WHO Food Add 70.37 Eng

Ji'" r ~ •
-~
'
SPECIFICATIONS FOR THE IDENTITY :1

AND PURITY OF SOME FOOD COLOURS,
.•
EMULSIFIERS, STABILIZERS,
ANTI-CAKING AGENTS
AND CERTAIN OTHER FOOD ADDITIVES
•,.
• •
Food and A1riculture Or1ani2ation of the United Nations
World Health Or1anization
1970
FAQ Nutrition Meetings
Report Series No. 468
WHO/Food Add./70. 37
SPECIFICATIONS FOR THE IDENTITY AND PURITY

OF SOME FOOD COLOURS, .
EMULSIFIERS, STABILIZERS, ANTI-CAKING AGENTS
AND CERTAIN OTHER FOOD ADDITIVES
Issued jointly by FAO and WHO
The contents of this document are the result

of the deliberations of the Joint F AO/WHO
Expert Committee on Food Additives which met
in Rome, 27 May-4 June 1969/!
!/ Thirteenth Report of the Joint

FAO/WHO Expert Committee
on Food Additives. FAQ Nutrition Meetings Report Series,
No. 46; Wld Hlth Org. Techn. Rep. Ser., 1970, No. 445.
110363
- ill -
TABLE OF CONTENTS
Page
Introduction v
List of Participants iv
- Specifications for -
Some Food Colours 1

Annatto Extracts 2
Chlorophyll 7
Chlorophyll Copper Complex 10
Chlorophyllin Copper Complex, 12
Sodium or Potassium Salts 12
Erythrosine 14
Chemically Treated Starches 18

Annex on Analytical Methods for
Chemically Treated Starches 25
Some Emulsifiers and Stabilizers 37

Carragheenan 38
Furcellaran 41
Arabic Gum 43
Carob Bean Gum 45
Guar Gum 47
Karaya Gum 49
Tragacanth Gum 51
Identification and O~her Tests for
Stabilizers and Thickeners (Annex) 53
Polyglycerol Esters of lnteresterified
Ricinoleic Acid 55
Propylene Glycol Esters of Fatty Acids 57
Stearoyl lactylate, Calcium Salt and
Stearoyl lactylate, Sodium Salt 61
Stearyl Citrate 64
Sucrose Esters of Fatty Acids 66
Hydroxylated Lecithin 69
Ammonium Salts of Phosphatidic Acids 73
- iv -·
Page
Hydroxypropyl Cellulose 80
Pectin 85
Propylene Glycol Alginate 89
Identification Tests for Emulsifiers (Annex) 91
Some Anti-caking Agents 96

Ferrocyanide, Calcium , )
Ferrocyanide, Sodium ~nd ) 97
Ferrocyanide, Potassium , )
Phosphate, Calcium, tribasic 99
Phosphate, Magnesium, tribasic 101
Salts of Myristic, Palmitic and Stearic
Acids with Bases Accepted for Food Use 104
Silicon Dioxide, Amorphous 106
Aluminium Silicate 109
Calcium Silicate 112
Magnesium Silicate 114
Sodium Alumina Silicate 117
Certain Other Food Additives 119

Monosodium L-Glutamate 120
Calcium Sulphate 122
Potassium Chlorate 124
Ascorybl Stearate 125
Dimethylpolysiloxane 128
Oxystearin 130
Isoamyl Gallate 132
Ethyl Protocatechuate 134
General References 136

- v -
INTRODUCTION
The specifications and identity tests appearing in this

publication were developed at the Thirteenth Session of the Joint
FAO/WHO Expert Committee on Food Additives for the sub-
stances for which, in the consideration of the experts, adequate
data was available. Readers are requested to consider these
specifications only in conjunction with the Report of the above
mentioned meeting, F AO Nutrition Meetings Report Series No .46;
World Health Organization Technical Report Series 1970, No .445.
In particular attention is drawn to the following paragraphs,
namely, the principles governing establishment of chemical
specifications ( Section l), and comments on substances on the
agenda ( Section 3). Here are explained the reasons for the
absence of specifications for certain compounds which were on
the agenda and the tentative nature of others or their presence as
identification tests rather than full specifications. The test
solutions mentioned in the specifications are those appearing in
earlier publications on specifications listed in the General
References.
For detailed monographs on toxicological evaluation of

these substances, a reference is invited to the publication
"Toxicological Evaluation of Some Food Colours, Emulsifiers,
Stabilizers, Anti-caking Agents and Certain Other Substances"
FAO Nutrition Meetings Report Series No.46A; WHO/Food Add/
70.36.
- vi -
LIST OP PARTICIPANTS
Members:
Professor F. Bar, Director, Toxicology and

Nutrition, c/o Federal Ministry of Health, Bad
Godesberg, Fed. Rep. of Germany
Dr. J. D. Brandner, Atlas Chemical Industries Inc.,

Wilmington, Del. , U.S. A.
Dr. J.M. Coon, Professor and Chairman, Department

of Pharmacology, Jefferson Medical College,
Philadelphia, Pa., U. S A. (Chairman)
Mr. A. Eisenberg, Ministry of Health, Jerusalem,

Israel
Dr. L. Golberg, Research Professor of Pathology,

Albany Medical College of Union University, Albany,
N. Y., U. S A. (Rapporteur)
Dr. H. Lange, Chairman of Food Chemistry Section,

Society of German Chemists, c/o Sarotti AG,
Hattersheim, Fed. Rep. of Germany
Professor R. Monacelli, Department of Chemistry,

Institute of Public Health, Rome, Italy
Professor M. J. Rand, Department of Pharmacology,

University of Melbourne, Victoria, Australia
Professor J. F. Reith, Department of Food Chemistry

and Toxicology, University of Utrecht, Netherlands
(Vice-Chairman)
Professor A. I. Stenberg, Head, Department of Hygiene,

Institute of Nutrition, U.S. S. R. Academy of Medical
Sciences, Moscow, U. S S. R.
- vii -
P .. ofessor R. Truhaut, Director, Toxicological Research

Centre, Faculty of Pharmacy of the University of Paris,
France
Observers (invited by FAO):
Mr. Durward F. Dodgen, Special Consultant, Food

Chemicals Codex, National Academy of Sciences,
Washington D. C., U S. A.
Professor M. J. L. Dols, Chairman of Codex Committee

on Food Additives, Wassenaar, Netherlands
Dr. W. Hausheer, International Union of Pure and

Applied Chemistry, c/o Hoffman-La Roche and Co. Ltd.,
Basle, Switzerland
Mr. L. Heckman, German Research Council,

Bad Godesberg, Fed. Rep. of Germany
Consultants :
Dr. H. Blumenthal, Public Health Service, Food and

Drug Administration, Washington D. C., U.S. A.
(Temporary Adviser, WHO)
Dr. P. S. Elias, Department of Health and Social

Security, Ministry of Health, London, England
(Temporary Adviser, WHO)
Mr. H.P. Mollenhauer, Reg. Dir., Federal Ministry

of Health, Bad Godesberg, Fed. Rep. of Germany
(Consultant, FAO)
Dr. D. M. Smith, Office for International Standards,

Food and Drug Directorate, Department of National
Health and Welfare, Ottawa, Canada (Consultant, FAO)
- viii -
Secretariat:
Dr. C. Agthe, Senior Scientist, Food Additives Unit,

WHO
Dr. F. C. Lu, Chief, Food Additives Unit, WHO

(Joint Secretary)
Dr. L. G. Ladomery, Food Standards Officer, F AO/WHO
Food Standards Programme, FAQ
Mr. R. K. Malik, Chief, Food Standards, Additives and
Regulations Section, Food Science Branch, Nutrition
Division, FAO (Joint Secretary)
SOME FOOD COLOURS
- 2 -.
ANNATTO EXTRACTS
Synonyms: C. I. Natural Orange 4; Lebensmittel

Orange No. 3; Rocou; Or lean;
Terre Orellana
Code (Index) Numbers· C. I. (1956} No. 75120

Schultz (1931} No. 1387
Definition: Annatto extracts are obtained by

extraction of the colour of the
pericarp of the fruit of Bixa orellana
J . Annatto extract in oil is prepared by heating the finely

divided pericarp with edible vegetable oil without addition of
other solvents or reagents under vicuum of about 20 mm Hg
at temperatures no higher than 130 C and filtering the
mixture to obtain the extract. It contains several
carotenoids, of which bixin is the principal colouring
matter. Bixin is the mono methyl ester of norbixin:
Bixin
H CH 3 H CH 3 H H H H H
I
H 3 COOC - C
I
C
I
C
I
C
I
C
I
C
I
C
I
C
I
C
\l\l\l\l~/\I\I\I\
C C C C 'C C C C CHCOOH
I
H
I
H
I
H
I
H H
I I
CH 3
I
H
I
CH 3
Annatto extracts in oil contain 0. 2 up to 2. 6 % of carotenoids

expressed as bixin. (see Assay) At least 30 % of the total
carotenoid content is bixin. It is probably present in the
a - form.
- 3 -
Annatto extract in oil is a red oily solution - or suspension.
2. Aqueous annatto extract is obtained by heating the

finely divided pericarp with a solution of sodium hydroxide
or cPotassium hydroxide at temperatures not higher than
70 C and filtering off the aqueous solution. In this process
bixin being an ester is hydrolysed to norbixin, a symmetrical
dicarboxylic acid.
Norbixin
H CH 3 H CH 3 H H H H H
I I I I ! I I I I
HOOC - - C C C C C C C C C
\/\/\/\/\/\/\!\!\
C C C C C C C C CHCOOH
I I I I I I I l
H H H H H CH 3 H CH
The principal colouring matter of aqueous annatlo extract

is the alkali salt of norbixin. Aqueous annatto extract is a
red alkaline solution.
Functional Use in Food: Colour
Identification
A. Absorption spectrophotop1etry
1. Annatto extract in oil, diluted with chloroform, shows

an absorption curve as in Fig. 1, a and b.
2. Aqueous annatto extract, diluted with water, shows an

absorption curve as in Fig. 1, c.
- 4 -·
70 2
8
6 I
I
' - -..... '
...-r-, :'
.ff
lf
I
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,,,. ./7
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"- - '\ l '\

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............
t::
,<:::, 70 1
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8
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ir I \\ \
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t
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I I I I
1/-00
I I I
'150
I I
' I I
\\
I I
J\
550mµ
,......._
B. Column Chromatography and Carr-Price Reaction:
1. Annatto extract in oil: Dissolve enough of the extract in

benzene to obtain a liquid having about the same colour as
a 0. J % potassium dichromate solution. Add 3 ml of the
solution at the top of the alumina-column (App. A, 1, under a and b)
and elute slowly with benzene. Bixin is very strongly
absorbed on the alumina surface and forms a brilliant
orange-red zone {difference from crocetin). A very pale
yellow-coloured zone migrates in general very quickly
through the column and is eliminated by washing with
benzene. Bixin cannot be eluted with benzene. Displace
the benzene in the column with chloroform, previously
dried with potassium carbonate, by washing the column
three times with chloroform. When the last chloroform
washing has been eluted add 5 ml of the Carr-Price T. S. on
the top of the column. The bixin-zone becomes immediately
blue-green {differentiates bixin from crocetin).
2. Aqueous annatto extract: Put 2 ml of the extract in a

50-ml separatory funnel. Add enough 2 N sulfuric acid to
give strongly acid reaction. Norbixin separates as a red
precipitate. Add 50 ml of benzere and shake vigorously.
- 5 -
After separation discharge the water layer and wash the

benzene phase with water until the elimination of the acid
reaction. Centrifuge for 10 minutes at 2500 rpm the generally
emulsified solution of norbixin in benzene. Decant the
clear norbixin solution and dry with anhydrous sodium
sulfate. Add 3-5 ml of this solution on the top of the alumina
column (App. A, 1, under a and b). Norboxin forms an orange-
red zone at the surface of the column and gives the same
Carr-Price reaction as bixin does.
C. Paper Chromatography
1. Annatto extract in oil: Impregpate Whatman 3 MM filter

paper or another paper having the same properties with a
mixture of 1 part liquid PBraffin and 9 parts of petroleum
ether boiling range 40-60 C. Place an adequate quantity of
the extract on the starting line and develop with a mixture
acetone: water : diethylamine (49:49:2). The annatto
extract in oil produces at least 3 or 4 red or yellow spots,
two red spots being clearly more intensive than the other
spots. Dissolve the red spots in benzene and identify that
one of the spots is bixin by the method given in B. 1.
2. Aqueous annatto extract: Prepare a chromatogram as

indicated under C. 1. At least 4 coloured spots are
obtained, of which 2 spots have an orange colour and the
others an orange-yellow colour. Dissolve the orange spots
in benzene and identify that one of the spots is norbixin.
Specification
Assay:
l. Annatto extract in oil: 0. 2 to 2. 6 % of carotenoids

expressed as bixin. Dissolve annatto extract in oil in
chloroform. Determine the pigment content expressed as
bixin by measuring the absorption at nm 500 using the
value E 10, . CHCl _ 2826 for bixin at nm 500.
,o 1n 3, 1 cm -
- 6 -
2. Aqueous annatto extract: Proceed as indicated in 1 ,

using glacial acetic ac1a as a solvent.
Arsenic (as As): not more than 5 mg/kg

Lead (as Pb): not more than 20 mg/kg
- 7 -
CHLOROPHYLL
Synonyms and varieties: C. 1. Natural Green 3;

Lebensmittel-Grlin Nr. 1
Class: Phorbin (• dihydroporphin)
Colour: Green
Code Numbers: C. 1. (1956) No. 75810

C. 1. (1924) No. 1249a
Schultz (1931) No. 1403
Chemical Name: Chlorophyll a: Magnesium

complex of 1, 3, 5, 8-tetramethyl-
4-ethyl-2-vinyl-9-keto-1 O-
carbomethoxyphorbinphytyl-7-
propionate
Chlorophyll b: Magnesium
complex of 1, 5, 8-trimethyl-3-
formy 1-4-ethyl-2-vinyl-9-keto-
l 0-car bomethoxyphor binphyty 1-
7 -propionate
Empirical Formula:
- 8 - .
Structural Formula:
~ (chlorophyll bi
(chlorophyll al
Molecular Weight: a 893. 54

b 907. 52
Definition:
Chlorophyll is the green pigment of plants. The

product to be used as a food colour should be produced from
alfalfa, lucerne, clover or spinach. It is a mixture of three
parts of chlorophyll a and one part of chlorophyll band
enzymatic degradation products thereof. It is the ester of
chlorophyllin with phytol. By the action of the enzyme
chlorophyllase phytol is split off. Chlorophyll a crystallizes
in green hexagonal plates, chlorophyll b in dark-green
needles.
Description
The chlorophyll of commerce is an intensely dark

green, aqueous, ethanolic, or oily solution of chlorophyll
degradation products. It is soluble in ethanol, ether,
chloroform and benzene; insoluble in water.
- 9 -
Identificat_:,.-911 Tests
A. Comparison for blue intensity vs. yellow intensity in

tintometer (Lovibond). Note: Method required.
B. A solution of chlorophyll in ethanol is blue-green with

deep red fluorescence.
C. Brown-phase reaction may be useful for characterization

of chlorophyll, when this has not been previously treated
with alkalies. Treat green ether or petroleum ether
solution of colouring matter with a small quantity of a
10% solution of potassium hydroxide in methanol. Colour
becomes brown, quickly returning to green.
Purity Tests
Lead (as Pb): not more than 20 mg/kg.

Arsenic (as As): not more than 5 mg/kg.
- 10 ...
CHLOROP!-~LL COPPER COMPLEX
DEFINITION
Functional Use in Foods: Food Colour Class: Porphyrin

Colour: Green
Synonym: Lebensmittel-Grtin No. 2
Code (Index) Numbers: C. I. (1956) No. 75810
Chemical Name: Chlorophyll copper complex
DESCRIPTION
Source: Chlorophyll copper complex is

obtained from chlorophyll by
partial replacement of
magnesium by copper and
usually contains between
4 and 6 % total copper.
Appearance: Blue-green powders, pastes or

viscous liquids, having a slight
amino-like odour.
CHARACTERISTICS
Identification
A. Solubility: Water: insoluble

Ethanol, Ester, Chloroform: soluble
B. Dissolve 0. 2 g of the sample in l 00 ml ether and add

J ml of a solution of 1 g of sodium hydroxide in
5 ml methanol, stir and allow to stand for 30 minutes.
Extract the solution three times with 10 ml each of water,
and dilute to 1 OOO ml with water. This solution exhibits
absorption maxima at the wavelengths of 405 nm and
630 nm, the ratio of the absorbances 405/ 630 being
3. 7 ± 0. 3.
- 11 -
C Dis,;· '"~ the residue on ignition of Chlorophyll Copper

complex L, 10 ml of diluted hydrochloric acid by heating
1m a wate:r bath, filter if the solution is not clear, and
dilute to 10 ml with water. Use this solution as the test
preparation for the following tests: (i) To 5 ml of this
preparaU(j;J add ammonia T. S. to make the solution alkaline:
a blue colour appears; (ii) To 5 ml of thls preparation
add 0. 5 ml of sodium diethyldithiocarbamate solution
(1 to 1000): a brown precipitate is formed.
Specification
Assay: Accurately weigh about 10 mg of Chlorophyll

Copper complex with the micro-chemical balance, dissolve
in 50 ml of ether, add 1 ml of a solution of sodium
hydroxide in methanol (1 to 5), stir, and heat for 30 minutes
on a water bath with a r~flux condenser. Cool, extract
3 times with 10 ml each of water, combine the extracts,
and add the phosphate buff er (pH 7. 5) to produce
100 ml solution: the absorbance E1 % is not less than
1 cm
249 at the wavelength of 405 nm.
Additional criteria: The article of commerce can be

further specified by loss on drying and residue on ignition
and total copper content.
LIMITS OF IMPURITIES AND ABSENCE OF ADULTERANTS
Arsenic (as As):. not more than 5 mg/kg.

Basic Coal Tar Dyes: To 5 ml of a 0. 5 % ethereal solution

of Chlorophyll copper add 1 ml acetic acid and 5 ml water
and stir. Filter by a gravity filter paper moistened with
water, allowing the ether layer to remain in the paper filter ;
the filtrate is colourless.
Free Ionizable Copper: not more than 200 mg/kg (IUPAC

Method for Copper on the aqueous extract).
- 12 ·-
CHLOROPHYLLIN COPPER COMPLEX,
SODIUM OR POTASSIUM SALT
(Tentative}
DEFINITION
Functional Use in Foods: Food Colour Class: Porphyrin

Colour: Green
Synonym: Lebensmittel-Grlin No. 2
Code (Index) Numbers: C. I. (1956} No. 75810
Chemical Name: Sodium or Potassium salt of

Chlorophyllin copper complex.
DE SCRIP TION
Source: Chlorophyllin copper complex
salts are obtained from
chlorophyll by partial replace-
ment of magnesium by copper
and replacing the methyl and
phytyl ester groups with alkali
and usually contains between
4 and 6 % total copper.
Appearance: Blue-black powders having

a slight amino-like odour.
CHARACTERISTICS
!dent ification
A. Solubility: Water: soluble

Ethanol, Ether, Chloroform: insoluble
B. Dissolve 0. 1 g of product in water and dilute to

100 ml with water. This solution exhibits absorption maxima
- 13 -
at the wavelengths of 405 nm and 530 nm. The ratio of the

ab~uruances 405/.330 being 3. 7 ± 0. 3.
C. Dissolve the residue on ignition of this material in

10 ml of diluted hydorchloric acid by heating on a water
bath, filtre if the solution is not clear, ~,nd dilute to
10 ml with water. Use this solution as the test preparation
for the following tests: (i) To 5 ml of this preparation add
ammonia T. S. to make the solution alkaline: a blue colour
appears. (ii) To n ml of this preparation add 0. 5 ml of
sodium diethyldithiocarbamate solution (1 to 1000): a
',Jrown precipitate is formed. (iii) Test in the usual manner
for sodium and/or potassium.
Specification
Assay: Accurately weigh about 10 mg of this material dried

at 105° for 1 h with the micro-chemical balance, dissolve
in phosphate buffer (pH 7. 5) to produce a 100 ml solution:
the absorbance
of 405 nm.
El'?o is not less than 249 at the wavelength
cm
Additional criteria: The article of commerce can be further

specified by loss on drying and residue on ignition, nitrogen,
pH, iron and total copper.
LIMITS OF IMPURITIES

Basic Coal Tar Dyes: To 5 ml of a 0. 5 % aqueous solution

of this material add 1 ml acetic acid and extract three times
with 5 ml of ether. Filter by a gravity filter paper moistened
with water, allowing the ether layer to remain in the paper
filter; the filtrate is colourless.
Free Ionizable Copper: not more than 2500 mg/kg (IUPAC

Method).
Note Information needed on level used for colouring foods

and on actual free ionizable copper content.
- 14 - .
ERYTHROSINE *
Synonyms: C. I. Food Red 14; FD and

C Red No. 3; LB - Rot 1
Class: Xanthene
Colour: Red
Code Numbers: C. I. (1956) No. 45430

C. I. (1924) No. 773
Schultz ( 1931) No. 887
Chemical Name: Disodium or dipotassium salt

of tetra-iodofluorescein.
Emnirical Formula:
* Tl1i s supersedes the specification contained in,

"Specifications, Identity and Purity of Food Additives" -
Vol. II Food Colours 19G3; published under the auspices of
the FAQ and the WHO.
- 15 -
Structural Formula
,,...a
NaO __ o
#
c
COON a
Molecular Weight: 879.9
Description
Erythrosine is a water-soluble food colour conforminl{

to the following specifications.
Identification Tests
3ee Appendix A, 1. of "Specifications for Identity and

Purity of Food Additives - Vol. II - Food Colours. "* The
tests should not indicate any presence of fluorescein.
* Published in 1963 under the auspices of foe Food and

Agriculture Organization of the United Nations and the
World Health Organization
- 16 -·
Purity Tests
Dye content: not less than 85 % by the method described

below. Dissolve 1 OOO g of Erythrosine in 250 ml of water,
transfer to a clean 500-ml beaker, add 8. 0 ml 1. 5 N nitric
acid and stir well.
Filter through a sintered glass crucible (porosity 3,

diameter 5 cm) which has been weighed containing a small
glass stirring rod. Wash thoroughly with 0. 5% nitric acid
until the filtrate gives no turbidity with silver nitrate T. S.
and then wash with 30 ml water. Dry to constant weight at
135° ~ 5°, carefully breaking up the precipitate by means
of the glass rod. Cool in desiccator and weigh.
%
O
d t t _ weight of residue x 105. 3
ye con en - weight of the sample
Loss on drying at 135° ) ~~~n8in

Chlorides and sulfates calculated as sodium salts) 15 %
Water-insoluble matter: not more than 0. 2 %.
Ether-extractable matter (from alkaline solution only):
not more than 0. 2% .
Lead (as Pb}: not more than 10 mg/kg.
Arsenic (as As): not more than 3 mg/kg.
Inorg·anic iodides: not more than 1 OOO mg/kg as sodium
iodide.
Place 5 OOO g of Erythrosine in 400 ml beaker and

add 150 ml of water; add just enough 10% NaOH solution to
give complete solution. Heat nearly to boiling and add
5 ml of H PO . Digest solution until precipitation is well
3 4
coagulated, cool to room temperature, transfer to 250 ml
volumetric flask, and make to volume. Mix thoroughly,
filter through dry fluted filter, and discard first few
ml of filtrate. Transfer 100 ml of filtrate to 500 ml tall-
form beaker, add 2. 5 ml of 30 NaOH solution, few glass
beads, and 15 ml of 7% KMn04 solution. Cover with watch-
glass, boil 5 minutes, and remove from heat. \Vhen boiling
ceases, add carefully 10 ml of HN03 and boil 5 minutes
more.
- 17 -
Remove beaker from heat and wash down cover glass

and sides (excess KMn04 must be present). Add 5 ml of
10% NaN02 solution quickly with swirling (KMn04colour will
be destroyed, and brown suspension of Mn02 left). Continue
addition of the NaN02 solution cautiously, allowing each
drop to react before next is added. When solution appears
colour less by transmitted light, but some particles of solid
Mn02 remain, do not attempt to destroy these, but
immediately add 1% KMn04 solution in 1 ml portions until
solution becomes pink.
If more than 2 ml is required or if brown colour

appears, add at once 10 ml of the diluted KMnO solution
and again heat to boiling. Repeat dropwise addi1ion of the
NaN02 solution and again add the diluted KMn04 solution
to pink colour.
Filter solution rapidly with suction through a

sintered-glass filter (medium porosity) into wide-mouthed
500 ml flask. Wash beaker and filter thoroughly with water
(solution must remain pink after filtration). Add the
NaNo 2 solution dropwise with shaking until 1 drop has
been added in excess of that required to decolourize
solution. Add 5 ml of 10 % sulfamic acid solution, wash
down sides of flask, and swirl contents. Cool solution to
room temperature, add 2-3 g of solid Kl, and titrate
liberated iodine with the standard sodium thiosulfate
solution.
1 ml of 0. 05 N N32 S203 0. 002498 g of Nal.

Subsidiary dyes: not more than 3 % •
Intermediates: not more than 0. 5% .
- 18 -·
CHEMICALLY TREAT~:) STARCHES

- 19 -
CHEMICALLY TREATED STARCHES
PEFINITION
Functional Use in Foods Thickening agents.
Structural Formula and Molecular Weight
Chemically treated starch is a macromolecule

composed of anhydro glucose units in linear and branched
form and modified as indicated below under "Chemical
Description. " Its molecular weight varies greatly depending
upon botanical origin and is generally lower than that of the
native starch from which it is obtained.
DESC~IPTION
Appearanc~
Most chemically treated starches are white or off-

whit,', tasteless and odourless powders. According to the
drying method these powders can consist of whole granules
having the appearance of the original native starch, of
ag::;regates consisting of a number of g:--anulcs (pearl starch,
starch-grits) or, if pregelatinized, of flakes, amorphous
powder OT coarse particles .
.('J1err1.i.QJ Description.
Chemically treated starches are food starches which

have one or more of their original characteristics altered
by chfm1ical treatment in ac~ordance with good manufacturing
practice with one or more of the agents listed in Table I.
In the case of starches treated with heat in the presence of
acid or with alkali, ( 1, 2 and 3 in Table I) the alteration is
a minor fragmentation of the chains.
When the starch is bleached, (4 in Table I) the change

is in the colour only. Oxidation (5 in Table I) involves the
production of carboxyl groups.
- 20 - ·
The treatments indicated under 6 and 7 of Table I

result in partial substitution in the 6, 3 or 2-position of
the anhydro glucose unit unless the 6-position is occupied
for branching.
In case of crossbonding, where a polyfunctional

substituting agent as mentioned under 6 in Table I connects
two chains, the structure can be represented by:
Starch-0-R-0-Starch,
where R = cross bonding group and Starch refers to the
linear and/or branched structure.
CHARACTERISTICS
Identification
A. Soiubility Water (cold): insoluble (if not pregelatinized)

Water (hot): form typical colloid with
viscous properties
Ethanol: insoluble
. B. Chemically treated starches which have not been

pregelatinized retain their granular structure and can be
identified as starches by macroscopic observation. Shape,
size and sometimes striations are characteristics of the
botanical origin. In polarized light under crossed nicols
the typical polarization cross will be observed.
C. Add a few drops of 0. 1 N 1/KI solution to an aqueous

suspension of the product. These starches stain with
iodine in the same way as native starches. The colour
can range from dark blue to red.
D. Boil 2. 5 g of the treated starch with 100 ml of

hydrochloric acid solution (3 % w/w) under reflux for
3 hours. Glucose can be identified in the hydrolysate by
any usual method.
- 21 -
E. The appropriate tests to differentiate between the

various treatments of the starches appear in the section
entitled "Analytical Methods for Chemically Treated
Starches" appearing immediately below this specification.
Specifications
The article of commerce can be specified by the

parameter specific for the particular type of modification
indicated in Column 3 of Table 1, and further as to loss on
drying, ash, chloride, pH, protein and fat.
Arsenic: not more than 3 mg/kg

Lead: not more than 5 mg/kg
Heavy metals: not more than 40 mg/kg
Sulphur dioxide: not more than 80 mg/kg
TABLE I
Column 1 Column 2 Column 3 Column 4
Treatment Maximum amount of substance Limitation and maximum Commercial Name
reasonably required to residual traces in the
accomplish the intended finished product
physical or technical effect
1. Roasted 0. 15 % acid, calculated as hydro- Final pH 2. 5 to 7. 0

starch with chloric acid anhydrous (100 %)
addition of acid and based on dry starch
2. Acid treatment 7 % hydrochloric acid or 2. 0 % Final pH 4. 8 to 7. 0 Acid-thinned Starches

in slurry sulphuric acid
3. Alkaline Sodium or potassium hydroxide Final pH 5. 0 to 7. 5 Alkali-treated Starches

treatment with not to exceed 1. 0 % .,.:,
.,.:,
4. Bleached Sodium hypochlorite, sodium No residues, no carboxyl Bleached Starches

starch chlorite, hydrogen peroxide, groups, except 50 mg/kg Mn
potassium permanganate, per- when permanganate used
acetic acid, ammonium per-
sulphate, sulphur dioxide in
amounts not more than that
sufficient to bleach the material.
5. Oxidized by Chlorine, as sodium hypochlorite, 0. 5 % sodium chloride Oxidized Starches

treatment with not to exceed 5. 0 % of chlorine, Carboxyl groups not more
based on dry starch than 1 ~. corresponding to
1 carboxyl group per
25 glucose units
TABLE J

(may be reasonably required to accomplish residual traces in the
combined) the intended physical or technical finished product
effect
6. Esterified by Acetic anhydride, not to exceed 10 %) Acetyl groups, not to Starch Acetate; Acetylated
treatment with Vinyl acetate, not to exceed 7. 5 % ) exceed 2. 5% Distarch phosphate* (POCl3);
Acetylated Di starch glycerol*;
Acetylated Distarch Adipate.
Adipic anhydride, not to exceed Acetylated Distarch Adipate.

0.12 %
I
Sodium tripolyphosphate and/or Residual phosphate, not to Mono-Starch Phosphate; ~
sodium trimetaphosphate and/or exceed O. 4 % , calculated Distarch Phosphate (sodium c.>
orthophosphoric acid and/or as phosphorus trimetaphosphate) Phosphated

sodium or potassium salts thereof Distarch Phosphate.
7. Etherified by Propylene oxide, not to exceed 5 Propylene oxide, 5 mg/kg. Hydroxypropyl Starch;
treatment with Propylene chlorohydrine, Hydroxypropyl Distarch
5 mg/kg. Phosphate* (POCl3).
TENTATIVE* due to lack of adequate data for toxicological evaluation
succinic anhydride, not to exceed 4 % Starch sodium succinate*

TABLE I

(may be reasonably required to accomplish residual traces in the
combined) the intended physical or technical finished product
effect
Phosphorus oxychloride, not to Residual phosphate not to Distarch phosphate•
exceed 0. 1% exceed 0. 04 % calculated (phosphorus oxychloride)
as phosphorus
Epichlorohydrin, not to exceed (Epichlorohydrin, 5 mg/kg Di starch glycerol*;

0. 3% (Glycerol monochlorohydrin, Acetylated Distarch
(5 mg/kg glycerol*; Hydroxypropyl
~
(Glycerol dichlorohydrin Distarch glycerol. ....
(5 mg/kg.
Identification tests for the various treatments ar.e given following this specification.
- 25 -
ANNEX
ANALYTICAL METHODS FOR CHEMICALLY
TREATED STARCHES
Tests to Differentiate, Identify and Assay the Treatment
and/or Limits of Residues of the Treatment Agent left in
the Product.
1. Hypochlorite oxidized starch

(Not for slightly oxidized potato starch)
Because of the carboxyl group content, hypochlorite-

oxidized starch has anionic properties. It can be dyed with
positively charged dyes such as methylene blue: 50 mg of
hypochlorite-oxidized starch is kept in suspension for 5-10
minutes in 25 ml of a 1 percent aqueous dye solution and
stirred occasionally. After decantation of the excess solution
the starch is washed in distilled water. Microscopic
inspection clearly shows the colouring. By this test
hypochlorite-oxidized starch is distinguished from native
and acid modified starch of the same botanical origin.
2. Specific reaction of acetyJ groups

Acetate is liberated upon saponification of acetylated
starch. After concentration the acetate is converted to
acetone by heating with calcium hydroxide. The acetone thus
produced strains blue with 0-nitro-benzaldehyde. About 10 g
of the test substance is suspended in 25 ml water to which
is added 20 ml 0. 4 N NaOH. After shaking for 1 hour the
starch is filtered off and the filtrate evaporated in an oven
at 110oc. The residue is dissolved in a few drops of water
a.nd transferred to a test tube. Calcium hydroxide is added
and the tube heated thereby giving off acetone vapours.
Thef;e produce a blue colour on a paper strip soaked in a
fresh saturated solution of 0-nitrobenzaldehyde in 2 N NaOH.
The blue colour is more distinct when the original yellow
colour of the reagent is removed with 1 drop of HCl 1: 10.
- 26 -·
3. Test for Ester Groups

The infrared spectrum of a thin film gives a typical
absorption band at about 1720 cm-1 which is an indication
for ester groups. The limit of detection is about: 0. 5 percent
acetyl odipyl a succinyl groups in the product.
4. Assay; Determination of Acetyl Groups and

Succinyl Groups
See "starch; Chemistry and Teclmology. Vol. II
Industrial Aspects. pp 612 and 634 Characterization and
Analysis of Starches. " Whistler, R. L. and Paschall, E. F.
Academic Press, 1967.
For succinyl the calculation of the degree of

substitution of the sodium salt is following:
(Blanktiter-Sampletiter) xacidnormx0.123x100
%succinyl:
sample weight in grams (dry basis)
162 A
DS:-----
12300-122 A (where A= succinyl)
5. Phosphor™
Method 1
Principle
The sample is ignited in the presence of an added

electrolyte to convert phosphorous to a stable form which
is not volatilized during ignition. The residual phosphate
is taken up in acid and determined spectrophotometrically
as the molybdivanadophosphoric acid complex (Note 1).
Scope
The procedure is applicable to determination of

phosphorus in unmodified and modified corn starches
(Note 2).
- 27 -
Special Apparatus
1. Platinum or Silica Dishes: About 100-ml capacity.
2. Muffle Furnace: Equipped with a pyrometer and

capable of operating at controlled temperatures up to 650°C.
3. Spectrophoton::eter: The rrethod requires an

instrument capable of continously-variable wavelengths in
the visible spectrum and equipped with with matching
cuvettes having a cell depth not over 2. 0 cm (Note 2).
Reag:ents
1. Calcium Acetate solution, 2 percent: Dissolve 20 g
of reagent grade calcium acetate monohydrate
(Ca(C2H302)2. H20) in 980 ml of distilled water.
2. Ammonium Vanadate solution, 0. 25 percent: Dissolve

2. 5 g of ammonium metavanadate (NH 4 vo 3) in 600 ml of
boiling water. Cool to 60-700C and add 20 ml of concentrated
nitric acid. Cool to room temperature and dilute to 1-liter
volume with distilled water.
3. Ammonium Molybdate solution, 5 percent: Dissolve

50 g of arnmonium molybdate tetrahydrate ((NH4)6Mo70 24 .4
H2o) in 900 ml of warm water. Cool to room temperature
and dilute to 1-liter volume with distilled water.
4. Standard Phosphorus solution, 0.1 mg P/ml:

Dissolve 0. 4387 g of reagent grade potassium dihydrogen
phosphate (KH 2 P04) in water and dilute to 1-liter volume
with distilled water.
5. Nitric Acid solution, 29 percent: Add 300 ml of

concentrated nitric acid (sp. gr. 1. 42) to 600 ml of distilled
water, and mix.
Erocedure
Standardization Curve: Pipet 5. O, 10. O, and 15. 0 ml
of standard phosphorus solution into respective 100 ml
- 28 -·
volumei.ric flasks, and use another flask for a blank. To

each flask add, in order, 10 ml of 29 percent nitric acid,
10 ml of O. 25 percent ammonium vanadate, and 10 ml of
5 percent ammonium molybdate, mixing thoroughly after
addition of each reagent (Note 3). Dilute to volume with
distilled water, mix thoroughly, and allow to ~tand for
10 minutes. Using the blank as a reference solution at
100 percent transmission, determine the transmission of
each standard at 460 m ,- . Plot log % transmission
versus mg of phosphorus per 100 ml (Note 4).
Analysis of Corn Starch: Weigh accurately 10 g of

corn starch into a platinum or silica dish; add 10 ml of
2 percent calcium acetate solution in a fine stream,
distributing the solution uniformly in the sample (Note 5).
Place the dish on a hot plate and carefully evaporate to
dryness, then increase heat and carbonize the sample on
the hot plate or over a gas flame. Place the dish in a muffle
furnace at 600-650°C until the ash is free of carbon
(1-2 hours, Note 6).
Cool to room temperature and wet the ash with 15 ml

of water. Slowly wash down the sides of the dish with 5 ml of
29 15ercent nitric acid; quantitatively transfer to a 200-ml
volumetric flask, rinsing the dish with three 20-ml portions
of distilled water. Dilute to volume with distilled water and
mix thoroughly. (If not clear, gravity filter through a
retentive paper.). Transfer an aliquot selected to contain
not more than 1. 5 mg of phosphorus to a 100-ml volumetric
flask, and add 50 ml of water to another flask to serve as a
blank. To each flask add, in order, 10 ml of 29 percent
nitric acid, 10 ml of 0. 25 percent ammonium vanadate, and
10 ml of 5 percent ammonium molybdate, mixing thoroughly
after addition of each reagent (Note 3). Dilute to volume
with water, mix thoroughly, and allow to stand for 10 minutes.
Determine% transmission of the sample at 460 m 1, , using
the blank as a reference solution at 100 percent transmission
(Note 7). Read mg of phosphorus in the aliquot from the
standardization curve.
- 29 -
Ca JcuJation
Total Phosphorus, ppm (as is) =
_ mg Phosphorus (From Graph) x 200 x 1 OOO
Aliquot Volume x Sample Wt. (g)
Notes and Precautions

1. The chemistry of the chromophore is in dispute;
for a discussion of some of the possibilities, see Kitson
and Mellon, Anal. Chem. 16, 379 (1944).
2. The procedure as written is not recommended for

starches having phosphorus contents below 40 ppm. For
phosphorus levels in such a low range, adequate precision
can be attained by tenfold dilution of the phosphorus
standard and use of 10-cm cuvettes.
3. To avoid interference from precipitation and side

reactions, reagents must be added in the order stated.
Since the vanadate and molybdate are present in large
excess, volumes of reagents need not be controlled more
closely than ± 1 ml.
4. The standardization curve is reproducible and need

be checked only when fresh reagents are prepared.
5. If desired, 3 ml of a saturated solution of magnesium

nitrate and 7 ml of water may be substituted for the
calcium acetate. The two systems give comparable results;
calcium acetate is recommended principally because it
yields a higher-density ash and because an ignited
magnesium nitrate blank must be included when using that
salt.
6. If difficulty is experienced in obtaining a carbon-

free ash, the dish may be removed from the muffle,
cooled, and the residue moistened with several drops
of 29 percent nitric acid. ~eating is then continued.
- 30 . :.
7. Calcium acetate has not been observed to contribute

apparent phosphorus to the system; however, it would be
advisable to check an ignited calcium acetate blank against
a water blank whenever a new lot of calcium acetate hydrate
is opened.
Method 2
Principle
The sample is digested with a mixture of sulphuric
and nitric acids, and the phosphorus content is determined
spectrophotometrically by the metal method.
Scope
The procedure is applicabie to determination of
phosphorus in unmodified and modified starches.
Special Apparatus:
Spectrophotometer: The method requires an instrument
with good susceptibility in the red
part of the visible spectrum.
Reaa-ents
a. Sulphuric acid 96 percent.
b. Nitric acid 65 percent.
(P20 5 content must be less than 0. 1 mg per 50 ml).
I. Metal solution:
Dissolve 1 g of monomethyl-p-aminophenolsulphate
(metal), 5 g sodiumsulphite 7 aq. and 150 g sodium
bisulphite in about 700 ml of distilled water and dilute
to 1 OOO ml.
Filter if the solution is not clear.
This solution is stable for several months.
- 31 -
IT. Molybdate solution:

Dissolve 50 g ammonium molybdate pro analysis in
about 450 ml of distilled water.
Pour this solution gradually in 500 ml 10 N sulphuric
acid while stirring.
Dilute to 1 OOO ml.
III. Sodium acetate solution:

Neutralize 1 OOO ml 5 N sodiumhydroxide solution
with acetic acid.
Dilute to 2 OOO ml.
IV. Standard Phosphorus solution:

Dissolve 1. 9 166 g of reagent grade potassium
dihydrogen phosphate (dried over sulphuric acid if
necessary) in distilled water and dilute to 1 OOO ml.
If the solution has to be stored for a prolonged
period of time, add a few drops of chloroform.
1 ml contains 1 mg P205.
For measurement dilute 50 ml to 1 OOO ml with
distilled water.
10 ml of this solution contains 0. 5 mg P205.
Procedure
Weigh:
Potato starch 2 grams
Other starches 5 grams
Starch with monostarch
phosphate 1 gram
Bring in 500 ml. Kohlrausch flask 10 ml of the

concentrated sulphuric acid and 10 ml of the concentrated
nitric acid solution.
- 32 -·
Add the starch. Heat mixture until development of

nitrous fumes becomes violent, then stop heating. If
development of brown fumes diminishes, repeat the heating
procedure, and add as long as contents of the flask obtain
a dark colour, a few drops of nitric acid. This procedure
is continued until the solution becomes colourless. (A faint
yellow or green colour sometimes disappears on cooling).
Dilute after cooling with 20 ml of distilled water, bring to
the boil to remove nitrous fumes. Bring contents in
calibrated flask of 200 ml, and dilute to mark.
Pipette 10 ml in calibrated flask of 100 ml, and add
successively 35 ml distilled water, 10 ml of solution I,
and 10 ml of solution II and shake.
After 15 minutes add 20 ml of solution m and dilute
to mark. Determine extinction at 600-610 m 1, •
For comparison a blank and a standard are measured.
a. Biank
Take for every series of determinations a Kohlrausch
flask of 500 ml, with 10 ml sulphuric and 10 ml nitric acid,
without starch addition. Treat by same procedure as the
sample.
b. · Also determine extinction of a standard, in which in

a calibrated flask of 100 ml, 10 ml of the blank solution
under a., and 10 ml of the phosphorus solution
(0. 5 mg P 2o 5 ) are treated with solutions I - m (Eb).
Calculation:
Subtract from extinction of the solution obtained

from the samples extinction of the blank.
Potato starch: _.E_ x 500 = mg P205 per 100 gram of starch

Eb
Other _E._ x 200 = mg P205 " " II II
starches: Eb
Starch with
monostarch : ___E_x 1 OOO = mg P205 11 II II II
phosphate Eb
- 33 -
Note: If solutions I - III are made correctly, pH before

addition of solution III is about 1. 0, after addition
slightly above 3. 0. If the latter value is too low
the intensity of the colour will continue to increase
rapidly.
6. Propylene Oxide, Epichlorohydrins

Benedict, J. H., J. Am. Oil Chemist Soc. 34, 450
(1957).
7. Propylene Chlorohydrins, Glycerol monochlorhydrin

and dichlorhydrin.
Determined by gas chromatography according to
Ragelis, E. P., Fishes, B. S., Klimeck, B.A. and Johnson,
C., J. A.O. A. C., ..5.1, 709 (1968) after extraction of the
product with acetone-water (5+1, V/V) according to Heuser,
S. G. and Scudamore, K. A. Analyst .9..3., 252-258 (1968).
8. Sulphur Dioxide
Principle
The sulfur dioxide is released from the sample in a

boiling acid medium and is removed by a stream of carbon
dioxide. The separated gas is collected in dilute peroxide
where it is oxidized to sulfuric acid and titrated with
standard alkali. Alternatively, the sulfuric acid may be
determined gravimetrically.
Scope
The method is applicable, with minor modifications,

to liquid or solid samples even in the presence of other
volatile sulfur compounds.
Special Apparatus
"Monier-Williams" apparatus for the determination

of sulfurous acid, constructed with standard-taper glass
connections, can be obtained from Scientific Glass Apparatus
Company, Bloomfield, N.J. It is customary however to
- 34 -·
construct the apparatus with regular laboratory glassware

using rubber stopper connexions (see sketch).
SULFUR DIOXIDE - Apparatus
Scrubber
Reaction Flask
Receivers
(See also: A. 0. A. C. 11 Methods of Analysis", Seventh

Edition, 1950, p. 471).
The 11 Monier-Williams 11 sulfurous acid apparatus is
constructed from standard laboratory glassware and
connected with rubber stoppers a..ric. rubber tubing in
accordance with diagram. In operation, carbon dioxide
is passed through the scrubber and bubbled through the
heated reaction mixture, sweeping sulfur dioxide through
the condenser and into the receivers where it is absorbed
quantitatively.
- 35 -
The assembly consists of a 1 OOO-ml two-neck round-bottom

boiling flask to which a gas-inlet tube, 60-ml dropping
funnel having a 2-mm bore stopcock, and sloping Allihn
reflux condenser are attached. A delivery tube connects
upper end of condenser to bottom of a 250-ml Erlenmeyer
receiving flask, which is followed by a Peligot tube.
Reagents
1. Sodium Carbonate solution: Dissolve approxi-

mately 15 g of Na2C0 3 or 40 g of Na 2co 3 .
10 H 2o in distilled water, and dilute to
100-ml volume.
2. Hydrogen Peroxide, 3 percent: Dilute 10 ml
of C. P. neutral 30 percent hydrogen peroxide
(H202) with distilled water to 100-ml volume.
3. Sodium Hydroxide, 0. 1 N: Standardize using
bromphenol blue indicator.
4. Hydrochloric Acid, concentrated: Reagent
grade.
5. Bromphenol Blue Indicator.
Procedur~
Pass carbon dioxide from a generator or cylinder

through a sodium carbonate scrubber solution to remove
chlnrine, thence into the gas-inlet tube of the boiling flask.
Place 15 ml of 3 percent hydrogen peroxide in the receiving
flask and 5 ml in the Peligot tube. Connect the apparatus
and introduce into the boiling flask, by means of the
dropping funnel, 300 ml of distilled water and 20 ml of
concentrated hydrochloric acid. Boil contents approximately
10 minutes in a current of carbon dioxide.
Weigh 100 g (± 1 g} of starch sample and disperse

in approximately 300 ml of recently-boiled distilled water.
Trar.sfer slurry to boiling flask by means of the dropping
funnel, regulating sample ·addition rate and gas flow rate
through the apparatus to prevent drawback of hydrogen
peroxide, inclusion of air, or burning of sample. Boil
mixture gently for 1 hour in a slow current of carbondioxide.
- 36
Stop flow of water in condenser just before end of

distillation. When delivery tube just above receiving flask
becomes hot, remove tube from condenser immediately.
Wash delivery tube and Peligot tube contents into receiving
flask, and titrate with 0.1 N sodium hydroxide, using
bromphenol blue indicator (see Note).
Determine a blank on the reagents, and correct

results accordingly.
Calculation
Sulfur Dioxide (as is) :
= (ml 0.1 N NaOH - Blank) x 0. 0032 x 100

~~~~~~~~~~~~~~~~~
100 g
Note:
A gravimetric determination may be made after

titration. Acidify with HC 1, precipitate with
BaC l 2, settle, filter, wash, ignite, and weigh
as BaS04.
- 37 -
SOME EMULSIFIBRS AND STABILIZERS

- 38 -
CARRAGHEENAN
S,y_nonvms
Carragheenan, Irish moss gelose, chondrus extract.
Chemical Descri,ptton
Carragheenan consists chiefly of the calcium,

potassium, so-dium, ammonium and magnesium salts of
polysaccharide sulphate esters which on hydrolysis yield
galactose and anhydrogalactose.
Definition
Carragheenan is obtained by extraction with water

of members of the Gigartinaceae and Solieriaceae families
of the class Rhodophyceae (red seaweed). Members of
these families used in the production of carragheenan include
Chondrus crispus; .c. ocellatus; Eucheuma cottooii;
E. spinosum; Gigartina acicularis; .G. pisti11ata; .G. radula;
and .G. stellata.
Description
Carragheenan occurs as a yellowish to colourless,

coarse to fine, powder which is practically odourless.
Thickening agent and stabilizer.
Identification
A. Solubility Water: soluble
Ethanol: insoluble
- 39 -
B. Add 4 g to 200 ml of water and heat the mixture in a

water bath at a temperature of about 80°, with constant
stirring, until a viscous solution results. Replace any
water lost by evaporation and allow it to cool to room
temperature. It becomes more viscous and may form
a gel.
To 50 ml of the solution or gel add 100 mg of potassium
chloride and 50 mg of sodium chloride; mix well,
reheat and cool. A short-textured gel forms.
C. To a solution of 100 mg of sample in 20 ml water add

3 ml IN barium chloride and 5 ml 2 N hydrochloric
acid and filter if there is a precipitate. Boil filtrate
for 5 minutes. A white, crystalline precipitate is
formed.
D. Identify galactose and anhydrogalactose as indicated

in Annex.
Pux.:ity Tests
Ash (Total): Between 20 to 37 percent on the dry
weight basis. Transfer about 2 g,
accurately weighed, into a previously
ignited, tared silica or platinum
crucible. Heat the sample with a suitable
infrared heat lamp, increasing the
intensity gradually, until it is completely
charred and then continue for an additio-
nal 30 minutes. Transfer the crucible
and charred sample into a muffle furnace
and ignite at about 550° for 1 hour, then
cool in a dessicator and weigh. Repeat
the ignition in the muffle furnace until a
constant weight is attained. If a carbon-
free ash is not obtained after the first
ignition, moisten the charred spot with
a 1 in 10 solution of ammonium nitrate
and dry under an infrared heat lamp
before igniting again.
- 40 -
Ash (Acid- Not more than 2 percent.

insoluble):
Sulphate Between 20 and 40 percent on the dry
(as so 4): weight basis. The sulphates are precipitated
as barium sulphate in hydrochloric acid,
after hydrolysis under reflux. Weigh
accurately 1 g of sample in 100 ml long
neck round bottom flask. Add 50 ml N/5
hydrochloric acid. Fit a cooler consisting
preferably of 5 round bulbs and heat to
boiling under reflux for 1 hour. Add 25 ml
110-volume hydrogen peroxide solution
and continue boiling under reflux for
5 hours when the solution becomes
completely clear.
Transfer the solution to 600 ml beaker.
Bring to boil and stir in dropwise 10 ml
of 10 percent solution of barium chloride.
Let stand for 2 hours on a boiling water
bath. Filter the precipitate through ash
free filter paper meant for slow filtration
(blue ribbon) and wash it with boiling
distilled water till the filtrate is free
from chloride. Dry the filter paper in a
drying oven and ash at 1 ooooc in a tared
silica crucible. Let cool when the ash
is white. Weigh and calculate the weight
(P) of the barium sulphate obtained.
% sulphate: P x 0. 04116
Arsenic: Not more than 3 mg/kg.
Heavy metals: Not more than 40 mg/kg.
Lead: Not more than 10 mg/kg.

specified by viscosity and loss on drying.
- 41 -
FURCELLARAN
Svnonvm
Danish agar, Furcellaran
Chemical Description
Furcellaran consists chiefly of salts ofpolysaccharide

sulphate esters which on hydrolysis yield galactose and
anhydro galactose.
Definition
Furcellaran is obtained by extraction with water of

the red alga Furcellaria fastia;iata (Fam. Florideae) and
is precipitated from the extract with potassium chloride.
Potassium chloride may be added to the gelling properties
of the product.
Description
Furcellaran occurs as a yellowish to colourless,

coarse to fine, powder, which is practically odourless and
has a mild, salty taste due to the potassium chloride.
As thickening agent and stabilizer.
Identification
A. Solubility: Water: Soluble

Ethanol: Insoluble
B. Add four grams to 200 ml of water and heat the mixture

in a water bath at a temperature of about 80°, with
constant stirring, until a viscous solution results.
Replace any water lost by evaporation and allow it to
cool to room tempera~re. It forms a gel.
- 42 -
C. To a solution of 100 mg of sample in 20 ml water add

3 ml 1N barium chloride and 5 ml 2N hydrochloric acid,
filter if precipitate is formed. Boil filtrate for 5
minutes. A white crystalline precipitate is formed.
D. Identify galactose, glucose and xylose as indicated in

the Annex.
Purity Tests
Ash (Total): As: Not more than 3 mg/kg.

Heavy metals: not more than 30 mg/kg.
Ph: not more than 10 mg/kg.
Sulphate (as S04): Between 14 and 18 percent on the dry
weight basis. Method as indicated under
carragheenan.
Additional criteria; The article of commerce can be

further specified by viscosity, loss on drying, and
potassium chloride content. ·
- 43 -
ARABIC GUM
Synonym
Acacia gum
Chemica,l Description
Gum arabic consists chiefly of a high molecular

weight polysaccharides and their calcium, potassium and
magnesium salts which on hydrolysis yield arabinose,
galactose, rhamnose and glucuronic acid.
Definition
Gum arabic is a dried gummy exudation obtained

from the stems and branches of Acacia Sengal (L)
Willdenow or of related species of Acacia (Fam. Legum
inosae).
Description
Unground gum arabic occurs as white or yellowish

white spheroidal tears of varying size or in angular fragments
and is sometimes mixed with darker fragments and pieces
of bark. This crude material must be cleaned before use
in foods. It is also available commercially in the form of
white to yellowish white flakes, granules or as a powder.
Identification
A. Solubility of the Water: soluble. One gram dissolves
powder: in 2 ml of water forming a solution
which flows readily and is acid to
litmus.
Ethanol: insoluble.
- 44 -
B. A 1 in 10 solution filtered through diatomaceous

earth is slightly levorotatory.
C. Identify arabinose, rhamnose, galactose and glucuronic

acid as indicated in the Annex.
Purity Tests
Ash (Total): Not more than 4 percP.:1t.

Ash (Acid-insoluble): Not more than 0. 5 percent.
Acid (Insoluble matter): Not more than 1 percent.
Starch, or dextrin: Boil a 1 in 50 solution of the
gum, cool and add a few drops
of iodine TS. No bluish or
reddish colour is produced.
Tannin-bearing gums: To 10 ml of a 1 in 50 solution
add about 0. 1 ml of ferric chloride
TS. No blackish colouration
or blackish precipitate is formed.

further specified by loss on drying and viscosity.
- 45 -
CAROB BEAN GUM

Synonyms
Carob, Locust bean gum, St. John's bread, Algaroba.
CHEMICAL DESCRIPTION
Carob bean gum consists chiefly of high molecular

weight polysaccharides composed mainly of galactomannans.
Definition
Carob bean gum is obtained from the ground

endosperms of Ceratonia siliQ.ua (L.) Taub., (Fam.
Le'™minosae).
Description
Carob bean gum is a white to yellowish white,

nealy odourless powder.
Identification
A. Solubility: Water: Forms a solution in hot water.
Ethanol: Insoluble
B. A water solution of carob bean gum may be converted

to a gel by the addition of small amounts of sodium
borate.
C. Transfer a 2 gram sample into a 400 ml beaker,

moisten it thoroughly with about 4 ml of isopropanol
add with vigorous stirring 200 ml of water and
continue the stirring until the gum is completely and
uniformly dispersed. An opalescent, slightly viscous
- 46 -
solution is formed. Transfer 100 ml of the solution

prepared as above into another 400 ml beaker, heat
the mixture in a boiling water bath for about 10 minutes
and then cool to room temperature. There is an
appreciable increase in viscosity (differentiating
from gu.ar gum).
D. Identify sugars as indicated in the Annex. Only mannose

and galactose are present.
E. Place some ground carob bean gum in an aqueous solution

containing 0. 5 percent iodine and 1 percent potassium
iodide ein a glass slide for microscopic examination.
Carob bean meal contains long stretched tubiform cells,
separate or slightly interspaced; their brown contents
are much less regularly formed than in guar gum.
(Guar gum shows close groups of round to pear formed
cells; their contents are yellow to brown). ·
Purity Tests
Ash (Total): Not more than 1. 2 percent.
Acid-insoluble matter:Not more than 5 percent. See the
Annex for method.
Protein: Not more than 8 percent. Determine
nitrogen by Kjeldahl method. The
percent of nitrogen determined
multiplied by 5. 7 gives the percent
of protein in the sample.
Starch: To a 1 in 10 solution of the gum add
a few drops of iodine T. S. No blue
colour is produced.

further specified by viscosity and loss on drying.
- 47 -
GUARGUM
Synonym§
Gum cyamopsis, guar meal, guar flour.
Guar gum consists chiefly of polysaccharides of high

molecular weight composed mainly of galactomannans.
Definition
Guar gum is obtained by grinding the endosperm of
Cyamopsis tetr3,i'aoolobus (L) Taub., (Fam. Leguminosae).
Identification
A. Solubility: Forms a solution in cold or hot water.
B. A water solution of guar gum may be converted to a

gel by the addition of small amounts of sodium borate.
C. Transfer a 2 gram sample into a 400 ml beaker,

moisten it thoroughly with about 4 ml of isopropanol,
add with vigorous stirring 200 ml of water and continue
the stirring until the gum is completely and uniformly
dispersed. An opalescent, viscous solution is formed.
Transfer 100 ml of the solution into another 400 ml
beaker, heat the mixture in a boiling water bath for
about 10 minutes and then cool to room temperature.
There is no substantial increase in viscosity
(differentiating guar from locust bean gum).
D. Identify sugars as indicated in Annex. Only mannose

and galactose are pre~ent.
- 48 -
E. Place some ground ~ar gum in an acqueous solution

containing 0. 5 percent iodine and 1 percent potassium
iodide c,n a glass slide for microscopic examination.
Guar gu.m shows close groups of round to pear formed
cells; their contents are yellow to brown. (Locust bean
meal contains long stretched tubiform cells, separate
or slightly interspaced; their brown contents are much
less re1~larly formed than in guar gum.).
Purity Tests
Ash (Total): Not more than 1. 5 percent.

Acid-insoluble matter: Not more than 7 percent. See
Annex for method.
Protein: Not more than 10 percent. Determine
nitrogen by Kjeldahl method. The
percent of nitrogen in the sample
multiplied by 5. 7 gives the percent
of protein in the sample.
Starch: To a 1 in 10 solution of the sample
add a few drops of iodine T. S. No
blue colour is produced.
Additional criteria; The article of commerce can be further

specified by viscosity and loss on drying.
- 49 -
KARAYA GUM
Synonyms
Sterculia, Kadaya, Katilo, Kulla, Kuteera, mu,;_;cara

miscalled Indian tragacanth.
Chemical Desc;ciption
Karaya gum consists chiefly of high molecular

weight polysaccharides which on hydrolysis yield galactose,
rhamnose and galacturonic acid.
Definition
Karaya gum is a dried gummy exudation obtained

from Sterculia urens Roxburgh and other species of
Sterculia (Fam. Sterculiaceae), or from Cochlospermum
g:ossypium A. P. De Condolle, or other species of
Cochlospermum Kunth (Fam. Bixaceae).
Descrintion
Unground gum karaya occurs in tears of variable

size or in broken irregular pieces having a somewhat
crystalline appearance. It is pale yellow to pinkish brown,
translucent and horny and is sometimes admixed with
darker fragments and pieces of bark. This crude material
must be cleaned before use in foods. Powdered karaya
gum is light grey to pinkish grey. The gum has a slight
odour and taste of acetic acid.
Identification
A. Solubility of the Water: Two grams added to 50 ml

powder: of .water swells to form a granular,
stiff, slightly opalescent gel which
is acid to litmus.
- 50 -
~thanol: Insoluble
B. The gum swells in 60 percent ethanol as distinct from

other gums.
C. Boil 1 gram with 20 ml of water until a mucilage is

formed, add 5 ml of hydrochloric acid and again boil
the mbcture for 5 minutes. A permanent pink or red
colour develops.
D. Identify rhamnose, galactose and galacturonic acid

as indicated in the Annex.
Purity Tesi;.e
Ash (Acid, Insoluble): Not more than 1 percent.
Acid-insoluble matter: Not more than 3 percent. See
Annex for the method.
Starch: To a 1 in 10 solution of the gum
add a few drops of iodine T. S. No
blue colour is produced.
Additional criteria; The article of commerce can be

further specified by colour, size, viscosity and loss
on drying.
- 51 -
TRAGACANTH GUM
Synonym
Tragacanth
Tragacanth gum consists chiefly of high molecular

weight polysaccharides composed of galacto-arabans and
acidic polysaccharides containing galacturonic acid groups.
Definition
Tragacanth gum is a dried gummy exudation obtained

from Astral~s ~mmifer Labillardiere, or other Asiatic
species of Astral3.iJ.ls (Fam. Le~minosae).
Description
Unground Tragacanth occurs as flattened, lamellated,

frequently curved fragments or straight or spirally
twisted linear pieces from O. 5 to 2. 5 mm in thickness.
It is white to pale yellow in colour, translucent, horny in
texture and having a short fracture. It is odourless and has
an insipid, mucilaginous taste. It is rendered more easily
pulverizable if heated to a temperature of 50°. Powdered
Tragacanth is white to yellowish white in colour.
Identification
A. Solubility of the Water: One gram in 50 ml of water

powder: swells to form a smooth, stiff,
opalescent mucilage free from
cellular fragments.
Ethanol: Insoluble
- 52 -
B. Examine microscopically as a suspension in water.

Numerous angular fragments with circular or irregular
lamella.e and starch grains up to 25 in diameter are
visible and there should be very few or no fragments
of lignified tissues.
C. Identify arabinose, xylose, fucose, galactose and

galacturonic acid as indicated in the Annex.
Purity Tesi~
Ash: (Total) Not more than 3 percent.
Ash (Acid-:insoluble): Not more than 0. 5 percent.
Additional ,criteria; The article of commerce can be

further specified by viscosity and loss on drying.
- 53 -
ANNEX
IDENTIFICATION AND OTHER TESTS FOR
STABILIZERS AND THICKENERS
1. Chromatographic Identification of Sugars in the
Hydrolysate from ~ms,
Boil a mixture of 100 mg of the sample and 20 ml of

10 percent sulphuric acid for 3 hours. Allow to cool and add
excess barium carbonate (about 10 mg). Mix with a magnetic
stirrer until the solution is of pH 7, and filter. Evaporate
the filtrate in a rotary evaporator at 30°- 50° in vacuum
until a crystalline (or syrupy) or residue is obtained.
Dissolve it in 10 ml 40 percent methanol. This is the
hydrolysate used below.
Prepare thin layer chromatoplates with a mixture of

15 g cellulose powder (e. g. Camag Cellulose-D) and 90 ml
water and dry them for 10 minutes at 1000.
Place 1 to 10 microlitre spots of hydrolysate on the

starting line of two chromatoplates and spots containing 1 to
10 micrograms of the sugars which could be present in the
hydrolysate. Use two solvents: A. a mixture or formic
acid, methyl ethyl ketone, tertiary butanol and water
(15:30:40:15 by volume) and B. a mixture of isopropanol,
pyridine, acetic acid and water (40:40:5:20 by volume) to
develop the plates.
After development, spray with a solution of 1. 23 g

anisidine and 1. 66 g phthalic acid in 100 ml ethanol and
heat the plates at 100° for 10 minutes. A greenish yellow
colour is produced with hexoses, a red colour with pentoses
and a brown colour with uronic acids.
2. Method for Acid Insoluble Matter: Transfer a 2 g

sample, accurately weighed, into a 250 ml beaker containing
150 ml of water and 15 ml of 1 percent sulphuric acid.
Cover the beaker with a watch glass and heat the mixture
on a steam bath for 6 hours rubbing down the wall of the
- 54 -
beaker frequently with a rubber-tipped stirring rod and

replacing any water lost by evaporation. Then add about
500 mg of a suitable filter aid, accurately weighed, and
filter throu1?;h a tared Gooch crucible provided with an
asbestos pad. Wash the residue several times with hot
water, dry the crucible and its contents at 1050 for 3 hours,
cool in a desiccator and weigh. The difference between
the weight of the filter aid plus crucible and pad and the
total weight is the weight of the Acid Insoluble Matter.
Calculate as percentage.
- 55 -
POLYGLYCEROL ESTERS OF
INSTERESTERIFIED RICINOLEIC ACID
DEFINITION
Functional Use in Foods: Emulsifier
Synonyms: Glyceran esters of condensed

castor oil fatty acids
Structural formula:
The major components have the general structure
OR
I
RO - (CH2 - CH - CH20)n - R
Where the average value of n is about 3 and R is

hydrogen or a condensation polymer of ricinoleic acid with
itself (average 5 to 8 units) thus:
0 (CH 2 ) 5CH3 0
11 I II
R' - C - 0 - CH - CH2 - CH = CH - (CH2l7C -
(R' : aliphatic chain of ricinoleic acid)
DESCRIPTION
Polyglycerol esters of interesterified ricinoleic

acid are prepared by the esterification of polyglycerol with
condensed castor oil fatty acids. The product is a highly
viscous liquid.
Characteristics
Identification
A. Solubility Water: _ insoluble
Ethanol: insoluble
- 56 -
Hydrocarbons, ethers, halogenated hydrocarbons: soluble
B. Hydrolyse the material as indicated in the Annex.
C. Test for ricinoleic acid. The fatty acids liberated in B

should have a hydroxyl value corresponding to that for
castor oil fatty acids (about 150 to 170).
D. Spot 5 to 20 µ 1 of the residue obtained in B alongside

control spots of glycerol on paper such as Whatman No. 3
and develop using descending chromatography for 36 h with
isopropanol: water 90:10. The glycerol spot moves 40 cm
and the polyglycerols are revealed in succession below that
for glycerol when the paper is sprayed with either
permanganate in acetone or ammoniacal silver nitrate.
Specification
Not less than three-quarters of the polyols should
be glycerol, diglycerol and triglycerol as determined by
the gas chromatography method referred to below l/.
The article of commerce may be specified further as to
saponification value, solidification point of the free fatty
acids, iodine value, acid value, hydroxyl value and ash
content.
Limits of Impurities
other polyols: Polyols other than polyglycerol and

glycerol should be absent by the tests indicated in the
Annex.
l/ Sen, Keating and Barrett, J. Gas Chromatography

1967, ..5., 269.
- 57 -
PROPYLENE GLYCOL ESTERS OF FATTY ACIDS*
DEFINITION
Functioaal Use in Foods; Emulsifier
Structural Formula: CH a-CH-OR

I
CHrOR
Where R or R' represents the fatty acid moiety; R or R'

is hydrogen in the mono-esters.
DESCRIPTION
Propylene glycol esters of fatty acids are mixtures

of propylene glycol mono- and di-esters of fatty acids of
food fats. They are mainly the mono-esters with some di-
esters. The commercial products contain mono- and di-
glycerides when fats are used for transesterification with
propylene glycol and are white to yellowish white beads, or
flakes having a bland odour and taste.
CHARACTERISTICS
Identification
A. Solubility: Water: Insoluble
E thaaol: Soluble
B. Identify fatty acids and propylene glycol and glycerol
as in Annex.
Specification
Propylene glycol: The total propylene glycol content in
the commercial product must be indicated.
* Published previously as Food Additive Specification

FAS/IV/35 through the Joint FAO/WHO Food Standards
Programme.
- 58 -
Additional criteria for the commercial product:

The composition of tht:: a.rticle of commerce can be furtht
specified by saponification value, iodine value, solidifica·
tion point, free propylene glycol, soap content, hydroxyl
Acids other than fatty acids and polyols other than

propylene glycol and glycerol must be absent (See Methods
in Annex).
Arsenic: Not more than 3 mg/kg
Heavy metals: Not more than 10 mg/kg
EXAMINATION
The following is an international referee method to

be used in cases of dispute:
Assay for progylene glycol and glycerol;

Transfer about 15 g of sample, accurately weighed
into a 500-ml flask, add 250 ml of ethanol and 7. 5 of
potassium hydroxide and mix. Reflux the solution for 2 hours,
transfer into an 800-ml beaker rinsing the flask with about
100 ml of water and adding the rinse water to the beaker.
Heat on a steam or water bath, adding water occasionally
to replace the ethanol and evaporate until the odour of
ethanol can no longer be detected. Adjust the volume to
about 250 ml with hot water, neutralize with dilute sulphuric
acid (1 in 2), add a slight excess of acid, heat with gentle
stirring until the fatty acid layer separates. Transfer the
fatty acids into a warm 500-ml separatory funnel, wash
with four 20 ml portions of hot water and combine the
washings with the original aqueous layer from the saponi-
fication. Extract the combined aqueous layer with three
20 ml portions of petroleum ether. Neutralize the aqueous
layer with sodium hydroxide T. S. to pH 7. Transfer the
solution to a 500-ml volumetric flask and dilute to the
mark with water.
- 59 -
Determination of propylene glycol:

Pipette 5. 0 ml of the solution into a 125 ml
Ehrlenmeyer flask, add 5. 0 ml of 1 .M periodic acid, swirl
and let stand 15 minutes. Add 10 ml of a saturated solution
of sodium bicarbonate, followed by 15. 0 ml of 0.1 .Nsodium
arsenite and 1 ml of potassium iodide solution (1 in 20) and
mix. Add enough sodium bicarbonate so that at the end
point some remains undissolved, and titrate with 0.1 .N
iodine, using a 10-ml microburet and continuing the titration
to a faint yellow colour. Perform a blank determination
and make the appropriate corrections. Each ml of 0.1 N
iodine is equivalent to 3. 805 mg of propylene glycol.
_ 38.05xml 0.1.NSodium sol.
g propy1ene g1yea1/ 100 g es t er - · · ht mg
samp1e we1g ·
If the qualitative test for polyols included under

Identification showed the product to be a mixture of propy-
lene glycol and glycerol esters of fatty acids it becomes
necessary to determine the glycerol content of the polyol
solution obtained after saponification and separation of
liberated fatty acids.
Determination of glycerol:
Pipette 50 ml of the solution into a 600-ml beaker,
add bromothymol blue T. S. and acidify with 0. 2 N H2S04
to a definite greenish-yellow colour. Neutralize with 0. 05
N sodium hydroxide to a definite blue end-point free of
green colour. Prepare a blank containing 50 ml of water
and neutralize in the same manner. Pipette 50 ml of
sodium periodate solution T. S. (see Annex) into each
beaker, mix by swirling, cover with a watch glass and
allow to stand for 30 minutes at room temperature (not
above 35°) in the dark or in subdued light. Add 10 ml of a
mixture of equal volumes of ethylene glycol and water and
allow to stand 20 minutes. Dilute each solution to about
300 ml and titrate with O. 1 N sodium hydroxide to
pH 8.1 ± 0.1 for the sample and 6. 5 ± 0.1 for the blank
using a calibrated pH meter. Each ml of 0. 1 N sodium
hydroxide after correctioIJ. for the blank is equivalent to
9. 210 mg. of glycerol.
- 60 -
_ 9. 210 x ml 0.1 N NaOH

g glycerolI 100 g of esb•rs - sample weight in g
The true propylene glycol content in g/100 g of
ester is equal to the apparent propylene glycol content in
g/100 of ester - 1. 65 x g glycerine/100 g of ester.
- 61 -
CALCIUM STEAROYL LACTYLATE

and
SODIUM STEAROYL LACTYLATE
(Tentative)
DEFINITION
Structural Formula:
lactate: lactylate:
O
Ii
R -C-0-CH-COOCa
CH 3
\
O
\\ ' CH3 0
CH3
\\\
R-C-0-CH-C-O-CH-COOCa
2 2
Where R is the hydrocarbon chain of the fatty acid moiety.
DESCRIPTION
Calcium or sodium stearoyl lactylate consists mainly of the

calcium or sodium salts of lactic acid and its dimer which
have been esterified with the fatty acids of food fats such as
commercial stearic acid. Some esters of other fatty acids
such as myristic acid may be present and there may also
be some free fatty acids.
Appearance: Calcium or sodium stearoyl lactylate as a

white or slight yellowish white powder or brittle solid
depending on lactic/fatty acid ratio. It has a characteristic
odour.
CHARACTERISTICS
ldentif ication
A. Solubility: Water: inaoluble

Ethanol: soluble
- 62 -
B. Add 10 ml of dilute hydrochloric acid to 2 g of sample,

heat for 5 minutes in a water bath, filter and neutralize the
filtrate with ammonia T. S.
(a) Calcium salt: Add 5 ml of ammonium oxalate T. S.

A white precipitate is formed, soluble in dilute
hydrochloric acid T. S. but insoluble in dilute acetic
acid T. S.
(b) Sodium salt: Add uranyl zinc acetate T. S.; a yellow

crystalline precipitate appears within a few minutes.
C. Take the residue from the filter in test B, add 30 ml of

sodium hydroxide T. S. , heat for 30 minutes on a steam bath
and filter. Add 20 ml of dilute hydrochloric acid to the fil-
trate after cooling, extract twice. with 30 ml of ether, wash
the ether solution with 20 ml of water, dehydrate with
anhydrous sodium sulfate and evaporate the ether. The
residue melts between 54. 5° and 69°.
D. Add dilute suliuric acid T. S. and potassium perman-

ganate T. S. to calcium or sodium stearoyl lactylate. The
odour of acetaldehyde is produced on heating.
Spee if ication
The article of commerce can be specified as to loss on dry-

ing, calcium or sodium contents, pH of saturated solution,
ester value, acid value, lactic acid and fatty acid content.
For more complete analysis, see (1).
Acids: Acids moieties other than lactic, lactylic and fatty

acids should not be present.
Acrylic Acid: not more than 100 mg/kg (Method needed)
- 63 -

Reference: (1) G. Jurriens et al. "Analysis of Calcium

Salts of Fatty Acid - Lactic Acid Conden-
sate," Cereal Chem. 43, 669-74 (1966).
- 64 -
STEARYL CITRATE
Predominantly citric acid ester of n-octadecanol

and n-hexandecanol.
Structural Formulfl.
COOR
R O O C - CH - C - CH - C O O R
2 , 2
OH
Where R = stearyl, up to 50 % palmityl or

hydrogen.
Definition
Stearyl citrate is formed by esterifying citric acid with

commercial stearyl alcohol, which consists essentially of
n-octadecanol and n-hexadecanol.
Description
Stearyl citrate occurs as a cream-coloured unctuous

substance.
Emulsifier.
Identification
A. Solubility: Water: Insoluble

Ethanol: Insoluble cold; soluble hot
B. Hydrolize approximately 2 g of the ester by heating with

50 ml sodium hydroxide T. S. under reflux for 1 hour. Cool
and extract the aqueous solution with petroleum ether,
- 65 -
evaporate the petroleum ether in an evaporating dish. The

residue has a melting range of 43 to 58°.
C. To part of the aqueous solution obtained according to B,

add 5 ml of a 10 % solution of sodium citrate, 1 ml of cal-
cium chloride T. S. and 3 drops of bromothymol blue T. S.,
and slightly acidify with dilute hydrochloric acid T. S. Add
sodium hydroxide T. S. until the colour changes to a clear
blue, then boil the solution for 3 minutes, agitating it gently
during the heating period: a white crystalline precipitate
appears which is insoluble in sodium hydroxide T. S. but is
soluble in acetic acid T. S.
D. To part of the aqueous solution obtained according to B,

add 10 ml of a 10 % solution of sodium citrate, 1 ml of
mercuric sulfate T. S. Heat the mixture to boiling, and add
a few drops of potassium permanganate T. S. : a white
precipitate of the acetone dicarboxylic acid salt of mercury
is formed.
Purity Tests
Chloroform insoluble material: Not more than O. 5
Dissolve about 50. 0 g of sample in 400 ml chloroform.
Filter the solution through a sintered glass filter of porosity
3 previously weighed to the nearest 0. 001 g. Keep filter
warm and wash the residue in the filter with chloroform,
then dry at 1OOO.
Other acids and alcohols: Acids other than citric and alco-
hols other than those present in stearyl alcohol must not be
present.

specified by saponification value, total content and composi-
tion of stearyl alcohol, iodine value, acid value and citric
acid content.
- 66 -
SUCROSE ESTERS OF FATTY ACIDS
DEFINITION
Synonyms: Sucroesters, sucrose ester surfactants
Chemical Definition and Preparation: Sucrose fatty acid

esters are the mono-, di and triesters of sucrose with
edible fatty acids. They may be prepared as such from
sucrose and the methyl and ethyl esters of edible fatty acids
usually in the presence of a solvent. Another procedure is
to react edible fats or oils and sucrose to produce a mixture
of sucrose esters of fatty acids and mono- and diglycerides
called II sucroglycerides. 11 Both are usually produced in the
presence of a solvent.
DESCRIPTION
Sucrose fatty acid esters occur as stiff gels, soft solids

or white to slightly greyish white powders and are odourless.
CHARACTERISTICS
Identification:

Ethanol: soluble
B. Add 1 ml of alcohol to 0. 1 g of sucrose fatty acid ester,

dissolve by warming, add 5 ml of diluted sulfuric acid, heat
in a water bath for 30 min and cool. A yellowish white solid
or oil is form1:;J, which is soluble in 3 ml of ether.
C. Take 2 ml of the solution separated from the solid in

Test B and add 1 ml of anthrone T. S. carefully down the inside
of the test tube: the boundary surface of the two layers turns
to blue or green.
- 67 -
Specification
The article of commerce may be specified as to total fatty

acid, total sucrose, total glycerol, ash, chloride, soap and
moisture.
Dimethyl formamide: not more than 50 mg/kg

Propylene glycol: content to be determined as described
in Assay
EXAMINATION
Assay:
Sucrose Ester Content: Accurately weigh about 2 g of

sucrose fatty acid ester or sucroglyceride, previously dried
over sulfuric acid, in a vacuum desiccator for 4 hours, and
dissolve by warming in 100 ml of chloroform. Extract the
chloroform solution four times with 50 ml portions of aque-
ous 5 ~ sodium sulfate solution. The combined aqueous ex-
'
tract may be concentrated to 5 ml and, if propylene glycol
was used as the solvent, the content of free propylene may
be estimated by gas-liquid chromatography. Evaporate the
chloroform solution of the sucrose ester to incipient dryness,
dissolved with warming in 80 ml of water, add 10 ml of 10 N
sodium hydroxide solution. Allow to stand 24 hours at 35-
400, add 10 ml of concentrated hydrochloric acid and extract
three times with 50 ml, 30 ml and 30 ml of ether. Evaporate
the aqueous layer to about 50 ml. Allow to cool, add 2-3
drops of phenolphthaleinT.S., neutralize with sodium hydro-
xideT.S., add water to 100 ml and use this solution as test
solution. Take 20 ml of this solution, add 20 ml of Bertrand
A T.S. and 20 ml of Bertrand B T.S., boil gently for 3 min. and
let stand to allow cuprous oxide to precipitate. The upper
solution should remain deep-blue. Filter the upper solution
with a glass filter, wash the precipitate with hot water until
the washings are no longer alkaline and filter the washings
- 68 -
using a glass filter. The cuprous oxide precipitate must

not come in contact with air. Dissolve the precipitate in
the flask by adding 20 ml of Bertrand C T.S filter this
solution with the glass filter used before, wash with water,
combine the washing with filtrate, titrate with Bertrand
D T.S.and calculate quantity of copper from the result of the
titration; obtain the quantity of invert sugar and calculate
quantity of sucrose as follows:
% Sucrose = mg invert sugar from Bertrand Table (1) x
O. 95 x 500 weight of sample (mg).
The content of sucrose esters is calculated as sucrose
distearate as follows:
% Sucrose Esters % Sucrose
0.391
If propylene glycol was used as solvent for the preparation
of the sucrose ester, another 20 ml portion of the neutralized
aqueous layer may be concentrated to 5 ml, and the content
of combined propylene glycol may be estimated by gas-liquid
chromatography.
Dimethyl formamide: By hydrolysis to dimethylamine and

calorimetric estimation (M. Roussos, Parfumerie, Cos-
metiques et Savons, Vol. 4. No. 9, p. 362-363, September
1961).
- 69 -
HYDROXYLATED LECITHIN
(Tentative)
Synonyms
Hydroxylated phosphatides, dihydroxy phospholipids.
Chemical Names
Commercial hydroxylated lecithin is a mixture of phos-

phatides including:
CH 0COR
2
ICHOCOR
0
II II
CH 0POCH CH N+ (CH )
2 2 2 3 3
0
phosphatidy1 choline phosphatidyl ethanolamine

("lecithin fraction") (" cephalin fraction")
CH 0COR
2
ICHOCOR
0
II II
HO OH
CH - OP - 0 - OH
2
HO OH
OH
phosphatidic acid phosphatidyl inositol

where R = various saturated and unsaturated hydroxylated
fatty acid groups.
- 70 -
Definition
Commercial hydroxylated lecithin is a rr:ixture of phos-

phatides prepared from vegetable oils and seeds which is
treated with hydrogen peroxide, benzoyl peroxide, lactic
acid and sodium hydroxide or with hydrogen peroxide, acetic
acid and sodium hydroxide, under controlled conditions,
whereby the separated fatty acid fraction of the resultant
product has an hydroxyl value of 30 - 38. The commercial
product is specified by its acetone-insoluble fraction which
is usually in the range of 55 to 60 % •
Description
Comwercial hydroxylated lecithin is a very light amber

free -flowing liquid with a bland odour.
As an emulsifier.
Identification
A. Solubility: Water: Insoluble but hydrates characteris-

tically with swelling.
Acetone: Insoluble
Chloroform: Soluble
Benzene: Soluble
The "lecithin fraction" is soluble.
in ethanol, the 11 cephalin fraction"
insoluble.
B. Ignite 1 g of hydroxylated lecithin with 2 g of anhydrous

sodium carbonate. Cool and dissolve the residue in 5 ml of
water and 5 ml of nitric acid. Add 5 ml of ammonium
molybdate T. S. and heat to boiling. A yellow precipitate is
obtained.
C. Fuse about 0. 5 g of hydroxylated lecithin with about

0. 05 g of sodium in a soft glass tube, and heat to redness.
Plunge while hot into about 10 ml of distilled water, heat to
boiling and filter. Add a few crystals of ferrous sulphate to
- 71 -
the filtrate, boil and add dilute sulphuric acid until just acid.
Allow to stand for 15 minutes, filter and wash. A blue pre-
cipitate is obtained.
D. Reflux 1 g of hydroxylated lecithin for 1 hour with 25 ml

of 0. 5 N ethanolic potassium hydroxide. When cooled to o0 ,
a precipitate of potassium soap is obtained.
E. Determine the hydroxyl value *, it should be 30 - 38.

Purity Tests
Assay: Not less than 6
The hydroxylated lecithin content may be estimated by

determination of the quantity of acetone-insoluble material.
Weigh 2 OOO g of well-mixed sample into a centrifuge

tube which has been previously tared together with a stirring
rod. Add 15 rol of saturated acetone a/ from a burette.
Warm in a water bath until the lecithin melts, but avoid
evaporation of the acetone. Stir until the material is corn -
pletely disintegrated. Place in an ice-water bath and chill
for 5 minutes. Remove the tube from the bath and add
about one-half of the volume of chilled (0° - 5°) saturated
acetone required to make up to the final volume of 45 ml.
Stir well to complete dispersion of remaini~ particles.
Make to volume of 45 ml with chilled (0° - 5 ) saturated
acetone, stir, and return tube and contents to ice bath at
o0 - 5° for 15 minutes. Then stir again while in the bath,
remove rod and centrifuge immediately at 1 900 ±.100 r.p.m.
for 5 minutes. Decant the acetone -soluble material into a
clean beaker. Break up the centrifuged solids with the
previously tared stirring rod and refill the centrifuge tube
to the 40 ml mark with chilled (0° - 5°) saturated acetone;
stir well and repeat as directed above. Centrifuge, pour
off, return the stirring rod to the tube and break up the
* Ref. Seventh Report p. 182.

- 72 -
solids. Place the tube and contents in a horizontal position

on a laboratory bench until the excess of acetone evaporates.
Mix again and place in a forced-draft oven at 105° ± 2° until
constant weight is obtained, usually 30 to 45 minutes. Cool
to room temperature in an efficient desiccator and weigh
immediately.
Limits of Impurities
Ether insolubles: Not more than 1. 0 %. Weigh 10 g of well-

mixed sample into a 250 ml flask. Add 100 ml of ether and
shake until dissolved. Filter through a tared filter funnel.
Wash the flask with 25 ml portions of ether and pour the
washings through the funnel. Allow all ether to evaporate.
Place the funnel in a forced-draft oven and weigh for drying
for 1 hour at 105° ± 2°.

specified by the saponification value, acid value and loss on
drying.
a/ Saturated acetone: Add purified lecithin to acetone at

5°; about 5 g of lecithin are sufficient for 16 litres of
acetone. Maintain at 5° for two hours, shaking vigor-
ously at 15 minute intervals. Decant through a rapid
filter paper avoiding as far as possible transfer of any
of the solids to the paper and conducting the filtration
under refrigerated conditions (0° - 5°) so as to maintain
the same conditions for saturation as described under
procedure.
- 73 -
AMMONIUM SALTS OF PHOSPHATIDIC ACIDS
(Tentative)
DEFINITION
Synonyms: Emulsifier YN
Structural Formula: R P = 0 where R •mono-or digly-

3
ceride moiety or -OH or M-ONH
4
DESCRIPTION
The product consists essentially of a mixture of the

ammonium compounds of phosphatidic acids derived from
the edible fat (usually partially hardened rapeseed oil). One,
two or three glyceride moieties may be attached to phos-
phorus as indicated in the structural formula above. More-
over, two phosphorus esters may be linked together as
phosphatidyl phosphatides. The product is prepared by the
glycerolysis of the fat, phosphorylation by means of phos-
phorus pentoxide, and neutralization with ammonia.
CHARACTERISTICS
Identification
A. Solubility: Water: (
Ethanol: ( lnf ormation
Acetone: ( needed
Fats: (
B. Ignite 1 g of the product with 2 g of anhydrous sodium

carbonate. Cool and dissolve the residue in 5 ml of water
and 5 ml of nitric acid. Add 5 ml of ammonium molybdate
T. S. and heat to boiling. A yellow precipitate is obtained.
- 74 -
C. Reflux 1 g of the product for 1 hour with 25 ml of 0. 5 N

ethanolic potassium hydroxide. Ammonia is evolved from
the end of the reflux condenser, recognizable by its odour
and by its reaction on moist, red litmus paper. When cooled
to o0 c, a precipitate of potassium soap is obtained.
D. Test for glycerol as in Annex on Identification of Emulsi-

fiers.
Spee ification
Assay: Phosphorus content: 3. 0 to 3. 4 by weight

Ammonium content: 1. 2 to 1. 5 calculated as N.
The article of commerce can be specified further as to

water content, hexane insoluble matter, inorganic hexane
insoluble matter, pH and triglyceride content.
Arsenic: not more than 3 mg/kg.

Heavy metals: not more than 10 mg/kg.
EXAMINATION
Determination of phosphorus
Reagents The reagents used shall be of recognized ana -

lytical reagent quality and water refers to dis-
tilled water.
a. Vanadate - molybdate solution
Separately dissolve in water 20 g of ammonium

molybdate and 1 g of ammonium vandate. Mix
the two solutions, add 140 ml of concentrated
nitric acid and dilute to 1 OOO ml with water.
Mix well.
- 75 -
b. Standard phosphate solution
Stock solution: Dissolve 3. 8346 g of potassium

dihydrogen phosphate, previously dried at 110°C,
in water and dilute to 1 OOO ml. 1 ml of this
solution= 2.0 mg P o .
2 5
Working solution: Dilute 50 ml of the stock
solution to 500 ml with water. 1 ml of this
solution• 0. 20 mg P o .
2 5
c. Sulphuric acid, sp. gr. 1. 84
d. Nitric acid, sp. gr. 1. 42
e. Perchloric acid 60 % , sp. gr. 1. 54
Procedure
Weigh accurately 1. 5 to 1. 6 g of a representative sample

into a small glass capsule and transfer to a 300 ml Kjeldahl
flask containing 5 ml of concentrated sulphuric acid and 10 ml
of concentrated nitric acid. Heat the flask, gently at first,
with continual swirling, and later more strongly over a bare
flame. Add further measured amounts of nitric acid from
time to time, cooling the flask prior to addition, and continue
the heating until the stage where the digest is clear and
assumes a golden colour. Cool, add 5 ml of 60 % perchloric
acid and continue the oxidation until white acid fumes form in
the flask. Cool again and add 5 ml of water and continue
heating until white fumes are again driven off. Cool, dilute
carefully with water, cool again and transfer quantitatively
to a 500 ml volumetric flask. Dilute to volume with water
and mix well (-Test solution).
Carry out a blank digestion exactly as above but omit the

sample and use the same volume of acid as required to wet
oxidize the sample (-Blank digest solution).
- 76 -
Into separate 100 ml volumetric flasks, add by burette
(a) 25. 0 ml of dilute phosphorus standard ( = 5. 0 mg P o

2 5
)
(b) 30. 0 ml of the same solution ( • 6. 0 mg P 0 ), and

2 5
(c) a 25 ml aliquot of the test solution which will contain
the equivalent of between 5 and 6 mg P o .
2 5
Into each of the flasks containing the phosphorus standards,

i. e. (a) and (b) transfer an aliquot of the blank digest solu-
tion equal in volume to (c), in order to compensate for
possible traces of phosphorus derived from the acid digest
reagents and which may be present in the Test Solution.
To each add 25 ml of the vanadate-molybdate reagent, mix,

dilute to nearly 100 ml with water, mix well, adjust the
temperature of the solution to 20°c, dilute to the mark with
water and re-mix.
After 10 minutes measure the optical densities of both the

6 mg P o solution and the test solution against the 5 mg
2 5
standard contained in the blank cell. Use optically matched
1 cm cells and measure at a wavelength of 420 mu, or with
an Ilford 604 filter if using a photo-electric colorimeter.
Calculation
( OD test ) O. 873
5·; Phosphorus = ( 5 + OD 6 mg) x W
Where OD test = Optical density difference

between the 5 mg standard
and the test solution
OD 6 mg = Optical density difference

between the 6 mg and 5 mg
standards
W = Sample weight taken (g)

- 77 -
Determination of ammonium salt nitrogen in neutral YN
1. Apparatus for steam distillation
The apparatus consists of a 2 litre flask fitted with a

rubber bung through which pass an approximately 3' length
of glass tubing arranged so that the lower end is near the
bottom of the flask; and a shorter L shaped piece of tubing
arranged such that the tube projects about 1/4" below the
lower surface of the bung, to act as a steam outlet tube.
The flask should be approximately 2/3 filled with distilled
water made slightly acid with dilute sulphuric acid, and
contain a few pieces of sintered glass to prevent bumping
when the contents of the flask are vigorously boiled to act
as a steam generator. A tap funnel may be fitted to the flask
if desired to facilitate replenishing the water in the flask
between determinations.
The steam outlet tube is connected via a condensation

trap to the inlet of a steam distillation head, fitted to a short
necked 1 litre round bottomed 834 necked flask. The distil-
lation head should be such that the steam inlet tube reaches
almost to the bottom of the 1 litre flask and the outlet should
be fitted with two splash traps, one near the top of the 1 litre
flask and the other near the top of a 819 jointed vertical,
single -surface condenser to which the distillation head con-
nects. The vertical condenser should be fitted with an ex-
tended outlet tube, able to reach to the bottom of a 500 ml
conical flask.
2. Reagents
Reagents a, b, c should be prepared using chemicals of

analytical reagent grade.
(a) Boric acid, 2 % w. v. aqueous solution.
(b) 40 % w. v. aqueous solution of sodium hydroxide.
(c) 0. 02N, Hydrochloric acid.

- 78 -
{d) Mixed indicator. Mix 5. 0 ml of a 0. 1 % w. v. alco-

holic solution of bromocresol green and 2. 0 ml of
a 0.1 % w. v. alcoholic solution of methyl red and
dilute the mixture to 30 ml with 95 % alcohol.
{e) Silicone fluid 200/50 MS.
3. Procedure
Assemble and thoroughly steam out the apparatus.

Accurately weigh about 0. 2 gm. of a representative sample
of neutral YN into a small glass phial (approx. 3/ 4" diam. ,
1/2" deep). Add approximately 250 ml distilled water and
the phial and weighed contents to the distillation flask.
Connect the distillation head and splash traps to the distilla-
tion flask and vertical condenser, and arrange the condenser
such that the outlet dips below the surface of 10 ml of 2 %
boric acid and 1 ml mixed indicator contained in a 500 ml
conical flask. Add to the distillation flask, via a funnel
attached by means of a short piece of rubber tubing to the
steam inlet tube, 75 ml 40 % aqueous sodium hydroxide,
and wash in with distilled water *. Detach the funnel and
connect the steam inlet to the steam supply. Vigorously
steam distil the contents of the distillation flask and collect
200 ml distillate in the boric acid. During the distillation
gently agitate the distillation flask if necessary, to avoid
the sample being deposited around the upper surfaces of the
flask. When the required amount of distillate has been
collected, lower the receiving flask, stop the steam supply,
and wash down the inside of the condenser, and the outside
of the lower end, with a small quantity of distilled water,
collecting the washings in the receiving flask. Titrate the
contents of the receiving flask with 0. 02N hydrochloric acid.
* The sodium hydroxide may be added to the flask through

a tap funnel fitted to the distillation flask if preferred
and washed in with distilled water. If so a liquid seal
should be maintained in the funnel during the addition and
distillation.
- 79 -
Carry out at least one blank determination in exactly the

same way but omitting the sample.
During the distillation difficulty may be experienced

with frothing of the contents of the distillation flask. If so,
2 drops of silicone fluid should be added to the distillation
flask at the time of adding the sample; and a similar amount
included in the blank determination.
Calculation: 1 ml 0. 02N HCl = 0. 2802 mg of nitrogen
% Nitrogen = (sample titre - blank titre) x 28. 02

(sample wt. in mg)
- 80 -
HYDROXYPROPYL CELLULOSE
DEFINITION
Functional Use in· Foods_: Emulsifier, stabilizer, thickener

and suspending agent.
Chemical name: Eydroxypropy] ether of ceUulose;

cellulose hyc:l.roxypropyl ether.
Chemical formula: OH
I
0-CH-CH-CH
I 2 3
(OCH -CH-CH ) 2 _~
2 3
where x = less than 3. 0

y = not greater than 3. 0
z = not ~reater th:1r1 ( 4. 6 - y)
Structural formula:
OH
I
!
?--CH 0- ,- - C H - - - - C H 3
? - - C H , - - - C H -CH 3
'
CH 2
H ;-----0. ------·--·- 0 ----i-
i / H \
iI \
i\
i \ OH H I
/
o ...,...__ _.I , ~____ /
H
H 0---·CH_----CH - - - - CH
, I 3
I
I
I_ _ OH _ __Jn
- 81 -
Possible structural formula for repeating unit of a hydroxy-

propyl cellulose with molar substitution of 3. 0 and a degree
of polymerization of n.
Molecular weight:
Unsubstituted structural unit: 162. 14

Trisubstituted structural unit: 336. 37
Macromolecules: from about 30 OOO (n about 100)
up to 1 OOO OOO (n about 2 500)
DESCRIPTION
Chemical Description: liydroxypropyl cellulose is an ether

of cellulose containing hydroxypropyl substitution. It is
prepared from cellulose by treatment with alkali and propy-
lene oxide.
Appear 4 nce: Hydroxypropyl cellulose is a white or a slightly

yellowish, almost odourless and tasteless, granular or
fibrous powder.
CHARACTERISTICS
Identification
A. Solubility: Water: The product swells in water and

produces a clear to opalescent,
viscous colloidal solution.
Ethanol: Soluble
Ether: Insoluble
Spee ification
Assay: The dry product contains not more than 4. 6

hydroxypropyl groups per anhydroglucose unit. The article
of commerce can be specified further by viscosity and loss
on drying.
- 82 -

Sulphated ash content: not more than O. 2
EXAMINATION
Assay:
Hydroxypropyl Content
Principle: The sample is oxidized with chromic acid; the

acetic acid from the hydroxypropyl groups plus some chromic
acid are distilled over. The acids are titrated with base
and a correction for the chromic acid is obtained by iodo-
metric titration which is substracted from the total acid titre
to give the hydroxypropyl value.
c
nitrogen~ 50ml.
D
2mm.
18 /9 spherical }Oif}t
B
24/40T'
I 50ml.
Fig. 7-Apparatus for
Hydroxypropoxyl
Group Determination
- 83 -
Apparatus: (See Fig. 1)
The boiling flask A is fitted with a distilling head B.

The distilling head is fitted with a gas delivery tube, Q for
the introduction of nitrogen, a 50-ml graduated dropping
funnel C for the introduction of water, and a condenser E.
The boiling flask is immersed in an oil bath equipped with
a thermoregulator so that the oil can be maintained at a
temperature of 155° and the distillate is collected in a
50-ml graduate.
Procedure:
Transfer about 50 mg of hydroxypropyl methylcellulose

previously dried at 105° for 3 h and accurately weighed into
a flask and add 10 ml of chromium trioxide solution (430 g/1).
Immerse the flask in the oil bath to two thirds of its height,
assemble the apparatus and pass nitrogen gas through it at
the rate of ninety bubbles per minute. Raise the temperature
until the end of the determination. Distillation begins between
135° and 140° and as successive 5 ml volumes of distillate
collect in the 50-ml graduate, add 5 ml portions of water to
the flask from the 50-ml graduated dropping funnel. Con-
tinue this procedure until 50 ml of water has been added and
55 ml of distillate, which should be faintly yellow in colour,
has been collected. Detach the condenser from the stillhead
and wash both of its arms with distilled water, collecting the
washing in a 250-ml flask.
Transfer the contents of the 50-ml graduate to the flask

containing the washings. Add a few drops of phenolphthalein
and titrate the combined solution with 0. 02 N sodium hydro-
xide until the end point just begins to fade. Heat the solution
nearly to the boiling point to remove carbon dioxide, cool to
room temperature and continue to titrate until the pink colour
remains stable for 10 sec. Record the volume (x) of the
0. 02 N sodium hydroxide used, and then add 500 mg of
sodium hydrogen carbonate and 10 ml of dilute sulfuric acid
T. S. After evolution of carbon dioxide has ceased, add
1 g of potassium iodide, stopper the falsk, shake the mix-
ture and allow the solution to stand in the dark for 5 min.
- 84 -
Titrate the liberated iodine with 0. 02 N sodium thiosulfate,

adding a few drops of starch T. S. as the end point is
approached and record the volume (y) required. Correct
the volume (x) of 0. 02 N sodium hydroxide required for the
initial titration in order to obtain the equivalent of acetic
acid, by the formula x. - ~, in which IS: represents the ratio
(ml 0. 02 N sodium hydroxide from the blank)/ (ml 0. 02 N
sodium thiosulfate from the blank), obtained by performing
a blank determination with the same quantities of the same
reagents and in the same manner. Each ml of the corrected
volume of 0. 02 N sodium hydroxide is equivalent to 1. 5 mg
of hydroxypropoxyl groups (-OCH CHOHCH ).
2 3
- 85 -
PECTIN
CHEMICAL DESCRIPTION
Pectin consists of the partial methyl-esters of poly-

galacturonic acid and their salts.
DEFINITION
Function Use in Foods: Jelling, thickening and stabili-

zing agent. Pectin is a purified carbohydrate product
generally obtained from the dilute acid extract of the inner
portion of the rind of citrus fruits or from apple pomace.
DESCRIPTION
Pectin is a white, yellowish, light greyish or light

brownish powder.
The commercial product, pectin, is usually diluted with

15-35 % sugars and mixed with up to 40 % buffer salts, cal-
culated on the final product; buffer salts normally used are:
Calcium citrate
Calcium monophosphate
Potassium tartrate
Sodium citrate
Sodium hexametaphosphate
Sodium pyrophosphate
Buff er salts are required for pH utilization and for

obtaining different setting temperatures.
CHARACTERISTICS
Identification
A. Solubility: Water: soluble, forming a colloidal,

opalescent solution
Ethanol: insoluble
- 86 -
B. Heat 1 g of pectin with 9 ml of water on a steam bath

until a solution is formed, replacing water lost by evapora-
tion: it yields a gel upon cooling.
C. To a solution of pectin (1 in 100) add an equal volume of

alcohol: a translucent, gelatinous precipitate is formed.
D. To 10 ml of a solution of pectin (1 in 100) add 1 ml of

thorium nitrate T. S., stir, and allow to stand for 2 minutes:
a stable precipitate or gel forms.
E. To 5 ml of a solution of pectin (1 in 100) add 1 ml of a

solution of potassium hydroxide (1 in 50) and allow to stand
at room temperature for 15 minutes: a translucent gel or
semi-gel forms.
F. Acidify the gel from the preceding test with diluted

hydrochloric acid and shake well: a voluminous, colourless,
gelatinous precipitate forms, which upon boiling becomes
white and flocculent (pectic acid).
Specifications
Moisture: not more than 12
Ash: Low Methoxyl Pectins: not more than 12 %

Medium and High Methoxyl Pectins: not more than
6%
Acetyl groups: not more than 1%
Galacturonic acid: not less than 70 % ±. 5 % calculated on

the ash and moisture free basis.
Methoxyl content: Low Methoxyl Pectins: from Oto

7 % ± 2%
Medium and High Methoxyl Pectins:
not less than 7 % ±. 2 %
The article of commerce may be further specified as to
grade strength and pH.
- 87 -
Copper: not more than 60 mg/kg
Sulphur dioxide: not more than 50 mg/kg
Alcohol (methyl, ethyl or isopropyl): not more than 10 mg/kg
EXAMINATION
Determination of moisture content: drying at 105° for 2 h.
Determination of ash content: 3 h at 600°.
Determination of acetyl groups: The pectin is saponified

for 3 h in nitrogen current by 5 % H so at 100°, is dis-
2 4
tilled, and the distillate is titrated after elimination of co
2
by heating again with 0. 1 N NaOH.
Determination of alcohol contents: gas chromatography
Determination of methoxyl and galacturonic acid content:

Transfer exactly 5 g of pectin to a suitable beaker and stir
for 10 min with a mixture of 5 ml of hydrochloric acid and
100 ml of 60 % alcohol.
Transfer to a fritted glass filter tube (30 to 60 ml) and wash
with six 15 ml portions of the hydrochloric acid - 60 % al-
cohol mixture, fallowed by 60 % alcohol until the filtrate is
free of chlorides. Finally wash with 20 ml of alcohol and
dry for 1 h in an oven at 100°, cool and weigh.
Transfer exactly one -tenth of the total net weight of the
dried sample (representing 0. 5 g of the original unwashed
sample) to a 250 ml Erlenmeyer flask and moisten the
sample with 2 ml of alcohol.
Add 100 ml of recently boiled and cooled distilled water,
stopper and swirl occasionally until the pectin is completely
dissolved.
- 88 -
Add 5 drops of phenolphthalein T. S., titrate with 0. 5 N

sodium hydroxide and record the results as the initial titre.
Add exactly 20 ml of 0. 5 N sodium hydroxide, stopper,
shake vigorously and let stand for 15 minutes.
Add exactly 20 ml of 0. 5 N hydrochloric acid and shake until
the pink colour disappears.
After adding 5 drops of phenolphthalein T. S., titrate with
0. 5 N sodium hydroxide. to a faint pink colour which persists
after vigorous shaking: record this value as the saponifica-
tion titre.
Each ml of 0. 5 N sodium hydroxide used in the saponifica-
tion titre is equivalent to 0. 0155 g of OCH .
3
Assay for galacturonic acid: Each ml of 0. 5 N sodium
hydroxide used in the total titration (the initial titre added
to the saponification titre) is equivalent to 0. 09707 g of
C H 0 COOH.
5 9 5
- 89 -
PROPYLENE GLYCOL ALGINATE
Chemical Name
1, 2 propane-dial ester of alginic acid.
Definition
Propylene glycol alginate consists of esters of alginic

acid in which the car boxyl groups are partially esterified
with propylene glycol and partly neutralized with alkalis
approved in the Ninth Report.
Description
Propylene glycol alginate is a white to yellowish fibrous

or granular powder. It is almost odourless and tasteless.
As a stabilizer, thickener and emulsifier.
Identification
A. Solubility: Water: Soluble to give viscous colloidal

solution.
Ethanol: Soluble in up to 60 % aqueous
ethanol depending upon degree
of esterification.
B. To 10 ml of a 1 % solution of the sample add 1 ml of

sodium hydroxide T. S. Heat in a boiling water bath for
about 5 minutes, cool and add 1 ml of dilute sulphuric acid
T. S. A gelatinous precipitate is formed.
C. To 5 ml of a 1 % solution add 1 ml lead acetate T. S.

A gelatinous precipitate is formed.
D. Identify propylene glycol as indicated in Annex I to

"Specifications for the Identity and Purity of Food Additives:
Some Emulsifiers and stabilizers and certain other sub-
stances."
- 90 -
Purity Tests
Ash: Not more than 10 % •
Insoluble matter: Not more than 0. 2 %

specified by viscosity, acid value, loss on drying, free
esterified and neutralized carboxyl content and percentage
carbon dioxide produced on acid decarboxylation.
Note: Assay of propylene glycol in this product is required.

- Sl -
ANNEX
Identification Tests for Emulsifiers
The tests are specific for the component mentioned in

the heading.
1. Fatty acids
Reflux 1 g of sample with 15 ml of 0. 5 N ethanolic

potassium hydroxide for 1 h. Add 15 ml of water, acidify
with dilute hydrochloric acid TS (about 6 ml). Oily drops
or a white to yellowish-white solid is produced which is
soluble in 5 ml of hexane.
Remove the hexane layer, extract again with 5 ml of

hexane and remove again the hexane layer. The fatty acids
thus extracted may be identified by gas-liquid chromato-
graphy.
The aqueous layer is used for tests 2 to 9.
2. Acetic acid
Transfer about 5 ml of the aqueous layer resulting from

test 1 into a dish, add excess calcium carbonate and evaporate
until dry. Transfer the major part of the residue into a glass
tube. Place a filter paper, moistened with Reagent for
acetone, on top of the tube. Heat as indicated in the figure.
' ;
metal spiral
asbestos _..,.·===!11====
micro flame ~ ~
Figure 1
- 92 -
The yellow colour of the paper changes into greenish blue

by reaction with acetone, formed from calcium acetate.
Reagent for acetone: a saturated solution of c-nitro-

benzaldehyde in sodium hydroxide TS, freshly prepared.
3. Succinic acid
Transfer one drop of the aqueous layer resulting from

test 1 and a drop of a O. 5 % solution of ammonium chloride
and several mg of zinc powder into a micro test tube. The
mouth of the tube is covered with a disk of filter paper
moistened with a solution in benzene of 5 % p-dimethylamino-
benzaldehyde and 20 % trichloroacetic acid. The bottom of
the test tube is heated vigorously with a micro flame (Fig. 1)
for about 1 minute. Depending on the amount of succinic acid
or succinimide, a red-violet or pink stain appears on the
paper.
4. Fumaric acid
Transfer 1 ml of the aqueous layer resulting from test 1

with 1 ml of 2 N sodium carbonate into a test tube. Add 2 or
3 drops of O. 1 N potassium permanganate. The solution is
promptly discoloured.
- 93 -
5. Tartaric acid
Evaporate about 5 ml of the aqueous layer resulting

from test 1 in a porcelain dish until dry. Add 2 ml of
concentrated sulfuric acid containing 0. 5 % of pyrogallol
and heat on a steam bath. An intense violet colour is pro-
duced.
6. Citric acid
6. 1 To 3 ml of the aqueous layer resulting from test 1

add a few drops of 1 % potassium permanganate and warm
until the colour has disappeared. Then add an excess of
bromine TS. A white precipitate (pentabromoacetone) is
formed immediately or on cooling.
6. 2 Evaporate 1 ml of the aqueous layer resulting

from test 1 in a porcelain dish, add 1 ml of a mixture of
1 vol. acetic anhydride and 5 vol. of pyridine into the warm
dish. A red colour is produced. (Tartaric acid produces a
green colour).
Transfer 0. 2 ml of the aqueous layer resulting from

test 1 and 2 ml of cone. sulfuric acid into a test tube and
place for 2 min in boiling water. Cool and add 1 or 2 drops
of a 5 % guajacol solution in ethanol. A red colour is
immediately produced.
If tartaric acid is present according to test 5, it must

be removed as follows: transfer 3 ml of the aqueous layer
resulting from test 1 and an excess of calcium hydroxide as
a powder into a test tube, place in boiling water for 5 min,
shaking several times, cool and filter.
8. Calcium
Dissolve a grain (about 1 mg) of solid murexide in one

drop of 2 N sodium hydroxide and 2 drops of water on a
porcelain spot-plate. Add a drop of the solution resulting
from test 1. The colour changes from violet into red.
- 94 -
9. Glycerol
Transfer 5 ml of the aqueous layer resulting from test 1

into a test tube. Add excess calcium hydroxide as a powder,
place in boiling water for 5 min. shaking several times, cool
and filter.
Transfer one drop of the filtrate into a tube as indicated

in the figure in Test 1 and add about 50 mg of potassium
hydrogen sulfate. Place a filter paper, moistened with
Reagent for acrolein, on the top of the tube. Heat as indi-
cated in the figure. A blue colour of the filter paper indi-
cates the presence of glycerol. The colour changes into
light red after addition of sodium hydroxide TS.
The test cannot be employed in the presence of ethylene

glycol or lactic acid, since they decompose under the pre-
scribed conditions yielding acetaldehyde which reacts with
the reagents in the same manner as acrolein.
Reagent for acrolein: Prepare a 5 % solution of

disodium pentacyanonitrosylferrate in water and a 20 %
piperidine solution in water. Mix solutions 1 : 1 immediately
before use.
10. Polyols
Princig_le: The product is hydrolized. Fatty acids are

removed by ion-exchange in combination with hexane extrac-
tion. The filtrate is separated by thin layer chromatography.
Procedure: Reflux 1 g of sample with 15 ml of 0. 5 N

ethanolic potassium hydroxide for 1 h. Add 25 ml of
Amberlite Resin ffi-120 (H), 50 ml of hexane and 25 ml of
water. Stir the mixture for about 1 h. Filter and separate
the layers of the filtrate and use the aqueous layer.
Prepare a sililic acid G (Merck) plate and allow it to

dry at room temperature in the air. Place 2 to 5 1t 1 of the
aqueous layer on the plate and also 2µ 1 of 5 % solutions of
glycerol, ethylene glycol and 1. 2-propylene glycol.
- £5 -
Use a mixture of chloroform, acetone and 5 N ammonia

(10: 80: 10) as a solvent. After chromatographing dry in a
stream of air until water and ammonia have disappeared.
Spray with 0. 1 % aqueous sodium periodate solution. When
the plate is half dry spray with a solution of 2. 8 g benzidine
in 80 ml of 96 % ethanol, to which 70. ml of water, 30 ml of
acetone and 1. 5 ml of N hydrochloric acid are added.
1. 2-Diols show a yellow or white spot on blue back-

ground with following Rf-values:
According to Stahl
glycerol 0. 35
ethylene glycol 0. 70
1.2-propyleneglycol 0.85
Tartaric acid produces a white spot with Rf 0.

- 96 -
SOME ANTICAKING AGENTS

- 97 -
FERROCYANIDES OF
CALCIUM*, POTASSIUM AND SODIUM
DEFINITION
Functional Use in Foods: Anticaking agent in the manufac-

ture of salt
Synonyms: Yellow prussiate of lime, soda or

potash, hexacyanoferrate of cal-
cium, sodium or potassium
Formulae: Ca Fe(CN) . 12H 0;

2 6 2
K Fe(CN) . 3H 0;
4 6 2
Na Fe(CN) • tOH 0.
4 6 2
Molecular Weights: Calcium salt 508. 3
Potassium salt 422. 4
Sodium salt 484. 1
DESCRIPTION
Yellow crystals or crystalline powder.
CHARACTERISTICS
Identification
A. Solubility: Water: soluble (Na, K salt); insoluble

(Ca salt)
Ethanol: insoluble
B. Test for Ferrocyanide: To 10 ml of a 1 % solution of

the ferrocyanide add 1 ml of ferric chloride T. S. A dark
blue precipitate is formed. This test should be supplemented
by a negative test for cyanide.
* The specification for calcium f errocyanide is incomplete

due to lack of a test for calcium.
- 98 -
C. 1. Test for Calcium*: to be developed
C. 2. Test for Potassium: Potassium compounds impart a

violet colour to a non-luminous flame if not masked by the
presence of small quantities of sodium. In neutral, concen-
trated or moderately concentrated solutions of potassium
salts, sodium hydrogen tartrate T. S. slowly produces a
white, crystalline precipitate which is soluble in ammonia
T. S. and in solutions of alkali hydroxides or carbonates.
The precipitation may be accelerated by stirring or rubbing
the inside of the test tube with a glass rod or by the addition
of a small amount of glacial acetic acid or ethanol.
C. 3. Test for Sodium: Sodium compounds, after conver-

sion to chloride or nitrate, yield with cobalt-uranyl acetate
T. S. a golden-yellow precipitate, which forms after several
minutes agitation. Sodium compounds impart an intense
yellow colour to a non-luminous flame.
Spee ification
Assay: Not less than 99 percent of the respective ferro-

cyanide.
The article of commerce can be specified further by limits

on chloride, free moisture, insoluble matter and sulphate.
EXAMINATION
Assay:
Transfer about 3 grams, accurately weighed, into a

400-ml beaker, dissolve in 225 ml of water, and add
cautiously about 25 ml of sulphuric acid T. S. Add, with
stirring 1 drop of orthophenanthroline T. S. , and titrate with
0. 1 N eerie sulphate until the colour changes sharply from
orange to pure yellow. Each ml of 0. 1 N eerie sulphate is
equivalent to 101. 66 mg of Ca Fe(CN) . 12H 0; 84. 48 mg of
2 6 2
K Fe{CN) . 3H 0 or 96. 81 mg of Na Fe{CN) . 10H 0.
4 6 2 4 6 2
* The specification for calcium ferrocyanide is incomplete
due to lack of a test for calcium.
- 99 -
CALCIUM PHOSPHATE, TRIBASIC
DEFINITION
Functional Use in Foods: Anticaking Agent, Buffer
Synonyms: Tricalcium phosphate. Precipi-

tated Calcium Phosphate
'::>ESCRIPTION
Tribasic calcium phosphate consists of a variable mix-

ture of calcium phosphates having the approximate composi-
tion of lOCaO. 3P o5. H o. It occurs as a white, odourless,
2 2
tasteless powder which is stable in air.
CHARACTERISTICS
Identification
A. Solubility: Water: almost insoluble

Ethanol: insoluble
Dilute hydrochloric acid: soluble
Dilute nitric acid: soluble
B. To a warm solution of the sample in a slight excess of

nitric acid add ammonium molybdate T. S. A yellow pre-
cipitate forms.
C. Dissolve about 100 mg by warming with 5 ml of diluted

hydrochloric acid T. S. and 5 ml of water, add 1 ml of
ammonia T. S., dropwise, with shaking, and then add 5 ml
of ammonium oxalate T. S. A white precipitate forms.
Assay: not less than the equivalent of 90 percent of

Ca (PO ) calculated on the ignited basis.
3 42
The article' of commerce may be specified further as to
titration value and loss on ignition.
- 100 -

Fluoride (F): not more than 50 mg/kg
Heavy metals (as Pb): not more than 30 mg/kg
EXAMINATION
Arsenic: A solution of 1 gram in 25 ml of diluted hydrochloric

acid T. S. meets the requirements of the Arsenic Test.
Fluoride: Weigh accurately 1. 0 gram, and proceed as

directed in the Fluoride Limit Test.
Heavy Metals: Warm 1. 33 grams with 7 ml of diluted hydro-

chloric acid T. S. until no more dissolves, dilute to 50 ml
with water, and filter. A 25-ml portion of the filtrate meets
the requirements of the Heavy Metals Test, using 20 mcg of
lead ion (Pb) in the control (Solution A).
Lead: A solution of 250 mg in 5 ml of diluted hydrochloric

acid T. S. meets the requirements of the Lead Limit Test,
using 1. 25 mcg of lead ion (Pb) in the control.
Assay:
Weigh accurately about 200 mg, and dissolve it in a

mixture of 25 ml of water and 10 ml of diluted nitric acid
T. S. Filter, if necessary, wash any precipitate, add
sufficient ammonia T. S. to the filtrate to produce a slight
precipitate, then dissolve the precipitate by the addition of
1 ml of diluted nitric acid T. S. Adjust the temperature to
about 50°, add 75 ml of ammonium molybdate T. S. , and
maintain the temperature at about 50° for 30 minutes, stir-
ring occasionally. Wash the precipitate once or twice with
water by decantation, using from 30 to 40 ml each time.
Transfer the precipitate to a filter, and wash with potassium
nitrate solution (1 in 100) until the last washing is not acid
to litmus paper. Transfer the precipitate and filter to the
precipitation vessel, add 40. 0 ml of 1 N sodium hydroxide,
agitate until the precipitate is dissolved, add 3 drops of
phenolphthalein T. S. , and then titrate the excess alkali with
1 N sulphuric acid. Each ml of 1 N sodium hydroxide corres-
ponds to 6. 743 mg of Ca (P0 ) .
3 42
- 101 -
MAGNESIUM PHOSPHATE, TRIBASIC
DEFINITION
Functional Use in Foods: Anticaking Agent
Synonyms: Trimagnesium phosphate
Chemical Formula: Mg (PO ) (various hydrates)

3 42
DESCRIPTION
Tribasic magnesium phosphate may contain 4, 5, or 8

molecules of water of hydratioP.. It occurs as a white, odour-
less, tasteless crystalline powder.
CHARACTERISTICS
Identification
A. Solubility: Water: almost insoluble

Ethanol: insoluble
Dilute mineral acids: soluble
B. To a warm solution of the sample in a slight excess of

nitric acid add ammonium molybdate T. S. A yellow preci-
pitate of ammonium phosphomolybdate forms which is sol-
uble in ammonia T. S.
C. Dissolve about 100 mg in 0. 7 ml of diluted acetic acid

T. S. and 20 ml of water. Add 1 ml of ferric chloride T. S. ,
let stand for 5 minutes, and filter. The filtrate gives a
positive test for Magnesium.
Specifications
As§s:Y: not less than 98 percent and not more than the
equivalent of 101. 5 percent of Mg (PO ) after ignition.
3 4 2
- 102 -
The article of commerce may be specified further as to

titration value and loss on ignition.

Fluoride (F): not more than 10 mg/kg
EXAMINATION
Weigh accurately about 200 mg, previously ignited at

about 425° to constant weight, and dissolve it in a mixture
of 25 ml of water and 10 ml of diluted nitric acid T. S.
Filter, if necessary, wash any precipitate, then dissolve
the precipitate by the addition of 1 ml of diluted nitric acid
T. S. Adjust the temperature to about 50°, add 75 ml of
ammonium molybdate T. S. , and maintain the temperature
at about 50° for 30 minutes, stirring occasionally. Wash
the precipitate once or twice with water by decantation,
using from 30 to 40 ml each time. Transfer the precipitate
to a filter, and wash with potassium nitrate (1 in 100) until
the last washing is not acid to litmus paper. Transfer the
precipitate and filter to the precipitation vessel, add 40. 0 ml
of 1 N sodium hydroxide, agitate until the precipitate is
dissolved, add 3 drops of phenolphthalein T. S., and then
titrate the excess alkali with 1 N sulphuric acid. Each ml
of 1 N sodium hydroxide corresponds to 5. 715 mg of
Mg3(P04)2.
Arsenic: A solution of 1 gram in 10 ml of diluted hydro-

chloric acid T. S. meets the requirements of the Arsenic
Test.
Fluoride: Proceed as directed in the Fluoride Limit Test.

- 103 -
Heavy metals: Suspend 1. 33 grams in 20 ml of water, and

add hydrochloric acid, dropwise, until the sample just
dissolves. Adjust the pH to between 3 and 4, filter, and
dilute the filtrate to 40 ml with water. For the control
(Solution A), add 20 mcg of lead ion (Pb) to 10 ml of the
filtrate, and dilute to 40 ml. For the sample (Solution B),
dilute the remaining 30 ml of the filtrate to 40 ml. Add
10 ml of hydrogen sulphide T. S. to each solution, and allow
to stand for 5 minutes. Solution B is no darker than Solu-
tion A.
Lead: Dissolve 1 gram in 20 ml of diluted hydrochloric acid

T. S. , evaporate the solution to a volume of about 10 ml on
a steam bath, dilute to about 20 ml with water, and cool.
This solution meets the requirements of the Lead Limit
Test, using 5 mcg of lead ion (Pb) in the control.
- 104 -
SALTS of MYRISTIC, PALMITIC AND STEARIC ACID

with BASES ACCEPTED FOR FOOD USE
DEFINITION
~: The sodium, potassium, ammonium,

calcium, magnesium and aluminium
salts of commercial myristic, pal-
mitic and stearic acids produced
from edible fats and oils.
Functional Use in Food§: Anticaking agents.
DESCRIPTION
The products occur as hard, white or faintly yellowish

somewhat glossy and crystalline solids or as white or
yellowish white powder.
CHARACTERISTICS
Identification
A. Solubility: Sodium, Potassium and ammonium

salts:
Water: solu0h..
Ethanol: soluble
B. Heat 1 g with a mixture of 25 ml water and 5 ml hydro-

chloric acid. Fatty acids are liberated, floating as a solid
or oil layer on the surface which is soluble in hexane. After
cooling, the aqueous layer is decanted, evaporated to dry-
ness and the residue gives positive tests for the appropriate
cation.
Ammonium: Add sodium hydroxide T. S. ; the odour of

ammonia is observed.
Sodium: Add uranyl zinc acetate T. S.; a yellow crystalline

precipitate appears within a few minutes.
- 105 -
:potassium: Add 1 volume of saturated sodium hydrogen

tartrate solution and 1 volume of ethanol and shake. A
white crystalline precipitate is formed.
Calcium: Add ammonium oxalate T. S. The white precipi-

tate formed is soluble in hydrochloric acid, but insoluble in
acetic acid.
Magnesiurr.: Add ammonium chioride T. S. and ammonium

carbonate T. S. A white crystalline precipitate is formed
which is insoluble in ammonia T. S.
Aluminium: Add ammonia T. S. A white gelatinous preci-

pitate is formed which is insoluble in excess ammonia but
dissolves in sodium hydroxide T. S.
Spee ification:
The article of commerce can be specified by gas or thin

layer chromatography of the fatty acids obtained from the
salts, saponification value, freE, fatty acids, solidification
point for the QCids obt:i.ined frnn· the salts, iodine value,
residue on ignition ir,duding assay of the cation, unsaponifi-
able matter and moisture content.

Heavy metals (as Pb}: not more than 10 mg/kg
EXAMINATION
Arsenic: Prepare test solution as directed for organic

compounds.
Heavy metals: Prepare and test a 2 g sample as directed

under Method II in Heavy Metals Test, using 20 mcg lead
ion in the control (Solution A}.
- 106 -
SILICON DIOXIDE, AMORPHOUS
DEFINITION
Functional Use in Foods: Anticaking agent
Scope: The products included under this

specification are: Silica Aerogel
(precipitated silicon dioxide);
Hydrated Silica; "Silicic Acid";
Dehydrated silica gel.
Chemical name: Silicon dioxide
Empirical Formula:
DESCRIPTION
Silica aerogel is a microcellular silica occurring as a

fluffy powder or granules. Hydrated silica is a precipitated,
hydrated silicon dioxide occurring as a fine, white, amor-
phous powder, or as beads or granules.
CHARACTERISTICS
Identification

Ethanol: insoluble
HF: soluble (CAUTION *)
Alkali (80Q100°): soluble
B. Test for silica - Volatility of SiF4 - see Assay of silicon

dioxide.
Spee ification
Assay: On the ignited basis: Silica aerogel: not less than

90 % of Si0 . Hydrated silica: not less than 89 % of Si0 .
2 2
* Toxic, corrosive, must not contact skin.
- 107 -
The article of commerce may be further specified as to loss

on drying, loss on ignition and soluble ionizable salts.

EXAMINATION
Transfer about 2 grams, accurately weighed, into a

tared platinum crucible, ignite at 1000° for 1 hour, cool in
a dessicator, and weigh. Moisten the residue with 7 or 8
drops of ethanol, add 3 drops of concentrated sulphuric acid,
and, with CAUTION*, add enough hydrofluoric acid to cover
the wetted sample. Evaporate to dryness on a hot plate,
using medium heat (95-105°). then add 5 ml of hydrofluoric
acid, swirl the dish carefully to wash down the sides, and
again evaporate to dryness. Ignite the dried residue to a
red heat over a Meker burner, cool in a dessicator, and
weigh. The difference between the total weight loss and the
weight loss after ignition at 1000° represents the weight of
Si0 in the sample taken.
2
Sample Solution for the Determination of Arsenic, Heavy
Metals and Lead: Transfer 10. 0 grams of the sample into
a 250-ml beaker, add 50 ml of 0. 5 N hydrochloric acid,
cover with a watch glass, and heat slowly to boiling. Boil
gently for 15 minutes, cool, and let the undissolved material
settle. Decant the supernatant liquid through a Whatman
No. 3 filter paper, or equivalent. into a 100-ml volumetric
flask, retaining as much as possible of the insoluble material
in the beaker. Wash the slurry and beaker with three 10-ml
portions of hot water, decanting each washing through the
filter into the flask. Finally, wash the filter paper with
15 ml of hot water, cool the filtrate to room temperature,
dilute to volume with water, and mix.

- 108 -
Arsenic: A 10-ml portion of the Sample Solution meets the

requirements of the Arsenic Test.
Heavy Metals: Dilute 20. 0 ml of the Sample Solution to

30. 0 ml with water. A 10-ml portion of the dilution meets
the requirements of the Heavy Metals Test, using 20 mcg
of lead ion (Pb) in the control (Solution A).
Lead: A 10-ml portion of the Sample Solution meets the

requirements of the Lead Limit Test, using 10 mcg of lead
ion (Pb) in the control.
- 109 -
ALUMINIUM SILICATE
{KAOLIN)
DEFINITIO:N
runctional Use in Foods: Antieaking agent
Synonyms: Kaolin, light or heavy
DESCRIPTION
Kaolin is a native hydrated aluminium silicate, freed

from most of its impurities by elutriation and dried. It is
a soft, whitish powder free from gritty particles, odourless
and tasteless.
CHARACTERISTICS
Identification

Ethanol: insoluble
Mineral acids: insoluble
B. Mix 0. 2 g of Kaolin with 1. 5 g of a mixture of anhydrous

sodium carbonate and anhydrous potassium carbonate {1: 1),
and heat in a platinum or nickel crucible until the mixture
melts completely. Cool, add 5 ml of water, and allow to
stand for 3 minutes. Heat the bottom of the crucible gently
to detach the melt easily, and transfer the melt into a beaker
with water. Add gradually hydrochloric acid until no eff er -
vescence is observed, and add another 10 ml of the acid.
Evaporate the mixture to dryness on a water bath. Add
200 ml of water, boil and filter. Reserve the filtrate for
test C. Transfer the gelatinous residue into a platinum dish,
and add, with CAUTION* 5 ml of hydrofluoric acid. The
precipitate dissolves and on heating it volatilizes almost
completely.

- 110 -
C. Add ammonia TS to the filtrate from B above. A white

gelatinous precipitate is formed which is insoluble in excess
ammonia but dissolves in sodium hydroxide TS.
D. To 8 g of aluminium silicate (Kaolin) add 5 ml water and

mix well. The mixture is plastic.
Specification
Water soluble substances not more than 0. 3 %

Hydrochloric acid soluble substances: not more than 2 %
The commercial product may be further specified as to
chloride, foreign substances, particle size, loss on drying,
loss on ignition and pH.

Heavy Metals: not more than 10 mg/kg
Asbestos: absent - microscopic test needed
EXAMINATION
Water soluble substances: To 10 g of Kaolin, add 100 ml of

water, and boil for 30 minutes, supplementing water
occasionally. Cool, add water to 100 ml, and filter through
a glass filter. Evaporate a 50 ml portion of the filtrate to
dryness, and dry the residue at 105° for 1 hour. The weight
of the residue does not exceed 15 mg.
Hydrochloric acid soluble substances: Boil 2. 0 g with

100 ml of 0. 2N hydrochloric acid under a reflux condenser
for five minutes, cool, and filter; evaporate 50 ml of the
filtrate to dryness; the residue, after gentle ignition to
constant weight, weighs not more than 10 mg.
Arsenic: To 0. 5 g of Kaolin, add 5 ml of diluted hydrochloric

acid, and heat at 70° for 15 minutes, shaking well. Cool
immediately, and filter. Wash the residue with 5 ml of
diluted hydrochloric acid, and subsequently with 10 ml of
- 111 -
water. Combine the washings to the filtrate, and add water

to 20 ml. A 10 ml portion of the solution meets the require-
ments of the test for Arsenic, as directed in General Tests.
Heavy Metals: To 10 g of Kaolin, add 70 ml of water, 10 ml

of hydrochloric acid and 5 ml of nitric acid, and heat on a
water bath for 15 minutes with shaking. Cool, add water to
100 ml, and filter. Evaporate a 50 ml portion of the filtrate
to dryness on a water bath, Add 2 ml of diluted acetic acid
and 20 ml of water to dissolve and filter if necessary. Pro-
ceed as directed under Heavy Metals Test in General Te.sts.
- 112 -
CALCIUM SILICATE
DEFINITION
Synonyms and Varieties: Calcium silicates and poly-

silicates
DESCRIPTION
A synthetic hydrous calcium silicate or polysilicate

prepared by various reactions between siliceous material
(e. g. diatomaceous earth) and natural calcium compounds
(e. g. lime with varying proportions of other elements, such
as magnesium, etc.).
It is a very fine, white or off white powder with low

bulk density and high physical water absorption.
CHARACTERISTICS
Identification

Ethanol: insoluble
B. Mix 500 mg with about 200 mg of anhydrous sodium

carbonate and 2 g of anhydrous potassium carbonate, and
heat the mixture in a platinum crucible until fusion is corn -
plete. Cool, and transfer the fused mixture to a dish or
beaker with the aid of about 50 ml of hot water. Add hydro-
chloric acid to the liquid until effervescence ceases, then
add 10 ml more of the acid, and evaporate the mixture on a
steam bath to dryness. Cool, add 20 ml of water, boil, and
filter the mixture: an insoluble residue of silica remains.
Transfer the gelatinous residue into a platinum dish, and
add, with CAUTION*, 5 ml of hydrofluoric acid. The preci-
pitate dissolves and on heating it volatilizes almost com-
pletely.

- 113 -
C. Neutralize the filtrate from B above with ammonia T. S.

using 2 drops methyl red T. S. as indicator, then add
diluted hydrochloric acid T. S. dropwise until the solution
is acid. Upon the addition of ammonium oxalate T. S. a
white granular precipitate of calcium oxalate forms. This
precipitate is insoluble in acetic acid but dissolves in
hydrochloric acid.
3pecification
The commercial product may be specified as to

calcium and silicon dioxide contents, loss on drying, loss
on ignition, pH of 10% water slurry, bulk density, moistur~
sulphate and chloride.

Fluorine: not more than 50 mg/kg
Asbestos: absent. microscopic test needed.
- 114 -
MAGNESIUM SILICATE
(Talc and Magnesium trisilicate)
DEFINITIU:N
Synonyms and Varieties: Talc; Magnesium trisilicate
I)ESCRIPTIUN
Sources: Talc is a native, hydrous magnesium silicate,

sometimes containing a small portion of aluminium silicate.
Magnesium trisilicate also coming under this specification
may be a synthetic product.
f!.ppearance: Very fine, white or greyish white, odourless

crystalline powder. Is unctuous, adheres readily to the
skin, and is free from grittiness.
CHARACTERISTICS
Identification:

Ethanol: insoluble
B. Mix 500 mg with about 200 mg of anhydrous sodium

carbonate and 2 g of anhydrous potassium carbonate, and
heat the mixture in a platinum crucible until fusion is com-
plete. Cool, and transfer the fused mixture to a dish or
beaker with the aid of about 50 ml of hot water. Add hydro-
chloric acid to the liquid until effervescence ceases, then
add 10 ml more of the acid, and evaporate the mixture on
a steam bath to dryness. Cool, add 20 ml of water, boil,
and filter the mixture; an insoluble residue of silica remains.
Transfer the gelatinous residue into a platinum dish, and
add, with CAUTION*, 5 ml of hydrofluoric acid. The pre-
cipitate dissolves and on heating it volatilizes almost com-
pletely.
- 115 -
C. In the filtrate from B above. dissolve about 2 g of

ammonium chloride, and add 5 ml of ammonia T.S Filter
if ,eccss::-..ry, and add sodium phosphate T.S. tothe filtrate:
a white, crystalline precipitate of magnesium ammonium
phosphate separates.
Spee ification
pH: Neutral to litmus

Water soluble substances: not more than 0. 2
Hydrochloric acid-soluble substances: not more than 2
The article of commerce can be specified further by mag-
nesium oxide and silicon dioxide contents, loss on ignition,
chloride, sulphate and iron.
LIMITS OF lMPlJHlTlli~

Fluoride: not more than 20 mg/kg
Asbestos: absent, microscopic test needed
EXAMINA TICN
Hydrochloric Acid-soluble substances: Digest 1. 00 g with

20 ml of diluted hydrochloric acid at 50° for 15 minutes,
add water to restore the original volume, mix, and filter.
To 10 ml of the filtrate add 1 ml of diluted sulfuric acid,
evaporate to dryness, and ignite to constant weight.
pH and water-soluble substances: Boil 10 g with 50 ml of

water for 30 minutes, adding water from time to time to
maintain approximately the original volume, and filter.
The filtrate is neutral to litmus paper. Evaporate one-half
of the filtrate to dryness, and dry at 105° for 1 hour.
- 115 -
Arsenic: Mix 0. 5 g of Talc in 5 ml of diluted hydrochloric

acid, and boil while stirring. Cool the mixture, and filter.
Wash the residue with 5 ml of diluted hydrochloric acid,
and subsequently with 10 ml of water. A mixture of the
filtrate and the washings meets the requirements of the
test for Arsenic.
Heavy metals: Mix 2 g of Talc with 8 ml of diluted

hydrochloric acid and 10 ml of water, shake well, and
boil gently. Cool, add water to produce a 50 ml solution,
and filter.· To 25 ml of the filtrate, add ammonia T. S. to
adjust the pH to 5. 2, and add 2 ml of diluted acetic acid:
it meets the heavy metals limit, as specified above using
the General Test procedure.
- 117 -
SODIUM ALUMINO SILICATE
DEFINITION
Functional Use in Foods: An~icaking agent
Synonyms: Socfr1m silicoaluminate
DESCRIPTION
Sodium silicoaluminate is the name for a series of

hydrated sodium aluminum silicates. It occurs as a fine,
white, amorphous powder, or a:: beads. It is odourless
and tasteless.
CHARACTERISTICS
Identification

Ethanol: insoluble
Strong acids: partially soluble
Alkali Hydroxides: partially soluble
Band C. Mix 0. 2 g of material with 1. 5 g of a mixture of

anhydrous sodium carbonate and anhydrous potassium
carbonate (1: 1), and heat in a platinum or nickel crucible
until the mixture melts completely. Cool, add 5 ml of
water, and allow to stand for 3 minutes. Heat the bottom
of the crucible gently to detach the melt easily, and transfer
the melt into a beaker with water. Add gradually hydro-
chloric acid until no effervescence is observed, and add
another 10 ml of the acid. Evaporate the mixture to dryness
on a water bath. Add 200 ml oi water, boil and filter.
Reserve the filtrate for test C. Transfer the gelatinous
residue into a platinum dish, and add, with CAUTION*,
5 ml of hydrofluoric acid. The precipitate dissolves and on
heating it volatilizes almost completely.

- 118 -
C. Add ammonia TS to a part of the filtrate from B above.

A white gelatinous precipitate is formed which is insoluble
in excess ammonia but dissolves in sodium hydroxide TS.
D. Add uranyl zinc acetate TS to a part of the filtrate from

B above; a yellow crystalline precipitate appears within a
few minutes.
Specifications
The commercial product may be specified as to silicon

dioxide, aluminum oxide, and sodium oxide content, loss on
drying, loss on ignition and pH of the slurry with water.

EXAMINATION
Sample Solution for the Determination of Arsenic and Heavy

Metals: Transfer 10. 0 grams of the sample into a 250-ml
beaker, add 50 ml of 0. 5 N hydrochloric acid, cover with a
watch glass, and heat slowly to boiling. Boil gently for 15
minutes, cool, and let the undissolved material settle.
Decant the supernatant liquid through Whatman No. 4, or
equivalent, filter paper into a 100-ml volumetric flask,
retaining as much as posi::;ible of the insoluble material in
the beaker. Wash the slurry and beaker with three 10-ml
portions of hot water, decanting each wa~hing through the
filter into the flask. Finally, wash the filter paper with
15 ml of hot water, cool the filtrate to room temperature,
dilute to volume with water, and mix.
Arsenic: A 10-ml portion of the Sample Solution meets the

requirements of the Arsenic Test.
:t{eavy metals: A 20-ml portion of the Sample Solution meets

the requirements of the Heavy Metals Test, using 20 µg
of lead ion (Pb) in the control (Solution A).
- 119 -
CERTAIN OTHER FOOD ADDITIVES

- 120 -
MONOSODIUM L-GLUTAMATE
(Tentative)
DEFINITION
Functional Use in Foods: Flavour enhancer
Synonym CL: Monosodium Glutamate;

Sodium Glutamate; MSG
Empirical Formula: C5HgNNa04. H20
Structural Formula: HOOC - CH - CH2CH2 COONa. H20

I
NH2
Molecular Weight: 187. 1
DESCRIPTION
White, practically odourless, free-flowing crystals

or crystalline powder, with either a slightly sweet or a
slightly salty taste.
CHARACTERISTICS
Identification

Ethanol: insoluble
B. To 1 ml of a 1 in 30 solution add 1 ml of ninhydrin T. S.

and 100 mg of sodium acetate, and heat in a boiling water
bath for 10 minutes. An intense violet-blue colour is
formed.
C. To 10 ml of a 1 in 10 solution add 5. 6 ml of 1 N hydro-

chloric acid. A white crystalline precipitate of glutamic
acid forms on standing. When 6 ml of 1 N hydrochloric
acid is added to the turbid solution, the glutamic acid
dissolves on stirring.
- 121 -
D. To 1 ml of a 1 in 100 solution add uranyl zinc acetate

T. S; a yellow crystalline precipitate appears within a few
minutes.
Specifications
Assay: Not less than 99 percent of C5HaNNa04. H20.

0
Specific rotation: [ a J ;5 + 24. 2° to + 25. 5°
Loss on drying: not more than 0. 2 % on drying at

98 ± 1°c for 5 hours.
The article of commerce can be further specified

by nitrogen content, pH and chloride content.

Pb: not more than 10 mg/kg
EXAMINATION
Assay:
Dissolve about 250 mg, accurately weighed, in

100 ml of glacial acetic acid. A few drops of water may be
added prior to the addition of the acetic acid to effect
faster dissolution of the sample. Titrate with 0. 1 N
perchloric acid in glacial acetic acid, determining the
end-point potentiometrically. Each ml of 0.1 N perchloric
acid is equivalent to 9. 356 mg of C5HaNNa04. H2 0.
S:gecif ic Rotation
0
5
[ a ] ~ Determine in a 4 dm tube on a solution
containing 400 mg in each 10 ml of 2. 3 N hydrochloric acid.
122 -
CALCIUM SULPHATE
DEFINITION
Functional Use in Foods: Firming Agent
Chemical Formula: CaS0 4. 2H 20 and Caso 4

(anhydrous)
Molecular Weight: 172. 2; 136. 1 (anhydrous)
DESCRIPTION
Calcium sulphate is a fine, white to slightly yelle>w-

white odour less powder.
CHARACTERISTICS
Identification
A. Solubility: Water: slightly soluble

Ethanol: insoluble
B. Warm 0. 2 g calcium sulphate with 4 ml dilute

hydrochloric acid T. S. and 16 ml water. (Solution A).
To 10 ml of Solution A add 5 ml ammonium oxalate T. S.
A white precipitate forms.
C. To the remaining 10 ml of Solution A add barium

chloride T. S. A white precipitate forms which is insoluble
in hydrochloric and nitric acids.
6l)ecif ication
Assay: not less than 99% CaS04 after drying at 250° to

constant weight.
- 123 -

Fluoride: not more than 30 mg/kg
Selenium: not more than 30 mg/kg
EXAMINATION
Arsenic: Mix 1 g with 10 ml of water, add 12 ml of diluted

hydrochloric acid T. S., and heat to boiling to dissolve the
sample. Cool, filter, and dilute the filtrate to 35 ml with
water. This solution meets the requirements of the Heavy
Metals Test, using 20 mcg of lead ion (Pb) in the control
(Solution A).
Selenium: A solution of 2 grams in 40 ml of dilute

hydrochloric acid ( 1 in 2) meets the requirements of the
Selenium Limit Test.
Assay: Dissolve about 350 mg of the dried sample accurately

weighed, in 100 ml of water and 4 ml of diluted hydrochloric
acid T. S. While stirring, preferably with a magnetic
stirrer, add about 30 ml of 0. 05 N disodium ethylene-
diaminetetraacetate from a 50-ml buret, then add 15 ml of
sodium hydroxide T. S. and 300 mg of hydroxy naphthol
blue indicator, and continue the titration to a blue end-
point. Each ml of 0. 05 N disodium ethylenediaminetetra-
acetate is equivalent to 6. 807 mg of CaS04.
- 124 -
POTASSIUM CHLORATE
(Identification only)
Chemical Formula: KCl03
DESCRIPTION
Colourless crystals, white granules or powder.
CHARACTERISTICS
Caution
In admixture with organic or readily oxidizable

substances, it is liable to explode if heated or subjected to
percussion or trituration.
Identification

Ethanol: insoluble
B. To a 1 % aqueous solution of potassium chlorate add

silver nitrate T. S. No precipitate is formed. Reduce
chlorate in this solution to chloride with urea. Cool and
add 10 ml of water and silver nitrate T. S. A flocculent
white precipitate is formed.
C. To an aqueous solution of potassium chlorate add

1 volume of saturated sodium hydrogen tartrate solution
and 1 volume of ethanol and shake. A white crystalline
precipitate is formed.
- 125 -
ASCORBYL STEARATE
DEFINITION
Sy:nonyms: Vitamin C stearate
Structural Formula:
O=C -----.1
HO-C O
HO-C I
H-C--~-
HO-C-5
CH200CR
Where R is the aliphatic chain of stearic acid.

Ascorbyl stearate contains not less than 93 % of the product
and conforms to the following specifications.
DESCRIPTION
Ascorbyl stearate is a white or yellowish-white solid

with a citrus-like odour.
CHARACTERISTICS
Identification

Ethanol: soluble
. 0
B. Melting range 115-118
C. A solution of ascorbyl stearate in ethanol will decolourize

a solution of 2. 6-dichlorophenol-indophenol T. S.
- 126 -
PURITY TESTS
Sulphated ash
Not more than G. 1 % •
Heavy metals : Add 1 ml of hydrochloric acid and 0. 2 ml of nitric

acid to the residue obtained at the test for residue on
ignition, and evaporate to dryness on a water bath. Dissolve
the residue in 1 ml of diluted hydrochloric acid and 15 ml
of water by heating. Cool, add 1 drop of phenolphthalein T. S.,
add ammonia T. S. dropwise until a slightly pink colour
appears, and add 2 ml of diluted acetic acid. Proceed as
directed under Heavy Metals Test of Section 10 in General
Tests using the solution. The heavy metals limit for
L-Ascoryl Stearate is not more than O. 001 percent .
. ~ : P l a c e 2 g of L-Ascorbyl Stearate in a digestion

flask, aad 20 ml of nitric acid, and liquefy by heating
gently. Cool, add 5 ml of sulfric acid, and heat until
white fume evolves. If the solution is still brown, add
5 ml of nitric acid after cooling, and heat. Repeat this
process, until the colour of the solution turns into
colourless or pale yellow. Cool, add 15 ml of saturated
ammoniumoxalate solution, and heat until white fumes
evolve. Cool, and add water to 25 ml. A 5 ml portion of
the solution meets the requirements of the test for Arsenic,
as directed under Arsenic Test of Section 19 in General
Tests. For the control, place 2 ml of arsenic standard
solution in the digestion flask, add 20 ml of nitric acid,
and proceed in the same manner as the sample.
Loss on drying: not more than 2 % after drying

over ,sulphuric acid in a vacuum
dessicator for 4 hours.
- 127 -

Assay
Titrimetric method: Add 0. 800 g of ascorbyl stearate to

a mixture of 50 ml of carbon dioxide-free water, 50 ml of
chloroform and 25 ml of dilute sulphuric acid T. S. Titrate
the mixture at once with 0. 1 N iodine making sure that the
mixture is well shaken. Add a few drops of starch T. S.
as indicator, as the end point is reached. Each ml 0. 1 N
iodine is equivalent to 0. 0214 g of ascorbyl stearate.
- 128 -
DIMETHYPOL YSILOXANE
DEFINITION
Functional Use in Foods: Defoa.ming agent
Synonyms: Polydimethylsilo:xane,
Silicon~, Silicone fluid, oil
Structural Formula:
CH 3 CH 3 CH 3
CH 3
I . I I
Si--0-- Si---0 Si --CH 3
I I I
CH 3 CH 3 CH 3
n
The average value for n is 200 to 300.
DESCRIPTION
Dimethylpolysilo:xane frequently is used in commerce

as such, as a liquid containing silica gel and as an aqueous
emulsion formulation containing in addition to silica gel,
emulsifying agents and preservatives. The pure substance
treated here can be isolated by centrifuging from the silica
gel containing liquid. Dimethylpolysilo:xane is a clear,
colour less, viscous liquid.
CHARACTERISTICS
Identification
Ethanol: insoluble
Carbon tetrachloride: soluble
- 129 -
B. Ash O. 1 to O. 2 gram of sample in a platinum crucible

with H2S04 and HN03. After ashing is complete, transfer
a portion of the silica residue to a nickel crucible, add
about 1 gram of NaOH pellets and heat the mixture until
fusion is completed. Cool and dissolve in about 50 ml water
and filter out any residue. Place a drop of the filtrate on
a sheet of filter paper, followed by a drop of ammonium
molybdate T. S. * and a drop of benzidine T. S. ** Let the
test paper come into contact with ammonia fumes. If .
silicon is present, a blue spot will be developed.
* Ammonium Moly!xlate T. 3. 5 g ammonium molybdate

dissolved in 100 ml H20,
and this solution added to
35 ml of 25 % HNQ3.
** Benzidine T. 3. 0. 0!5 g benzidine hydro-

chloride is dissolved in
100 cc of 10 % acetic acid.
C. Infrared 3pectrum: Characteristic for dimethylpolysiloxane,

and shall not indicate presence of phenyl compounds.
$.pecif ication
Specific gravity 2!1°/23°: 0. 972 ± 0. 004

Refractive index at 25 : 1. 400 to 1. 404
Viscosity: 300 to 600 centistokes at 25°
Loss on heating for 4 hat 200°: not more than 18%
Dimethylpolysiloxane in the article of commerce used

as an antifoaming agent can be specified further as to total
silicon.
LIMIT3 OF IMPURITIES
Arsenic: 3 mg/kg
Lead: 10 mg/kg
Heavy metals: 40 mg/kg
- 130 -
OXYSTEARIN
DEFINITION
Functional Use in Foods:
Crystallization inhibitor in vegetable oils.
DESCRIPTION
"Oxystearin" is a mixture of glycerides of partially

oxidized stearic and other fatty acids. The product is
obtained by heating hydrogenated cottonseed or soybean oil
under controlled conditions in the presence of air and a
suitable catalyst (not present in the final product). It occurs
as a tan to light brown fatty or wax-like substance having
a bland taste. (Information needed from manufacturer on
nature of the oxidation products, ketonic and polymeric
acids}.
CHARACTERISTICS
Identification

Ethanol: soluble
B. Identify fatty acids as indicated in Annex to FA3/IV.
C. Oxystearin can be acetylated with acetic anhydride and

pyridine (see hydroxyl number under Assay, below).
Specification
Assay: Hydroxyl number between 30 and 45.
The article of commerce can be specified as to acid

number, iodine number, saponification number, unsaponifiable
material, refractive index.
- 131 -

Epoxides: not more than 50 mg/kg
EXAMINATION
Hydroxyl Numher: see 6th Report of the Joint

FAO/WHO Expert Committee
on Food Additives, p. 182
Arsenic and heavy metals: as for fatty acids.
Epoxides: Ref. Fiorite, et al, J. A. 0. C. S.
43. 37 (1966); 43, 487 (1966).
- 132 -
ISOAMYL GALLA TE
(Tentative)
DEFINITION
Functional Use in Foods: Antioxidant
Chemical Name: iso-Amyl ester of 3, 4, 5 trihydroxy

benzoic acid
Empirical Formula:
Structural Formula:
HO--.
OH
DESCRIPTION
Isoamyl gallate is a white to pale brownish yellow

crystalline solid, odourless, with a slightly bitter taste.
CHARACTERISTICS
Identification

Ethanol: soluble
- 133 -
B. Melting range: 140-145°
C. Dissolve 0. 5 g of isoamyl gallate in 10 ml of sodium

hydroxide T. S. , distil. and take 4 rr.l of the distilled
portion. This solution is separated in two layers, emitting
a strong isoamyl alcohol odour.
D. Dissolve 0. 1 g of isoamyl ~allate in 5 ml of ethanol,

and add 1 drop of diluted ferric chloride T. S. ; a violet
colour is produced.
E. Add 1 ml of ammonium hydroxide to 5 ml of a 1 %

ethanolic solution of isoamyl galiate. A pink to red colour
appears.
3pecif ication
Assay: Method needed
The commercial product may be further specified as

to limits for chloride. sulphate. colour of solution, loss on
drying, residue on ignition.

Lead: not more than 10 mg/kg.
- 134 -
ETHYL PROTOCATECHUATE
(Identification only)
DEFINITION
Chemical Name: Ethyl ester of 3, 4-dihydroxy-benzoic

acid
Empirical Formula: C9H1004
Structural Formula:
HO
HO--
DESCRIPTION
Ethyl protocatechuate is a white or pale brownish

yellow, crystalline powder. It is odourless or has a faint
phenol-like odour with a slight bitter taste.
CHARACTERISTICS
Identification

Ethanol: soluble
B. Melting range: 132-135°
C. Dissolve 0. 5 g of ethyl protocatechuate in 10 ml of

sodium hydroxide T. S. , distil, and take 4 ml of the initial
distillate. This is clear. To this initial distillate add an
- 135 -
equal volume of sodium hydroxide T. S. and 2 or 3 drops

of iodine T. S., and heat the solution: iodoform odour
evolves.
D. Dissolve 0. 1 g of ethyl protocatechuate in 5 ml of

ethanol, and add 1 drop of diluted ferric chloride T. s~
a green colour develops.
E. Acidify the residue from the distillation in C, to

precipitate an acid which on recrystallization has a melting
range of 190-194° with decomposi:::ion.
- 136 -
ANNEX
General References
REPORTS AND OTHER DOCUMENTS RESULTING FROM

PREVIOUS MEETINGS OF THE JOINT FAO/WHO EXPERT
COMMITTEE ON FOOD ADDITIVES
1. General Principles Governing the Use of Food Additives:

First Report, FAO Nutrition Meetings Report Series,
1956, No 11: Wld Hlth Org. techn. Rep. Ser., 1956,
.w..
2. Procedures for the Testing of International Food
Additives to Establish their Safety for Use; Second
Report, FAO Nutrition Meetings Report Series, 1958,
No. 17; Wld Hlth Org. techn. Rep. Ser., 1958, 144.
3. Specifications for Identity and Purity of Food Additives

(Antimicrobial Preservatives and Antioxidants): Third
Report. These specifications were subsequently
revised and published as Specifications for Identity and
Purity of Food Additives, vol. I. Antimicrobial
Preservatives and Antioxidants, Rome, Food and
Agriculture Organization of the United Nations, 1962.
4. Specifications for Identity and Purity of Food Additives

(Food Colours): Fourth Report. These specifications
were subsequently revised and published as
Specifications for Identity and Purity of Food Additives,
vol. II. Food Colours, Rome, Food and Agriculture
Organization of the United Nations, 1963.
5. Evaluation of the Carcinogenic Hazards of Food

Additives: Fifth Report, FAQ Nutrition Meetings
Report Series, 1961, No. 29; Wld Hlth Org. techn. Rep.
Ser., 1961, .2.2.Q.
6. Evaluation of the Toxicity of a Number of Antimicrobials

and Antioxidants: Sixth Report, F AO Nutrition Meetings
Report Series, 1962, No. 31; Wld Hlth Org. techn. Rep.
Ser. , 1962, .2..2.a.
- 137 -
7. Specifications for the Identity and Purity of Food

Additives and their Toxicological Evaluation:
Emulsifiers, Stabilizers, Bleaching and Maturing
Agents: Seventh Report, FAQ Nutrition Meetings
Report Series, 1964, No. 35; Wld Hlth Org. techn.
Rep. Ser., 1964~ .2.81..

Additives and their Toxicological Evaluation: Food
Colours and Some Antimicrobials and Antioxidants:
Eighth Report, FAQ Nutrition Meetings Report Series,
1965, No. 38; Wld Hlth Org. techn. Rep. Ser., 1965,
309.
Specifications for Identity and Purity and Toxicological

Evaluation of some Antimicrobials and Antioxidants,
FAQ Nutrition Meetings Report Series, 1965, No. 38A;
WHO/Food Add/24. 65.
10;1' Specifications for Identity and Purity and Toxicological

Evaluation of some Food Colours. FAQ Nutrition
Meetings Report Series, 1966, No. 388; WHO/Food
Add/66. 25.
11. Specifications for the ldenti':y and Purity of Food Additives

and their Toxicological Evah.ation: Some Antimicrobials,
Antioxidants, Emulsifiers, Stabilizers, Flour-treatment
Agents, Acids and Bases: Ninth Report, FAQ Nutrition
Meetings Report Series, 1966, No. 40: Wld Hlth Org.
techn. Rep. Ser., 1966, .3.3.9_.
* These documents can be obtained on request from:

Food Additives, World Health Organization,
Avenue Appia, 1211 Geneva. Switzerland or: Food
Science Branch, Food and Agriculture Organization of
the United Nations, 00100 Rome, Italy.
- 138 -
12;1' Toxicological Evaluation of Some Antimicrobials,

Antioxidents, Emulsifiers, Stabilizers, Flour-Treatment
Agents, Acids and Bases, F AO Nutrition Meetings
Report Series, 40 A, B, C; WHO/Food Add/67. 29.

Additives and their Toxicological Evaluation: Some
Emulsifiers and Stabilizers and Certain Other
Substances: Tenth Report, F AO Nutrition Meetings
Rep. Ser. , 1967, m.

Flavouring Substances and Non-Nutritive Sweetening
Agents: Eleventh Report, FAO Nutrition Meetings
Rep. Ser., 1968, .a.a.a..
15!" Toxicological Evaluation of Some Flavouring Substances

and Non-Nutritive Sweetening Agents, FAO Nutrition
Meetings Report Series, 1968, No. 44A; WHO/Food
Add. /68. 33.
16!" Specifications and Criteria for Identity and Purity of

Some Flavouring Substances and Non-Nutritive
Sweetening Agents. F AO Nutrition Meetings Report
Series No. 44B; WHO/Food Add. /69. 31.

Antibiotics: Twelfth Report, FAO Nutrition Meetings
Report series 1968 No. 45; Wld Hlth Org. techn. Rep.
Ser., 1969, .ia.Q.
* These documents can be obtained on request from:

Food Additives, World Health Organization,
Avenue Appia, 1211 Geneva, Switzerland, or: Food
Science Branch, Food and Agriculture Organization of
the United Nations, 00100 Rome, Italy.

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Ji'" r ~ •

SPECIFICATIONS FOR THE IDENTITY :1

SPECIFICATIONS FOR THE IDENTITY AND PURITY

Issued jointly by FAO and WHO

The contents of this document are the result

!/ Thirteenth Report of the Joint

Some Food Colours 1

Chemically Treated Starches 18

Some Emulsifiers and Stabilizers 37

Some Anti-caking Agents 96

Certain Other Food Additives 119

General References 136

The specifications and identity tests appearing in this

For detailed monographs on toxicological evaluation of

Professor F. Bar, Director, Toxicology and

Dr. J. D. Brandner, Atlas Chemical Industries Inc.,

Dr. J.M. Coon, Professor and Chairman, Department

Mr. A. Eisenberg, Ministry of Health, Jerusalem,

Dr. L. Golberg, Research Professor of Pathology,

Dr. H. Lange, Chairman of Food Chemistry Section,

Professor R. Monacelli, Department of Chemistry,

Professor M. J. Rand, Department of Pharmacology,

Professor J. F. Reith, Department of Food Chemistry

Professor A. I. Stenberg, Head, Department of Hygiene,

P .. ofessor R. Truhaut, Director, Toxicological Research

Observers (invited by FAO):

Mr. Durward F. Dodgen, Special Consultant, Food

Professor M. J. L. Dols, Chairman of Codex Committee

Dr. W. Hausheer, International Union of Pure and

Mr. L. Heckman, German Research Council,

Dr. H. Blumenthal, Public Health Service, Food and

Dr. P. S. Elias, Department of Health and Social

Mr. H.P. Mollenhauer, Reg. Dir., Federal Ministry

Dr. D. M. Smith, Office for International Standards,

Dr. C. Agthe, Senior Scientist, Food Additives Unit,

Dr. F. C. Lu, Chief, Food Additives Unit, WHO

Synonyms: C. I. Natural Orange 4; Lebensmittel

Code (Index) Numbers· C. I. (1956} No. 75120

Definition: Annatto extracts are obtained by

J . Annatto extract in oil is prepared by heating the finely

Annatto extracts in oil contain 0. 2 up to 2. 6 % of carotenoids

Annatto extract in oil is a red oily solution - or suspension.

2. Aqueous annatto extract is obtained by heating the

The principal colouring matter of aqueous annatlo extract

Functional Use in Food: Colour

1. Annatto extract in oil, diluted with chloroform, shows

2. Aqueous annatto extract, diluted with water, shows an

"- - '\ l '\

B. Column Chromatography and Carr-Price Reaction:

1. Annatto extract in oil: Dissolve enough of the extract in

2. Aqueous annatto extract: Put 2 ml of the extract in a

After separation discharge the water layer and wash the

1. Annatto extract in oil: Impregpate Whatman 3 MM filter

2. Aqueous annatto extract: Prepare a chromatogram as

l. Annatto extract in oil: 0. 2 to 2. 6 % of carotenoids

2. Aqueous annatto extract: Proceed as indicated in 1 ,

Arsenic (as As): not more than 5 mg/kg

Synonyms and varieties: C. 1. Natural Green 3;

Class: Phorbin (• dihydroporphin)

Code Numbers: C. 1. (1956) No. 75810