalein solution is added and titration is made with 1 M sodium hydroxide until the red colour stays for 5 minutes.
7. Expression of the Results
7.1. Formula and calculation method
The amount of acetic acid, as sample percentage is calculated with the formula given below:
6.005 x V x c
------------------
mo
V= the volume of sodium hydroxide that is spent, in milliliters.
c= the concentration of sodium hydroxide solution, M.
mo= the amount of sample, in grammes.
7.2. Repeatability
The difference between the two analyses made under the same conditions, by the same analyst, at the same time or rapidly on after the other for the same sample must not be more than 500 milligrammes for each 100 grammes of the sample.
8. Note:
When 3 g sample containing 40% acetic acid is titrated with 1 M sodium hydroxide, 20 ml is used.
Method 7: The Determination of Sodium Acetate in Sodium Diacetate (E262)
1. Scope and Field of Application
This method is used for the determination of sodium acetates and their hydrates in sodium diacetate (E262) in terms of sodium acetate.
2. Definition
Sodium acetate content: is the determination of sodium acetate and its hydrates through the defined method, in terms of sodium acetate.
3. Principle
The sample is dissolved in glacial acetic acid. It is titrated by perchloric acid accompanied by crystal violet indicator.
4. Reagents
4.1. Glacial acetic acid, at 20oC, d= 1, 049 grammes/millilitres.
4.2. Crystal violet indicator solution, Cl No: 42555, in 0.2 % mass/volume glacial acetic acid.
4.3. Potassium hydrogen phthalate, C8 H5 KO4
4.4. Dehydrated acetic acid, (CH3 CO)2O
4.5. Perchloric acid solution, in 0.1 M glacial acetic acid. Perchloric acid solution is prepared and adjusted according to the method mentioned below:
P g perchloric acid solution is weighed in 1000 milliliters of balloon flask and completed to its volume. P amount is calculated from the formula mentioned below:
1004.6
P = ----------
m
m= concentration (as % mass/mass) of perchloric acid determined by alkalimetric titration (the most appropriate %70-72 mass/mass).
1000 milliliters glacial acetic acid is added and Q grams dehydrated acetic acid is added in the form of small pieces; while adding, the solution is stirred continuously and is cooled. Q amount is calculated by the formula mentioned below:
(567 x P) - 5695
Q = ------------------
a
P= the amount of perchloric acid measured
a= concentration of dehydrated acetic acid (% mass/mass)
“The mouth of balloon flask is closed and left in a dark place for 24 hours. Sufficient amount of glacial acetic acid is added to obtain 1000 milliliter of solution. In practice this solution is anhydrous. This solution is set against potassium hydrogen phthalate.
0.2 grams of potassium hydrogen phthalate dried at 110oC for 2 hours is weighed with 0.1 milligrams of sensitivity. The amount weighed is dissolved in an erlenmeyer by heating slightly with 25 milliliters glacial acetic acid. It is cooled off and 2 drops of 0.2% crystal violet is added. Afterwards, it is titrated until perchloric acid solution indicator’s color turns to light green. Blank titration is made by using solutions of the same volume. Blank value is subtracted from the real value: 20-42 milligrams potassium hydrogen phthalate = 1 milliliters 0.1 M perchloric acid.
5. Apparatus
5.1. Analytical balance.
6. Procedure
0.2 a gram of sample is weighed with 0.5 milligrams sensitivity and is dissolved in 50 milliliters of glacial acetic acid. A few drops of crystal violet indicator solution are added and by using 0.1 M adjusted perchloric acid, titration is made until pale green color end point.
7. Expressions of the Results
7.1. Formula and method of calculation:
Sodium acetate amount is calculated with the following amount as sample percentage:
8.023 x V x c
---------------
m0
V= the amount of adjusted perchloric acid used, in milliliters
c= molarity of the perchloric acid solution
mo= amount of sample, grams
7.2. Repeatability
The difference between results of two analyses made under the same conditions, by the same analyst, at the same time or rapidly one after the other for the same sample must not be more than 1.5 grams for each 100 grams of the sample.
8. Note
The reagents used within the scope of this method are toxic and explosive; therefore they should be used carefully.
Method 8: Determination of Aldehydes in Sorbic Acid (E200); Sodium, Potassium and Calcium Sorbates (E201, E202, E203) and Propionic Acids (E280).
1. Scope and Field of Application
This method, is used for the determination of aldehydes as formaldehydes in sorbic acid (E200), sodium, potassium and calcium sorbates (E201, E202, E203), and propionic acid (E280)
2. Definition
Aldehyde concentration: the determination of aldehyde concentration according to the specified method.
3. Principle
The aldehydes in the experiment solution and the formaldehydes in comparison solutions go into reaction with Schiff’s reagent and form red colored complexes. The intensities of color formation is compared.
4. Reagents
4.1. The standard formaldehyde solution of 0.01 milligrams/milliliters is prepared by diluting concentrated formaldehyde solution (400 milligrams/milliliters).
4.2. Schiff’s reagent
5. Procedure
5.1. 1 gram sample is weighed with 1 milligram sensitivity; is agitated after adding 100 milliliters waters. If necessary, the solution is filtered. 1 milliliter of Schiff’s reagent is added into 1 milliliter of filtrate or sample solution. At the same time comparison solution is prepared by adding 1 milliliter Schiff’s reagent into the 1 milliliter of formaldehyde standard solution.
5.2. The color of the sample is compared to the comparison solution.
6. Expression of the Results
6.1. Interpretation of the results
If the red color change in the sample solution is more intense than in the comparison solution, the experiment is positive and the sample contains at least 0.1 % aldehyde in the form of formaldehyde.
6.2. Sensitivity
The experiment limit is 30 milligrams of formaldehyde for every 100 grams of sample.
6.3. Repeatability
The results of the two analyses, made under the same conditions, by the same analyst, at the same time or rapidly one after the other for the same sample should to the same.
Method 9: The Determination of Peroxide Number in Lecithin (E322)
1. Scope and Field of Application
This method is used for the determination the peroxide number in lecithin (E322)
2. Definition
The number of peroxides in lecithin: the determination of the numbers of peroxides in the lecithin according to the specified method.
3. Principle
Oxidation of potassium iodide by lecithin peroxides and titration of iodine liberated using standard sodium thiosulfate solution
4. Reagents
4.1. Glacial acetic acid
4.2. Chloroform
4.3. Potassium iodide
4.4. Sodium thiosulfate solution, 0.1 M or 0.0.1 M
4.5. Starch solution, approximately 1 % mass/volume
5. Apparatus
5.1. Analytical balance
5.2. Special glass material, Figure 1.
5.2.1. Balloon round based, 100 milliliters
5.2.2. Back cooler
5.2.3. Glass tube, 250 millimeters long and with 22 millimeters internal diameter, united with glass joints.
5.2.4. Micro beaker, with 20 millimeter outer diameter and 35-50 millimeter high.
6. Procedures
6.1. 10 milliliters of glacial acetic acid and 10 milliliters of chloroform are put into a balloon (5.2.1) of 100 milliliters. The glass column (5.2.3) is placed into the back cooler and the mixture is boiled slightly for two minutes to eliminate the dissolved air. 1 gram of potassium iodide is dissolved in 1.3 milliliters of water and this solution is added (5.2.1), caring not to prevent boiling of the solution in the balloon. At this stage, if a yellow color change is observed, the procedure is cancelled and repeated with new reagents.
6.2. 1 gram sample is weighed with 1 milligram sensitivity. It is boiled two more minutes. The sample is added into the flask, while the contents of the flask are still boiling. For this purpose, the sample must be emptied from the glass tube with the help of a glass rod, bottom of which is as seen in the figure, being placed in the micro beaker (5.2.4). The back cooler can be taken out for a short while. Boiling is continued for 3-4 minutes. Heating is stopped and back cooler is taken out immediately. 50 milliliters of water is added from the glass tube rapidly. The glass tube is taken out and the flask is cooled under tap water until room temperatures. The diluted layer is titrated with 0.1 M or 0.0.1 M thiosulphate until its color turns to pale yellow. 1 milliliter of starch solution is added and titration is continued until the blue color disappears. During titration, in order to provide for the full extraction of the iodine from the dehydrated layer the solution is stirred thoroughly.
6.3. Comparison titration measurement is made without adding any sample, by repeating the procedures in 6.1 and 6.2.
7. Expression of the Results
7.1. Formula and method of calculation:
The number of peroxides is calculated with the following formula in terms of milliequivalent for one kg :
1000 x a x (V1 – V2)
---------------------------------
mo
V1= The volume of thiosulfate needed for titration of the sample (6.2), milliliters
V2= The volume of thiosulfate needed for blank titration, milliliters
a= Concentration of thiosulfate solution in terms of M
mo= Amount of sample, grams
7.2. Repeatability
The difference between the two analyses made under the same conditions, by the same analyst, at the same time or rapidly, one after the other for the same sample should not exceed 0.5 as peroxide number in terms of milliequivalent per kilogram sample
8. Notes
8.1. The sodium thiosulfate concentration to be chosen depends on the foreseen titration value. If less than 0.5 milliliters of 0.1 M sodium thiosulfate is required, the procedure shall be repeated by using 0.01 M sodium thiosulfate.
8.2. The procedure must not be performed under strong light.
Method 10: The Determination of Toluene-insoluble matter in lecithin (E322)
1. Scope and Field of Application
This method is used for the determination of toluene-insoluble matter in lecithin (E322).
2. Definition
The amount of toluene-insoluble matter: determination of the amount of toluene-insoluble matter according to this method.
3. Principle
Sample is dissolved in toluene, filtered and determined by drying and weighing the residue.
4. Solutions
4.1. Toluene
5. Apparatus
5.1. Sintered glass crucible, with a capacity of 30 milliliters, with G3 or equivalent porosity.
5.2. Oven that can be kept at 103±2oC thermostatically.
5.3. Water bath that can function without surpassing 60oC.
5.4. Desiccators that contain newly activated silica gel or an equivalent desiccant and a water-content indicator.
5.5. Erlenmeyer flask, 500 milliliter
5.6. Vacuum pomp
5.7. Analytical balance
6. Procedure
6.1. 30 milliliters capacity sintered glass crucible is kept in the oven of 103±2oC. Crucible is placed in the dessicator and weighed when reaches a stable weighed.
6.2. If necessary, lecithin sample is heated in a water bath (5.3). 10 grams of sample is weighed in the Erlenmeyer flask with 1 milligram sensitivity. 100 milliliters of toluene is added. It is stirred until the whole lecithin is dissolved. The solution is filtered through the crucible (5.1). Erlenmayer flask is washed twice with 25 milliliters toluene and filtered through the crucible. Excess toluene is taken from the crucible with vacuum pump.
6.3. Crucible is kept in the oven of 103±2oC for 2 hours. It is placed in the desiccator and left to cool. When it is cold, the crucible and the residue are weighed.
6.4. The procedure is repeated until the difference between the two consecutive measurements in 6.3 is less than 0.5 milligrams. If any increase in weight is observed, the lowest value read is used in the calculation.
7. Expression of the Results
7.1. Formula and method of calculation:
The amount of toluene-insoluble matter is calculated using the formula given below:
100 (m2 – m1)
---------------------
mo
m1= Mass of the empty crucible, grams
m2= Mass of the crucible and the residue, grams
mo= Amount of the sample, grams
7.2. Repeatability
The difference between the results of the two analyses, made under the same conditions, by the same analyst, at the same time or rapidly one after the other for the same sample should not be more than 30 milligrams of every 100 grams of the sample.
Method 11: Determination of Reducing Agents in Sodium, Potassium and Calcium Lactates (E325, E326 and E327)
1. Scope and Field of Application
This method is used for the determination of reducing agents in sodium, potassium and calcium lactates (E325, E326 and E327).
2. Definition
Determination of concentrations of reducing agents: determination of concentrations of reducing agents according to the specified method.
3. Principle
Fehling solution is reduced by substances that show reducing properties. These substances are normally the reducing sugars.
4. Solutions
4.1. Fehling A solution: 6.93 grams of copper sulfate pentahydrate is weighed, dissolved in water and completed to 100 milliliters with water.
4.2. Fehling B solution: 34.6 grams of potassium sodium tartarate and 10 grams of sodium hydroxide are weighed, dissolved in water and completed to 100 milliters with water.
5. Procedure
1 gram sample is weighed with 1 milligram sensitivity and dissolved in 10 milliliter hot water. 2 milliliters Fehling A solution and 2 milliliters Fehling B solution are added. The mixture is boiled for 1 minute and checked for color change. Probable calcium sulfate sedimentation does not affect the result.
6. Expression of the Results
6.1. Interpretation of th e results:
If there is a color change after boiling, the experiment is positive and shows the existence of reducing agent.
6.2. Sensitivity
The limit of the reducing agents that go into interaction is 100 milligrams glucose for 100 grams of sample
6.3. Repeatability
The results of the two analyses made under the same conditions, by the same analyst, at the same time or rapidly one after the other for the same sample should be the same.
7. Note
If there is 2% glucose in the sample, all Fehling solutions go into reaction.
Method 12: Determination of Volatile Acids in Ortho-phosphoric Acids (E338)
1. Scope and Field of Application
This method is used for the determination of volatile acids in ortho-phosphoric acid (E338) in terms of acetic acid.
2. Definition
Amount of volatile acid: is the determination of volatile acid amount in terms of acetic acid according to the specified method.
3. Principle
Water is added into the sample and the solution is distilled. Distillate is titrated against the standard sodium hydroxide solution; the acidity is calculated and defined as acetic acid.
4. Solutions
a) Phenolphthalein solution, 1% mass/volume in ethanol
b) Sodium hydroxide solution, 0.01 M
5. Apparatus
a) Distillation apparatus including a spray trap.
6. Procedure
60 grams sample is weighed with 50 milligrams sensitivity. The weighed sample and freshly boiled and cooled 75 milliliters of water, is placed into distillation flask in which a spray trap is situated. It is stirred and distilled into approximately 50 milliters.
Distillate is titrated with 0.01 M sodium hydroxide by using phenolphtalein indicator. Titration is continued until the first red color change in the solution goes on for 10 seconds.
7. Expression of the Results
7.1. Formula and method of calculation
The volatile acid amount is calculated in milligram for kilogram of acetic acid by using the following formula:
600 x V
---------------
mo
V= The volume of 0.01 M sodium hydroxide used, milliliters
mo= Amount of the sample, grams
7.2. Repeatability
The difference between the two analyses made under the same conditions, by the same analyst, at the same time and rapidly, one after the other, for the same sample should not be more than 1 milligram of every 100 grams of the sample.
Method 13: Determination of Nitrate in Orthophosphoric Acids (E338)
1. Scope and Field of Application
This method is used for the determination of nitrates in orthophosphoric acid (E338).
2. Definition
Determination of nitrate concentration in terms of sodium nitrate: determination of nitrate concentration in terms of sodium nitrate according to the specified method.
3. Principle
The sample is added into the indigo carmine solution in a concentrated sulfuric acid medium. It is destroyed by oxidized agents just like the blue colored nitrates.
4. Solutions
4.1. Indigo carmine solution, 0.18 % mass/volume, 0.18 grams of sodium indigotina, is dissolved in disulphonated water and completed to 100 milliliters
4.2. Sodium chloride solution, 0.05 % mass/volume
4.3. Concentrated sulfuric acid, density 1.84 grams/milliliters.
5. Procedure
2 milliliters of sample, is completed to 10 milliliters with sodium chloride solution. 0.1 milliliter indigo carmine solution is added. 10 milliliters of concentrated sulfuric acid is added slowly by cooling. Attention should be paid that the blue color formation.lasts for 5 minutes
5. Expression of the Results
6.1. Interpretation of the results
If the blue-color formation in the sample solution disappears in 5 minutes, the experiment is positive and oxidized agent content mentioned as sodium nitrate is bigger than 5 mg/kg.
6.2. Repeatability
The results of the two analyses made under the same conditions, by the same analyst, at the same time or rapidly one after the other for the same sample should be the same.
7. Notes
7.1. Comparison experiment should be applied.
7.2. The indigo carmine solution that is prepared should not be used more than 60 days.
If the result of the experiment is positive, the sample may contain nitrates or other oxidized agents. The experiment should be repeated by using ISO Method 3709 (1976) “The determination of oxides of phosphoric acid-nitrogen content for industrial use (including food products) –3, 4-xylenol spectrophotometric method”
Method 14: The Determination of water-insoluble substances
Inside Mono-, Di-and Tri-Sodium Orthophosphate as well as Mono-, Di- and Tri- Potassium Orthophosphate (E339 (i), E339 (ii), E339 (iii), E340 (i), E340 (ii), E340 (iii).
1. Scope and Field of Application
This method is used for the determination of water-insoluble substances, in mono-sodium orthophosphate (E399 (i), di-sodium orthophosphate (E399 (ii)), tri-sodium orthophosphate (E399 (iii)), mono-potassium ortho-phosphate (E340 (i)), di-potassium orthophosphate (E340 (ii)), tri-potassium orthophosphate (E340 (iii))
2. Definition
The amount of water-insoluble substances: The determination of water-insoluble substances with the specified method.
3. Principle
The sample is dissolved in water and filtered through a porcelain crucible. After the washing and drying process, the residue is measured and determined by calculating the water-insoluble.substance
4. Apparatus
4.1. Porcelain crucible, G3 or porous
4.2. Desiccators, that contains, newly activated silica gel with a water-content indicator, or equivalent desiccator.
4.3. Oven kept at 103±2oC thermostatically.
4.4. Polypropylene beaker, 400 milliliters
4.5. Boiling water bath.
5. Procedure
10 grams of phosphate sample is weighed with 10 milligrams sensitivity. For boiling, it is kept in polypropylene erlenmeyer flask, in hot water bath for 15 minutes and dissolved in 100 milliliters hot water. It is filtrated in the crucible, which is cleaned, dried and weighed beforehand. The residue that is not dissolved is washed with hot water. The container that has the residue is placed in the oven and dried for two hours at 103±2oC. At the end of this period, the container is taken into the desiccator, left to cool and weighed.
The procedures of drying, cooling and weighing are repeated until the difference between two consecutive measurements is less than 0.5 milligrams. If any increase is seen in the mass, the lowest reading is used in the calculation.
6. Expression of the Results
6.1. Formula and method of calculation
The amount of water-insoluble substance in the sample is calculated by the following formula:
m1
-------- x 100
mo
m1= the residue mass after the drying process, grams
mo= amount of the sample, grams
6.2. Repeatability
The difference between the two analyses made under the same conditions, by the same analyst, at the same time or rapidly one after the other for the same sample should not be more than 10 milligrams for every 100 grams of the sample.
Method 15: Determination of Ph in Food Additives
1. Scope and Field of Application
This method includes general methods concerning pH determination in food additives.
2. Definition
The pH of food additives; is the determination of the pH values of the food additives through the specified method.
3. Principle
The pH value of water-soluble solutions is determined by using glass electrodes, reference electrodes and pH meter.
4. Reagents
4.1. The instrument is calibrated using the following buffer solutions.
4.1.1. The pH: 6-88 buffer solution at 20oC: Contains 0.05 M potassium hydrogen phosphate (KH2PO4) and 0.05 M disodium hydrogen orthophosphate dehydrate (Na2PO4.2H2O).
4.1.2. pH: 4 buffer solution at 20oC: Contains 0.05 M potassium hydrogen phthalate. (C8H5O4).
4.1.3. pH: 9.22 buffer solution at 20oC: contains 0.05 M sodium borate (Na2B4O7.10H2O).
4.2. Another solution to be used to fill the reference electrode advised by the producing firm or 3 M or saturated potassium chlorine solution
4.3. Distilled water which is between pH 5-6 and which does not contain carbon dioxide
5. Apparatus
5.1. pH meter, with 0.01 sensitivity
5.2. Appropriate clamps to hold the electrodes
5.3. Magnetic mixer with heater element
5.4. Thermometer, 0-100oC
6. Procedure
6.1. Standardization of pH meter
Glass electrodes are placed in relevant parts of the equipment. Electrodes are checked by buffer solution the pH of which is known. Before the next use, electrodes are washed with water, should be wiped with a soft piece of cloth or passed through the new sample or standard solution after washing.
If the pH is acidic, the pH of the buffer solution to be used should be those of pH 4 and pH 6.88.If the pH is alkali, the pH of the buffer solution to be used should be those of pH 9.22 and pH 6.88.
6.2. Measurement of the sample solution
The sample solution is prepared by using distilled water and its temperature is adjusted to 20oC while as stirring. The glass electrode is placed inside the solution and 2 minutes later the pH value in the pH meter is read.
7. Expression of the Results
7.1. Repeatability
The difference between the two analyses carried out under the same conditions, by the same analyst, at the same time or rapidly, one after the other, for the same sample should not be more than 0.05 pH unit.
8. Note
This method can be applied to the water-soluble food additives