DEAS 995: 2019  
ICS 67.220.20  
 
© EAC 2019                                                                                                                               First Edition 2019   
 DRAFT EAST AFRICAN STANDARD  
Food grade saccharin  — Specification  
EAST AFRICAN COMMUNITY  
 
 
 
 
 
 
  DEAS 995: 2019  
ii © EAC  2019  – All rights reserved  
 Copyright notice  
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© East African Community  2019  — All rights reserved  
East African Community   
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Reproduction for sales purposes may be subject to royalty payments or a licensing agreement.  
Violators may be prosecuted . DEAS 995: 2019  
© EAC 2019  – All rights reserved  iii 
 Contents  Page 
Foreword  ................................ ................................ ................................ ................................ ............................  iv 
1 Scope  ................................ ................................ ................................ ................................ ......................  1 
2 Normative references  ................................ ................................ ................................ ............................  1 
3 Terms and definitions  ................................ ................................ ................................ ...........................  1 
4 Requirements  ................................ ................................ ................................ ................................ .........  2 
4.1 General requirements  ................................ ................................ ................................ ...........................  2 
4.2 Specific requirements  ................................ ................................ ................................ ...........................  2 
5 Hygiene  ................................ ................................ ................................ ................................ ...................  2 
6 Contaminants  ................................ ................................ ................................ ................................ .........  2 
7 Packaging  ................................ ................................ ................................ ................................ ...............  3 
8 Weights and measures  ................................ ................................ ................................ .........................  3 
9 Labelling  ................................ ................................ ................................ ................................ .................  3 
10 Sampling  ................................ ................................ ................................ ................................ .................  3 
Annex  A (normative)  Assay: Test for purity  ................................ ................................ ................................ ... 4 
Annex  B (normative)  Loss on drying  ................................ ................................ ................................ ...............  5 
Annex  C  (normative)   Test for acidity and alkalinity  ................................ ................................ .....................  6 
Annex  D (normative)  Determination of the melting range  ................................ ................................ ............  7 
Annex  E (normative)  Test for benzoic and salicylic acid  ................................ ................................ ..............  8 
Annex  F (normative)  Test for readily carbonizable substances  ................................ ................................ ... 9 
Annex  G (normative)  Test for Selenium  ................................ ................................ ................................ ........  10 
Annex  H (normative)  Test for toluene sulfonamides  ................................ ................................ ...................  11 
Bibliography  ................................ ................................ ................................ ................................ ......................  14 
 DEAS 995: 2019  
iv © EAC  2019  – All rights reserved  
 Foreword  
Development of the East African Standards has been necessitated by the need for harmonizing requirements 
governing quality of products and services in the East African Community. It is envisaged that through 
harmonized  standardization, trade barriers that are encountered when goods and services are exchanged 
within the Community will be removed.  
The Community has established an East African Standards Committee  (EASC)  mandated to develop and 
issue East African Standards  (EAS) . The Committee is composed of representatives of the National 
Standards Bodies in Partner States, together with the representatives from the public and private sector 
organizations  in the community .  
East African Standards are developed through  Tech nical Committees that are representative of key 
stakeholders including government, academia, consumer groups, private sector and other interested parties. 
Draft East African Standards are circulated to stakeholders through the National Standards Bodies in the 
Partner States. The comments received are discussed and incorporated before finalization of standards, in 
accordance with the Principles and procedures for develop ment of East African Standards . 
East African Standards are subject to review, to keep pac e with technological advances. Users of the East 
African Standards are therefore expected to ensure that they always have the latest versions of the standards 
they are implementing .  
The committee responsible  for this document is Technical Committee EASC /TC 005, Food additives . 
Attention is drawn to the possibility that some of the elements of this document may be subject of patent 
rights. EAC shall not be held responsible for identifying any or all such patent rights.  DEAS 995: 2019  
© EAC 2019  – All rights reserved  v 
 Introduction  
Saccharin (1,2 -Benzisoth iazole -3(2H) -one-1,1-dioxide, 3 -oxo-2,3-dihydrobenzo[d]isothiazol -1,1-dioxide) and 
its sodium, calcium and potassium salts are widely utilised in the food industry as non -nutritive artificial 
sweeteners. They are used as a substitute for cane sugar in the production of diet and low -calorie foods . They 
are colourless crystals or white crystalline powders with faint aroma and about 300 – 500 times sweeter than 
sucrose (table sugar). In the Codex Alimentarius Commission International Numbering System, sacchari ns are 
assigned as INS 954 and the FAO/WHO Joint Experts Committee on Food Additives (JECFA) established the 
Acceptable Daily Intake (ADI) for saccharin and its salts at 0  mg/kg – 5 mg/kg body weight.  
There are two main approaches to making saccharin: the Remsen -Fahlberg process (named after the two 
scientists who discovered the compound) and the Maumee or Sherwin -Williams process.  
The Remsen -Fahlberg process requires reacting toluene, which has a natural sweet smell, with chlorosulfonic 
acid, which is a c olourless liquid. This acid compound is then reacted with a series of compounds, including 
potassium permanganate and ammonia and heated to yield saccharin. Because this process takes a lot of 
compounds to produce a relatively low yield, improvements were sought -which is why the Maumee process 
was created.  
The Maumee process begins with converting phthalic anhydride, an industrial compound used in creating 
plastics to anthranilic acid. The acid is reacted with several compounds, including nitrous acid, sulp hur 
dioxide, chlorine and ammonia to produce saccharin.  
Common food products containing saccharin and/or its Na, Ca or K salts include: soft drinks (sodas), low -
calorie jams or jellies, biscuits, candies and dessert toppings such as flavo ured syrups for ic e cream, and 
salad dressings.  These sweeteners are also used in pharmaceutical and oral hygiene products . 
This standard has been developed to ensure that the saccharin used in the food industry  is safe for human 
consumption.  
 DEAS 995: 2019    
 
© EAC 2019  – All rights reserved  
  
 Food grade saccharin  — Specif ication  
1 Scope  
This Draft East African Standard specifies requirements, sampling and test methods  for food grade saccharin 
intended  for use in food products .  
2 Normative references  
The following documents are referred to in the text in such a way that so me or all of their content constitutes 
requirements of this document.  For dated references, only the edition cited applies. For undated references, 
the latest edition of the referenced document (including any amendments) applies.  
AOAC 952.13, Arsenic in fo od. Silver diethyldithiocarbamate method  
AOAC 972.25 , Lead in food. Atomic absorption spectrophotometric method  
CAC/GL 50 , General guidelines on sampling  
CODEX STAN 107, General standard for the labelling of food additives when sold as such  
3 Terms and def initions  
For the purposes of this document, the following terms and definitions apply.  
ISO and IEC maintain terminological databases for use in standardization at the following addresses:  
— ISO Online browsing platform: available at http://www.iso.org/obp   
3.1 
food grade material  
material, made of substances that  are safe and suitable for their intended use and which will not impart any 
toxic substance or undesirable odour or flavour to the product  
3.2 
artificial swee tener  
synthetic substance used as a sugar substi tute to sweeten food and drink  
 DEAS 995: 2019  
2 © EAC  2019  – All rights reserved  
 4 Requirements  
4.1 General  requirements  
Food grade saccharin shall be:  
a) white crystals or white  crystalline powder;  
b) odourless or with a faint  aromatic odour ; and  
c) slightly solubl e in water, soluble in basic solutions and sparingly soluble in ethanol.  1 g of product 
shall be soluble in 1.5 ml of water or basic solutions  and in about 50 ml of ethanol . 
4.2 Specific requirements  
Food grade saccharin shall comply with the specific req uirements given in Table 1  when tested in accordance 
with the test methods  specified therein.  
Table 1 — Specific r equirements for food grade  saccharin  
S/N Characteristic  Requirement  Test method  
i) Percentage purity, as C7H5NO 3S, % m/m (dry 
matter basis)  99 – 101  Annex A  
ii) Loss on drying, % m/m, max.   1 Annex B  
iii) Acidity and alkalinity  at 25 ºC To pass test  Annex C  
iv) Melting range, ºC 226 – 230  Annex D  
v) Benzoic and salicylic acid  To pass test  Annex E  
vi) Readily carbonizable substances  To pass test  Annex F  
5 Hygiene  
Food grade saccharin shall be manufactured and han dled in accordance with EAS 39.  
6 Contaminants  
Food grade saccharin shall  comply with the maximum levels of contaminants  given in Table 2 when tested in 
accordance with the test  methods specified therein.   
Table 2 — Maximum Limits for cont aminants in food grade saccharin   
S/N Contaminant  Maximum level  
mg/kg  Test method  
i) Lead (Pb)  1 AOAC 972.25  
ii) Arsenic (As)  2 AOAC 952.13  
iii) Selenium (Se)  30 Annex G 
iv) Toluenesulfonam ides 25 Annex H  DEAS 995: 2019  
© EAC 2019  – All rights reserved  3 
 7 Pack aging 
 Food grade saccharin shall be packaged in clean, sound and airtight food grade materials. The packages 
shall preserve the quality and safety of the product and preclude contamination from the external environment.  
8 Weights an d measures  
The product s shall comply with the Weights and Measures Regulations  of the respective Partner States  
9 Labelling  
In addition to the requirements of CODEX STAN 107,  the product packages shall be legibly and indelibly 
labelled with the following i nformation:  
a) name of the product  as “Food grade saccharin”;  
b) name and physical address of the processor/packer/importer;  
c) date of manufacture;  
d) expiry date;  and 
e) net weight  of the product in metric  units . 
10 Sampling  
Representative samples of the product shal l be drawn in accordance with CAC/GL 50.  
 DEAS 995: 2019  
4 © EAC  2019  – All rights reserved  
 Annex  A 
(normative)  
 
Assay: Test for purity  
A.1  Apparatus  
A.1.1  Analytical balance   
A.1.2  Hot air oven  
A.2  Reagents  
A.2.1 0.1N sodium hydroxide  
A.2.2 Phenolphthalein indicator  
A.2.3 Distilled water  
A.3  Procedure  
Dry 10 g of product in a hot air oven at 120 °C for 4h. Accurately weigh about 0.5 g ( M) of the dried sample 
and dissolve it in 75 ml of hot water. Quickly cool the mixture and add two to three drops of phenolphthalein 
indicator. Titrate the mixture with 0. 1N sodium hydroxide till the endpoint and record the volume of NaOH 
used ( V).   
Each millilitre of 0.1N sodium hydroxide is equivalent to 18.32 mg of C 7H5NO 3S. 
The purity (as C7H5NO 3S), expressed as percentage m/m (dry matter basis), shall be calculated as  follows:  
100
1000 18.32

 MV
 
where  
V is the volume, in millilitres, of 0.1N NaOH used and  
M is the mass, in grams, of dried sample dissolved in 75 ml of hot water.   
 
 
 DEAS 995: 2019  
© EAC 2019  – All rights reserved  5 
 Annex  B 
(normative)  
 
Loss on dryin g  
B.1  Apparatus  
B.1.1 Weighing bottle  with a stopper  
B.1.2 Air oven  
B.1.3 Desiccator  
B.1.4 Weighing scale   
B.2  Sample preparation  
Weigh 1 to 2 g of sample (M 1). Tare a glass -stoppered, shallow weighing bottle that has been dried for 30 
minutes at 105  ºC. Transfer the sample into the bottle, replace the cover, and weigh the bottle and the sample 
(M2). Distribute the sample as evenly as practicable to a depth of about 5 mm, and not over 10 mm.  
B.3  Procedure   
Place the bottle with its contents in the d rying chamber, removing the stopper and leaving it also in the 
chamber, and dry the sample at the 105 ºC for 2 hours. Upon opening the chamber, close the bottle promptly 
and allow it to come to room temperature in a desiccator. Weigh the cool bottle and it s contents (M 3).  
Calculate the loss on drying from the following equation:  
Loss on drying (%w/w) = 
  
where:  
 M1 is the mass of sample in grams;  
 M2 is the mass of sample and weighing bottle in grams before drying; and  
 M3 is the mass of samp le and weighing bottle in grams after drying and cooling in a desiccator.  
If the sample melts at a temperature lower than 105 ºC, prepare the sample as described above, then place it 
in a vacuum desiccator containing sulfuric acid. Evacuate the desiccator  to 130 Pa (1 mm of mercur y), 
maintain this vacuum for 24 h, and then weigh the dried sample. Calculate the loss on drying using the same 
equation above.  DEAS 995: 2019  
6 © EAC  2019  – All rights reserved  
 Annex  C  
(normative)  
 
Test f or acidity and alkalinity  
C. 1 Apparatus  
Beaker  
Measuring cylinder  
C. 2 Reagents  
Phenolphthalein indicator  
Sodium hydroxide  
C. 3 Procedure  
Dissolve 1 g of sample in 10 ml of freshly boiled and cooled water. Add one drop of phenolphthalein indicato r. 
No pink colour shall appear. Add one drop of 0.1 M sodium hydroxide. A pink col our shall appear.  
C.1  Procedure  
Dissolve 1 g of sample in 10 ml of freshly boiled and cooled water. Add one drop of phenolphthalein indicator. 
No pink colour shall appear.  Add one drop of 0.1 M sodium hydroxide. A pink colour shall appear.  
 DEAS 995: 2019  
© EAC 2019  – All rights reserved  7 
 Annex  D 
(norma tive) 
 
Determination of the melting range  
D.1 Apparatus  
D.1.1 Desiccator  
D.1.2 10cm C apillary -tube   
D.1.3 Standard Thermometer  
D.1.4  Auxiliary Thermometer  
D.2 Procedures  
Dry the samples under the condition s specified for Loss on Drying under Annex B or 
alternatively, dry the sample for 24 h in a desiccator.  
Transfer a quantity of the dried powder to a dry capillary -tube about 10 cm long and sealed 
at one end (thickness of the wall, 0.10 -0.15 mm; i.d. 0.9 -1.1 mm) and pack the powder by 
tapping the tube on a hard surface so as to form a tightly -packed column 2 -4 mm in height.  
Attach the capillary -tube and its contents to a standard thermometer so that the closed end 
is at the level of the middle of the bulb, a nd heat in a suitable apparatus containing an 
appropriate liquid (liquid paraffin or silicone oil) and fitted with a stirring device and an 
auxiliary thermometer. Regulate the rise in temperature during the first period to 3° per min.  
When the temperature has risen to 5° below the lowest figure of the range for the 
substance being tested, heat more slowly: if no other directions are given, the rate of rise in 
temperature should be 1 -2° per min.  
Unless otherwise directed, read the temperature at which the su bstance is observed to form 
droplets against the side of the tube and the temperature at which it is completely melted, 
as indicated by the formation of a definitive meniscus.  
 
D.4 Calculations  
To the temperature readings, apply the emergent -stem correctio n determined as follows: 
Before starting the determination of the melting range, adjust the auxiliary thermometer so 
that the bulb touches the standard thermometer at a point midway between the graduation 
for the expected melting range and the surface of t he heating material. When the substance 
has melted, read the temperature on the auxiliary thermometer.  
 
Calculate the correction to be added to the temperature reading of the standard 
thermometer from the following formula: 0.00015 N(T - t) in which T is the temperature 
reading of the standard thermometer, t is the temperature reading of the auxiliary 
thermometer and N is the number of degrees of the scale of the standard thermometer 
between the surface of the heating material and the level of the mercury.  
 
 
 DEAS 995: 2019  
8 © EAC  2019  – All rights reserved  
 Annex  E 
(normative)  
 
Test for benzoic and salicylic acid  
E.1 Procedure  
Add ferric chloride dropwise to 10 ml of a hot, saturated solution of the sample. No precipitate or violet colour 
shall appear.  DEAS 995: 2019  
© EAC 2019  – All rights reserved  9 
 Annex F 
(normative)  
 
Test for readily carbonizable substances  
F.1  Reagents  
F.1.1 Sulphuric acid  
F.1.2 Matching fluid  composed of 0.1 ml of  cobalt chloride solution (see F .1.2.1), 0.4 ml of ferric chloride 
solution (see E1.2.2), 0.1 ml cupric sulphate solution (see F .1.2.3) and 4.4 ml of distilled water.  
F.1.2.1  Cobalt chloride solution:  Dissolve 65 g of cobaltous chloride (CoCl 2.6H 2O) in a mixture of 25 ml of 
hydrochloric acid and 975 ml of distilled water. Pl ace exactly 5 ml of this solution in a 250 -ml iodine 
flask, add 5 ml of 3 % hydrogen peroxide solution and 15 ml of 20 % sodium hydroxide solution. Boil 
for 10 minutes, cool, and add 2 g of potassium iodide and 20 ml of 25 % sulphuric acid. When the 
precip itate has dissolved, titrate the liberated iodine with 0.1N sodium thiosulphate solution, adding 
starch as indicator. Each millilitre of 0.1N sodium thiosulphate is equivalent to 23.8 mg of cobaltous 
chloride. Adjust the final volume of the solution by add ing an adequate amount of the hydrochloric 
acid and distilled water mixture so that each millilitre contains 59.5 mg of cobaltous chloride.  
F.1.2.2  Ferric chloride solution:  Dissolve 55 g of ferric chloride (FeCl 3.6H 2O) in a mixture of 25 ml 
hydrochloric a cid and 975 ml of distilled water. Place 10 ml of this solution in a 250 -ml iodine flask. 
Add 15 ml of distilled water and 3 g of potassium iodide, and allow the mixture to stand for 15 
minutes. Dilute with 100 ml of distilled water, and titrate the libera ted iodine with 0.1N sodium 
thiosulphate solution, adding starch as indicator. Each millilitre of 0.1N sodium thiosulphate is 
equivalent to 27.03 mg of ferric chloride. Adjust the final volume of the solution by adding enough of 
the hydrochloric acid and d istilled water mixture to make each millilitre contain 45.0 mg of ferric 
chloride.  
F.1.2.3  Cupric sulphate solution:  Dissolve  65 g of cupric sulphate (CuSO 4.5H 2O) in a mixture of 25 ml of 
hydrochloric acid  and 975 ml of distilled water. Place 10  ml of  this solution in a 250 -ml iodine flask, 
add 40 ml of distilled water,  4 ml of acetic acid and 3 g of potassium iodide. Titrate  the liberated iodine 
with 0.1 N sodium thiosulphate  solution, adding starch  as indicator. Each millilitre of 0.1 N sodium  
thiosulphate is equivalent to 24.97 mg of cupric sulphate.  Adjust the final volume of the solution by 
adding  enough of the  hydrochloric acid and  distilled  water  mixture to make each millilitre contain 62.4 
mg of cupric sulphate.  
F.2  Procedure  
Dissolve 0.2 g of the sam ple in 5 ml of sulphuric acid. Keep the solution at 48 °C – 50 °C for 10 min. The 
colour of the resultant solution shall not be darker than that of the matching fluid in F .1.2 (a very light 
brownish -yellow).  DEAS 995: 2019  
10 © EAC  2019  – All rights reserved  
 Annex  G 
(normative)  
 
Test for Selenium  
G.1 Reagents  
G.1.1 Selenium stock solution : Transfer 120 mg of metallic selenium (Se) into a 1 000 -ml volumetric flask, 
add 100 ml of dilute nitric acid (1 in  2). Gently warm the solution on a steam -bath and  dilute  to volume with 
distilled water. Transfer 5 ml of this solution into a 200 -ml volumetric flask, dilute to volume with distilled water, 
and mix. Each millilitre of this solution contains 3 µg of selenium ions (Se).  
G.1.2 Standard selenium solution : Just pri or to the test, transfer 20 ml of selenium stock  solution (60 µg 
Se) into a 200 mm x 25 mm test tube,  add 20 ml of hydrochloric acid, and mix.  
G.1.3 Sample solution : Transfer 2 g of the sample to a 250 -ml Erlenmeyer flask,  and cautiously add 10 ml 
of 30 % hydrogen  peroxide solution. After the initial reaction has subsided, add  6 ml of 70 % perchloric acid, 
heat slowly until  white fumes of perchloric acid are copiously evolved and continue heating gently for a few 
minutes to ensure  decomposition of any exces s peroxide. If the solution  is brownish in colour due to non -
decomposed organic  matter, add a small amount of hydrogen peroxide solution  and heat again to white 
perchioric acid fumes, repeating,  if necessary until complete decomposition of the organic matt er when a 
colourless solution is obtained.  Cool, add 10 ml of distilled water and filter into a 200 mm x 25  mm test tube. 
Wash the filter paper with hot water until  the filtrate measures 20 ml, add 20 ml of hydrochloric  acid and mix.  
G.2 Procedure  
Place th e test tubes containing the standard selenium solution and the sample solution in a water -bath, and 
heat until the temperature of the solution reaches 40 °C. To each tube, add 400 mg of ascorbic acid, stir until 
dissolved and maintain at 40 °C for 30 min. Cool the solution, dilute with distilled water to 50 ml and mix.  
Any pink colour produced by the sample shall not exceed that produced by the standard selenium solution.  DEAS 995: 2019  
© EAC 2019  – All rights reserved  11 
 Annex  H 
(normative)  
 
Test for toluen e sulfonamides  
H.1 General  
Two methods have been specified. Any method may be used depending upon the facilities available.  
H.2 Method 1 – Gas Chromatography  
H.2.1 Reagents  
H.2.1.1 Methvlene chloride : Use a suitable pure grade, equivalent to the product  obtained by distillation in 
all glass apparatus.  
H.2.1.2 Internal standard stock solution: Transfer 100 mg of 95 % n-ticosane into a 10-ml volumetric flask. 
Dissolve in n -heptane, dilute to volume  with the same solvent, and mix.  
H.2.1.3 Stock standard prepar ation: T ransfer 20 mg each of reagent grade  o-toluenesulfonamide and  p-
toluenesulfonamide into  a 10 -ml volumetr ic flask. Dissolve in methylene chloride,  dilute to volume with the 
same solvent, and mix.  
H.2.1.4 Diluted standard preparation:  Pipette into five 10-ml volumetric flasks, 0. 1 ml, 0.25 ml, 1.0 ml, 2.5 
ml and 5.0 ml respectively, of the ‘stock standard preparation’ prepared in G.2.1.3. Pipette 0.25 ml of the 
‘internal standard stock solution’ into each flask, dilute each to volume with methylene chl oride and mix. These 
solutions contain 250 µg of n -tricosane, plus respectively, 20, 50, 200, 500 and 1000 µg per ml of each 
toluenesulfonamide, plus 250 mg of n -tricosane.  
H.2.1.5 Test preparation:  Dissolve 2 g of the sample in 8 ml of 5 percent  sodium bi carbonate solution. Mix 
the solution  thoroughly with 10 g of chromatographic siliceous  earth (Celite 545 or equivalent). Transfer the 
mix into  a 25 mm x 250 mm chromatographic tube having a  fritted glass disk and a Teflon stopcock at the 
bottom,  and a rese rvoir at the top. Pack the contents of the  tube by tapping the column on a padded surface, 
followed by tamping firmly from the top. Place 100 ml of  methylene chloride in the reservoir and adjust the  
stopcock so that 50 ml of eluate is collected in 20 min t o 30 min. To the eluate, add 25 µl of ‘internal standard 
stock solution’. Mix, and then concentrate the solution to a volume of 1 ml in a suitable concentrator tube fitted 
with a modified Snyder column, by using a Kontes tube heater maintained at 90 °C.  
H.2.2 Procedure  
H.2.2.1 Inject 2.5 µl of the test preparation (see G.1.1.5) into a suitable gas chromatograph equipped with 
a flame -ionization detector. The column should be of glass, approximately 3 m in length and 2 mm in inside 
diameter and packed with 3 % phenylmethyl silicone on 100 - to 120 -mesh equivalent to 150 - to 125 -micron 
IS test sieve and silanised calcined diatomaceous silica.  
NOTE  The glass column should extend into the injector for on -column injection and into the detector base to avoid 
contac t with metal.  
The carrier is helium flowing at a rate of 30 ml per min. The injection port, column, and detector are 
maintained at 225 °C, 180 °C and 250 °C respectively. The instrument attenuation setting should be such that 
2.5 µl of the ‘diluted standa rd preparation’ containing 200 µg per ml of each toluenesulfonamide gives a 
response of 40 % to 80 % of full -scale deflection. Record the chromatogram, note the peaks for o -
toluenesulfonamide, p -toluenesulfonamide, and the n -tricosane internal standard, an d calculate the areas for 
each peak by suitable means. The retention times for o -toluenesulfonamide, p -toluenesulfonamide, and n -
tricosane are about 5 min, 6 min and 15 min, respectively.  DEAS 995: 2019  
12 © EAC  2019  – All rights reserved  
 H.2.2.2 In a similar manner, obtain the chromatograms for 2.5 -µl por tions of each of the live ‘diluted 
standard preparations’ and for each solution, determine the areas of the o -toluenesulfonanude, p -
toluenesulfonamide, and n -tricosane peaks.  
From the values thus obtained, prepare standard curves by plotting concentration  of each toluenesulfonamide, 
in micro grams per millilitre versus the ratio of the respective toluenesulfonamide peak area to that of n -
tricosane. From the standard curve determine the concentration, in µg per ml, of each toluenesulfonamide in 
the ‘test pre paration’. Divide each value by 2 to convert the result to parts per million of toluenesulfonamide in 
the 2 g sample taken for analysis.  
NOTE: If the toluenesulfonamide content of the sample is greater than 500 mg/l, the impurity may crystallize out of the  
methylene chloride concentrate (see G.1.1.1). Although this level of impurity exceeds that permitted by the specification, 
the analysis may be completed by diluting the concentrate (usually 1: 10 is satisfactory) with methylene chloride containing 
250 µg of n-tricosane per ml, and by applying appropriate dilution factors in the calculation. Care shall be taken obtain a 
homogeneous solution by completely dissolving any crystalline toluenesulfonamide.  
H.3 Method 2 – Thin Layer Chromatography  
H.3.1 Apparatus  
H.3.1.1  Flat glass plates  (200 mm x 100 mm)  
H.3.1.2  Micropipette  
H.3.1.3  Developing chamber  lined with filter paper  
H.3.2 Reagents  
H.3.2.1  Silica gel  
H.3.2.2  Chloroform  
H.3.2.3  Methyl alcohol  
H.3.2.4  Ammonia solution , strong  
H.3.2.5  4–Sulphamoylbenzoic ac id reference material  
H.3.2.6  Toluene –2–sulfonamide acid reference material  
H.3.2.7  Sodium hypochloride solution  diluted with water to contain 0.5 % (m/v) of available chlorine  
H.3.2.8  Potassium iodide  
H.3.2.9  Starch mucilage  
H.3.2.10  Glacial acetic acid  
H.3.2.11  Solution A: 4 volumes of methyl alcohol plus 1 volume of acetone plus 0.5 % (m/v) of the sample  
H.3.2.12  Solution B: 4 volumes of methyl alcohol plus 1 volume of acetone plus 0.005 % (m/v) of the 4 -
sulphamoylbenzoic acid  
H.3.2.13  Solution C: 4 volu mes of methyl alcohol plus 1 volume of acetone plus 0.005 % (m/v) of the 
toluene -2-sulfonamide . 
 DEAS 995: 2019  
© EAC 2019  – All rights reserved  13 
 H.3.3 Procedure  
Prepare suspension of silica gel G . Spread the suspension on the plates about 0.25 mm thick. Allow to stand 
until the coating sets and then dry  the plates at 105 °C to 110 °C for one hour. Protect the plates from 
moisture. Pour into the developing chamber sufficient quantity of mobile phase (100 volumes of chloroform + 
50 volumes of methyl alcohol + 11.5 volume of strong ammonia solution) to form  a layer about 15 mm deep. 
Close the tank for one hour at 20 °C to 27 °C. Remove the narrow strips of the coating, about 5 mm inside 
from the margins of the chromatoplate. Using micropipette apply separately to the chromatoplates 2 ml each 
of solutions A, B and C. These spots should be about 25 mm from the bottom of the plates and not less than 
25 mm from the sides of the plates. The diameter of the spots should not be more than 6 mm. Dry the spots 
and place the chromatoplates in the developing chamber at 2 0 °C to 27 °C until the mobile phase has 
ascended to the 150 -mm line.  
Remove the plates and dry them in current of warm air. Then heat at 105 °C for five minutes. Spray the hot 
plates with the sodium hypochlorite solution. Dry in a current of cold air unt il sprayed area of the plate below 
the line of application give at most a faint blue colour with a drop of a mixture, prepared by dissolving 0.5 % 
(m/v) of potassium iodide in starch mucilage containing 1 % (m/v) of glacial acetic acid. Avoid prolonged 
exposure to cold air. Spray the plates with the same mixture.  
The spots in the chromatograms obtained with solution (B) and (C) should be more intense than any 
corresponding spots in the chromatogram obtained with solution (A).  
 DEAS 995: 2019  
14 © EAC  2019  – All rights reserved  
 Bibliography  
[1] IS 5345: 199 6 (Reaffirmed in 2001) , Sodium saccharin  food grade — Specification  (Second revision)  
[2] JECFA (2001), Saccharin monograph  
[3] Food chemicals Codex  (5th Edition)   
[4] JECFA (2006), Analytical methods, test procedures and laboratory solutions used by and  referenced 
in the food additive specifications, Volume 4.