Topic. THE GROUP OF SUBSTANCES ISOLATED FROM THE

BIOLOGICAL MATERIAL BY STEAM DISTILLATION (VOLATILE

POISONS). THE METHODS OF ISOLATION AND ANALYSIS OF

DISTILLATES BY THE CHEMICAL METHOD

1. General description of volatile poisons

Volatile poisons are toxic substances, which are isolated from biological ma-

terial by steam distillation and other similar methods. Volatile poisons are acetic

acid; hydrocyanic acid; halocarbons (chloroform, tetrachloromethane, dichloroetha-

ne, chloral hydrate); aldehydes and ketones (formaldehyde, acetone); alcohols (me-

thanol, ethanol, isopentanol, ethylene glycol); esters (ethyl acetate, isopentyl ace-

tate); aromatic hydrocarbons and their derivatives (benzene, toluene, xylene, ani-

line); phenols and phenoloacids (phenol, salicylic acid); tetraethyl lead (TEL);

hydrogen sulfide; phosphorus and its products of oxidation (phosphoric acids) and

reduction (phosphine).

Lethal doses of some volatile poisons are as follows:

for hydrocyanic acid (HCN) the lethal dose is 0.05–0.1 g;

for potassium cyanide (KCN) the lethal dose is 0.15–0.25 g;

for methanol (CH3OH) the lethal dose is 25–100 g (7–8 g causes

blindness);

for chloroform (CHCl3) the lethal dose is 50–70 g;

for acetic acid (CH3COOH) the lethal dose is 15 g.

Substances of the group mentioned are widely used in industry, agriculture,

medicine. For example, chloroform, ethanol, phenol, diethyl ether are used in

medicine; cyclones A and B are used in agriculture (for pesticide control),

chloroform, ethanol, benzene, acetone, etc., are used as solvents in various

branches of industry.

The general property of poisons of this group is volatility and ability to be

distilled. The main isolation method of this group of poisons is steam distillation.

This method allows creating softer conditions then, for example, usual distillation

at atmospheric pressure when the given substances are isolated from the

biological material. This advantage is important for those compounds, which can

be destroyed at high temperature.

Dependence of pressure of saturated vapour of the mixture against

temperature is in the theoretical basis of this method. The liquid begins boiling

and can be distilled when pressure of vapour above the liquid is equal to

atmospheric pressure or exceeds it:

P1 + P2 + P3... = Pn > Patm

where P1 + P2 + P3 +... is the sum of partion pressures of water vapour (P1)

and vapours of liquids analysed (P2, P3, …) in the mixture;

Pn is the pressure of vapour above the mixture.

Consequently, the mixture is distilled at the temperature, which is below

then boiling temperatures of pure substances. For example:

T boil (C6H6) = 80.2ºC;

T boil (H2O) = 100ºC.

The mixture of equal volumes of benzene and water boils at 69.2ºC.

2. Methods of the volatile poison isolation from the biological material

In chemical toxicological analysis for the isolation of volatile poisons the

following methods are used:

steam distillation at atmospheric pressure;

distillation at increased pressure;

distillation at reduced pressure;

microdiffusion;

dry distillation;

vapour-phase method.

The method of steam distillation is the most commonly used. In accordance

with this method 100 g of the biological material is reduced to fine particles, mixed

up with distilled water (Vmixture= 1/3 Vretort ) and placed into the retort placed into a

cold water-bath. Then the vaporization flask is heated until water vapour appears,

the biological material examined is acidified by the saturated water solution of oxalic

acid to pH 2.0–2.5 and all parts of the device (vaporization flask, retort, refrigerator

and receiver) are connected immediately and only then the water-bath is heated.

Fig. 3. Apparatus for steam distillation:

1 – vaporization flask; 2 – retort with the sample; 3 – water refrigerator; 4 – receiver; 5 – water

bath

The choice of the pH value of the sample examined is due to the fact that

when pH is 2.0–2.5, the most complete destruction of bonds between proteins and

poisonous substances occurs.

The choice of the acids for acidification of the biological material is due to

the fact that mineral acids can decompose volatile poisons, for example, hydrocyanic

acid:

HCN + 2H2O + H+ HCOOH + NH4+

,

or hydrolyze the endogenous phenol, which as a result of protein consumption

appears in a living organism as phenol sulphate, a conjugate:

OSO

3H

OH

H2O

the conjugate is not volatile volatile

Weak organic acids do not destroy this conjugate, and only phenol coming to

a body is distilled.

In the case of acetic acid isolation the biological material is acidified with the

help of H2SO4 or H3PO4 solutions, in order to displace the acetic acid dissociation

equilibrium in the aqueous solution to the molecular form and increase the volatility

of the acid.

The distillates are collected into receivers. The first distillate, which contains

the most volatile poisons, for example HCN, is collected into the receiver with 2 ml

of 2% NaOH solution. The total volume of the first distillate is (V1) 5 ml.

Then two more distillates (V2; V3) are collected:

V2 =V3 =25 ml.

When the positive result is obtained when examining the distillate sample for

a particular volatile poison, the distillation is continued out until the negative result

of the corresponding reaction.

Some basic substances are distillated from the alkalized biological material:

aniline, pyridine, nicotine, anabasine, etc. In this case after distillation of substances

from the acid medium, the biological material is alkalized by the addition of 5 NaOH

solution to pH 8–9 and is distilled again collecting 2–4 distillates with 10–15 ml

each are collected in 0.1 M solution of hydrochloric acid.

All volatile poisons may be divided into groups according to their ability to

mix with water:

1. Substances, which are mixed with water in all ratios, but do not form

azeotropic mixtures (methanol, acetone, acetic acid, ethylene glycol, etc.);

2. Substances, which are not mixed or poorly mixed with water (benzene,

chloroform); in this case after distillation two clear layers appear, the water layer

and the organic solvent layer, which are easily separated.

3. Substances, which form azeotropic mixtures. Azeotropic mixture has

the identical composition of the vapour and liquid phases and cannot be divided

completely by distillation at atmospheric pressure (phenol, ethanol). For sepa-

ration of azeotropic mixtures distillation at increased or reduced pressure is used.

For example: the azeotropic mixture of ethanol with water (96 % C2H5OH and

4 % H2O) is distilled at atmospheric pressure at 78ºC. When reducing the

pressure to 100 mm mer. col. (13.3 kPa) the distilled mixture has the following

composition, C2H5OH 99.62 % and H2O 0.4 % and it is distilled at 34ºC.

With the purpose of concentration of the isolated poisons in distillates and

their purification from biological impurities these distillates should be subjected to

fraction distillation.

The method of distillation at reduced pressure is carried out by means of

rotary vaporizers and used most commonly for thermally unsteady substances.

The method of distillation at increased pressure is used for the isolation of

thermally unsteady substances with a high boiling temperature.

The method of microdiffusion is used for analysis of the blood, urine,

homogenized biological samples. This isolation method is carried out with the help

of special chambers; the sample examined and a crucible with the reaction solution

are placed at the bottom of this chamber and it is closed. Evaporation of poison into

the reaction solution is performed in the presence (or without) of salting-out, at the

room temperature or while heating to 37–50ºC. Then the reaction solution is

examined for the presence of volatile poisons.

The method of dry distillation is similar to `microdiffusion and differs only

by dry air which, is skipped through the sample examined. This method is used for

the analysis of substances with a low boiling temperature.

The Vapour phase method (or gaseous extraction) uses the transfer of the

volatile poison examined to the vapour phase followed by the analysis of the

resulting phase with the help of the gas chromatography (GC) method. The vapour

phase method is very important when laboratory express-analysis of biological

fluids in acute intoxication takes place.

3. The scheme of the distillate analysis by the chemical method

The first distillate is analysed for the presence of hydrocyanic acid.

Hydrocyanic acid

Physical properties. Hydrocyanic acid is a volatile liquid with the boiling

temperature of 25.6ºC and the characteristic almond odour.

It is very weak acid (Kd = 4.8 · 10-10), salts are unsteady in water:

KCN + H2O + CO2 HCN + KHCO3

HCN + 2H2O HCOONH4

KCN + 2H2O NH3 (g) + HCOOK

Toxic action. Hydrocyanic acid produces hypoxia by inhibiting

cytochromeoxidase.

Metabolism of hydrocyanic acid. Two basic metabolism pathways take place

– hydrolysis with formation of ammonium formiate and conversion via rhodanase,

the liver enzyme, to thiocyanate, which is subsequently excreted in the urine.

HCN

hydrolysis

rhodanase, S

HCOONH4

HSCN

Antidotes, which are used in hydrocyanic acid poisoning, are:

1. Substances containing sodium (potassium) thiosulphate:

CN + S2O32-

rhodanaseSCN + SO4

2-

2. Substances forming methemoglobin (salts and esters of nitrous acid):

NaNO2; KNO2; C5H11-O-NO; methylene blue:

Hb(Fe2+) MtHb(Fe3+) MtHb(Fe3+)-CN CN-NO2

-

cyanmethemoglobin

(not toxic)hemoglobin methemoglobin

But CN--ion can separate, therefore, it is necessary to give simultaneously

sulphur-contaning substances and carbohydrates to a patient.

3. Carbohydrates (e. g. glucose) bind hydrocyanic acid and its salts with

formation of glucose cyanhydrine.

OH

OH

H O

H

H

H OH

H OH

CH2OH

OH

OHH

H

H OH

H OH

CH2OH

H OH

CN

HCN

D-glucose cyanhydrineD-glucose

Peculiarities of hydrocyanic acid isolation from the biological material.

Taking into account that hydrocyanic acid is volatile well and dissociates poorly in

water solutions, it is transformed to salt when the first distillate is collected into

sodium hydroxide solution:

HCN + NaOH NaCN + H2O

The analysis of the distillate for hydrocyanic acid is started with the reaction

of Prussian blue formation; highly sensitive and specific:

2NaCN + FeSO4 Fe(CN)2+ Na2SO4

Fe(CN)2+ 4NaCN Na4[Fe(CN)6]

3Na4[Fe(CN)6]+ 4FeCl3 Fe4[Fe(CN6)3] s + 12NaCl

This reaction takes place in the alkaline medium; under this condition it is

possible to form Fe(OH)3 and Fe(OH)2, for their dissolution HCl is added:

Fe(OH)3+ 3HCl FeCl3 + 3H2O,

the excess of HCl can slow the precipitate formation.

Conclusion about the presence of cyanide is made in 24–48 hours, because in

the presence of HCN traces and protein admixtures the precipitate appears slowly.

For acceleration of the sedimentation solution of BaCl2 is added; Prussian blue

precipitate coprecipitates on the BaSO4 precipitate.

Prussian blue precipitate is considered to be a material proof for court and

investigation establishments.

Sensitivity of this reaction is 20 µg / ml.

In addition, for detecting hydrocyanic acid the following colour tests may be

used.

The reaction of ferric (III) thiocyanate formation; it is highly sensitive

(10 µg / ml), but non-specific:

KCN + (NH4)2 S2 KSCN + (NH4)2S ammonium polysulphide

H+

3KSCN + FeCl3 Fe(SCN)3+ 3KCl a red colour

The reaction of polymethine formation; it is highly sensitive (0.2 µg / ml),

non-specific:

HCN + Cl2 ClCN + HCl chlorocyane

N N

CN

Cl N

CN

OH

NH

CN

O NH

CN

NC

6H

5

ClCN H2O

-HCl

NH2-C6H5

pyridine N-cyanpyridine chloride

polymethine dye

(an orange colour)

+ +

The reaction of benzidine blue formation;it is sensitive, non-specific:

2HCN + Cu(CH3COO)2 Cu(CN)2+ 2CH3COOH

2Cu(CN)2 (CN)2 + 2CuCN dicyane

(CN)2 + H2O O + 2HCN

NH2

NH2

NH2

NH2

NH NH

2HX +

H2O

2

. 2HX +

O

benzidine (a blue colour)

Paper moistened by copper salt solution and benzidine turns blue in the

presence of hydrocyanic acid or its salts.

The analysis of the second distillate

The analysis of the second distillate is started with the reactions for

halogenated hydrocarbons.

Halogenated hydrocarbons

Lethal doses for some halogenated hydrocarbons are as follows:

for chloroform – CHCl3 the lethal dose is 50–70 g;

for tetraclormethane – CCl4 the lethal dose is 20-50 ml;

for chloralhydrate – CCl3-COH · H2O the lethal dose is 10 g and less;

for 1,2-Dichloroetane – CH2Cl-CH2Cl the lethal dose is 15–25 ml.

Toxic action. Chloroform and chloral hydrate are narcotics. At first, they

excite and then depress the nervous system. Acute ingestion of as little as 10 ml of

chloroform may result in death due to the central nervous system depression.

Tetrachloromethane acts on an organism like chloroform, but slower and

causes more considerable disorders in organs; the liver and kidneys are subjected to

fatty degenerative changes.

Dichloroethane according to the narcotic action is the strongest poison among

halocarbons.

Halocarbon poisonings are accompanied by vomiting, diarrhea, swelling of

the stomach, increasing and sickliness of the liver.

Metabolism has not been studied completely, carbon dioxide and

hydrochloric acid are the eventual results of the metabolic processes:

CHCl3 CO2 + HCl

CCl4 CHCl3 and CO2

The feature of halocarbon isolation is distillation to the first portions of the

distillate. With a great amount of the poisons (less than 1 g) drops of organic liquid,

which does not mix with water are observed in the distillate.

The analysis is started with general (non-specific) and little-sensitive reaction

on Cl- with AgNO3 after separation of the organic bonded chlorine. This reaction

is characteristic for all halocarbons:

CO

HCl

3C

C ONa

O

H

C ONa

O

H

CH2

CH2

OHOH

CH2

CH2

ClCl

NaOH

CHCl3

CCl4

NaCl + H2O +

NaCl + CO2 + H2O

CHCl3 + H2O +

NaCl +

NaOH

(the reaction mixture is heated in a soldered ampoule for 4 hours)

Cl- + Ag+ AgCl (s)

A white precipitate or white lees appear when Cl– -ion is present.

If the lees or the precipitate do not appear, Fujivara reaction (based on the

polymethine formation) is performed; it is sensitive, non-specific.

The reaction of isonitril formation; it is highly sensitive, non-specific

(among the halocarbons mentioned only dihloroethane does not give this reaction):

NH2

N+

CCHCl3

OH-

isonitril

(unpleasant odour appears)

With the positive result of this reaction the complete analysis for halocarbons

is performed.

The reaction with resorcinol in the alkaline medium; it is sensitive; non-

specific (aldehydes, formic acid give this reaction). The mechanism has been studied

incompletely:

OH

OH

O

OH

R

H

OO

OH

H

R

keto-form

A pink colour appears.

The reaction with Fehling’s reagent; it is little sensitive and non-specific (for

aldehydes), tetracloromethane and dichloroethane do not give this reaction.

CuSO4 + 2NaOH Cu(OH)2 + Na2SO4

COOK

CH

CH OH

OH

COONa

CH

CH

O

OH

O O

CH

CH

COOK

OH

O

OOCOONa

H

H

Cu2

Cu(OH)2

-KOH

-NaOH

CH

CH

COOK

OH

OH

OO

CH

CH

OH

OH

O O

COONa

R C

O

H

R C

O

ONa

COOK

CH

CH

OH

OH

COONa

Cu+

3NaOH

2KOH

+ 2CuOH + 2H2O + 4

2

2CuOHt

Cu2O + H2O

The reaction with Nessler`s reagent gives only chloralhidrate in the group

of halogenated hydrocarbons:

Cl3C C

O

H

Cl3C C

O

K

+ K2[Hg I4] + 3KOH + 4KI + Hg + 2H2O

An orange precipitate is observed, after a while it becomes green.

Dichloroetane gives some general reactions for halogenated hydrocarbons

(Fujivara’s reaction, separation of organic bonded chlorine):

The reaction of separation of organic bonded chlorine:

Cl CH2

CH2

Cl OH CH2

CH2

OH+ NaOHto

+ 2NaCl

A white precipitate of silver chloride appears after addition of argentum

nitrate in the nitric acid medium.

Then the detection of ethylene glycol is carried out by oxidation to formal-

dehyde followed by identification of formaldehyde with the help of the reactions

described for formaldehyde above:

HO–CH2–CH2–OH + KIO4 2HCHO + KIO3 + H2O

The reaction of copper acetylenide formation:

Cl CH2

CH2

Cl CH CH

CH CH CuC CCu

C2H5OH

t + 2NaOH + 2NaCl + 2H2O

+ 2CuNO3 + 2NH4OH + 2NH4NO3 + 2H2O

a pink or red colour

Then the second distillate is examined for the presence of formaldehyde.

Formaldehyde

The lethal dose is 15–25 ml.

Toxic action. Formaldehyde vapour disturbs respiration, causes sharp cough,

lacrimation. The oral administration of formaldehyde is accompanied by nausea,

cramps, faint, circulatory collapse, damage of the stomach and small intestine,

kidneys.

Formaldehyde metabolism occurs according to the following mechanism:

O

H

H

O

H

OH

[O] [O]CO2 + H2O

The analysis of the distillate for formaldehyde is started with the following highly

sensitive reactions:

The reaction with chromotropic acid, a violet or red-violet colour indicates

the presence of formaldehyde. The reaction is non-specific because substances,

which in hydrolysis, dehydration or oxidization form formaldehyde, give this

reaction too.

SO3H

OH

OH

SO3H

H C

O

H

SO3H

OH

OH

SO3H

CH

2

HSO3

OH

OH

HSO3

SO3H

OH

OH

SO3H

CH

HSO3

O

OH

HSO3

+2- H2O

[O]

+ H2O

The reaction with codeine and sulphuric acid, a blue-violet or red-violet

colour appears:

O

OH

OCH

3

NCH

3

O

OH

OH

NCH

3

H

H

O

O

OH

OH

NCH

3

CH

2

O

OH

OH

NCH

3

codeine morphine

H2SO4 conc

- CH3OH

The reaction with fuchsin-sulphurous acid; it is non-specific because

aldehydes (furfurol, acetaldehyde, etc.) and even oxidants of the air (chlorine,

oxygen, nitrogen oxides) give this reaction. A dark blue colour of the solution can

appear in 10–15 min. However, when colour appears in half an hour, it is not the

positive result for aldehydes.

It should be noted that under certain conditions this reaction can be specific

for formaldehyde – only formaldehyde gives this reaction in the strong acidic

medium (pH 0.7) and a lot of aldehydes give it at pH 2.7

Reactions with resorcinol, Fehling reagent, reduction of silver are less

sensitive and non-specific, but they are used when the positive results of the highly

sensitive reactions described above are obtained.

With the negative results of the reactions for formaldehyde the analysis for

alcohols (methanol and ethanol) and then for ketones (acetone) is carried out.

Methanol, ethanol and isopentanol have the most toxicological value in the

group of monobasic alcohols.

Alcohols are used in medicine, manufacture, food industry.