FOR INTERNAL USE ONLY 
COMMITTEE DRAFT DRAFT UGANDA STANDARD  
  DUS DEAS  995 
First  Edition  
2019-mm-dd 
 
 Reference number  
DUS DEAS  995: 2019 
© UNBS 2019 
   
Food grade saccharin — Specification  
 
 FOR INTERNAL USE ONLY 
COMMITTEE DRAFTDUS DEAS 995: 2019 
ii                © UNBS  2019 - All rights reserved 
 Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2019 
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
form or by any means, electronic or mechanical, including photocopying and microfilm, without prior written 
permission from UNBS. 
Requests for permission to reproduce this document should be addressed to 
The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
Kampala 
Uganda 
Tel: +256 417 333 250/1/2 
Fax: +256 414 286 123 
E-mail: info@unbs.go.ug  
Web: www.unbs.go.ug  
 FOR INTERNAL USE ONLY 
COMMITTEE DRAFTDUS DEAS 995: 2019 
© UNBS  2019 - All rights reserved                iii National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry of Trade, Industry and 
Cooperatives established under Cap 327, of the Laws of Uganda, as amended.  UNBS is mandated to co-
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex Alimentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT Agreement of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a given field or area and consists of representatives of 
consumers, traders, academicians, manufacturers, government and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council.  
This Draft Uganda Standard, DUS DEAS 995: 2019, Food grade saccharin  — Specification , is identical with 
and has been reproduced from an East African Standard, DEAS 995: 2019, Food grade saccharin  — 
Specification , and is being proposed for adoption as a Uganda Standard. 
The committee responsible for this document is Technical Committee UNBS/TC 2,  Food and agriculture.  
Wherever the words, “ East African Standard " appear, they should be replaced by "Uganda Standard." 
 
  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 
This EAC document is copyright-protected by EAC. While the reproduction of this document by 
participants in the EAC standards development process is permitted without prior permission from EAC, 
neither this document nor any extract from it may be reproduced, stored or transmitted in any form for 
any other purpose without prior written permission from EAC. 
Requests for permission to reproduce this document for the purpose of selling it should be addressed 
as shown below or to EAC’s member body in the country of the requester:  
© East African Community 2019  — All rights reserved 
East African Community  
P.O. Box 1096, 
Arusha 
Tanzania 
Tel: + 255 27 2162100 
Fax: + 255 27 2162190 
E-mail: eac@eachq.org 
       Web: www.eac -quality.net  
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 Technical 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 development of East African Standards. 
East African Standards are subject to review, to keep pace 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-Benzisothiazole-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, saccharins 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 colourless 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, sulphur 
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 flavoured syrups for ice 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 — Specification 
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 some 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 food. 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 definitions 
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 sweetener 
synthetic substance used as a sugar substitute to sweeten food and drink 
  
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 soluble 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 requirements given in Table 1 when tested in accordance 
with the test methods specified therein. 
Table 1 —  Specific requirements 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 handled 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 contaminants 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   
© 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 products 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 information: 
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. 
  
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 P henolphthalein 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 C 7H5NO 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.   
 
 
  
© EAC 2019  – All rights reserved  5 
 Annex B 
(normative) 
 
Loss on drying  
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 drying 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 its contents (M 3).  
Calculate the loss on drying from the following equation:  
Loss on drying (%w/w) = 
  
where: 
 M 1 is the mass of sample in grams; 
 M 2 is the mass of sample and weighing bottle in grams before drying; and  
 M 3 is the mass of sample 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 mercury), 
maintain this vacuum for 24h, and then weigh the dried sample. Calculate the loss on drying using the same 
equation above.   
6 © EAC  2019  – All rights reserved  
 Annex C  
(normative)  
 
Test for 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 indicator. 
No pink colour shall appear. Add one drop of 0.1 M sodium hydroxide. A pink colour 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. 
  
© EAC 2019  – All rights reserved  7 
 Annex D 
(normative) 
 
Determination of the melting range 
D.1 Apparatus 
D.1.1 Desiccator 
D.1.2 10cm Capillary-tube   
D.1.3 Standard Thermometer 
D.1.4  Auxiliary Thermometer  
D.2 Procedures 
Dry the samples under the conditions 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, and 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 substance 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 correction 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 the 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. 
 
 
  
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.  
© 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. Place 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 
precipitate 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 adding 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 acid 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 liberated 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 distilled 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 0.1N sodium thiosulphate solution, adding starch as indicator. Each millilitre of 0.1N 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 sample 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).   
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 prior to the test, transfer 20 ml of selenium stock  solution (60 µ g 
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 excess 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 matter 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 the 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.  
© 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 preparation: Transfer 20 mg each of reagent grade  o-toluenesulfonamide and p-
toluenesulfonamide into  a 10-ml volumetric 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 bicarbonate 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 reservoir 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 to 30 min. To the eluate, add 25 µ l 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 
contact 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 standard 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, and 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.  
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 micrograms 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 preparation’. 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 volumes of methyl alcohol plus 1 volume of acetone plus 0.005 % (m/v) of the 
toluene-2-sulfonamide. 
  
© 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 20 °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 until 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). 
  
14 © EAC  2019  – All rights reserved  
 Bibliography 
[1] IS 5345: 1996 (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.