EUROPEAN PHARMACOPOEIA 6.0 2.3.1.

Identification reactions of ions and functional groups

2.3. IDENTIFICATION solution R. The colour of the indicator changes to yellow.

On addition of 1 ml of a freshly prepared 100 g/l solution of
sodium cobaltinitrite R a yellow precipitate is formed.
01/2008:20301 AMMONIUM SALTS AND SALTS OF VOLATILE BASES
Dissolve about 20 mg of the substance to be examined in
2.3.1. IDENTIFICATION REACTIONS 2 ml of water R or use 2 ml of the prescribed solution. Add
OF IONS AND FUNCTIONAL GROUPS 2 ml of dilute sodium hydroxide solution R. On heating, the
solution gives off vapour that can be identified by its odour
ACETATES and by its alkaline reaction (2.2.4).
a) Heat the substance to be examined with an equal quantity
of oxalic acid R. Acid vapours with the characteristic odour ANTIMONY
of acetic acid are liberated, showing an acid reaction (2.2.4). Dissolve with gentle heating about 10 mg of the substance
b) Dissolve about 30 mg of the substance to be examined in to be examined in a solution of 0.5 g of sodium potassium
3 ml of water R or use 3 ml of the prescribed solution. Add tartrate R in 10 ml of water R and allow to cool : to 2 ml
successively 0.25 ml of lanthanum nitrate solution R, 0.1 ml of this solution, or to 2 ml of the prescribed solution,
of 0.05 M iodine and 0.05 ml of dilute ammonia R2. Heat add sodium sulphide solution R dropwise ; an orange-red
carefully to boiling. Within a few minutes a blue precipitate precipitate is formed which dissolves on addition of dilute
is formed or a dark blue colour develops. sodium hydroxide solution R.

ACETYL ARSENIC
In a test-tube about 180 mm long and 18 mm in external Heat 5 ml of the prescribed solution on a water-bath with
diameter, place about 15 mg of the substance to be examined, an equal volume of hypophosphorous reagent R. A brown
or the prescribed quantity, and 0.15 ml of phosphoric precipitate is formed.
acid R. Close the tube with a stopper through which passes BARBITURATES, NON-NITROGEN SUBSTITUTED
a small test-tube about 100 mm long and 10 mm in external
diameter containing water R to act as a condenser. On the Dissolve about 5 mg of the substance to be examined in 3 ml
outside of the smaller tube, hang a drop of lanthanum of methanol R, add 0.1 ml of a solution containing 100 g/l
nitrate solution R. Except for substances hydrolysable only of cobalt nitrate R and 100 g/l of calcium chloride R. Mix
with difficulty, place the apparatus in a water-bath for 5 min, and add, with shaking, 0.1 ml of dilute sodium hydroxide
then take out the smaller tube. Remove the drop and mix solution R. A violet-blue colour and precipitate are formed.
it with 0.05 ml of 0.01 M iodine on a tile. Add at the edge BENZOATES
0.05 ml of dilute ammonia R2. After 1 min to 2 min, a blue
a) To 1 ml of the prescribed solution add 0.5 ml of ferric
colour develops at the junction of the two drops ; the colour
chloride solution R1. A dull-yellow precipitate, soluble in
intensifies and persists for a short time.
ether R, is formed.
For substances hydrolysable only with difficulty heat the
b) Place 0.2 g of the substance to be examined, treated if
mixture slowly to boiling over an open flame and then
necessary as prescribed, in a test-tube. Moisten with 0.2 ml
proceed as prescribed above.
to 0.3 ml of sulphuric acid R. Gently warm the bottom of the
ALKALOIDS tube. A white sublimate is deposited on the inner wall of
the tube.
Dissolve a few milligrams of the substance to be examined,
or the prescribed quantity, in 5 ml of water R, add dilute c) Dissolve 0.5 g of the substance to be examined in 10 ml
hydrochloric acid R until an acid reaction occurs (2.2.4), of water R or use 10 ml of the prescribed solution. Add
then 1 ml of potassium iodobismuthate solution R. An 0.5 ml of hydrochloric acid R. The precipitate obtained,
orange or orange-red precipitate is formed immediately. after crystallisation from warm water R and drying in vacuo,
has a melting point (2.2.14) of 120 °C to 124 °C.
ALUMINIUM
BISMUTH
Dissolve about 15 mg of the substance to be examined in
2 ml of water R or use 2 ml of the prescribed solution. Add a) To 0.5 g of the substance to be examined add 10 ml of
about 0.5 ml of dilute hydrochloric acid R and about 0.5 ml dilute hydrochloric acid R or use 10 ml of the prescribed
of thioacetamide reagent R. No precipitate is formed. Add solution. Heat to boiling for 1 min. Cool and filter if
dropwise dilute sodium hydroxide solution R. A gelatinous necessary. To 1 ml of the solution obtained add 20 ml of
white precipitate is formed which dissolves on further water R. A white or slightly yellow precipitate is formed
addition of dilute sodium hydroxide solution R. Gradually which on addition of 0.05 ml to 0.1 ml of sodium sulphide
add ammonium chloride solution R. The gelatinous white solution R turns brown.
precipitate is re-formed. b) To about 45 mg of the substance to be examined add
10 ml of dilute nitric acid R or use 10 ml of the prescribed
AMINES, PRIMARY AROMATIC solution. Boil for 1 min. Allow to cool and filter if necessary.
Acidify the prescribed solution with dilute hydrochloric To 5 ml of the solution obtained add 2 ml of a 100 g/l
acid R and add 0.2 ml of sodium nitrite solution R. After solution of thiourea R. A yellowish-orange colour or an
1 min to 2 min, add 1 ml of β-naphthol solution R. An orange precipitate is formed. Add 4 ml of a 25 g/l solution
intense orange or red colour and usually a precipitate of the of sodium fluoride R. The solution is not decolorised within
same colour are produced. 30 min.
AMMONIUM SALTS BROMIDES
To the prescribed solution add 0.2 g of magnesium oxide R. a) Dissolve in 2 ml of water R a quantity of the substance
Pass a current of air through the mixture and direct the to be examined equivalent to about 3 mg of bromide (Br–)
gas that escapes just beneath the surface of a mixture of or use 2 ml of the prescribed solution. Acidify with dilute
1 ml of 0.1 M hydrochloric acid and 0.05 ml of methyl red nitric acid R and add 0.4 ml of silver nitrate solution R1.

General Notices (1) apply to all monographs and other texts 103
2.3.1. Identification reactions of ions and functional groups EUROPEAN PHARMACOPOEIA 6.0

Shake and allow to stand. A curdled, pale yellow precipitate over the opening of the test-tube. The paper turns violet-red.
is formed. Centrifuge and wash the precipitate with three The impregnated paper must not come into contact with the
quantities, each of 1 ml, of water R. Carry out this operation potassium dichromate.
rapidly in subdued light disregarding the fact that the
supernatant solution may not become perfectly clear. CITRATES
Suspend the precipitate obtained in 2 ml of water R and Dissolve in 5 ml of water R a quantity of the substance to be
add 1.5 ml of ammonia R. The precipitate dissolves with examined equivalent to about 50 mg of citric acid or use 5 ml
difficulty. of the prescribed solution. Add 0.5 ml of sulphuric acid R
b) Introduce into a small test-tube a quantity of the substance and 1 ml of potassium permanganate solution R. Warm
to be examined equivalent to about 5 mg of bromide (Br–) or until the colour of the permanganate is discharged. Add
the prescribed quantity. Add 0.25 ml of water R, about 75 mg 0.5 ml of a 100 g/l solution of sodium nitroprusside R in
of lead dioxide R, 0.25 ml of acetic acid R and shake gently. dilute sulphuric acid R and 4 g of sulphamic acid R. Make
Dry the inside of the upper part of the test-tube with a piece alkaline with concentrated ammonia R, added dropwise
of filter paper and allow to stand for 5 min. Prepare a strip until all the sulphamic acid has dissolved. Addition of an
of suitable filter paper of appropriate size. Impregnate it by excess of concentrated ammonia R produces a violet colour,
capillarity, by dipping the tip in a drop of decolorised fuchsin turning to violet-blue.
solution R and introduce the impregnated part immediately
into the tube. Starting from the tip, a violet colour appears ESTERS
within 10 s that is clearly distinguishable from the red colour To about 30 mg of the substance to be examined or the
of fuchsin, which may be visible on a small area at the top of prescribed quantity add 0.5 ml of a 70 g/l solution of
the impregnated part of the paper strip. hydroxylamine hydrochloride R in methanol R and 0.5 ml
of a 100 g/l solution of potassium hydroxide R in ethanol
CALCIUM
(96 per cent) R. Heat to boiling, cool, acidify with dilute
a) To 0.2 ml of a neutral solution containing a quantity hydrochloric acid R and add 0.2 ml of ferric chloride
of the substance to be examined equivalent to about solution R1 diluted ten times. A bluish-red or red colour is
0.2 mg of calcium (Ca2+) per millilitre or to 0.2 ml of the produced.
prescribed solution add 0.5 ml of a 2 g/l solution of
glyoxal-hydroxyanil R in ethanol (96 per cent) R, 0.2 ml IODIDES
of dilute sodium hydroxide solution R and 0.2 ml of
sodium carbonate solution R. Shake with 1 ml to 2 ml a) Dissolve a quantity of the substance to be examined
of chloroform R and add 1 ml to 2 ml of water R. The equivalent to about 4 mg of iodide (I–) in 2 ml of water R or
chloroform layer is coloured red. use 2 ml of the prescribed solution. Acidify with dilute nitric
acid R and add 0.4 ml of silver nitrate solution R1. Shake
b) Dissolve about 20 mg of the substance to be examined or and allow to stand. A curdled, pale-yellow precipitate is
the prescribed quantity in 5 ml of acetic acid R. Add 0.5 ml formed. Centrifuge and wash with three quantities, each of
of potassium ferrocyanide solution R. The solution remains 1 ml, of water R. Carry out this operation rapidly in subdued
clear. Add about 50 mg of ammonium chloride R. A white, light disregarding the fact that the supernatant solution may
crystalline precipitate is formed. not become perfectly clear. Suspend the precipitate in 2 ml
of water R and add 1.5 ml of ammonia R. The precipitate
CARBONATES AND BICARBONATES does not dissolve.
Introduce into a test-tube 0.1 g of the substance to be b) To 0.2 ml of a solution of the substance to be examined
examined and suspend in 2 ml of water R or use 2 ml of containing about 5 mg of iodide (I–) per millilitre, or to 0.2 ml
the prescribed solution. Add 3 ml of dilute acetic acid R. of the prescribed solution, add 0.5 ml of dilute sulphuric
Close the tube immediately using a stopper fitted with a acid R, 0.1 ml of potassium dichromate solution R, 2 ml of
glass tube bent twice at right angles. The solution or the water R and 2 ml of chloroform R. Shake for a few seconds
suspension becomes effervescent and gives off a colourless and allow to stand. The chloroform layer is coloured violet
and odourless gas. Heat gently and collect the gas in 5 ml of or violet-red.
barium hydroxide solution R. A white precipitate is formed
that dissolves on addition of an excess of hydrochloric IRON
acid R1.
a) Dissolve a quantity of the substance to be examined
CHLORIDES equivalent to about 10 mg of iron (Fe2+) in 1 ml of water R or
a) Dissolve in 2 ml of water R a quantity of the substance to use 1 ml of the prescribed solution. Add 1 ml of potassium
–
be examined equivalent to about 2 mg of chloride (Cl ) or ferricyanide solution R. A blue precipitate is formed that
use 2 ml of the prescribed solution. Acidify with dilute nitric does not dissolve on addition of 5 ml of dilute hydrochloric
acid R and add 0.4 ml of silver nitrate solution R1. Shake acid R.
and allow to stand. A curdled, white precipitate is formed. b) Dissolve a quantity of the substance to be examined
Centrifuge and wash the precipitate with three quantities, equivalent to about 1 mg of iron (Fe3+) in 30 ml of water R.
each of 1 ml, of water R. Carry out this operation rapidly in To 3 ml of this solution or to 3 ml of the prescribed solution,
subdued light, disregarding the fact that the supernatant add 1 ml of dilute hydrochloric acid R and 1 ml of potassium
solution may not become perfectly clear. Suspend the thiocyanate solution R. The solution is coloured red. Take
precipitate in 2 ml of water R and add 1.5 ml of ammonia R. two portions, each of 1 ml, of the mixture. To one portion
The precipitate dissolves easily with the possible exception add 5 ml of isoamyl alcohol R or 5 ml of ether R. Shake and
of a few large particles which dissolve slowly. allow to stand. The organic layer is coloured pink. To the
b) Introduce into a test-tube a quantity of the substance to be other portion add 2 ml of mercuric chloride solution R. The
examined equivalent to about 15 mg of chloride (Cl–) or the red colour disappears.
prescribed quantity. Add 0.2 g of potassium dichromate R c) Dissolve a quantity of the substance to be examined
and 1 ml of sulphuric acid R. Place a filter-paper strip equivalent to not less than 1 mg of iron (Fe3+) in 1 ml of
impregnated with 0.1 ml of diphenylcarbazide solution R water R or use 1 ml of the prescribed solution. Add 1 ml

104 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.0 2.3.1. Identification reactions of ions and functional groups

of potassium ferrocyanide solution R. A blue precipitate is formed. Add to the hot solution 0.05 ml of sodium sulphide
formed that does not dissolve on addition of 5 ml of dilute solution R. No precipitate is formed. Cool in iced water and
hydrochloric acid R. add 2 ml of a 150 g/l solution of tartaric acid R. Allow to
stand. A white crystalline precipitate is formed.
LACTATES
b) Dissolve about 40 mg of the substance to be examined in
Dissolve a quantity of the substance to be examined 1 ml of water R or use 1 ml of the prescribed solution. Add
equivalent to about 5 mg of lactic acid in 5 ml of water R or 1 ml of dilute acetic acid R and 1 ml of a freshly prepared
use 5 ml of the prescribed solution. Add 1 ml of bromine 100 g/l solution of sodium cobaltinitrite R. A yellow or
water R and 0.5 ml of dilute sulphuric acid R. Heat orange-yellow precipitate is formed immediately.
on a water-bath until the colour is discharged, stirring
occasionally with a glass rod. Add 4 g of ammonium SALICYLATES
sulphate R and mix. Add dropwise and without mixing
0.2 ml of a 100 g/l solution of sodium nitroprusside R in a) To 1 ml of the prescribed solution add 0.5 ml of ferric
dilute sulphuric acid R. Still without mixing add 1 ml of chloride solution R1. A violet colour is produced that
concentrated ammonia R. Allow to stand for 30 min. A dark persists after the addition of 0.1 ml of acetic acid R.
green ring appears at the junction of the two liquids. b) Dissolve 0.5 g of the substance to be examined in 10 ml
of water R or use 10 ml of the prescribed solution. Add
LEAD 0.5 ml of hydrochloric acid R. The precipitate obtained,
a) Dissolve 0.1 g of the substance to be examined in 1 ml of after recrystallisation from hot water R and drying in vacuo,
acetic acid R or use 1 ml of the prescribed solution. Add has a melting point (2.2.14) of 156 °C to 161 °C.
2 ml of potassium chromate solution R. A yellow precipitate
is formed that dissolves on addition of 2 ml of strong sodium SILICATES
hydroxide solution R. Mix the prescribed quantity of the substance to be examined
b) Dissolve 50 mg of the substance to be examined in 1 ml in a lead or platinum crucible by means of a copper wire
of acetic acid R or use 1 ml of the prescribed solution. Add with about 10 mg of sodium fluoride R and a few drops of
10 ml of water R and 0.2 ml of potassium iodide solution R. sulphuric acid R to give a thin slurry. Cover the crucible
A yellow precipitate is formed. Heat to boiling for 1 min with a thin, transparent plate of plastic under which a drop
to 2 min. The precipitate dissolves. Allow to cool. The of water R is suspended and warm gently. Within a short
precipitate is re-formed as glistening, yellow plates. time a white ring is rapidly formed around the drop of water.
MAGNESIUM SILVER
Dissolve about 15 mg of the substance to be examined in Dissolve about 10 mg of the substance to be examined in
2 ml of water R or use 2 ml of the prescribed solution. Add 10 ml of water R or use 10 ml of the prescribed solution.
1 ml of dilute ammonia R1. A white precipitate is formed Add 0.3 ml of hydrochloric acid R1. A curdled, white
that dissolves on addition of 1 ml of ammonium chloride precipitate is formed that dissolves on addition of 3 ml of
solution R. Add 1 ml of disodium hydrogen phosphate dilute ammonia R1.
solution R. A white crystalline precipitate is formed.
MERCURY SODIUM
a) Place about 0.1 ml of a solution of the substance to be a) Dissolve 0.1 g of the substance to be examined in 2 ml of
examined on well-scraped copper foil. A dark-grey stain that water R or use 2 ml of the prescribed solution. Add 2 ml of
becomes shiny on rubbing is formed. Dry the foil and heat in a 150 g/l solution of potassium carbonate R and heat to
a test-tube. The spot disappears. boiling. No precipitate is formed. Add 4 ml of potassium
pyroantimonate solution R and heat to boiling. Allow to cool
b) To the prescribed solution add dilute sodium hydroxide in iced water and if necessary rub the inside of the test-tube
solution R until strongly alkaline (2.2.4). A dense yellow with a glass rod. A dense white precipitate is formed.
precipitate is formed (mercuric salts).
b) Dissolve a quantity of the substance to be examined
NITRATES equivalent to about 2 mg of sodium (Na+) in 0.5 ml of
To a mixture of 0.1 ml of nitrobenzene R and 0.2 ml of water R or use 0.5 ml of the prescribed solution. Add 1.5 ml
sulphuric acid R, add a quantity of the powdered substance of methoxyphenylacetic reagent R and cool in ice-water
equivalent to about 1 mg of nitrate (NO3–) or the prescribed for 30 min. A voluminous, white, crystalline precipitate
quantity. Allow to stand for 5 min. Cool in iced water and add is formed. Place in water at 20 °C and stir for 5 min.
slowly and with mixing 5 ml of water R, then 5 ml of strong The precipitate does not disappear. Add 1 ml of dilute
sodium hydroxide solution R. Add 5 ml of acetone R. Shake ammonia R1. The precipitate dissolves completely. Add
and allow to stand. The upper layer is coloured deep violet. 1 ml of ammonium carbonate solution R. No precipitate
is formed.
PHOSPHATES (ORTHOPHOSPHATES)
a) To 5 ml of the prescribed solution, neutralised if necessary, SULPHATES
add 5 ml of silver nitrate solution R1. A yellow precipitate is a) Dissolve about 45 mg of the substance to be examined
formed whose colour is not changed by boiling and which in 5 ml of water R or use 5 ml of the prescribed solution.
dissolves on addition of ammonia R. Add 1 ml of dilute hydrochloric acid R and 1 ml of barium
b) Mix 1 ml of the prescribed solution with 2 ml of chloride solution R1. A white precipitate is formed.
molybdovanadic reagent R. A yellow colour develops. b) To the suspension obtained during reaction (a), add 0.1 ml
of 0.05 M iodine. The suspension remains yellow (distinction
POTASSIUM from sulphites and dithionites), but is decolorised by adding
a) Dissolve 0.1 g of the substance to be examined in 2 ml of dropwise stannous chloride solution R (distinction from
water R or use 2 ml of the prescribed solution. Add 1 ml of iodates). Boil the mixture. No coloured precipitate is formed
sodium carbonate solution R and heat. No precipitate is (distinction from selenates and tungstates).

General Notices (1) apply to all monographs and other texts 105
2.3.2. Identification of fatty oils by TLC EUROPEAN PHARMACOPOEIA 6.0

TARTRATES precipitate is formed. Add a further 2 ml of strong sodium

hydroxide solution R. The precipitate dissolves. Add 10 ml of
a) Dissolve about 15 mg of the substance to be examined ammonium chloride solution R. The solution remains clear.
in 5 ml of water R or use 5 ml of the prescribed solution. Add 0.1 ml of sodium sulphide solution R. A flocculent
Add 0.05 ml of a 10 g/l solution of ferrous sulphate R and white precipitate is formed.
0.05 ml of dilute hydrogen peroxide solution R. A transient
yellow colour is produced. After the colour has disappeared
add dilute sodium hydroxide solution R dropwise. A violet
or purple colour is produced.
01/2008:20302
b) To 0.1 ml of a solution of the substance to be examined
containing the equivalent of about 15 mg of tartaric acid per
millilitre or to 0.1 ml of the prescribed solution add 0.1 ml
2.3.2. IDENTIFICATION OF
of a 100 g/l solution of potassium bromide R, 0.1 ml of a FATTY OILS BY THIN-LAYER
20 g/l solution of resorcinol R and 3 ml of sulphuric acid R. CHROMATOGRAPHY
Heat on a water-bath for 5 min to 10 min. A dark-blue colour
develops. Allow to cool and pour the solution into water R. Examine by thin-layer chromatography (2.2.27), using as the
The colour changes to red. coating substance a suitable octadecylsilyl silica gel for high
performance thin-layer chromatography.
Test solution. Unless otherwise prescribed, dissolve
XANTHINES about 20 mg (1 drop) of the fatty oil in 3 ml of methylene
chloride R.
To a few milligrams of the substance to be examined or the
prescribed quantity add 0.1 ml of strong hydrogen peroxide Reference solution. Dissolve about 20 mg (1 drop) of maize
solution R and 0.3 ml of dilute hydrochloric acid R. Heat oil R in 3 ml of methylene chloride R.
to dryness on a water-bath until a yellowish-red residue is Apply separately to the plate 1 µl of each solution. Develop
obtained. Add 0.1 ml of dilute ammonia R2. The colour of twice over a path of 0.5 cm using ether R. Develop twice
the residue changes to violet-red. over a path of 8 cm using a mixture of 20 volumes of
methylene chloride R, 40 volumes of glacial acetic acid R
and 50 volumes of acetone R. Allow the plate to dry in air
ZINC and spray with a 100 g/l solution of phosphomolybdic
acid R in alcohol R. Heat the plate at 120 °C for about 3 min
Dissolve 0.1 g of the substance to be examined in 5 ml and examine in daylight.
of water R or use 5 ml of the prescribed solution. Add The chromatogram obtained typically shows spots
0.2 ml of strong sodium hydroxide solution R. A white comparable to those in Figure 2.3.2.-1.

1. arachis oil 6. rapeseed oil (erucic acid-free) 11. wheat-germ oil

2. sesame oil 7. linseed oil 12. borage oil
3. maize oil 8. olive oil 13. evening primrose oil
4. rapeseed oil 9. sunflower oil 14. safflower oil (type I)
5. soya-bean oil 10. almond oil 15. safflower oil (type II)

Figure 2.3.2.-1. – Chromatograms for the identification of fatty oils

106 See the information section on general monographs (cover pages)