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भारतीय मानक IS 1699 : 2024

Indian Standard

खाद्य रंग — नमूनाकरण और परीक्षण

पद्धतियााँ
( तीसरा पनु रीक्षण )

Food Colours — Methods of

Sampling and Test
( Third Revision )

ICS 67.220.20

 BIS 2024

भारतीय मानक ब्यरू ो

BUREAU OF INDIAN STANDARDS
मानक भवन, 9 बहादरु शाह ज़फर मार्ग, नई ददल्ली - 110002
MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI - 110002
www.bis.gov.in www.standardsbis.in

August 2024 Price Group 10

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Food Additives Sectional Committee, FAD 08

FOREWORD
This Indian Standard (Third Revision) was adopted by the Bureau of Indian Standards, after the draft finalized by
the Food Additives Sectional Committee had been approved by the Food and Agriculture Division Council.

These methods are intended to provide uniform procedure for sampling and testing of food colours permitted
under the Food Safety and Standards (Food Products Standards and Food Additives) Regulation, 2011. This
standard includes the general tests applicable to all the permitted food colours.

This standard was initially issued in two parts — (Part 1) in 1960 and (Part 2) in 1963. In the first revision issued
in 1974, (Part 1) and (Part 2) were amalgamated and the column chromatography method for determination of
dye intermediates was incorporated. In the second revision issued in 1995, the methods for determination of leuco
base in sulphonated triarylmethane colouring matters; chloride, sulphate, and heavy metals were incorporated.
The instrumental method for determination of metallic impurities were also incorporated.

In this revision, the following major changes have been made:

a) The spectrophotometric method for determination of total colouring matters content has been
incorporated as an alternate method;
b) The high performance liquid chromatography (HPLC) method for determination of dye intermediates
has been incorporated as an alternate method;
c) The inductively coupled plasma (ICP) technique for the measurement of limits of antimony, barium,
cadmium, chromium, copper, lead and zinc has been incorporated; and
d) The atomic absorption electro-thermal atomization (furnace atomization) technique for measurement of
limits of lead and cadmium has been incorporated.

The composition of the Committee responsible for the formulation of this standard is given in Annex A.

In reporting the result of a test or analysis made in accordance with this standard, is to be rounded off, it shall be
done in accordance with IS 2 : 2022 ‘Rules for rounding off numerical values (second revision)’.
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IS 1699 : 2024

Indian Standard
FOOD COLOURS — METHODS OF SAMPLING AND TEST
( Third Revision )

1 SCOPE 4.1.3 Precaution shall be taken to protect the sample,

the material being sampled, the sampling instrument
This standard prescribes the methods of sampling and the containers for samples from adventitious
and test for food colours. contamination.

2 REFERENCES 4.1.4 To draw a representative sample, the contents

of each container selected for sampling shall be
The standards given below contain provisions, mixed as thoroughly as possible by suitable means.
which through reference in this text, constitute
provision of this standard. At the time of publication, 4.1.5 The sample shall be placed in clean, dry,
the editions indicated were valid. All standards are air-tight glass containers or other suitable
subject to revision and parties to agreements based containers which do not react with the sample.
on this standard is encouraged to investigate the
possibility of applying the most recent edition of 4.1.6 The sample containers shall be of such a size
these standards: that they are almost completely filled by the sample.

IS No. Title 4.1.7 Each sample container shall be sealed air-tight

with a stopper after filling, and marked with full
IS 265 : 2021 Hydrochloric acid —
details of sampling, date of sampling, batch and code
Specification (fifth revision)
number.
IS 1070 : 2023 Reagent grade water —
Specification (fourth 4.1.8 The samples shall be stored in such a manner
revision) that the temperature of the material does not vary
unduly from the normal atmospheric temperature.
IS 2088 : 2023 Methods for determination of
arsenic (third revision)
4.1.9 The sampling shall be done by a person agreed
IS 4905 : 2015/ Random sampling and to between the purchaser and the supplier and in the
ISO 24153 randomization procedures presence of the purchaser (or his representative) and
: 2009 (first revision) the supplier (or his representative).

3 QUALITY OF REAGENTS 4.2 Scale of Sampling

Unless specified otherwise, pure chemicals and 4.2.1 Lot

distilled water (see IS 1070) shall be employed in
tests. All the containers of the same material produced
under the same conditions of manufacture shall be
NOTE — ‘Pure chemicals’ shall mean chemicals that do not grouped together to constitute a lot.
contain impurities which affect the results of analysis.
The sample shall be tested from each lot for
4 SAMPLING ascertaining the conformity of the material to the
requirements of the specification.
4.1 General Requirements of Sampling
4.2.2 The number of containers to be selected from
In drawing, preparing, storing and handling test the lot shall depend on the size of the lot and shall
samples; the precautions and directions as given be in accordance with col (2) and (3) of Table 1.
below shall be observed:
4.2.3 These containers shall be selected at a random
4.1.1 The samples shall be taken in a protected place from the lot. For this purpose, reference may be
not exposed to damp air, dust or soot. made to IS 4905.
4.1.2 The sampling instrument shall be clean and
dry.

To access Indian Standards click on the link below:

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IS 1699 : 2024

Table 1 Number of Containers to be Selected for 5 DETERMINATION OF TOTAL

Sampling COLOURING MATTERS CONTENT
(Clauses 4.2.2, 4.3 and 4.4.2)
5.1 General
Sl No. Lot Size No. of
Containers to be Two general methods are used for determination of
Selected total colouring matters: ‘colouring matters content
(1) (2) (3) by spectrophotometry’ and ‘colouring matters
i) 2 to 15 2 content by titration with titanium trichloride’. When
using the spectrophotometric method, the analyst
ii) 16 to 50 3 should take into account the accuracy and precision
iii) 51 to 150 5 of the spectrophotometer used for the analysis. All
colours present in the sample that absorb in the same
iv) 151 and above 8 region as that of the main colour will contribute to
the absorbance figure used to calculate the results;
4.3 Test Sample and Referee Sample subsidiary colouring matters of markedly different
hue will not be accounted for by this method. This
Draw with an appropriate sampling instrument, method uses accepted absorptivity figures obtained
small quantities of the material from different parts from purified standard colours for calculating the
of each container selected according to col (3) of total colouring matters content.
Table 1. Mix all the portion so drawn, thoroughly to
form a composite sample weighing not less than The titanium trichloride reduction method assumes
30 g. Divide the composite sample into three equal that isomers and subsidiary colouring matters have
parts to form test samples. Each part thus obtained the same titanium trichloride equivalent as the main
shall constitute the test sample weighing not less colouring matter.
than 10 g and shall be sufficient to conduct all the NOTE — The spectrophotometric method shall be the
tests. The test samples shall be transferred referee method in case of any dispute.
immediately to thoroughly clean and dry containers
which shall be sealed air-tight. These shall be 5.2 Method I — Colouring Matters Content by
labelled with the particulars given in 4.1.7. One Spectrophotometry
sample shall be for the purchaser, the second for the
supplier and the third sample bearing the seals of Three experimental procedures are described.
the purchaser and the supplier shall constitute the Procedure 1 is used for water-soluble colouring
referee sample to be used in case of dispute matters. Procedure 2 is used for organic
between the purchaser and the supplier and shall be solvent-soluble colouring matters, especially, the
kept in a place agreed to between the purchaser and synthetic carotenoids. (The solutions prepared in
the supplier. Procedure 2 are used in the identification tests for
the carotenoids). Procedure 3 is used for lakes.
4.4 Criterion for Conformity
5.2.1 Principle
4.4.1 The test for the various characteristics shall be
performed on the composite samples and shall meet The absorbance of a solution of the colouring matter
the corresponding requirements specified in the is determined at its wavelength of maximum
standard for that particular food colour. absorption and the total colouring matters content is
calculated using a standard absorptivity value given
4.4.2 In case any of the samples selected as per in the relevant Indian Standard.
col (3) of Table 1 fail in any of the tests, two more
samples shall be selected from the unopened 5.2.2 Apparatus
containers from the sample lot for retesting, if these
two samples satisfy the requirements of the 5.2.2.1 UV-visible range spectrophotometer —
specification, the lot shall be deemed to comply with capable of accurate (± 1 percent or better)
the requirements specified in the standard for that measurement of absorbance in the region of 350 nm
particular food colour. If either of the two samples to 700 nm with an effective slit width of 10 nm or
fail in the retests, the lot shall be deemed as not less .
conforming to the requirements specified in the
standard for that particular food colour. 5.2.2.2 Spectrophotometer cells — 1 cm path length

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5.2.3 Procedure 1 — Colouring Matters Content of 5.2.4.3 Calculation

Water-Soluble Colouring Matters
Total colouring matters, percent by mass
5.2.3.1 Accurately weigh 0.25 g (± 0.02 g) of the 𝐴 × 𝑉1 × 𝑉2 × 𝑉3
sample (M). Transfer to a 1 litre volumetric flask. = × 100
𝑣1 × 𝑣2 × 𝑀 × 𝐴1%
1𝑐𝑚
Add freshly distilled water or the solvent prescribed
where
in the specification monograph and swirl to dissolve.
Make up to volume and mix. Dilute to a solution of A = absorbance of the sample
suitable strength according to the details given in the solution at the wavelength
specification monograph. Measure the absorbance of maximum absorption;
(A) at the wavelength of maximum absorption in a
V1 = V2 = V3 = the volumes of the three
1 cm cell, using water or the prescribed solvent as
volumetric flasks (each
the blank.
100 ml); and
5.2.3.2 Calculation v1 = v2 = the volumes of the two
pipets (each 5 ml).
Total colouring matters, percent by mass 𝐴1%
1𝑐𝑚
= the specific absorbance of
the standard indicated in
𝐴 × 𝐹 the relevant Indian
= × 100 Standard;
𝑀 × 𝐴1%
1 𝑐𝑚
where
5.2.5 Procedure 3 — Colouring Matters Content of
A = the absorbance of the sample Lakes
solution at the wavelength of
maximum absorption; 5.2.5.1 Reagents

F = the dilution factor (volume a) Potassium dihydrogen phosphate — reagent

diluted/volume measured); and grade

𝐴1%
1𝑐𝑚
= the specific absorbance of the b) Sodium hydroxide — reagent grade
standard indicated in the
relevant Indian Standard. c) Phosphoric acid — reagent grade

5.2.4 Procedure 2 — Colouring Matters Content of d) Hydrochloric acid — reagent grade

Organic Solvent - Soluble Colouring Matters
5.2.5.2 Prepare pH 7 phosphate buffer as follows:
5.2.4.1 Reagents
a) Weigh 13.61 g of potassium dihydrogen
a) Chloroform, reagent grade, acid free; and phosphate into a 2 000 ml beaker, dissolve
in 200 ml of water, and dilute to 1 000 ml.
b) Cyclohexane, reagent grade. Add about 90 ml of 1 N sodium
hydroxide. Determine the pH using a pH
5.2.4.2 Accurately weigh 0.08 g (± 0.01 g) of the metre and adjust the pH to 7.0 using 0.1 N
sample (M) into a 100 ml volumetric flask (V1). Add sodium hydroxide or diluted phosphoric
20 ml of chloroform and dissolve by swirling acid.
briefly. Make sure that the solution is clear. Make up
to volume with cyclohexane and mix. Pipet
5.0 ml of the solution (v1) into a second 100 ml 5.2.5.3 Accurately weigh a quantity of lake which
volumetric flask (V2) and make up to volume with will give an absorbance approximately equal to that
cyclohexane. Pipet 5.0 ml of this diluted solution of the parent colour when the latter is tested
according to Procedure 1 above. Transfer to a 250 ml
(v2) into the final 100 ml volumetric flask (V3) and
beaker containing 10 ml hydrochloric acid previously
make up to volume with cyclohexane. Measure the diluted with water to approximately 50 ml. Heat with
absorbance (A) of the twice-diluted solution at the stirring to dissolve the lake, then cool to ambient
wavelength of maximum absorption in a 1 cm cell, temperature. Transfer to a 1 litre volumetric flask,
using cyclohexane as the blank. make up to volume with pH 7 phosphate buffer, and
mix. Proceed as detailed in Procedure 1 above, and
Perform this procedure promptly, avoiding exposure in the relevant Indian Standard, using pH 7 phosphate
to air insofar as possible and undertaking all buffer as the spectrophotometric blank.
operations in the absence of direct sunlight.

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IS 1699 : 2024

5.3 Method II — Titanium Trichloride Reduction 1 ml of TiCl3 = 0.013 36 g of tartrazine

Method
0.011 31 g of sunset yellow
5.3.1 Reagents 0.023 32 g of indigo carmine
0.015 11 g of ponceau 4R
5.3.1.1 Sodium citrate
0.012 56 g of carmoisine
5.3.1.2 Standard potassium dichromatic solution — 0.039 64 g of brilliant blue FCF
0.1 N 0.040 44 g of fast green FCF

5.3.1.3 Standard titanium trichloride solution — 6 DETERMINATION OF LOSS ON DRYING

0.1 N, prepared and standardized as described in
[see 5.3.1.3(a) and 5.3.1.3(b)] 6.1 Procedure

a) Prepare a 15 percent (m/v) solution of titanium Weigh accurately about 2 g of the material in a
trichloride. Take 200 ml of this solution, add 150 ml tared weighing bottle fitted with a ground-glass lid.
of concentrated hydrochloric (specific gravity 1.16) A weighing bottle of squat form about 50 mm in
and dilute to 2 000 ml so as to make the solution diameter and 30 mm in height is suitable. Heat for
approximately 0.1 N, place the solution in a 3 hours in an air-oven at 135 °C ± 2 °C. Cool in a
container provided with an arrangement to maintain desiccator and weigh.
it in an atmosphere of hydrogen and allow to stand
for two days for absorption of residual oxygen. 6.2 Calculation

(𝑀1−𝑀2) × 100
b) Standardization of titanium trichloride Loss on drying, percent by mass =
(𝑀1 − 𝑀)
solution
where
Weigh 3 g of ferrous ammonium sulphate M1 = mass, in g, of the weighing bottle
[FeSO4(NH4)2S04.6(H2O)] and transfer to a 500 ml with the material before heating;
flask. Introduce a stream of carbon dioxide and add
50 ml of freshly boiled water and 25 ml of sulphuric M2 = mass, in g, of the weighing bottle
acid (40 percent m/v). Then without interrupting the after heating; and
current of carbon dioxide, add rapidly 40 ml of the M = mass, in g, of the weighing bottle.
standard potassium dichromate solution, add the
titanium trichloride solution until the calculated end-
7 DETERMINATION OF WATER-
point is nearly reached. Then add quickly 5 g of
INSOLUBLE MATTER
ammonium thiocyanate (NH4CNS) and complete
the titration. Run a blank on 3 g of ferrous 7.1 Apparatus
ammonium sulphate using the same quantities of
water, sulphuric acid, ammonium thiocyanate and 7.1.1 Prepared Gooch Crucible
the current of carbon dioxide. From the net volume
of titanium trichloride, calculate the normality of Digest a good grade retentive asbestos with dilute
titanium trichloride as follows: hydrochloric acid (1 : 3), wash free from acid and
Normality of TiCl3 decant to remove fine particles. Prepare well-packed
asbestos mat of suitable thickness in a Gooch, wash
𝑚𝑙 𝑜𝑓 𝐾2𝐶𝑟2𝑂7 × 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝐾2𝐶𝑟2𝑂7 with hot water, dry. Ignite, rewash, redry at 135 °C,
= cool in a desiccator and weigh. Repeat washing,
𝑚𝑙 𝑜𝑓 𝑇𝑖𝐶𝑙3
heating and drying to constant mass. (Alternatively,
5.3.2 Procedure sintered glass filter Grade 4, may be used).

Prepare 1.0 percent (m/v) aqueous solution of the 7.2 Procedure

material. Take a quantity of the solution
corresponding to about 20 ml of the standard Dissolve 4.5 g to 5.5 g of the material in 200 ml of
titanium trichloride solution in a 500 ml Erlenmeyer hot water (80 °C to 90 °C) and allow the solution to
flask. Add 15 g of sodium citrate and dilute with cool to room temperature. Filter through the tared
water to a volume of 150 ml to 200 ml. Heat to boil Gooch or sintered glass filter, wash with cold water
and titrate with the standard titanium trichloride until the washings are colourless. Dry at
solution till colourless. Calculate percentage of pure 135 °C ± 2 °C for 3 hours. Cool in a desiccator and
dye in the material. weigh.

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7.3 Calculation the first separator with 10 ml of the ether, decanting

into the second separator. Reserve the aqueous
Water insoluble matter, percent by mass colour solution (see 8.3.2). Wash combined extracts
with 20 ml portions of water until the washings are
(𝑀2 − 𝑀1 ) × 100 colourless. Decant the ether into a beaker. Place the
= beaker on a water-bath or steam-bath in dust free
𝑀
where atmosphere and allow the ether to evaporate to a
volume of 50 ml. Transfer to a previously weighed
M2 = mass, in g, of the Gooch with the evaporating dish of 250 ml capacity. Rinse the
residue; beaker with 40 ml of ether and drain into the same
M1 = mass, in g, of the prepared dish. Evaporate the remaining ether, dry in a
Gooch; and desiccator and weigh. Repeat the process of
evaporating, drying and weighing till the difference
M = mass, in g, of the material taken between two successive weighing is less than a
for the test. milligram. Note the lowest mass. Calculate the
mass of the neutral ether extract.
8 DETERMINATION OF COMBINED
EXTRACTS NOTE — Do not fill the beaker or dish more than one-third
of the capacity and do not allow the ether to boil.

8.1 Apparatus 8.3.2 Alkaline Ether Extract

Separator or continuous extractor of 250 ml To the reserved aqueous solution (see 8.3.1), add
capacity. 2 ml of sodium hydrochloride solution and proceed
in the same manner as in 8.3.1 except to wash the
8.2 Reagents ether extract with sodium hydroxide wash solution
instead of water. Reserve the aqueous solution for
8.2.1 Isopropyl Ether — wash one litre of isopropyl use in 8.3.3. Calculate the mass of the alkaline ether
ether with: extract.
a) two 100 ml portions of sodium hydroxide
8.3.3 Acid Ether Extract
(0.5 N);
b) saturated solution of ferrous sulphate; and To the reserved aqueous solution (see 8.3.2) add
3 ml of dilute hydrochloric acid (1 : 1) and proceed
c) with three 100 ml portions of water. in the same manner as in 8.3.1 except to wash the
ether extract with hydrochloric acid wash solution
8.2.2 Sodium Hydroxide Solution — 10 percent instead of water. Discard the colour solution.
(m/v) Calculate the mass of the acid ether extract.
8.2.3 Sodium Hydroxide Wash Solution — 0.1 N
8.4 Procedure for Extraction in the Continuous
Extractor
8.2.4 Dilute Hydrochloric Acid (1 : 1)
8.4.1 Neutral Ether Extract
Prepared by diluting hydrochloric acid, specific
gravity 1.16 (see IS 265) with equal volume of Dissolve 5 g of the material in water and extract in
water. the continuous extractor with about 100 ml of the
ether for 5 hours. Transfer the extract to the
8.2.5 Hydrochloric Acid Wash Solution — separator, rinse the flask with 10 ml of ether and add
concentrated hydrochloric acid diluted 200 times the rinsing to the main extract. Proceed in the same
manner as in 8.3.1 from the washing stage onwards.
8.3 Procedure for Extraction in the Separator
8.4.2 Alkaline Ether Extract
8.3.1 Neutral Ether Extract
To the aqueous solution in the extractor add 2 ml of
Place the aqueous, solution containing 10 g of the sodium hydroxide solution and proceed in the same
material in a separator and dilute to 200 ml. Extract manner as in 8.4.1 except to wash the ether extract
with two 100 ml portions of the washed ether, with sodium hydroxide wash solution instead of
shaking for one minute during each extraction. water.
Decant ether into another clean separator and rinse

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IS 1699 : 2024

8.4.3 Acid Ether Extract 9.2.2 Microsyringe — capable of delivering 0.1 ml

with a tolerance of ± 0.002 ml
Add 3 ml of dilute hydrochloric acid solution to the
alkaline aqueous solution of the material in the 9.2.3 Spectrophotometer
extractor and proceed in the same manner as in
8.4.1 except to wash the ether extract with 9.3 Reagents
hydrochloric acid wash solution instead of water.
9.3.1 Chromatography Solvents
9 SUBSIDIARY DYES
a) Water : ammonia (specific gravity 0.880) :
9.1 Principle trisodium citrate (95 ml : 5 ml : 2 g);

The subsidiary dyes are separated from the main b) n-butanol : water : ethanol : ammonia
dye by ascending paper chromatography and are (specific gravity 0.880) (600 : 264 : 135 :
extracted separately from the paper. The optical 6);
densities of the extracts are measured at their c) Butan-2-one : acetone: water (7 : 3 : 3);
wavelengths of maximum absorption in the visible
spectrum and are used to calculate the content of d) Butan-2-one : acetone : water : ammonia
subsidiary dyes as a percentage by mass of the (specific gravity 0.880) (700 : 300 : 300 :
sample. 2);
e) Butan-2-one : acetone: water: ammonia
9.2 Apparatus (specific gravity 0.880) (700 : 160 : 300 :
2); and
9.2.1 Chromatography Tank and Ancillary
Equipment f) n-butanol : glacial acetic acid : water (4 :
1 : 5).
Suitable apparatus as shown in Fig. 1 and
comprising the following: Shake for 2 minutes, allow layers to separate. Use
the upper layer as the chromatography solvent. The
a) a glass tank (A) and glass cover (B); particular solvent to be used as given in individual
specifications.
b) a supporting frame (C) for the
chromatography grade paper sheets; 9.3.2 Extracting Solvent — a mixture of equal
c) a tray (D) for developing solvent; volumes of acetone and water
d) a secondary frame (E) supporting drapes of 9.3.3 Sodium Bicarbonate — 0.05 N
filter paper; and
e) sheets of chromatography grade paper, not
less than 200 mm × 200 mm
(Whatman No. 1 chromatography grade is
suitable).

FIG. 1 CHROMATOGRAPHY APPARATUS

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9.4 Procedure sodium bicarbonate solution and shake the tube to

ensure mixing. Filter the coloured extract, and
9.4.1 Not less than 2 hours before carrying out the blanks through a 9 cm filter paper of open texture
determination, arrange the filter-paper drapes in the and determine wavelengths of maximum absorption,
glass tank and pour over the drapes and into the using cells of suitable light path, against a filtered
bottom of the tank sufficient of the atmosphere mixture of 5.0 ml of extracting solvent and 15.0 ml
saturating solvent to cover the bottom of the tank to of the sodium bicarbonate solution. Measure the
a depth of approximately 1 cm. Place the solvent tray optical densities of the extract of the blank strips at
D in position and fit the cover to the tank. the wavelengths at which those of the corresponding
coloured extracts were measured.
9.4.2 Mark out a sheet of chromatography grade
paper as shown in Fig 2. Apply 0.10 ml of a 1.0 9.5 Calculation
percent aqueous solution of the dye as uniformly as
possible within the confines of the 180 mm × 7 mm The content of the subsidiary dye, expressed as a
rectangle, holding the nozzle of the micro syringe percentage (S) of the sample is given by:
steadily in contact with the paper. Allow the paper
to dry at room temperature for 1 hours to 2 S = F [(D1 + D2 + ……) – (b1 + b2 + ········)]
hours, or at 50 °C for 5 minutes followed by 15
where
minutes at room temperature. Mount the sheet,
together with a plain sheet to act as a blank, in
F = the mean conversion factor
frame C. Pour sufficient of the chromatography
and is equal to 11.4;
solvent into the tray D to bring the surface of the
solvent about 1cm below the base line of the sheet D1 = D2 = are the optical densities of
of chromatography paper. The volume necessary the subsidiary dye extracts;
will depend on the dimensions of the apparatus and and
should be predetermined. Put the frame C into
position and replace the cover. Allow the solvent b1 = b2 = the optical densities of the
front to ascent the full height of paper, development extracts of the corresponding
being continued for 1 hour afterwards, then remove blanks.
the frame C and transfer it to a drying cabinet at 50
°C to 60 °C for 10 minutes to 15 minutes. Remove The conversion factor F in the above expression is
the sheets from frame C. derived from the extraction coefficient of the main
colour, not that of the subsidiaries, and from the
NOTE — If required, several chromatograms may be other constants of the determination.
developed simultaneously.

10 DETERMINATION OF DYE
INTERMEDIATES (ORGANIC COMPOUNDS
OTHER THAN COLOURING MATTERS)

Two general methods are used for determination of

dye intermediates: Determination by high
performance liquid chromatography and
determination by column chromatography. The high
performance liquid chromatographic (HPLC)
method shall be the referee method in case of any
dispute.

10.1 Method I — Determination by High

Performance Liquid Chromatography

FIG. 2 METHOD OF MARKING OUT 10.1.1 Principle

CHROMATOGRAPHY PAPER
The organic compounds other than colouring
9.4.3 Cut each subsidiary band from the sheet as a matters are separated by HPLC using gradient
strip, and cut an equivalent strip from the elution and are quantitatively determined by
corresponding position of the plain sheet. Place each comparison of their peak areas against those
strip, subdivided into a suitable number of obtained from standards. The conditions prescribed
approximately equal portions in a separate test-tube. must be treated as guidelines and minor
Add 5.0 ml of extracting solvent to each test-tube, modifications might be needed to achieve
swirl for 2 minutes to 3 minutes, add 15.0 ml of the the separations. Deviations from the prescribed

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IS 1699 : 2024

conditions, such as a different column length, other h) Integration — peak area.

types of column packing and solvent system, and the
use of paired ion procedures, can result in elution 10.1.5 Procedure
characteristics different from those for the
conditions given here, such as order of elution and Prepare 0.5 percent (m/m) colouring matter sample
resolution. solutions in 0.02 M ammonium acetate. Prepare
calibration solutions from standards of impurities
10.1.2 Apparatus named in the specification monograph.

10.1.2.1 High–performance liquid chromatograph Analyse, following the instructions given for the
— HPLC capable of gradient elution with: HPLC chromatograph and detector.
a) controller/integrator; 10.2 Method II — Determination by Column
b) pump(s), flow rate 1 ml/min; Chromatography
c) auto-sampler with a 20 μl injector ; 10.2.1 Apparatus
d) detector, UV-visible absorption; and
10.2.1.1 Chromatographic tube — see Fig. 3
e) printer/plotter.
10.2.1.2 Suitable spectrophotometer for use in the
10.1.2.2 Chromatography column — C-18 on silica ultra violet range
gel, 5 μm particle size, 250 × 4.6 mm

10.1.2.3 Guard column — C-18 on silica gel, 5 μm

particle size, 15 × 4.6 mm

10.1.3 Reagents

10.1.3.1 Methanol — HPLC grade

10.1.3.2 Ammonium acetate — HPLC grade

10.1.3.3 Reference standards as required

10.1.4 Instrument Parameters

a) Injection volume — 20 μl;

b) Eluents:
1) A — 0.2 N ammonium acetate; and
2) B — methanol.
c) Gradient:
1) 0.0 (sample injection);
FIG. 3 CHROMATOGRAPHIC APPARATUS
2) 0 min to 35 min — 0 percent to
40 percent B (analysis); 10.2.1.3 Column preparation
3) 35 min to 41 min — 100 percent B
Prepare a slurry of Whatman powdered cellulose (or
(wash); and
equivalent) in a 25 percent ammonium sulphate
4) 41.1 min to 55 min — 100 percent to (very low in iron) solution. If other cellulose is used,
0 percent B (return to initial gradient the iron content should be very low. Prepare the
composition and equilibrate column). column and pass 200 ml of 25 percent ammonium
sulphate solution through it. The ultra-violet
d) Flow rate — 1.0 ml per min;
absorption of the solution shall be sufficiently low to
e) Temperature — Ambient; avoid interference with the intended analysis. Use
about 75 g of cellulose to 500 ml of liquid, place
f) Pump pressure — minimum 300 psi, a small disc of stainless steel gauze in the
maximum 4000 psi; constriction above the tip of the tube. Pour
g) Detector wavelengths — as required; and sufficient slurry into the tube to give a column to a

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IS 1699 : 2024

height of about 5 cm in the mouth of the tube. Tap 11 DETERMINATION OF UNSULPHONATED

the tube occasionally to ensure a well-packed PRIMARY AROMATIC AMINES
column. Wash the column with 200 ml of the
eluent. 11.1 Principle

10.2.2 Procedure Unsulphonated primary aromatic amines are

extracted into toluene from an alkaline solution of
10.2.2.1 Place 0.200 g of the dye sample in a beaker the sample, re-extracted into acid and then
and dissolve in 20 ml of water, Add approximately determined spectrophotometrically after
5 g of powdered cellulose. Add 50 g of ammonium diazotisation and coupling. They arc expressed as
sulphate to the dye. Transfer the mixture to the aniline unless they are known to be some other
column, rinse the beaker with 25 percent amine.
ammonium sulphate solution and add washings to
the tube. Allow the column to drain until flow 11.2 Apparatus
ceases or nearly so. Add the ammonium sulphate
solution to the column at the rate equivalent to the 11.2.1 Visible Range Spectrophotometer
rate of flow through the column. Collect the
effluent in 100 ml fractions. Continue until 11.3 Reagents
12 fractions have been collected. Reserve the
column and contents until the last fractions have The reagents shall be of a recognized analytical
been examined. Mix each fraction well, and obtain reagent quality. Distilled water or water of at least
the ultra-violet absorption spectra of each solution equal purity shall be used.
from 220 nm to 400 nm. The specific spectra may
be chosen depending on the nature of the dyes. If 11.3.1 Toluene
the spectrum of the twelfth fraction shows the
presence of any intermediate, continue collecting 11.3.2 Hydrochloric Acid — 1 N solution (approx)
fractions until the intermediate present are eluted.
Usually only one intermediate is encountered. 11.3.3 Hydrochloric Acid — 3 N solution (approx)
Identification and quantitative determination shall
be accomplished by comparison of the absorption 11.3.4 Potassium Bromide — 50 percent solution
spectra of the eluted material with the spectra of (approx)
solutions of the pure intermediaries in the solvent.
When more than one intermediate is present in 11.3.5 Sodium Carbonate — 2 N solution (approx)
quantities in any fractions, the spectrophotometric
data shall indicate this. In such cases, the amounts, 11.3.6 Sodium Hydroxide — 1 N solution (approx)
of the various intermediaries, should be determined
by the procedure customarily used in 11.3.7 Sodium Hydroxide — 0.1 N solution (approx)
spectrophotometric analysis of mixtures of
absorbing materials. 11.3.8 R Salt (2-naphthol-3,6-disulfonic acid,
disodium salt) — 0.05 N solution (approx)
10.2.2.2 Some samples contain small amounts of
various materials, particularly inorganic salts, that 11.3.9 Sodium Nitrite — 0.5 N solution (approx)
contribute ‘background absorption’. Correction for
this is made as follows: 11.3.10 Standard Aniline Solution

a) Determine the amount of such absorption a) Solution A — Into a small weighing beaker,
of the fraction collected from the column weigh 0.100 g of redistilled aniline, then
immediately before and after the fraction wash it into a 100 ml one-mark volumetric
immediately following those fractions in flask, rinsing the beaker several times with
which the intermediates are encountered. water. Add 30 ml of approx. 3 N
Subtract one-half of the sum of these two hydrochloric acid solution and dilute to the
determinations from the observed mark with water at room temperature; and
absorbance of the fractions containing the
intermediates. The remainder should be b) Solution B — Dilute 10.0 ml of Solution A
taken as the absorbance due to the with water to 100 ml in a one-mark
intermediate present.

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volumetric flask and mix well. 1 ml of this test solution, 10 ml of the sodium carbonate
solution will be equivalent to 0.000 01 g of solution and 2.0 ml of the solution, diluted to
aniline. 25.0 ml with water.

NOTE — Prepare solution when freshly when

required.
Read from the calibration graph the mass of aniline
corresponding to the observed optical density of the
11.4 Procedure test solution.

11.4.1 Preparation of Calibration Graph 11.5 Calculation

Measure 5 ml, 10 ml, 15 ml, 20 ml and 25 ml of Percentage of unsulphonated primary aromatic

standard aniline Solution B into a series of 100 ml amine (as aniline) in sample
one-mark volumetric flasks.
Mass of aniline × 100
=
Dilute to 100 ml with approximately 1 N Mass of sample taken
hydrochloric acid solution and mix well. Pipette
10 ml of each mixture into clean, dry test tubes and 12 DETERMINATION OF LEUCO BASE IN
cool for 10 minutes by immersion in a beaker of SULPHONATED TRIARYLMETHANE
ice/water mixture. To each test tube add 1 ml of the COLOURING MATTER
potassium bromide solution and 0.05 ml of the
sodium nitrite solution. Mix and allow to stand for 12.1 Principle
10 minutes in the ice/water bath. Into each of the five
25 ml volumetric flasks, measure 1 ml of the R salt Air is blown through an aqueous solution containing
solution, and 10 ml of the sodium carbonate the chloride and dimethylformamide. Under these
solution. Pour each diazotised aniline solution into a conditions the leuco base is oxidized to colouring
separate flask containing R salt solution, rinsing the matters and the increase in absorptivity is a measure
test tubes with a few drops of water. Dilute to the of the amount of leuco base originally present.
mark with water, stopper the flasks, mix the contents
well and allow to stand for 15 minutes in the dark. 12.2 Reagent
Measure the absorbance of each coupled solution at
510 nm in 1 cm cells, using as a reference a mixture 12.2.1 Dimethylformamide (DMF)
of 10.0 ml of 1 N hydrochloric acid solution, 10.0 ml
of the sodium carbonate solution, and 2.0 ml of the a) Solution A — Weigh 10.0 g of CuCl2.2H2O
R salt solution, mass of aniline in each 100 ml of and dissolve in 200 ml of DMF. Transfer to
aniline solution. a 1 ml volumetric flask and make up to the
mark with DMF; and
11.4.2 Preparation and Examination of Test
Solution b) Solution B — Accurately weigh the
specified quantity of sample, dissolve in
Weigh to the nearest 0.01 g about 2.0 g of the colour approximately 100 ml water, transfer
sample into a separating funnel containing 100 ml of quantitatively to a 10 litre volumetric flask
water, swirl down the sides of the funnel with further and make up to the mark with water.
50 ml of water. Swirl to dissolve the sample, and add
5 ml of 1 N sodium hydroxide solution. Extract with 12.3 Procedure
two 50 ml portions of 0.1 N sodium hydroxide
solution to remove traces of colour. Extract the 12.3.1 Prepare the following solutions:
washed toluene with three 10 ml portions of 3 N
hydrochloric acid solution and dilute the combined a) Solution A — Pipette 50 ml DMF into a
extract to 100 ml with water. Mix well. Call this 250 ml volumetric flask. Cover with
solution T. Pipette 10.0 ml of solution T into a clean, parafilm and place in the dark;
dry test tube, cool for 10 minutes by immersion in a b) Solution B — Accurately pipette 10 ml of
beaker of ice/water mixture, add 1 ml of the Solution B into a 250 ml volumetric flask.
potassium bromide solution and proceed as Add 50 ml DMF. Cover with parafilm and
described above for the preparation of the place in the dark;
calibration graph, starting with the addition of
0.05 ml of the sodium nitrite solution. c) Solution C — Pipette 50 ml of Solution A
into a 250 ml volumetric flask. Bubble air
Use as the reference solution in the measurement of through this solution for 30 minutes, in the
absorbance, a solution prepared from 10.0 ml of the

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Table (Concluded)
1) Invert a 5 ml pipette into a box Sl Curve Cell Cell Comments
attached to a bench air flow source. No.
Turn on the air, slowly. Stick the (1) (2) (3) (4) (5)
pipette down into the solution in the
flask and adjust the air flow to a rapid ii) 2 a b Run curve
but controlled rate. After 30 minutes without
pull the pipette out of the solution and readjusting zero
rinse the side of the pipette into the setting; record
flask with water from a wash bottle. absorbance at
Then turn off the air flow. maximum
d) Solution D — Accurately pipette 10 ml iii) 3 c c Set zero at
Solution B into two separate 250 ml 700 nm;
volumetric flask, in the same manner as record
used for Solution B. Add 50 ml Solution A absorbance at
to each flask. Bubble air through the absorption
solutions for 30 minutes, using the above standard for
method. colouring matter

12.3.2 After 30 minutes of rapid bubbling of air iv) 4a c d1 Run curve

through the solutions, dilute all 5 flasks nearly to without
volume with water. Heat is evolved when DMF and readjusting zero
water are mixed, so place the flask in a water bath of setting; record
tap water until they have cooled to room absorbance at
temperature. Do not leave them for longer than maximum
necessary: 5 minutes to 10 minutes is normally long v) 4b c d2 Run curve
enough. Bring accurately to volume with water. without
Run the solutions on the spectrophotometer readjusting zero
immediately. The entire procedure should be selling; record
completed as quickly as possible. absorbance at
maximum
12.4 Spectrophotometric Determination NOTES
1 d1 and d2, are duplicate determination.
12.4.1 Draw the following curves from 700 nm to
500 nm using the absorbance range of 0.1 cm and 2 Cells must be thoroughly rinsed before each run for
the flow through cell, use 3 separate rinses of at least
1 cm cells. Run all the curves on same spectrogram, 40 ml of the sample solution to be run.
and (for maximum accuracy) take readings for the
numerical display at the maximum between 620 nm 12.5 Calculation
and 635 nm by cranking back after the curve is
drawn. Leuco base, percent by mass =

12.4.2 Reference Sample [(4 − 3) − (2 − 1)] × 25 × 100

𝑎 × 1 𝑐𝑚 × 𝑀 × 𝑟𝑎𝑡𝑖𝑜
Sl Curve Cell Cell Comments
where
No.
(1) (2) (3) (4) (5) a = absorptivity of 100 percent
i) 1 a a Set zero at colouring matters;
𝑡𝑒𝑟
700 nm, run M = mass, in mg, of sample taken for
curve; record test; and
absorbance at
absorption ratio Molecular weight of colouring matter
=
standard for Molecular weight of leuco base
colouring matter

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13 DETERMINATION OF CHLORIDE AS sodium sulphate.

SODIUM CHLORIDE
Mass of sodium sulphate (Na2SO4) =
13.1 Apparatus
Express the result as a percentage of the mass of
Potentiometric titration apparatus, with silver sample taken.
indicator electrode, calomel reference electrode, and
saturated potassium sulphate bridge. 15 DETERMINATION OF METALLIC
IMPURITIES
13.2 Procedure
Both instrumental and chemical methods have been
Accurately weigh 0.5 g to 1.0 g of the dye sample, given for determination of metallic impurities. For
dissolve in 100 ml of water, and acidify with 5 ml of certain metallic impurities, namely; antimony,
1.5 N nitric acid solution. Place the silver electrode barium, cadmium, zinc and mercury only
in the colour solution and connect the calomel instrumental methods have been given. For lead,
electrode to the solution by means of the saturated arsenic, copper and chromium both instrumental and
potassium sulphate bridge. The saturated potassium chemical methods have been given. In cases where
sulphate bridge may be eliminated by using a glass more than one method has been given, any of these
electrode as the reference electrode; this simplifies may be used. However, in case of dispute,
the apparatus considerably, and the glass electrode instrumental method shall be used as referee
is sufficiently constant to be used as a reference for method.
this type of titration.
15.1 Instrumental Methods
Determine the chloride content of the solution by
titration against the 0.1 N silver nitrate solution
(AgNO3) and calculate the result as sodium 15.1.1 Principle
chloride. [1 ml of 0.1 N silver nitrate
The samples are dissolved in acid or digested in a
solution = 0.005 85 g of sodium chloride (NaCl)].
mixture of sulphuric, nitric and, in some cases per
Express the result as a percentage of the mass of
sample taken. chloric acids. Metals (barium, cadmium, lead,
copper, chromium, and zinc) in solution are
14 DETERMINATION OF SULPHATE AS determined by suitable atomic absorption
SODIUM SULPHATE spectrophotometry (AAS) or inductively coupled
plasma (ICP) methods. The choice of flame/furnace
Accurately weigh about 5.0 g of the sample, AAS or ICP methods depend on the concentration of
transfer it to a 250 ml conical flask and dissolve in the analyte in the prepared sample solution (its
about 100 ml of water by heating on a water bath. concentration in the sample and limitations
Add 35 g of sulphate-free sodium chloride, stopper associated with the sample preparation). Furnace
the flask, and swirl at frequent intervals during technique, offers better sensitivity, may be preferred
1 hour. Cool, transfer with saturated sodium over flame technique, when dealing with low levels
chloride solution to a 250 ml measuring flask and of impurities in complex matrices. Antimony and
dilute to the mark at 20 °C. Shake the flask, and arsenic may be determined by using a hydride
filter the solution through a dry filter paper. Pipette generation AAS or ICP.
100 ml of the filtrate into a 500 ml beaker, dilute to
300 ml with water and acidify with hydrochloric Alternatively, antimony may be determined by
acid, adding 1 ml in excess. Heat the solution to flame atomic absorption but the hydride generation
boiling, and add an excess of 0.25 N barium technique is more sensitive.
chloride solution, drop by drop, with stirring. Allow
the mixture to stand on a hot plate for 4 hours, or 15.1.2 General Precautions
leave it overnight at room temperature and then
bring it to about 80 °C and allow the precipitate to Because of the minute amounts of metals involved
settle. Filter off the precipitated barium sulphate, special care must be taken to reduce the reagent
wash with hot water, and ignite at a dull red heat in blanks to as low a value as possible. Contamination
a tared crucible until a constant mass is obtained. in the laboratory is a major concern in trace metal
analysis. All apparatus should be thoroughly cleaned
Carry out a blank determination, apply any with a mixture of hot dilute acids (1 part
necessary correction to the mass of barium hydrochloric acid, 1 part concentrated nitric acid,
sulphatefound in the test, and calculate the result as and 3 parts water) followed by thorough washing

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IS 1699 : 2024

with water immediately before use. All operations 15.1.4 Reagents

involving acids shall be carried out in the specified
fume cupboards. 15.1.4.1 Reagents shall be of an order of purity
higher than accepted analytical reagent grade
NOTE — Special care must be taken while using per quality. Metal free water (see below) shall be used
chloric acid. throughout:
15.1.3 Apparatus
a) Nitric acid — specific gravity 1.42;
15.1.3.1 Kjeldahl flasks — of silica or borosilicate b) Perchloric acid — 60 percent (m/m)
glass fitted with an extension to the neck by means solution;
of a B24 ground joint, as shown in Fig. 4. The
extension serves to condense the fumes and carries c) Sulphuric acid — 98 percent;
a tap funnel through which the reagents are d) Hydrochloric acid — specific gravity 1.16
introduced. to 1.18;
15.1.3.2 Atomic absorption spectrophotometer — e) Hydrochloric acid — 5 N solution prepared
any commercial instrument operating in the by dilution of reagent (d) with metal-free
absorption mode may be used provided it has distilled water;
required accessories (furnace and vapour
generation) and facilities for the selection of the f) Water (metal-free) — distilled water may
required oxidant/fuel combination from a choice of be re-distilled from an all glass apparatus or
air, argon, nitrous oxide, hydrogen and acetylene may be passed down a column or cation
and has a wavelength range from 180 nm to exchange resin, for example,
600 nm. Amberlite IR 120 (H);
g) Sodium sulphate;
All automated instruments have the facility of
instrument control (selection of lamp, pre warm up h) Sodium borohydride pellets; and
of lamp, wavelength and slit width and optimization)
data acquisition and processing through a suitable j) Potassium chloride.
software in a data station. However, with classical
instruments, these need to be set manually and for 15.1.4.2 Preparation of standard solutions
operations in emission mode and measurements of
absorption involving the generation of a gaseous Use commercially available standard solutions or
hydride, a potentiometric recorder is necessary, prepare solutions as follows:
preferably a multi-range type covering the range
1 mV to 20 mV. a) Standard copper solution

15.1.3.3 Inductively coupled plasma-atomic Dissolve 3.928 g of pure copper sulphate

emission spectrophotometer — any commercial CuSO4.5H2O in water, dilute to 1 000 ml at 20 °C
instrument, sequential or simultaneous system, with water in a one-mark graduated flask. Dilute
operating in axial or radial mode may be used. 10 ml to 100 ml with water in a one-mark graduated
flask as required. 1 ml contains 100 μg Cu.

b) Standard zinc solution

Dissolve 1.000 g of pure zinc powder in a mixture of

10 ml water and 5 ml hydrochloric acid [reagent (d)]
and dilute to 1 000 ml at 20°C with water, in a one-
mark graduated flask. Dilute 10 ml to 100 ml with
water in a one mark graduated flask as required.
1 ml contains 100 μg Zn.

c) Standard chromium solution — dilute 5.80 ml of

0.1 N potassium dichromate solution to 100 ml at
20 °C with water in a one-mark graduated flask as
required. 1 ml contains 100 μg Cr.

FIG. 4 MODIFIED KJELDAHL FLASK (OPEN TYPE)

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d) Standard antimony solution 15.1.5.1 Method I

Dissolve 2.668 g potassium antimony tartrate Accurately weigh about 2.5 g of the sample and
K(SbO)C4H4O6 in distilled water, dilute to dissolve in a mixture of 4 ml of sulphuric acid and
1 000 ml at 20 °C with water in a one-mark 5 ml of hydrochloric acid. Transfer the solution to a
graduated flask. Dilute 10.0 ml to 100 ml with 50 ml one-mark graduated flask. If barium is to be
distilled water in a one–mark graduated flask as measured from the solution, add 0.095 4 g of
required. 1 ml contains 100 μg Sb. potassium chloride. Dilute to the mark with water.
Call this ‘Solution A’.
e) Standard lead solution
15.1.5.2 Method II
Dissolve 1.60 g of lead nitrate, Pb(NO3)2, in nitric
acid (10 ml of concentrated nitric acid diluted with Accurately weigh about 2.5 g of the sample into a
20 ml water, boiled to remove nitrous fumes, and 100 ml to 150 ml Kjeldahl flask, and add 5 ml of
cooled) and dilute to 1 000 ml with water in a dilute nitric acid. As soon as any initial reaction
one-mark graduated flask. Dilute 10.0 ml of this subsides, heat gently until further vigorous reaction
solution to 500 ml at 20 °C with water in a ceases and then cool. Add gradually 4 ml of
one-mark graduated flask as required. 1 ml contains concentrated sulphuric acid at such a rate as not to
20 μg Pb. cause excessive frothing on heating (5 min to
10 min are usually required) and then heat until the
f) Standard barium solution liquid darkens appreciably in colour, that is, begins
to char.
Dissolve 1.779 g barium chloride BaCI2.2H2O in
distilled water, dilute to 1 000 ml at 20°C with Add concentrated nitric acid slowly in small
water in a one-mark graduated flask. Dilute 10.0 ml portions, heating between additions until darkening
to 100 ml with water in a one–mark graduated again takes place. Do not heat so strongly that
flask as required. 1 ml contains 100 μg Ba. charring is excessive or loss of arsenic may occur;
small but not excessive amount of free nitric acid
g)Standard arsenic solution should be present throughout. Continue this
Dissolve 1.320 g of arsenous oxide, As2O3, by treatment until the solution is only pale yellow in
warming at a temperature not exceeding 60 °C with colour and fail to darken in colour on prolonged
14 ml of 5 N sodium hydroxide solution in a heating. If the solution is still coloured run in
100 ml beaker. Cool, add 0.2 ml of phenolphthalein 0.5 ml of the per chloric acid solution and a little
indicator and neutralize with 6 N sulphuric acid. concentrated nitric acid and heat for about
Transfer the solution to a 1 000 ml one-mark 15 minutes, then add a further 0.5 ml of the per
graduated flask containing 10 g of sodium hydrogen chloric acid and heat for few minutes longer. Note
carbonate dissolved in water, washing out the beaker the total amount of concentrated nitric acid used.
with water. Dilute to the mark with water at 20 °C Allow to cool somewhat and dilute with 10 ml of
and mix. Dilute 5 ml of this solution to 1 000 ml at water. The solution should be quite colourless (if
20 °C with water in a one-mark graduated flask as much iron is present, it may be faintly yellow). Boil
required. 1 ml contains 5 μg As. down gently, taking care to avoid bumping, until
while fumes appear. Allow to cool, add a further
h) Standard cadmium solution 5 ml of water and again boil down gently to fuming.
Finally, cool, add 10 ml of 5 N hydrochloric acid and
Dissolve 2.282 g 3CdSO4.8H2O in distilled water, boil gently for a few minutes. Cool and transfer the
dilute to 1 000 ml at 20 °C with water in a solution to a 50 ml one-mark graduated flask
one-mark graduated flask. Dilute 10.0 ml of this washing out the Kjeldahl flask with small portions
solution to 500 ml at 20 °C with water in a of water. Add the washings to the graduated flask
one-mark graduated flask. 1 ml contains 20 μg Cd. and dilute to the mark with water. If barium is to be
measured from the solution, add before dilution
15.1.5 Preparation of Test Solutions 0.095 4 g of potassium chloride, as an ionizing
buffer to prevent ionization of barium. Call this
Method I is applicable to substances soluble in dilute ‘Solution A’.
acids or mixtures of acids. Method II is used for
other substances. The choice of method for the Prepare a reagent blank using the same quantities of
pre-treatment of a substance can also follow that reagents as used in the sample oxidation.
given in the relevant Indian Standard.

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IS 1699 : 2024

15.1.6 Measurement of Antimony, Barium, time of use to obtain the best results. Instruments
Cadmium, Chromium, Copper, Lead and Zinc by should therefore be adjusted as described in the
Atomic Absorption Flame Technique manufacturer's instructions using the type of flame
and wavelength settings specified above.
15.1.6.1 Preparation of calibration curve solutions
To a series of 100 ml one-mark volumetric flasks, 15.1.6.3 Procedure
pipette 0 ml, 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of the
appropriate standard solution [standards (a) to (f) Set the atomic absorption spectrophotometer to the
and (h) (see 15.1.4.1)] and dilute to about 50 ml. appropriate conditions. Aspirate the strongest
Add 8 ml concentrated sulphuric acid standard containing the element to be determined
[see 15.1.4.1(c)] and 10 ml concentrated and optimize the instrument settings to give
hydrochloric acid [see 15.1.4.1(d)]. Shake to full-scale or maximum deflection on the chart
dissolve. In the case of barium [see 15.1.4.2 (e)], recorder. Measure the absorbance of the other
add 0.191 g of potassium chloride as an ionization standards and plot a graph showing the net
buffer. When solution is complete, dilute to the absorbance against the concentration of the element
mark with metal free water. in the standard solutions. Aspirate the Solution A
obtained from dissolution or the wet oxidation of the
These solutions then contain 0 μg/ml, 1.0 μg/ml, sample and the corresponding blank solution and
2.0 μg/ml, 3.0 μg/ml, 4.0 μg/ml and 5.0 μg/ml for determine the net absorbance. Using the graph
lead; 0 μg/ml, 2.0 μg/ml, 4.0 μg/ml, 6.0 μg/ml, prepared above, determine the concentration of the
8.0 μg/ml and 10.0 μg/ml for barium and clement in the sample solution:
antimony; , 0 μg/ml, 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/
ml, 0.4 μg/ml and 0.5 μg/ml of cadmium and zinc Element in the sample, mg/kg
or 0 μg/ml, 0.50 μg/ml, 1.0 μg/ml, 1.5 μg/ml,
2.0 μg/ml, 2.5 μg/ml for copper and chromium. Concentration of element (μg/ml) × 50
=
Mass of sample taken (g)
15.1.6.2 Instrumental conditions
15.1.7 Measurement of Antimony, Barium,
Select the wavelength and gases to be used for the Cadmium, Chromium, Copper, Lead and Zinc by
particular element under consideration from the Inductively Coupled Plasma (ICP) Technique
table below:
15.1.7.1 Preparation of standard curve solutions
Sl Element Wavelength Gases
No. (nm) The standard curve solutions given below are
nominal in nature. The concentration of standard
(1) (2) (3) (4) curve solutions differ based upon the operation
i) Antimony 217.6 Air/ mode of the torch (axial or radial) of the ICP
acetylene instrument. The analyst may alternatively prepare
appropriate standard curve solutions following the
ii) Barium 553.6 Nitrous instrument operation manual.
oxide/
acetylene To a series of 100 ml volumetric flasks pipette
iii) Cadmium 228.8 Air/ 0 ml, 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of the
acetylene appropriate standard solution [see 15.1.4.2(a) to
iv) Chromium 357.9 Nitrous 15.1.4.2(e) and 15.1.4.2(g)] and dilute to about
oxide/ 50 ml.Add 8 ml concentrated sulphuric acid
acetylene [see 15.1.4.1(c)] and 10 ml concentrated
hydrochloric acid [see 15.1.4.1(d)]. Dilute to the
v) Copper 324.8 Air/ mark with metal free water. These solutions then
acetylene contain 0 μg/ml, 1.0 μg/ml, 2.0 μg/ml, 3.0 μg/ml,
vi) Lead 283.3 Air/ 4.0 μg/ml and 5.0 μg/ml for lead; 0 μg/ml, 2.0 μg/
acetylene ml, 4.0 μg/ml, 6.0 μg/ml, 8.0 μg/ml and 10.0 μg/ml
vii) Zinc 213.9 Air/ for barium and antimony; 0 μg/ml, 0.1 μg/ml,
acetylene 0.2 μg/ml, 0.3 μg/ml, 0.4 μg/ml and 0.5 μg/ml of
cadmium and zinc or 0 μg/ml, 0.50 μg/ml, 1.0 μg/
The recommended settings for the various ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml for copper and
instrumental parameters vary from model to model, chromium.
and certain parameters require optimization at the

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IS 1699 : 2024

15.1.7.2 Instrumental conditions available commercially. One or more of the

following modifiers may be used for the
Select appropriate emission wavelengths to be used determination of lead and cadmium in different food
with each element under consideration. The additives:
recommended settings for the various instrumental
parameters vary from model to model, and certain a) Palladium solution — 1 000 μg/l to
parameters require optimization at the time of use to 2 000 μg/l;
obtain the best results. Instruments should therefore b) Ascorbic acid — 5 000 μg/l;
be adjusted as described in the manufacturer's
instructions. c) Monobasic ammonium phosphate —
5 000 μg/l; and
15.1.7.3 Procedure d) Orthophosphoric acid — 1 000 μg/l.
Set the ICP instrument as stated in the operation
manual. Activate the method and key in the 15.1.8.2 Preparation of standard curve solutions
standards data into the data station of the ICP.
Aspirate the blank solution and set the instrument to In a 100 ml volumetric flask, pipette 25 ml of lead
zero, aspirate the standards and determine a standard and 10 ml cadmium standards [standards (e) and (h)
curve for each element with emission intensity (see 15.1.4.2)] and dilute to the mark with water
plotted against the concentration of the element in (standard Solution A, 1 ml = 25 μg of Pb and
the standard solutions. Aspirate the Solution A 1.0 μg of Cd). Dilute 10 ml of A to 100 ml with water
obtained from dissolution or the wet oxidation of the (standard Solution B, 1 ml = 2.5 μg of pb and 0.1 μg
sample. If the concentration of the element in the Cd). Dilute 10 ml of B to 100 ml with water
solution is beyond the standard curve, dilute the (standard Solution C, 1 ml = 250 ng of Pb and
solution as required and read it again. Using the 10 ng Cd). Dilute 10 ml of C to 100 ml with water
standard curve, determine the element in the sample. (working standard Solution D, 1 ml = 25 ng of Pb
and 1 ng Cd).
15.1.7.4 Calculation
15.1.8.3 Instrumental conditions
Element in the sample, mg/kg
General instrumental conditions are provided in the
Concentration of element (μg/ml) × 50 table given below. The recommended settings for
=
Mass of sample taken (g) the various instrumental parameters vary from
model to model, and certain parameters require
15.1.8 Measurement of Lead and Cadmium by optimization at the time of use to obtain the best
Atomic Absorption Electro–thermal Atomization results. Instruments should therefore be adjusted as
(Furnace Atomization) Technique described in the manufacturer's instructions.

15.1.8.1 Chemical modifier solutions

Use of chemical modifier solutions in the furnace

atomization allows use of higher ashing
temperatures to reduce the background absorbance.
These solutions must be of very high purity and are

Sl No. Element Wavelength Slit Gases Maximum Ashing Atomization

(nm) (nm) Temperature Temperature

Without With
modifier modifier
(1) (2) (3) (4) (5) (6) (7) (8)
i) Cadmium 228.8 0.5 Argon 300 Argon 1 800
ii) Lead 283.3 0.5 Argon 400 Argon 2 100

16
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15.1.8.4 Procedure vessel and expel any air as described in the maker’s
instructions filling the apparatus with argon. Isolate
Place blank (1 percent nitric acid), working standard the vessel from the atomizer using the by-pass valve.
solution (Solution D), a suitable modifier solution (if Remove the atomizer and then quickly add 1 pellet
required) and sample solutions in the appropriate of sodium borohydride weighing approximately
locations provided in the furnace auto sampler. Set 0.2 g and replace the stopper. Ensure that all the
up the furnace parameters following the instruction joints are secure.
provided by the manufacturer to carry out triplicate
injections. Clean the graphite tube and inject blank. When the reaction slows (20 s to 30 s) open the
Program the auto sampler to inject 5 μl, 10 μl, 15 μl, appropriate taps to allow argon to drive the
20 μl of standard (5 μl of modifier and remaining generated hydroxide into the flame. When the
blank solution so that the total volume is 25 μl). hydride has all been expelled as shown by the
Construct the standard curve from the absorbance recorder trace, return the laps to their original
either from peak area or height. Inject 10 μl of position and empty the vessel.
sample solution and calculate the concentration in
the samples as follows: Optimize the instrument settings to give full scale
a) Injection volume of sample to furnace — deflection for the strongest standard. Measure the
10 μl; other standards, the sample and the blank solution
using the same procedure.
b) Volume made up — 50 ml;
c) Instrument reading (ng) — R; Plot a graph relating peak height on the recorder to
the concentration of the arsenic or antimony in the
d) Weight of sample, g — W; and standards. Using the net absorbance of the sample,
𝑅×5 read from the graph the concentration of arsenic or
e) Concentration in sample (mg/kg) = . antimony in the solution.
𝑊

15.1.9 Measurement of Arsenic and Antimony by 15.1.9.4 Calculation

Atomic Absorption Hydride Technique
Arsenic or antimony in the sample, mg/kg =
Arsenic and antimony are determined after μg
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝐴𝑟𝑠𝑒𝑛𝑖𝑐 𝑜𝑟 𝐴𝑛𝑡𝑖𝑚𝑜𝑛𝑦 ( )
preparation of their volatile hydrides which are 𝑚𝑙
× 50
collected either in the generation vessel itself or, in 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛 (𝑔)

some designs, in a rubber balloon attached to the

vessel. The gases are then expelled with argon into 15.1.10 Determination of Mercury by Atomic
a hydrogen flame. Absorption Cold Vapour Technique

15.1.9.1 Preparation of calibration curve solution 15.1.10.1 Principle

Into a series of 100 ml one-mark volumetric flasks, The sample is digested under closed conditions by
add from a burette, 0 ml, 1 ml, 2 ml, 3 ml, 4 ml and heating under reflux with sulphuric and nitric acids.
5 ml of standard arsenic or antimony solution [see The oxidation is completed by addition of potassium
15.1.4.2(d) and 15.1.4.2(g)] and dilute to about 50 permanganate solution. After successive additions
ml with distilled water. Add 8 ml concentrated of hydroxylamine hydrochloride solution and
sulphuric add [see 15.1.4.1(c)] and 10 ml stannous chloride solution, the mercury content is
hydrochloric acid [see 15.1.4.1(d)]. Shake to measured by cold vapour atomic absorption
dissolve, and when solution is complete, dilute to the spectrometry. Alternatively, closed vessel
mark with distilled water. microwave digestion system may be used for the
digestion of samples.
15.1.9.2 Instrumental conditions
15.1.10.2 Special reagents
Using the atomic absorption spectrophotometer with
the appropriate hollow cathode or electrode less a) Nitric acid — specific gravity 1.40
discharge lamp, select the wavelength for either
arsenic (193.7 nm) or antimony (217.6 nm). b) Sulphuric acid — specific gravity 1.84

15.1.9.3 Procedure c) Sulphuric acid — approximately 3.5 M. Prepare

by diluting 1 volume of concentrated sulphuric acid
Measure 5.0 ml of the strongest standard into the (b), with 4 volumes of water
generation vessel, add 25 ml water and 2 ml
5 N hydrochloric acid [see 15.1.4.1(e)]. Stopper the
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d) Sulphuric acid — approximately 1 M. Prepare by drop wise until a colouration persists.

diluting 1 volume of 3.5 M concentrated sulphuric Renew this solution daily.
acid (c), with 2.5 volumes of water
15.1.10.4 Apparatus
e) Hydrochloric acid — specific gravity 1.18
All the glassware must by cleaned with hot nitric
f) Potassium permangnate solution — 50.0 g/l acid [see 15.1.10.2(d)] and washed thoroughly with
water before use.
g) Hydroxylamine hydrochloride solution — 10.0g/l
a) Mineralization apparatus — fitted with
h) Stannous chloride solution — prepare by reflux condenser (see Fig 5);
dissolving 250 g of' stannous chloride b) Bubbler — with a ground glass stopper
(SnCl2.2H2O), in 50 ml hydrochloric acid (e). Make fitted with two tubes to permit entrainment
up to 250 ml with water and bubble nitrogen through of the mercury vapour and with a
the solution. Store over a few granules of metallic calibration mark at the required volume for
tin measurement. The capacity of the bubbler
and position of the mark depends on the
j) Chromic acid mixture atomic absorption spectrophotometer used.
Clean the bubbler successively with
Dissolve 4.0 g of potassium dichromate in 300 ml of chromic acid mixture [see 15.1.10.2(j)], tap
3.5 M sulphuric acid (c) and make up to 1 litre with water and double distilled water before use;
water. c) Water vapour absorption apparatus
containing magnesium perchlorate
k) Magnesium perchlorate — in granular form for [see 15.1.10.2(k)]; and
gas desiccation
d) Atomic absorption spectrophotometer —
m) Mercury chloride suitable for the cold vapour determination
of mercury in open or closed circuit, with
15.1.10.3 Standards recorder.

Use commercially available standard solutions, or

prepare the standards as follows:

a) Mercuric chloride solution — 0.5 mg

Hg/ml.

Weigh out, to the nearest 0.1 mg. 0.677 g

of mercury chloride. Dissolve in
approximately 250 ml 3.5 M sulphuric acid
in a l litre volumetric flask, add
approximately 700 ml water and then
potassium permanganate solution drop
wise until a colouration persists. Make up
to the mark with water and mix well.
Renew this solution every three months.

b) Mercuric chloride solution – 0.02 μg

Hg/ml.

Dilute the standard mercuric chloride FIG. 5 MINERALIZATION APPARATUS

solution 0.5 mg Hg/ml [see 15.1.10.3(a)] by
15.1.10.5 Procedure
a factor of 25 000 by successive dilution
with sulphuric acid [see 15.1.10.2(d)] for
a) Digestion of sample
example, 10 ml made up to 250 ml twice
followed by 10 ml made up to 400 ml.
Weigh out, to the nearest 2 mg, approximately 0.5 g
Before bringing up to the mark in the final
sample containing not more than 0.5 μg total
dilution, add potassium permanganate
mercury. Introduce the sample into the receiver flask
solution [see 15.1.10.2(f)].
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(M), and add a few glass beads. Connect the receiver mark either with double distilled water or with
flask to the condensate reservoir (D) and close the sulphuric acid [see 15.1.10.2(d)] in the case of
stopcock (R). standard solution. Add 5 ml of stannous chloride
solution [see 15.1.10.2(h)], assemble the bubbler,
Introduce into the reservoir 25 ml of nitric acid connect it to the water vapour absorption apparatus
(specific gravity 1.40) followed by 10 ml sulphuric and to the atomic absorption spectrophotometer set
acid (specific gravity 1.84). Mount and turn on the the latter in operation. Mix the contents of the
condenser (A). Open the stopcock carefully and bubbler well by gently shaking, pass air or nitrogen
allow small portions of the mixture of acids to run through, measure and record. Carry out
into the receiver flask. Interrupt the flow of acids if measurements as quickly as possible after the
the reaction becomes too vigorous. addition of stannous chloride If an open-circuit
system is used, wait 30 seconds before passing air or
Empty the reservoir into the receiver flask, mix the nitrogen.
contents of the latter well by careful shaking and
leave the stopcock open. d) Standard curve

Heat the receiver flask carefully. As soon as foaming Introduce respectively 2 ml, 5 ml, 10 ml, 15 ml and
has ceased, close the stopcock (R), continue heating 25 ml aliquots of the standard mercury solution
and let the condensate collect in the reservoir. [see 15.1.10.3(b)] into bubblers and 25 ml sulphuric
acid [special reagent (d)] into a sixth bubbler. Add
Discontinue heating when the contents of the potassium permanganate solution [special reagent
receiver flask begin to char. Allow a small portion (f)] drop wise, with agitation to each bubbler until a
of the condensate to run into the receiver flask, close colouration persists. Measure the mercury content as
the stopcock again and resume heating the receiver described above.
flask. Repeat this procedure for as long as the
contents display charring when heated. Plot the calibration curve with the measured
When charring has ceased, heat and add condensate absorption values as ordinates and the
as soon as white fumes appear. Continue alternately corresponding mercury contents in micrograms as
heating and adding condensate for one hour. Finally, abscissae. The working standards contain 0.04 μg,
heat the contents of the flask to white fumes. 0.10 μg, 0.20 μg, 0.30 μg and 0.50 μg of mercury
respectively.
Stop heating and allow to cool to approximately 40
°C. Open the stopcock and allow all the condensate e) Method of addition
to run into the receiver flask. Wash the apparatus out
from the top of the condenser with 5ml to 10 ml of The method of addition may be used if an open–
water, collect the washings in the receiver flask and circuit system is used. Place one of the working
disconnect it from the reservoir. standard solutions in a bubbler and add an aliquot
portion of the sample solution obtained after
b) Treatment of the solution treatment. The quantity of mercury in the bubbler
must lie in the range in which the photometer gives
Introduce the potassium permanganate solution a linear response. Measure the mercury content as
[see 15.1.10.2(f)] drop wise into the receiver flask, described above. If necessary, carry out several such
with agitation, until a pink coloration persists. Note determinations using different working standard
the volume of permanganate solution used (If this solutions.
quantity exceeds 10 ml, repeat the procedure
‘digestion of sample’ as above). f) Blank determination

Heat gently to boiling, then allow to cool. Pour the Carry out all the operations, from ashing to
contents of the receiver flask into a bubbler, wash measurement, except for introduction of the
the receiver flask with water and add the washings sample. When treating the solution, add a quantity
to the contents of the bubbler. of potassium permanganate solution
[see 15.1.10.2(f)] equal to that used for the
Measure the mercury content (see below) the same experimental sample.
day as the treatment of the solution.
15.1.10.6 Calculation
c) Measurement of mercury content
Read off from the calibration curve the quantities, in
μg, of mercury corresponding to the measured
Introduce 5 ml of hydroxylamine hydrochloride
absorption values.
[see 15.1.10.2(g)], into the bubbler and make up the
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Subtract the quantity of mercury found in the blank chloride in a 200 ml volumetric flask and add water
from that found in the sample: to volume

Hg in the sample, mg/kg = 15.2.1.3 Procedure

𝑁𝑒𝑡 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑚𝑒𝑟𝑐𝑢𝑟𝑦 (μg)
𝑆𝑎𝑚𝑝𝑙𝑒 𝑚𝑎𝑠𝑠 (𝑔) The limit test described for lead is designed to
show if a sample contains more than 10 mg/kg or
15.2 Chemical Methods 20 mg/kg of lead. The sample is digested with
nitric and sulphuric acids, and a clear solution of
Chemical methods have been given for the digest is prepared.
determination of lead (Pb), arsenic (As), copper (Cu)
and chromium (Cr). a) Digestion
15.2.1 Test for Lead Weigh 1.0 g of sample for substances with 10 mg/kg
limit or 0.5 g for those with 20 mg/kg limit. Place
15.2.1.1 Apparatus with 5 ml of water, 5 ml of 65 percent nitric acid and
5 ml of sulphuric acid in a digestion flask. Warm
a) Digestion funnel; and slightly. If foaming becomes excessive, add a little
b) Separatory funnel. water. Evaporate the mixture. Maintain strongly
oxidizing conditions in the flask during digestion by
15.2.1.2 Reagents adding cautiously small quantities of nitric acid
whenever the mixture begins to tum brown or dark.
a) Nitric acid — 65 percent Continue digestion until organic matter is destroyed
and sulphuric trioxide fumes are copiously evolved.
b) Sulphuric acid — specific gravity 1.84 The final solution should be colourless or at slightest
straw colour.
c) Ammonium acetate-citrate solution
To remove nitrosylsulphuric acid, after partial
Dissolve 12.5 g of ammonium acetate and 12.5 g of cooling transfer the residue to a dish, rinse with
ammonium citrate in water, add concentrated 25 ml of water, evaporate and heat again to fuming
ammonia until the solution is alkaline to thymol blue point. Add 25 ml of water and evaporate and heat
paper and add water to 100 ml. Purify with again.
0.002 percent (m/v) solution of dithizone in carbon
tetrachloride, and finally shake the solution with b) Solution of digest
carbon tetrachloride to remove excess of dithizone.
Allow 10 cool, add 20 ml of ammonium acetate-
d) Ammonia solution — 25 percent citrate solution, and allow again to cool, neutralize
to about pH 7 with 25 percent ammonia and boil if
e) Carbon tetrachloride; necessary to dissolve calcium sulphate. Cool the
clear solution. Shake the solution with 10 ml of
f) Ammonium hydroxide — 0.2 N carbon tetrachloride and discard the lower layer.

g) Potassium cyanide — 10 percent c) Test with dithizone — to a 100 ml separatory

funnel, add 10 ml of 0.2 N ammonium hydroxide,
h) Hydroxylamine hydrochloride solution — 2 ml of 10 percent potassium cyanide, 2 ml of
10 percent 10 percent hydroxylamine hydrochloride solution
and 2 ml of 20 mg per litre solution of dithizone in
j) Dithizone solution — 0.1 percent (m/v), purified carbon tetrachloride. Shake the separatory funnel for
by the following procedure a few minutes and discard the carbon tetrachloride
layer. Add 2 ml of carbon tetrachloride, shake and
Dissolve the dithizone in chloroform and treat it discard again the carbon tetrachloride layer. Add the
with ammonia. Add mineral acid. Precipitate shall solution of digest to the separatory funnel. Again
be pure dithizone. The aqueous ammonical solution add 5 ml of dithizone solution in carbon
obtained from chloroform solution of dithizone tetrachloride and shake vigorously for a few
should be colourless; otherwise further purification minutes. The carbon tetrachloride layer becomes red
as mentioned above should be carried out according to the amount of lead present

k) Buffer pH 2 — add 11.90 ml of 0.2 M Treat simultaneously a standard solution containing

hydrochloric acid and 88.10 ml of 0.2 M potassium 10 μg of lead in the same manner as the solution of
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the digest. Evaluate the quantity of lead in the digest

by comparing the colour of the carbon tetrachloride
layers.

Ensure that the red colour is due to lead by shaking

the carbon tetrachloride layer obtained from the
digest solution with 10 ml of a buffer pH 2. The red
colour will turn green if it is due to lead.

15.2.2 Test for Arsenic

Arsenic may be tested by either the method given

below for routine purposes or the modified Gutzeit
method as given in IS 2088.

15.2.2.1 Apparatus

a) Distillation apparatus — as shown in Fig. 6; and

b) Conical flask — 25 ml dose, as shown in Fig. 7.

15.2.2.2 Reagents

a) Sulphuric acid — specific gravity 1.84

b) Potassium permanganate solution — 0.1 N FIG. 7 CONICAL FLASK
c) Ferrous sulphate — freshly powdered
15.2.2.3 Procedure
d) Hydrochloric acid — 38 percent
e) Potassium bromide solution — 20 percent The limit test prescribed for arsenic shows whether
a sample contains more than 3 mg/kg of arsenic.
f) Aluminium strips — 8 mm × 8 mm × 1 mm
g) Tin chloride solution — 2 percent tin chloride in Digestion of 1 g sample and removal of nitrosyl
10 percent hydrochloric acid sulphuric acid as described in para 3 of 15.2.1.3.
Allow the digest with 5 ml sulphuric acid to cool.
h) Test paper — soak strips of filter paper in
saturated ethanolic solution of mercuric bromide a) Distillation (according to Snyder)
and allow to dry
Pour the digest into the distillation flask (see Fig 6).
Rinse the dish twice using each time 2.5 ml of water
and add the water to the digest. Cool the solution.
Add some drop of potassium permanganate solution
until the red colour persists. Add 0.250 g of freshly
powdered ferrous sulphate, 2.5 ml of 38 percent
hydrochloric acid and 0.1 ml of potassium bromide
solution.

Close the flask, place a reagent tube containing 8 ml

of water in a beaker with water as shown in Fig. 6.

Heat with micro burner. In the beginning a slow

stream of air bubbles appears, followed after 3 to
5 minutes by hydrochloric acid gas. Heat then a
little stronger so that the solution boils. After bulb
A has become very hot, boil for 40 seconds.
Thereupon, lower the reagent tube and remove the
flame. The hydrochloric acid content of the
distillate shall be 9 percent to 10 percent, only then
has arsenic been completely distilled over;
moreover, this acid concentration required for the
FIG. 6 DISTILLATION APPARATUS test given below.
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b) Test with mercury bromide paper (Mavercon- total volume of the solution should be about
Bergeret) 100 ml.

The distillate should be transferred to a conical flask Extract the copper by shaking with three successive
of 25 ml capacity. The flask should be dosed with portions of 5 ml of the solution of dithizone, shaking
the device as shown in Fig. 7 containing a small thoroughly for a minute for each extraction.
disk of test paper. Add 3 pieces of aluminium strips, Separate the dithizone layers and wash the combined
1 ml of tin chloride solution and immediately close dithizone extracts with about 10 ml of water.
the flask with the stopper. Allow the flask to stand Transfer the dithizone extract to a tube of heat-
in a water-bath of 25 °C to 30 °C for 50 minutes to resistant glass and evaporate the chloroform on a
60 minutes. At the same time carry out a parallel water bath.
test using a solution of 2, 4, 6 or 8 of arsenic in
10 ml of 10 percent hydrochloric acid in place of the Heat the copper-dithizone residue in the test tube
test sample. Compare the colour of the test paper for with one millilitre or concentrated sulphuric acid
evaluating arsenic content of the sample. and a little of nitric acid until all organic matter is
destroyed. Add 5 ml of water and re-heat to fuming
15.2.3 Determination of Copper stage. Cool, dilute with water and transfer the whole
of the solution or a measured volume of the
15.2.3.1 Reagents solution, depending upon the amount of copper
present, to a Nessler cylinder. Add one millilitre of
a) Citric acid — solid the citric acid solution and 4 ml of the ammonium
hydroxide solution followed by 5 ml of the gum
b) Ammonium hydroxide solution — specific gravity arabic solution and make up the volume to 50 ml
0.92 (not less than 27 percent ammonia) with water. Add 5 ml of the sodium diethyl
dithiocarbamate solution and match the colour by
c) Concentrated hydrochloric acid adding the standard dilute solution of copper
[see 15.2.3.1(k)] to a control cylinder containing the
d) Dithizone (diphenyl thiocarbazone) solution — same quantities of reagents as present in the test
0.1 percent (m/v) in chloroform solution. Calculate the copper content of the
material in parts per million from the known volume
e) Concentrated nitric acid of the standard dilute solution of copper required for
matching.
f) Concentrated sulphuric acid
15.2.4 Determination of Chromium
g) Citric acid solution — 5 percent (m/v) aqueous
15.2.4.1 Reagents
h) Gum arabic solution — one percent
a) Magnesium nitrate solution — 25 percent (m/v)
j) Sodium diethyl dithiocarbamate solution —
0.2 percent (m/v) aqueous, freshly prepared b) Strong sulphuric acid solution — 4 N

k) Standard strong solution of copper c) Potassium permanganate solution — 0.1 N

Dissolve 0.392 5 g of pure crystallized copper d) Sodium azide solution — 5 percent (m/v)
sulphate (CuSO4.5H20) in water and make up the
volume to 100 ml. This solution contains one e) Sodium dihydrophosphate — 4 M
milligram of copper per millilitre.
f) Diphenylcarbazide solution
m) Standard dilute solution of copper — dilute one
millilitre of the standard strong solution of copper to Dissolve 125 mg of diphenylcarbazide
100 ml in a graduated flask. One millilitre of this [(C6H5.NH2NH)2CO] in a mixture of 25 ml of
solution contains 0.01 mg of copper acetone and 25 ml of water. This should be prepared
immediately before use
15.2.3.2 Procedure
g) Standard chromium solution
Take a suitable aliquot of the test solution prepared
as described above and add 2 g of citric acid. Dissolve 0.0566 g of potassium dichromate
Neutralize exactly with the ammonium hydroxide (K2Cr2O7) in one litre of water. One millilitre of this
solution using a piece of litmus paper and acidify solution contains 0.02 mg of chromium
with one millilitre of concentrated hydrochloric
acid. Cool and transfer to a separating funnel. The
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h) Sucrose 16.1.4 Standard Lead Solution — dilute 10 ml of

lead nitrate stock solution, accurately measured with
15.2.4.2 Procedure water to 100 ml. Each millilitre of the solution so
prepared contains the equivalent of 10 mcg of lead
Weigh 1.0 g of the sample into a quartz dish. Char ion (Pb). Prepare the solution on the day of use
the material raising the temperature slowly. Allow to
cool. Add 10 ml of the magnesium nitrate solution 16.1.5 Nitric Acid — 10 percent
and evaporate, heating slowly until no more nitrous
vapour evolves. Heat the material in an oven at 16.1.6 Sulphuric Acid — 94.5 percent to
600 °C ± 20 °C until all black particles have 95.5 percent
disappeared (30 minutes to 60 minutes). Dissolve the
residue by adding 10 ml of the strong sulphuric acid 16.1.7 Hydrogen Sulphide — a saturated solution of
solution and 20 ml of water. Heat on a water-bath for hydrogen sulphide made by passing hydrogen
about 5 minutes. Add 0.5 ml of the potassium sulphide gas through cold water
permanganate solution, cover with a watch-glass and
beat on a water-bath for about 20 minutes. Add more 16.2 Procedure
potassium permanganate, if the solution
16.2.1 Solution A
decolourizes. Add sodium azide solution, one drops
every 10 seconds, until the excess potassium Take 1.25 ml of the standard solution in 50 ml
permanganate has been removed. (Avoid excess of Nessler tube and add 22.5 ml of water. Adjust the
sodium azide, 2 drops are usually sufficient.) Cool pH to between 3.0 to 4.0 by addition of acetic acid
the solution in running water and filler, if manganese or ammonia solution. Dilute with water to 40 ml and
dioxide is evident. Transfer the solution to a 50 ml mix.
graduated flask. Add 2.5 ml of sodium
dihydrophosphate and 2 ml of diphenylcarbazide 16.2.2 Solution B
solution and fill to the mark with water. Measure the
extinction at 540 nm, 30 minutes after adding the Place 500 mg of the sample, accurately weighed in
diphenylcarbazide solution, a blank with the last two a suitable crucible, add sufficient sulphuric acid to
reagents (sodium dihydrophosphate and wet the sample and carefully ignite at a low
diphenylcarbazide solutions) should show no colour temperature until thoroughly charred, covering the
or only a slight purple colour. At the same time, run crucible loosely with a suitable lid during the
a parallel test with 1.00 ml of the standard chromium ignition. After the substance is thoroughly
solution and a few milligrams of sucrose placed in a carbonized, add 2 ml of nitric acid and 5 drops of
second quartz dish. Treat the mixture exactly as the sulphuric acid, and cautiously heat until white fumes
sample and measure the extinction at the same are evolved, then ignite, preferably in muffle
wavelength. Calculate the chromium content of the furnace, at 50 °C to 60 °C until the carbon is
sample from the two extinction values observed. completely burned off. Cool and add 4 ml of dilute
hydrochloric acid, cover and digest on a steam-bath
16 DETERMINATION OF HEAVY METALS to dryness. Moisten the residue with one drop of
hydrochloric acid, add 10 ml of hot water and digest
16.1 Reagents for 2 minutes. Add drop wise ammonia solution until
the solution is just alkaline to litmus paper, dilute
16.1.1 Ammonia Solution — dilute 400 ml of with water to 25 ml and adjust the pH to between
ammonium hydroxide (28 percent) to 1 000 ml with 3.0 and 4.0 (pH indicator paper) by the addition of
water diluted acetic acid. Filter if necessary, wash the
crucible and the filter with 10 ml of water. Transfer
16.1.2 Hydrochloric Acid — 10 percent to a 50 ml Nessler tube. Dilute the combined filtrate
and washing with water to 40 ml and mix.
16.1.3 Lead Nitrate Stock Solution
16.2.3 To each tube add 10 ml of freshly prepared
Dissolve 159.8 mg of lead nitrate in 100 ml of hydrogen sulphide, mix and allow to stand for
water containing 1 ml of nitric acid. Dilute with 5 minutes and view over a white surface. The
water to 1 000 ml and mix. Prepare and store the colour of Solution B shall not be darker than that of
solution in lead free glass containers. Solution A.
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IS 1699 : 2024

ANNEX A
(Foreword)
COMMITTEE COMPOSITION
Food Additives Sectional Committee, FAD 08
Organization Representative(s)

CSIR - Indian Institute of Toxicology Research, DR YOGESHWAR SHUKLA (Chairperson)

Lucknow

All India Food Processors Association, New Delhi MS SHREYA PANDEY

SHRI KRISHNA KUMAR JOSHI (Alternate)

Bose Institute, Kolkata PROF GAOURISHNKAR

Confederation of Indian Food Trade and Industry, DR JASVIR SINGH

New Delhi MS VARSHA YADAV (Alternate)

Confederation of Indian Industry, New Delhi MS NEHA AGGARAWAL

MS MAMTA ARORA BUDHIRAJA (Alternate)

Consumer Education and Research Centre, MS ANINDITA MEHTA

Ahmedabad MS DOLLY A. JANI (Alternate)

Consumer Guidance Society of India, Mumbai DR SITARAM DIXIT

DR M. S. KAMATH (Alternate)

CSIR - Central Food Technological Research DR NGASEPPAM IBOYAIMA

Institute, Mysuruore DR ARUNA KUMAR (Alternate)

ICMR - National Institute of Nutrition, Hyderabad DR J. PADMAJA

Indian Institute of Chemical Technology, Hyderabad DR ASHOK KUMAR TIWARI

DR T. KUMARAGURU (Alternate)

Oleochemicals and Surfactants Technology, Mumbai DR UDAY S. ANNAPURE

PROF REKHA SINGHAL (Alternate)

Protein Foods and Nutrition Development DR SHASHANK BHALKAR

Association of India, Mumbai SHRI NIKHIL KAMAT (Alternate)

Roha Dye Chem Private Limited, Mumbai SHRI ZAINULABIDIN DHANSE

VR Food Tech Pvt Ltd, Mumbai DR ASHLESH PARCHURE

DR N. RAM (Alternate)

BIS Directorate General SHRIMATI SUNEETI TOTEJA, SCIENTIST ‘E’/DIRECTOR

AND HEAD (FOOD AND AGRICULTURE) [REPRESENTING
DIRECTOR GENERAL (Ex-officio)]

Member Secretary
SHRI KULDEEP MITTAL
SCIENTIST ‘B’/ASSISTANT DIRECTOR
(FOOD AND AGRICULTURE), BIS

24
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 Free Standard provided by BIS via BSB Edge Private Limited to FARE LABS Private Limited -
gurgaon([email protected]) 162.120.184.5.

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This Indian Standard has been developed from Doc No.: FAD 08 (23204).

Amendments Issued Since Publication

Amend No. Date of Issue Text Affected

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