Examination of the acid chloroform extract for derivatives of barbituric and salicylic acids, pyrazolone, purine

Table – Detection of derivatives of barbituric and salicylic acids, pyrazolone, purine by chemical tests
Specificity
No. Method Observation Chemical reaction equation(s) and sensitivity
of the reaction
Detection of salicylates (salicylic acid)

The reaction of tribromophenol formation Specificity:

Method. Add a few drops of distilled water to the
remainder obtained after evaporation of the chloroform
extract, mix and add 2–3 drops of bromine water saturated
1.
solution. Observe appearance of a precipitate sensitivity:
1:40000, the
limiting
dilution
The reaction with iron (III) chloride Specificity:
Method (the first variant of the reaction performing). Add
1 drop of iron (III) chloride solution to the residue
obtained after evaporation of the chloroform extract
placed in a porcelain cup. Observe appearance of colour.
2.
Method (the second variant of the reaction performing).
Apply 1 drop of iron (III) chloride solution (prepare fresh)
on the filtration paper and dry. Apply 1–2 drops of the
chloroform extract examined at the same place. Observe
appearance of colour
The reaction of methylsalycilate formation (study Specificity:
theoretically)
Method. Place a few drops of the chloroform extract
3. examined into a test tube, evaporate to dryness in a water
bath and add to the remainder 2–3 drops of concentrated
sulphuric acid, 2–3 drops of methanol and heat in a water
bath. Methyl salicylate shows a characteristic odour
 Detection of barbituric acid derivatives (barbital, phenobarbital)
The model solutions containing some substances Specificity:
imitaiting barbiturates are used
The general reaction for barbiturates
The reaction with cobalt nitrate or acetate and
ammonia
1. Method. Apply a drop of the chloroform extract on a
filter paper previously treated with 1 % cobalt nitrate
ethanol solution, dry out. Then fumigate the paper by
ammonia vapours (leave it over the surface of 25 %
ammonium hydroxide solution). Observe appearance of
colour
The formation of barbiturate characteristic crystals Specificity:
Method. Apply a few drops of the chloroform extract on
a slide, evaporate to dryness at room temperature. Repeat
this procedure. Dissolve the remainder obtained in a drop
of concentrated sulphuric acid. In 3–5 min. place one
drop of distilled water next to this drop, combine
2.
carefully the contents of the drops with the help of glass
rod. Characteristic crystalline precipitates are observed sensitivity:
for particular barbiturates in 10–20 min., the crystals can 800 µg per
appear in 1–2 hours when little amounts of barbiturates a sample
are analysed. Describe the form of the crystals obtained (barbital)
and draw them
The reaction with chlorine-zinc-iodine reagent Specificity:
Method. Apply a few drops of the chloroform extract on
a slide, evaporate to dryness at room temperature. Repeat
this procedure. Apply a drop of chlorine-zinc-iodine
3.
reagent on the remainder. In 10–15 min. the
characteristic crystals appear. When the crystals do not
appear for a long time, add 1–2 crystals of iodine to the sensitivity:
sample and in 10–15 min. observe them again with the 4 µg per
 help of a microscope. Describe the form of the crystals a sample
obtained and draw them (barbital)
The reaction with ferric-iodine reagent Specificity:
Method. Apply a few drops of the chloroform extract on
a slide, evaporate to dryness at room temperature. Repeat
this procedure. Apply one drop of ferric-iodine reagent
to the dry remainder. In 10–15 min. characteristic
crystals appear. With abundance of crystals evaporate the
4. reaction mixture placed on a slide using an alcohol
burner, then add a drop of distilled water to the dry
remainder. In 10–15 min. examine the crystals obtained
with the help of a microscope. Describe the form of the sensitivity:
crystals obtained and draw them 1.8 µg per
a sample
(barbamyl)
The reaction with copper-iodine reagent Specificity:
Method. Apply a few drops of the chloroform extract on a
slide, evaporate to dryness at room temperature. Repeat
this procedure. Add one drop of copper-iodine reagent to
the dry remainder. In 10–15 min. examine the crystals
5.
obtained with the help of a microscope. Describe the form
of the crystals obtained and draw them sensitivity:
2.1 µg per
a sample
(barbamyl)
Detection of purine alkaloids (caffeine)

The general reaction Specificity:

The reaction of murexide formation
Method. Place 5–6 drops of the chloroform extract
1.
examined in a porcelain cup and evaporate the solvent
without heating. Add 0.5–1 ml of bromine water to the
dry remainder and evaporate using a water bath to
 dryness. Apply a drop of 25 % ammonium hydroxide sensitivity:
solution on the remainder coloured in brown. Observe 0.05 mg per
appearance of colour a sample
(barbital)
Specificity:
The reaction with Nessler`s reagent
Method. Place 5–6 drops of the chloroform extract
examined in a porcelain cup and evaporate the solvent
2. without heating. Add Nessler`s reagent to the remainder
and heat the mixture to 100 °C in a water bath for 1–
2 min. Observe appearance of a coloured precipitate. The
blank experiment should be performed
Detection of pyrazolone derivatives (antipyrine, analgin)

Antipyrine Specificity:
The reaction with iron (III) chloride
Method. Place a few drops of the chloroform extract
1.
examined into a porcelain cup, evaporate it to dryness.
Add a drop of 5 % iron (III) chloride solution to the dry
remainder. Observe appearance of colour
The reaction of nitrosoantipyrine formation Specificity:
Method. Place 3–5 ml of the chloroform extract
examined into a porcelain cup, evaporate it to dryness.
Dissolve the dry remainder in 3–5 drops of water, add 2–
2.
4 drops of 10 % sulphuric acid solution and 2–3 drops of
sodium nitrite saturated solution to the solution obtained.
Observe appearance of colour

The reaction of azo dye formation (study theoretically) Specificity:

Method. Place 2–5 drops of the chloroform extract
3. examined into a test tube, evaporate it in an water bath to
dryness. Add 1–2 drops of water to the residue. Then add
a drop of acetic acid and a drop of 5 % potassium nitrite
 solution to the solution obtained. Shake periodically for
5 min. Then add a bit of sodium azide to the mixture.
When gas liberation stops, add 3–4 crystals of β-
naphtylamine and heat the test tube in a water bath for 1–
2 min. Observe appearance of colour
Analgin Specificity:
The reaction with iron (III) chloride
Method. Place a few drops of the chloroform extract
1. examined into a porcelain cup, evaporate it to dryness.
Add a drop of 5 % iron (III) chloride solution to the
residue. Observe appearance of colour
The lignin test Specificity:
Method. Apply 2–3 drops of the chloroform extract in the
same place of a newspaper piece. Observe appearance of
2. colour. It becomes more intensive after treatment of the
spot with hydrochloric acid diluted solution sensitivity:
5 µg per
a sample
Detection of cannabinoids
The reaction with Duquenois-Levine reagent Specificity:
the model solutions containing some substance imitaiting
cannabinoids is used)
Method. Place 4–5 drops of the chloroform extract
examined into a porcelain cup, evaporate it to dryness.
1.
Add a drop of Duquenois-Levine reagent (2 g of vaniline
in 100 ml of 1 % acetaldehyde ethanol solution) and a sensitivity:
drop of concentrated hydrochloric acid to the residue. 1 µg per
Observe appearance of colour a sample