AMMONIUM SALTS of PHOSPHATIDIC ACID

Prepared at the 55th JECFA (2000) and published in FNP 52 Add 8 (2000),
superseding tentative specifications prepared at the 17th JECFA (1973) and
published in FNP 4 (1978) and in FNP 52 (1992). An ADI of 0-30 mg/kg bw
was established at the 18th JECFA (1974).

SYNONYMS Ammonium phosphatides, Emulsifier YN, Mixed ammonium salts of

phosphorylated glycerides; INS No. 442

DEFINITION The product consists essentially of a mixture of the ammonium compounds of

phosphatidic acids derived from the edible fat (usually partially hardened
rapeseed oil). A mono- or diglyceride moiety may be attached to phosphorus.
Moreover, two phosphorus esters may be linked together as phosphatidyl
phosphatides. The product is prepared by glycerolysis of the fat, followed by
phosphorylation with phosphorus pentoxide, and neutralization with
ammonia.
The article of commerce may be further specified as to water content,
hexane-insoluble matter, inorganic hexane-insoluble matter, pH value and
triglyceride content.

Structural formula
(approximate composition)

where R may be a mono- or di-glyceride moiety

Assay The phosphorus content is not less than 3.0% and not more than 3.4% by
weight; the ammonium N content is not less than 1.2% and not more than
1.5%.

DESCRIPTION Unctuous semisolid

FUNCTIONAL USES Emulsifier

CHARACTERISTICS
IDENTIFICATION

Solubility (Vol. 4) Insoluble in water, partially soluble in ethanol and in acetone, soluble in fats

Test for phosphate Passes tests

See description under TESTS

Test for fatty acid Passes tests

See description under TESTS
Test for glycerol (Vol. 4) Passes test

PURITY
Lead (Vol. 4) Not more than 2 mg/kg
Determine using an atomic absorption technique appropriate to the specified
level. The selection of sample size and method of sample preparation may
be based on the principles of the method described in Volume 4,
“Instrumental Methods.”

TESTS

IDENTIFICATION TESTS

Test for phosphate Ignite 1 g of the sample 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 TS and heat to boiling. A yellow precipitate is
obtained.

Test for fatty acid Reflux 1 g of the product for 1 h with 25 ml of 0.5 N ethanolic potassium fatty
acid hydroxide. Ammonia is evolved from the end of the reflux condenser,
recognizable by its odour and by its reaction on moist, red litmus paper. On
cooling the residue to 0o, a precipitate of potassium soap is obtained.

METHOD OF
ASSAY
Determination of Absorption spectrophotometry
phosphorus
Reagents and working solutions
Sulfuric acid: sp gr 1.84
Nitric acid: sp gr 1.42
Perchloric acid: 60%, sp gr 1.54
Vanadate-molybdate solution: Separately dissolve in water 20 g of
ammonium molybdate and 1 g of ammonium vanadate. Mix the two solutions,
add 140 ml of concentrated nitric acid and dilute to 1000 ml with water. Mix
well.
Standard phosphate solution: Stock solution: Dissolve 3.8346 g of potassium
dihydrogen phosphate, previously dried at 110º, in water and dilute to 1000
ml; 1 ml of this solution = 2.0 mg P2O5.
Standard phosphate working solution: Transfer 50.0 ml of the standard
phosphate solution (1ml = 2.0 mg P2O5) to a 500 ml volumetric flask and
dilute to volume with water and mix well. 1 ml of standard phosphate working
solution = 0.2 mg equivalents of P2O5

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
sulfuric acid and 10 ml of 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).

Into separate 100-ml volumetric flasks, add by burette:

(a) 25.0 ml of Standard phosphate working solution (= 5.0 mg P2O5),
(b) 30.0 ml of Standard phosphate working solution (= 6.0 mg P2O5),
(c) a 25 ml aliquot of the test solution which will contain the equivalent of
between 5 and 6 mg P2O5

Into each of the flasks containing the phosphorus standards, i.e. (a) and (b),
transfer an aliquot of the blank digest solution 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 20º,
dilute to the mark with water and re-mix.

After 10 min measure the absorbance of both the 6 mg P2O5 solution and the
test solution against the 5 mg standard contained in the blank cell. Use
optically matched 1 cm cells and measure at a wavelength of 420 nm, or with
an Ilford 604 filter if using a photo-electric colorimeter.

Calculation

where
A test = absorbance difference between the 5 mg standard and the test
solution
A 6 mg = absorbance difference between the 6 mg and 5 mg standards
W = weight of sample taken (g)

Determination of Apparatus for steam distillation

ammonium nitrogen The apparatus consists of a 2-L 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 sulfuric acid TS 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-L round bottomed B34
necked flask. The distillation head should be such that the steam inlet tube
reaches almost to the bottom of the 1-L flask and the outlet should be fitted
with two splash traps, one near the top of the 1-L flask and the other near the
top of B19 jointed verticle, single-surface condenser to which the distillation
head connects. The vertical condenser should be fitted with an extended
outlet tube, able to reach to the bottom of a 500-ml conical flask.
Reagents
Boric acid solution (2% w/v in water)
Sodium hydroxide solution (40 w/v in water)
0.02N Hydrochloric acid
Mixed indicator: Mix 5.0 of 0.1% w/v alcoholic solution of bromocresol green
and 2.0 of a 0.1% w/v alcoholic solution of methyl red and dilute the mixture
to 30 ml with 95% alcohol.
Silicone fluid 200/50 MS

Procedure
Assemble and thoroughly steam out the apparatus. Accurately weigh about
0.2 g of a representative sample of neutral Ammonium salts of phosphatidic
acids into a small glass phial (approx. 3/4" diameter, 1/2" deep). Transfer the
phial and weighed contents to the distillation flask and add approximately 250
ml distilled water. Connect the distillation head and splash traps to the
distillation 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. (Alternatively, 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). 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. 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 of 0.02 N HCl = 0.2802 mg of nitrogen.