EAS nnnn-n: yyyy
DRAFT EAST AFRICAN STANDARD
Food grade saccharin  — Specification
EAST AFRICAN COMMUNITY
© EAC yyyy – All rights reservedISO/WD nnn-n
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© EAC 2019 – All rights reserved iiDEAS 995: 2019
Contents Page
Foreword..........................................................................................................................................................
1Scope....................................................................................................................................................
2Normative references ..........................................................................................................................
3Terms and definitions ..........................................................................................................................
4Requirements.......................................................................................................................................
4.1General requirements ..........................................................................................................................
4.2Specific requirements ..........................................................................................................................
5Hygiene.................................................................................................................................................
6Contaminants.......................................................................................................................................
7Packaging.............................................................................................................................................
8Weights and measures ........................................................................................................................
9Labelling...............................................................................................................................................
10Sampling...............................................................................................................................................
Annex A (normative)  Assay: Test for purity ...................................................................................................
Annex B (normative)  Loss on drying.............................................................................................................
Annex C  (normative)   Test for acidity and alkalinity ....................................................................................
Annex D (normative)  Determination of the melting range ............................................................................
Annex E (normative)  Test for benzoic and salicylic acid .............................................................................
Annex F (normative)  Test for readily carbonizable substances ..................................................................
Annex G (normative)  Test for Selenium.......................................................................................................
Annex H (normative)  Test for toluene sulfonamides ..................................................................................
Bibliography....................................................................................................................................................
© EAC 2019 – All rights reserved iiiDEAS 995: 2019
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.
iv © EAC 2019 – All rights reservedDEAS 995: 2019
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.
© EAC 2019 – All rights reserved vDEAS 995: 2019 
Food grade saccharin — Specification
1Scope
This Draft East African Standard specifies requirements, sampling and test methods for food grade saccharin
intended for use in food products. 
2Normative 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
3Terms 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
4Requirements
4.1General requirements
Food grade saccharin shall be:
© EAC 2019 – All rights reservedDEAS 995: 2019 
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.
4.2Specific 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 C 7H5NO3S, % 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
5Hygiene
Food grade saccharin shall be manufactured and handled in accordance with EAS 39.
6Contaminants
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/kgTest method
i) Lead (Pb) 1 AOAC 972.25
ii) Arsenic (As) 2 AOAC 952.13
iii) Selenium (Se) 30 Annex G
iv) Toluenesulfonamides 25 Annex H
7Packaging
 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.
© EAC 2019 – All rights reservedDEAS 995: 2019
8Weights and measures
The products shall comply with the Weights and Measures Regulations of the respective Partner States
9Labelling
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.
10Sampling
Representative samples of the product shall be drawn in accordance with CAC/GL 50.
© EAC 2019 – All rights reserved 3DEAS 995: 2019
Annex A
(normative)
Assay: Test for purity
A.1Apparatus
A.1.1Analytical balance 
A.1.2Hot air oven
A.2Reagents
A.2.10.1N sodium hydroxide
A.2.2Phenolphthalein indicator
A.2.3Distilled water
A.3Procedure
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 7H5NO3S.
The purity (as C7H5NO3S), expressed as percentage m/m (dry matter basis), shall be calculated as follows:
1001000 18.32
 MV
where
Vis the volume, in millilitres, of 0.1N NaOH used and
Mis the mass, in grams, of dried sample dissolved in 75 ml of hot water.  
4 © EAC 2019 – All rights reservedDEAS 995: 2019
Annex B
(normative)
Loss on drying 
B.1Apparatus
B.1.1Weighing bottle with a stopper 
B.1.2Air oven
B.1.3Desiccator
B.1.4Weighing scale 
B.2Sample 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.3Procedure 
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:
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 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. 
© EAC 2019 – All rights reserved 5DEAS 995: 2019
Annex C 
(normative) 
Test for acidity and alkalinity
C. 1Apparatus
Beaker
Measuring cylinder
C. 2Reagents
Phenolphthalein indicator
Sodium hydroxide
C. 3Procedure
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.1Procedure
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.
6 © EAC 2019 – All rights reservedDEAS 995: 2019
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.
© EAC 2019 – All rights reserved 7DEAS 995: 2019
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.
8 © EAC 2019 – All rights reservedDEAS 995: 2019
Annex F
(normative)
Test for readily carbonizable substances
F.1Reagents
F.1.1Sulphuric acid
F.1.2Matching 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.1.2.1Cobalt chloride solution:  Dissolve 65 g of cobaltous chloride (CoCl 2.6H2O) 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.1.2.2Ferric  chloride  solution:  Dissolve  55  g  of  ferric  chloride  (FeCl 3.6H2O)  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.1.2.3Cupric sulphate solution:  Dissolve 65 g of cupric sulphate (CuSO 4.5H2O) 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.2Procedure
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). 
© EAC 2019 – All rights reserved 9DEAS 995: 2019
Annex G
(normative)
Test for Selenium
G.1Reagents
G.1.1Selenium 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.1.2Standard selenium solution: Just prior 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.1.3Sample 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.2Procedure
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.
10 © EAC 2019 – All rights reservedDEAS 995: 2019
Annex H
(normative)
Test for toluene sulfonamides
H.1General
Two methods have been specified. Any method may be used depending upon the facilities available.
H.2Method 1 – Gas Chromatography
H.2.1Reagents
H.2.1.1Methvlene chloride: Use a suitable pure grade, equivalent to the product  obtained by distillation in
all glass apparatus.
H.2.1.2Internal 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.2.1.3Stock 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.2.1.4Diluted 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 chloride 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.2.1.5Test 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 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.2.2Procedure
H.2.2.1Inject 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.
NOTEThe 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.
© EAC 2019 – All rights reserved 11DEAS 995: 2019
H.2.2.2In a similar manner, obtain the chromatograms for 2.5- µl portions 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.3Method 2 – Thin Layer Chromatography
H.3.1Apparatus
H.3.1.1Flat glass plates (200 mm x 100 mm)
H.3.1.2Micropipette
H.3.1.3Developing chamber  lined with filter paper
H.3.2Reagents
H.3.2.1Silica gel 
H.3.2.2Chloroform
H.3.2.3Methyl alcohol
H.3.2.4Ammonia solution, strong
H.3.2.54–Sulphamoylbenzoic acid reference material
H.3.2.6Toluene–2–sulfonamide acid reference material
H.3.2.7Sodium hypochloride solution diluted with water to contain 0.5 % (m/v) of available chlorine
H.3.2.8Potassium iodide
H.3.2.9Starch mucilage
H.3.2.10Glacial acetic acid
H.3.2.11Solution A: 4 volumes of methyl alcohol plus 1 volume of acetone plus 0.5 % (m/v) of the sample
H.3.2.12Solution B: 4 volumes of methyl alcohol plus 1 volume of acetone plus 0.005 % (m/v) of the 4-
sulphamoylbenzoic acid
H.3.2.13Solution C:  4 volumes of methyl alcohol plus 1 volume of acetone plus 0.005 % (m/v) of the
toluene-2-sulfonamide.
H.3.3Procedure
12 © EAC 2019 – All rights reservedDEAS 995: 2019
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).
© EAC 2019 – All rights reserved 13DEAS 995: 2019
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.
14 © EAC 2019 – All rights reserved