Experiment 4: Respiratory Toxicants

I. Introduction
*see manual

II. Methods

CYANIDE
Preparation of Sample

III. Results and Discussion

Preparation of Standard
Prepare a 10% solution of sodium cyanide. Use this for qualitative tests

Qualitative Test for Nitrite

Quantitative Test for Carboxyhemoglobin

IV. Qualitative Tests

Test Procedure Reaction Theoretical Result Notes

Schonbein-Pagenstecher ● Saturate filter paper strip Forms ozone with CuSO4 and turns the paper will turn to blue or bluish green Interference: ammonia, ammonium salts, HCl, nitric
with guaiac solution guaiaconic acid of guaiac resin blue acid, and chlorine give positive results

● Soak to sample

Prussian blue ● 1-2gtt KOH (do not add in Ferric ferrocyanide / Prussian blue Prussian blue precipitate Alkalis will interfere with the formation of prussian
excess) blue (KOH should not be added in excess)
Fe4[Fe(CN)6]3
● FeSO4

● FeCl3

Sulfocyanate test / Leibig’s ● KOH Ferric sulfocyanate Reddish or blood-red solution

test
● (NH4)2S Fe(SCN)3

● Evaporate to half. Filter.

● FeCl3

Vortmann’s Sodium ● NaNO2 K2Fe(NO)(CN)5 violet color will appear and pass through blue, This test is the reverse of the nitroprusside test for
nitroprusside test green, and yellow hydrogen sulphide and is due to conversion of
● 10% H2SO4 hydrocyanic acid to potassium nitroprusside ,
which causes the color changes when ammonium
● FeCl3
sulphide is added . Very small quantities of
● Boil hydrocyanic acid give a bluish green to greenish
yellow color
● NH4OH

● Filter

Silver nitrate test
Picric acid test
Qualitative test for Cyanide
Qualitative test for Nitrite
● (NH4)2S to side of tilted test
tube
● HNO3 Silver cyanide (AgCN) – insoluble in
HNO3, retain its pure white color when
● 1% AgNO3 exposed to the rays of the sun
● KOH
● 1% Picric acid
● 10% NaOH Potassium ferrocyanide
● 10% FeSO4
● 10% HCl (to dissolve the NaOH + HCN → NaCN + H2O
ferrous hydroxide ppt)
FeSO4 + 2 NaCN → Fe(CN)2 +
Na2SO4
Fe(CN)2 + 4 NaCN → Na4Fe(CN)6
FeSO4 + NaOH → Fe(OH)2
6 Fe(OH)2 + ½O2 + 3 H2O →
6Fe(OH)3
3 Na4Fe(CN)6 + 4 Fe(OH)3 + 12HCl
→ Fe4(Fe(CN)6)3 + 12 NaCl + 12 H2O
● sample and distilled water
● sonicate 15 mins
White curdy precipitate
Yellow to brown reddish solution
Lemon-yellow precipitate (dissolved by HCl)
Formation of strong blue color which forms
immediately at the junction of two layers
Nitrate of silver detects hydrocyanic acid with which
it produces a white ppt of cyanide of silver even in a
very dilute soln of the acid. This ppt is distinguished
from other white ppt of silver by its being insoluble
in nitric acid at ordinary temperatures, but readily
soluble by boiling heat; and by its retaining its pure
whit color when exposed to the rays of the sun.
Cyanide of silver, when dried and heated, emits
cyanogen gas which is easily known by the
beautiful rose red color of its flame.
The test uses the reaction of picric acid (yellow
compound) changing into isopurpuric acid (brown-
reddish) in the presence of volatile hydrogen
cyanide (HCN).
However, Sulfur also results to a similar colored
solution when it forms Picraminic acid with Picric
acid -- Therefore, picric acid alone can’t conclude
the presence of cyanides .
•Ferrocyanide ions were produced in situ and the
oxidation of the Ferrous ion formed Ferric ions
• The reaction between the said compounds results
in the formation of the Ferric ferrocyanide.
Diphenylamine first converts to the colorless
compound Diphenylbenzidine and upon long
standing with nitrites, it irreversibly forms
 ● filter, 1ml in tube Diquinonediimine, which is blue in color

● 1 ml diphenylamine solution

In the presence of nitrates, diphenylamine is

oxidized, giving a blue coloration.

Quantitative test for ● 25 ml 0.1% (v/v) ammonium Addition of sodium dithionite Transparent colloidall solution in water
Carboxyhemoglobin hydroxide and blood deoxygenates hemoglobin (and
reduces methemoglobin if present) % COHb saturation: ● Quantitation is based on the difference
● Portion A – saturate with without affecting between two isobestic points obtained with
pure CO carboxyhemoglobin Pure CO > Patient sample > O2 oxidized and reduced hemoglobin

● Portion B – saturate with ● Total hemoglobin is measured, and this

oxygen to dissociate COHb value along with the isobestic absorbance is
Portion C – test sample used to calculate the percentage of
carboxyhemoglobin
● sodium dithionite.

● Mix and read absorbance at

500 – 600nm versus
reagent blank. Measure
absorbance at 540
(COHbmax), and 579nm
(isosbestic point)