Methods of organic analysis

G r o u p Analysis and Detailed Analysis Ion Exchangers in Analytical Chemistry Methods of Organic Analysis Chemical Microscopy Thermomicroscopy of Organic Compounds Gas and Liquid Analy

Translated by

Ildiko Egyed and Judit Gaal

JOINT EDITION P U B L I S H E D BY

ELSEVIER SCIENTIFIC P U B L I S H I N G C O M P A N Y , A M S T E R D A M , THE

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Mazor, Laszlo

Methods of organic analysis

(Wilson and Wilson's Comprehensive analytical

chemistry; v 15)

Rev translation of: Szerves kemiai analizis

Bibliography: p

Includes index

1 Chemistry, Analytic, 2 Chemistry, Organic

I Title II Series: Comprehensive analytical

chemistry; v 15

QD75.W75 vol 15 [QD271] 543s [547.3] 81-17371

A A C R 2 ISBN 0-444-99704-0 (Vol XV)

ISBN 0-444-41735-4 (Series)

© A K A D E M I A I K I A D O , B U D A P E S T 1983

All rights reserved N o part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher

Printed in H u n g a r y

Professor of Analytical Chemistry, University of Chemical Engineering, Veszprem

H.M.N.H Irving, M.A., D.PHIL., F.R.S.C

Professor of Inorganic Chemistry, University of Cape Town

Alan Robinson, B.A

Department of Pharmacy, The Queen s University of Belfast

Wilson and Wilson's

C O M P R E H E N S I V E ANALYTICAL C H E M I S T R Y

Edited by

G Svehla, PH D., D SC., F.R.S.C

Reader in Analytical Chemistry

The Queen s University of Belfast

WILSON A N D WILSON'S

C O M P R E H E N S I V E ANALYTICAL C H E M I S T R Y

V O L U M E S IN THE SERIES

Analytical Processes Gas Analysis Inorganic Qualitative Analysis Organic Qualitative Analysis Inorganic Gravimetric Analysis

Inorganic Titrimetric Analysis Organic Quantitative Analysis Analytical Chemistry of the Elements Electrochemical Analysis

Electrodeposition Potentiometric Titrations Conductometric Titrations High-frequency Titrations

Liquid Chromatography in Columns Gas Chromatography

Ion Exchangers Distillation Paper and Thin-Layer Chromatography Radiochemical Methods

Nuclear Magnetic Resonance and Electron Spin Resonance Methods

X-Ray Spectrometry Coulometric Analysis

Elemental Analysis with Minute Samples Standards and Standardization

Separations by Liquid Amalgams Vacuum Fusion Analysis of Gases in Metals Electroanalysis in Molten Salts

Instrumentation for Spectroscopy Atomic Absorption and Fluorescence Spectroscopy Diffuse Reflectance Spectroscopy

Emission Spectroscopy Analytical Microwave Spectroscopy Analytical Application of Electron Microscopy Analytical Infrared Spectroscopy

Thermal Methods in Analytical Chemistry Substoichiometric Analytical Methods

Enzyme Electrodes in Analytical Chemistry Molecular Fluorescence Spectroscopy Photometric Titrations

Analytical Applications of Interferometry Ultraviolet Photoelectron and Photoion Spectroscopy Auger Electron Spectroscopy

Plasma Excitation in Spectrochemical Analysis

Organic Spot Test Analysis The History of Analytical Chemistry

The Application of Mathematical Statistics in Analytical Chemistry

Mass Spectrometry Ion Selective Electrodes

Thermal Analysis Part A Simultaneous Thermoanalytical Examinations by Means of the Derivatograph Part B Biochemical and Clinical Applications of Thermometric and Thermal Analysis

Analysis of Complex Hydrocarbon Mixtures Part A Separation Methods

Part B G r o u p Analysis and Detailed Analysis Ion Exchangers in Analytical Chemistry Methods of Organic Analysis

Chemical Microscopy Thermomicroscopy of Organic Compounds Gas and Liquid Analysers

Kinetic Methods in Chemical Analysis

Vol XIII A

] ]

To my grandchildren Corinna and Dominique Nobilis

Preface

In Comprehensive Analytical Chemistry, the aim is to provide a work

which, in many instances, should be a self-sufficient reference work, but where this is not possible, it should at least be a starting point for any analytical investigation

It is hoped to include the widest selection of analytical topics that is possible in the compass of the work, and to give material in sufficient detail to allow it to be used directly, not only by professional analytical chemists, but also by those workers whose use of analytical methods is incidental to their work rather than continual Where it is not possible to give details of methods, full reference to the pertinent original literature is made

Volume XV covers one topic: organic analysis In earlier volumes there were some chapters devoted to this field The Author's Preface makes it clear that overlaps and repetitions have been avoided, as far as possible Tlje present text describes the subject in more depth and detail than the earlier chapters, and covers developments which have occurred since their publication The author has published very successful books on the subject in Hungarian and in English; we hope that this English version will be equally well received The present Editor remembers with affection those years which,

as a student and later as junior colleague, he spent in close association with Professor Mazor

Dr C L Graham of the University of Birmingham, England, assisted in the production of the present volume; his contribution is acknowledged with many thanks

July, 1982 G Svehla

Nowadays, not least because of skilful advertising by instrument manufacturers, instrumental methods seem to have taken over the traditional fields of qualitative and quantitative analysis, although experts often emphasize that classical chemical methods still retain a definite role even today Undoubtedly, routine-type quantitative determinations can be performed very well with automatic devices, in qualitative analysis the importance of ultraviolet, infrared, N M R and mass spectrometry steadily increases, and modern chromatographic methods also have their role to play Even the aforementioned chemical microanalytical methods do not require skilled, highly trained chemists, because all the devices, with their spare and optional parts, are of reliable quality, even though mass produced Their creation formerly needed the special skill of the microanalyst Thus, qualified chemists may be reserved for research, development or management, while routine work can be left to skilled technicians and assistants

Anyone who follows the vast amount of research and development now taking place in the field of organic analysis, but who at the same time is familiar with the work done in industrial laboratories, would agree that

because of the different requirements of industrial and research organic chemists, the science of organic analysis is slowly splitting into two distinguishable groups of techniques

The first group consists of methods that still can be called chemical, being

applied mainly in industrial laboratories, where the task is to identify, monitor the purity of, or determine the active ingredient content of raw materials, intermediates and final products This is partly done by determining certain physical characteristics and partly by chemical elemental and functional group analysis These techniques are often augmented by UV and IR spectrometry and by gas-liquid chromatography

Research in organic chemistry, extending to the preparation of new compounds, establishment of their structure and the examination of the kinetics and mechanisms of the reactions involved, also requires the methods mentioned in the first group, but most of the analytical work employs modern instrumental techniques, such as mass spectrometry, high-performance liquid and gas chromatography, nuclear magnetic resonance spectroscopy and so on Some of the chemical methods have already been discussed in earlier volumes of Comprehensive Analytical Chemistry Thus, a short, mainly a practical survey of qualitative organic analytical methods is found in Chapter

V of Volume IA, and quantitative micro-methods in some detail in Chapter VIII of Volume IIB A number of separation methods, such as gas, liquid and ion-exchange chromatography, are discussed in Volume IIA, spectroscopic instrumentation and diffuse reflectance methods in Volume IV, microwave spectroscopy and electron microscopy in Volume V, and Volume VI is devoted entirely to (mainly organic) infrared spectrophotometry When writing my text for Comprehensive Analytical Chemistry, I had these volumes

in mind, trying to avoid unnecessary repetition I was especially careful not to repeat what has been well described in Volume IIB, restricting the discussion

of elemental and functional group analysis to recent developments only I also avoided theoretical or practical aspects of the instrumental techniques mentioned, emphasizing only those details which are relevant to the particular case under discussion References to the literature are given at the end of each chapter Most of these are dated post-1960 to cover recent developments, the earlier literature being well listed in Chapter VIII of Volume IIB

I wish to acknowledge the assistance of my co-workers, who were helpful in testing some of the methods in the laboratory and in surveying the literature Thanks are also due to Elsevier Scientific Publishing Company and the printers of the Publishing House of the Hungarian Academy of Sciences for their efforts in producing this volume

Budapest, August, 1982 Laszlo Mazor

The identification of organic compounds with authentic samples becomes more difficult when the molecular weight of the compound is high, and when a distinction between compounds with similar structures and molecular weights (e.g., isomers) must be made

Organic chemists have dealt with the identification of organic compounds

by means of chemical reactions for many years One of the first researchers was H SchifT, who reported a sensitive reaction suitable for the identification

of urea in 1859 However, in the early days, only few really specific reactions were known, most being suitable only for the detection of a given group of compounds (e.g., alkaloids) Intuition often helped the recognition of new reactions, and the mechanisms of some of them are still not properly understood Systematic research work started only in the 1930s, with a knowledge of the chemical properties of the compound to be detected and the reagent, and a presumed course of the reaction In this field, Feigl [1] developed the method of spot test analysis, and also achieved outstanding

TABLE 1

Qassification of organic chemical analysis

(Separation if the material is not homogeneous)

Physical constants

Spectral analysis

ELEMENTAL ANALYSIS

Chemical methods formulae

GROUP ANALYSIS

Chemical met

Physical lods

of structure) Sometimes only some physical methods, e.g NMR

results in the development of specific reactions and in the elucidation of reaction mechanisms Today there are specific microreactions available that make possible the detection of characteristic functional groups or the substance itself in 0.1-1 jig samples by means of spot tests on slides or on filter-paper The reagents may be either inorganic or organic The disadvantage of organic reagents is that reactions between different organic compounds are usually slower and less complete than reactions between ions or between organic compounds and ions This difficulty led to attempts to decompose first the organic compounds of higher molecular weight, which are insoluble

in water, by applying thermal energy or oxidizing or reducing agents, in order

to obtain simple water-soluble or volatile simple inorganic substances (acids, ammonia, hydrogen sulphide, sulphur dioxide, etc.) or organic substances (e.g., aldehydes) that can be detected easily and sensitively These compounds can also be converted by oxidation or reduction or with appropriate reagents

in such a way that the product can be detected in the reaction mixture specifically and without interference

Instrumental methods have gained increasing importance in organic qualitative analysis Infrared spectrophotometry and gas chromatography make possible the detection of not only the functional group but also the entire structures of complex molecules The work is easier when standard compounds are also available and the spectra and chromatograms of the sample and the reference substance can be compared directly However, useful information can often be obtained from the appearance of bands characteristic of certain functional groups or bonds in the IR spectra, or from retention indices when using gas chromatography With homologous series of compounds, Kovats retention indices can be used for identification purposes The IR spectra can be compared with literature data and accurate identification is possible on the basis of the "fingerprint" pattern Pyrolysis gas chromatography and reaction gas chromatography provide possibilities not only for more accurate identification through decomposition or conversion of complex molecules, but also for the establishment of the composition of mixtures from the results

Even higher performance is offered by mass spectrometry owing to its much higher resolution The most modern technique, gas chromatography combined with mass spectrometry, is employed in the determination of the structures of complex molecules and the compositions of mixtures

The identification of a homogeneous pure organic substance is relatively simple by applying either chemical or instrumental methods However, the analysis of mixtures of organic compounds and the identification of the constituents is much more difficult and may be almost insoluble In almost all instances preliminary separation is necessary, as it is rarely possible that the components of a mixture (even with binary mixtures) can i>e detected specifically in the presence of each other When a large amount of sample is available, separation can be carried out by, e.g., fractional crystallization, sublimation, distillation, steam distillation or extraction; as quantitative separation is not necessary, only small amounts of pure fractions need to be obtained In suitable micro-scale apparatus these simple separation oper-ations can be accomplished with a few milligrams of sample

Today, in the separation of organic compounds for qualitative analytical purposes, paper, thin-layer and ion-exchange chromatographic techniques

predominate The suitable choice of the paper or layer material and developing mixtures to ensure optimal separations, and the use of the most sensitive detection reactions, make possible the identification of compounds with very similar chemical and physical properties in multicomponent mixtures The relative retention often offers useful information, but a more reliable method is

to effect simultaneous development of an authentic substance

Column, liquid and ion exchange chromatography are less important in qualitative analysis, and gel chromatography and ultracentrifugation are suitable primarily for the separation of high-molecular-weight biological substances and polymers

Systematic analysis of organic compounds should also be discussed here Starting with the work of H Staudinger, many attempts have been made to develop systematic analytical procedures for the identification of unknown organic substances similar to the system elaborated for particular groups of and elements in inorganic compounds However, owing to the fundamental difference in the nature of organic and inorganic compounds, such a system with general applicability could not be worked out The best known and most widely used procedure was described by R L Shriner, R C Fuson and D Y Curtin in their book "The Systematic Identification of Organic Compounds", published in 1956 The method consists of the following steps:

1 Preliminary tests;

2 Determination of physical constants;

3 Detection of elements in the compound;

4 Detection of functional groups by chemical and spectroscopic methods;

5 Checking literature data;

6 Preparation and examination of derivatives

Experts in organic chemistry and organic chemical analysis need not, of course, follow the steps listed above in all instances when an unknown organic substance is to be identified, as one or two of the tests will often provide firm evidence of the identity of the compound However, the above tests cannot be applied routinely like the tests in inorganic analysis, and careful consideration

of the individual results is necessary Any method may be useful and none should be regarded as out-of-fashion and, except for spectroscopic tech-niques, all of them will be dealt with in the first part of this book

A very practical although less systematic survey of qualitative organic chemical analysis was given in Volume IA of CAC (In: Wilson and Wilson's series: McGookin) [2] where, in addition to the preliminary tests and detection methods for elements and functional groups, specific reactions and the use of derivatives for identification purposes are also reported on the basis

of literature data published up to 1957

Chapter 2

Preliminary tests, identification of organic

compounds by sensory tests, simple physical

and chemical methods, and on the basis

of thermal decomposition products

It is possible to obtain some information on a sample simply by observing its appearance This is easier with inorganic substances, as certain inorganic ions [copper(II), chromium(III), chromium(IV), etc.] have characteristic colours, and some compounds have characteristic crystal forms (sodium chloride, alums), whereas most organic compounds are colourless liquids or white powders Literature data regarding the crystal forms may be available, but polymorphism often occurs Several compounds have different crystal forms when crystallized from different solvents, and heating may also give rise

to such changes (e.g., diethylbarbituric acid) Here not the crystal form itself, but the change brought about by thermal action or by the use of another solvent may be characteristic

The density of liquids may also be a characteristic property, often without instrumental determination of its actual value, as the mobility, viscosity, foaming, etc., of liquids are related to density The crystal forms can be observed under a microscope and the information obtained may be useful, but only when the solvent or solvent mixture used for crystallization is also known For example, 2,7-dihydroxynaphthalene is obtained as needles from water or aqueous ethanol, whereas plates appear on crystallization from glacial acetic acid However, when solutions of the same concentration ( 1 -

areas on a microscope slide under appropriate identical conditions, the shape

of the crystals formed can provide some information regarding the type of substance involved

Several different compounds crystallize in the same form from the same solvent This isomorphism can be eliminated by the use of solvent mixtures with suitable variations of the components and their concentration Misleading information can be obtained if polymorphism occurs; therefore,

when such an occurrence is suspected, the substance in question should be repeatedly crystallized (preferably under identical conditions) until the most stable crystal form is obtained A more effective method is to increase the number of components in the solvent mixture used

The microcrystallized product can be used for further physical and chemical tests In examining the crystals, a polarizing microscope is useful: the picture obtained with crossed Nicols is often very characteristic The so-called microcrystal test is based on the fact that certain compounds form precipitates with characteristic crystals' shape on addition of appro-priate reagents This is a very old method and can be regarded as one of the first tests in qualitative organic microanalysis Several systematic procedures have been developed for the recognition of certain compound groups (e.g., alkaloids) Most recently, Fulton [3] has dealt with this method and described microcrystal tests for 159 organic compounds, mainly drugs and drug precursors

The microcrystals obtained in this test were divided into nine groups by Fulton on the basis of the shape of crystals, the direction of growth, aggregation phenomena, the manner of branching of crystal aggregates, etc

In this system, 25 reagents are applied (acids, bases, complex salts, etc.) and the reactions taking place are divided into nine groups

This procedure is actually a chemical method similar to the spot tests suitable for the detection of elemental constituents and functional groups in organic compounds The difference is that in the microcrystal test the reactions are carried out on a microscope slide and the result is observed with

a microscope of 30-50-fold magnification In contrast to spot tests, which will

be dealt with at length in the chapter devoted to qualitative chemical analysis, the microcrystal test also makes use of the crystal shape, but identification is based exclusively on this feature and is somewhat uncertain because of other phenomena following crystallization

Organic compounds, unlike inorganic compounds may have very characteristic odours, usually indicating the group of compounds to which the substance belongs Some groups of compounds with characteristic odour are listed in Table 2

The smell of liquids is enhanced by heating, and the smell of solids will also become more intense when some crystals are rubbed in the hand There are compounds and compound groups with characteristic tastes, but such testing should be avoided, as several substances are toxic even in very small amounts

It must be emphasized that these sensory tests provide limited information, which depends strongly on the experience of the observer Chemists with a Hmited knowledge of materials cannot make use of these characteristics, and even for experts they represent initial guidance only

TABLE 2

Some compounds with characteristic smell (according to Linne-Zwaadenaker)

Character of smell Compound

Ether-type Ethyl acetate, ethanol, acetone, amyl acetate Aromatic, almond smell Nitrobenzene, benzaldehyde, benzonitrile

Aromatic, camphor smell Camphor, thymol, saffrole, eugenol, carvacrol Aromatic, lemon smell Citral, linalol acetate

Balsamic, flower smell Methyl anthranilate, terpineol

Balsamic, lily smell Heliotropine (piperonal), styrene

Balsamic, vanillin smell Vanillin, anisaldehyde

Musk smell Muscone, trinitroisobutyltoluene

Garlic smell Ethyl sulphide

Cacodyl oxide smell Cacodyl, trimethylamine

Tar smell Isobutanol, aniline, p-isopropylaniline, benzene,

cre-sol, guaiacol Rancid smell Valeric acid, capric acid, methyl heptyl ketone

Organic compounds are usually insoluble in water, some are slightly soluble in water and readily soluble in alcohols, while most are soluble mainly

in apolar solvents The dissolution of liquids in liquids, that is, mixing, may also be characteristic, but much more apolar liquids than solids can be dissolved in or mixed with water and alcohols

Dissolution of organic compounds in organic solvents is often not merely a simple physical process, but a certain interaction occurs between the molecules of the solute and the solvent; even chemical reactions can take place As a result of this interaction, the molecules of the solvent in the vicinity

of the solute molecules may possess properties different from those in the bulk

of liquid For example, a strongly polar solute exerts a polarizing action on the solvent molecules in its vicinity The solvent may facilitate reactions between solutes, and therefore the solvent cannot always be regarded simply

as an inert medium

In view of these facts, the earlier classification of solvents (polar and apolar) should be replaced by another system (Table 3):

1 Protic solvents;

2 Apolar or less aprotic solvents;

3 Dipolar aprotic solvents

Of the protic solvents, the most important are water, alcohols, amines and carboxylic acids The second group consists of hydrocarbons, chlorinated

TABLE 3

Physical constants of some solvents

Solvent Melting point Boiling point Relative

hydrocarbons, benzene, dioxane and pyridine The most important solvents

in G r o u p 3 are acetone, nitrobenzene, nitromethane and dimethylformamide Protic solvents (Group 1) possess both nucleophilic and electrophilic properties In these solvents the anions are strongly solvated and the mobile hydrogen atoms of these solvents are often capable of forming hydrogen bonds For example, with the bromide ion we have:

When such solvents also have a high permittivity (water, formamide), they greatly facilitate spontaneous ionization, that is, SN1 reactions The solvent molecules usually form associates through hydrogen bonds

Solvents in G r o u p 2 have relatively low dielectric constants Some of them, e.g., diethyl ether, dioxane and tetrahydrofuran, are strongly nucleophilic The solvents in G r o u p 3 have relatively high permittivity In dissolution reactions these behave like those in G r o u p 2, but ion aggregates are formed to

a lower extent in comparison with solvents with lower permittivities (less than 15) From the practical point of view, acetone, dimethylformamide, dimethylacetamide and dimethyl sulphoxide are the most important solvents; they are miscible with water Both strongly and weakly nucleophilic solvents (dimethyl sulphoxide and acetonitrile, respectively) appear in this group

For inorganic ions, by the middle of the last century a system had already been developed and used in which different reagents made possible the precipitation and complete separation of inorganic ions (e.g., the Fresenius method for separation of cations as sulphides) Later attempts were made to develop a similar system for the grouping and separation of organic compounds At that time, sufficient knowledge had been gathered on the easily determined properties of organic compounds (volatility, solubilities in different solvents, etc.) However, owing to the rapid increase in the number of known organic compounds, these trials, which had seemed to be successful at first, lost their importance In practice, the most modern methods can still be useful when an unknown organic substance is to be identified, but should be regarded as informatory data and preliminary tests only

At the beginning of the twentieth century, Th Mullikan and H Staudinger developed almost simultaneously a "solubility system" which has been used

up to the present time without substantial modifications The latest edition of Staudinger's book, modernized and supplemented, was published in 1968 Staudinger classified organic compounds according to their melting point, volatility (boiling point) and solubility, regarded as physical characteristics related to molecular weight With respect to volatility, the limiting temperature is 160°C At lower temperatures compounds can be distilled without decomposition (in some instances, distillation is carried out at reduced pressure) Measurement of the melting point and boiling point and their comparison with literature data provide useful information for identification However, neither of these data are suitable for use in the classification of organic compounds, as they are hardly related to their chemical properties

The solubility of a substance in certain solvents and reagents is more suitable for classification purposes because, as discussed in connection with

the mechanism of dissolution, this may indicate the chemical properties of a given compound

O n the basis of solubility, Staudinger divided organic compounds into the following five groups (today this system is obsolete and is primarily of historical importance):

1 Compounds soluble in diethyl ether and insoluble in water As the permittivity of diethyl ether is low (4.35), it belongs to the less polar aprotic solvent group, and therefore a larger number of compounds are soluble in it than in solvents with definitely apolar character, such as carbon tetrachloride and benzene Several solvents with similarly low relative permittivities are known (chloroform, dioxane, etc.), but diethyl ether was preferred because of its easy removal after the test, which allows the the same sample to be used in further examinations According to Staudinger, compounds soluble in diethyl ether and insoluble in water are definitely organic in character

2 Compounds soluble in both diethyl ether and water These are called compounds of mixed organic-inorganic nature The permittivity of water and diethyl ether differ greatly, the mechanisms of their dissolution are also different, and ionization may occur in water The relative permittivities of two frequently used alcohols, methanol (33.7) and ethanol (25.8), lie between those of water and diethyl ether They can dissolve most compounds with

"clearly organic character" in G r o u p 1 and also certain salts, and are particularly good solvents for compounds containing hydroxyl group(s)

3 Compounds soluble in water and insoluble in diethyl ether These are organic substances with a somewhat inorganic character Primarily salts of low-molecular-weight organic acids belong to this group, and are limited in number

4 Compounds insoluble in both diethyl ether and water This group consists of high-molecular-weight organic compounds, such as polycarbo-xylic acids, acid amides with an otherwise mixed organic-inorganic character and insoluble salts (e.g., alkaline earth metal and other metal salts of organic acids), which can be regarded as inorganic compounds, irrespective of their solubility

5 There are some groups of compounds that are soluble in diethyl ether, which fundamentally belong to G r o u p 1, but which undergo decomposition

in water This group includes acid halides and isocyanates

The members of the above five groups may be volatile, like the molecular-weight compounds in Groups 1, 2 and 5, or non-volatile (or volatilized only after decomposition), which are usually compounds with higher molecular weight in any of the five groups

low-With compounds that are soluble in diethyl ether, further information can

be obtained after the simple dissolution test by use of so-called reactive

solvents An example is 5% hydrochloric acid, which dissolves basic substances, such as amines, with the formation of hydrochlorides

Compounds with acidic character are soluble in 5% sodium hydroxide or 5% sodium hydrogen carbonate solutions while forming salts Organic acids, mainly those of low molecular weight with acid dissociation constants higher

than that of carbonic acid (4.3 x 1 0 "7) (e.g., acetic acid, 1.7 x 1 0 "5) undergo dissolution while releasing carbon dioxide Weaker acids, with acid dissociation constants higher than the second acid dissociation constant of

carbonic acid (5.6 x 1 0 "1 1) are dissolved without the formation of carbon

dioxide, like diethylbarbituric acid (3.7 x 10~8) Even weaker acids, if their sodium salts are soluble in water, are dissolved in 5% sodium hydroxide solution, which, of course, also dissolves the stronger acids When a freshly prepared solution of 5% sodium hydrogen carbonate is made pale pink with one drop of phenolphthalein, the colour disappears under the influence of weak acids, whereas it becomes enhanced in the presence of basic compounds

If the sample is soluble in water or water-alcohol (1:1), the reaction of the solution can be checked with a universal indicator solution or a p H indicator paper, and this can indicate the group to which the compound in question belongs

Neutral compounds, insoluble in water, and therefore insoluble in acids and bases also, can further be tested with concentrated sulphuric acid If the substance dissolves in it, a simple dissolution process explainable by the high permittivity of concentrated sulphuric acid (84.0) or sulphonation of the substance can be considered If the substance is soluble in concentrated sulphuric acid, the solution tests are continued with concentrated orthophos-phoric acid, which also has a high permittivity This is the most suitable solvent for alcohols with less than nine carbon atoms, aldehydes, methyl ketones, alicyclic ketones and esters Similar compounds with more than nine carbon atoms and also quinones and unsaturated hydrocarbons, are insoluble in this reagent

Saturated aliphatic hydrocarbons, aromatic hydrocarbons and their halogen derivatives are insoluble in both concentrated sulphuric acid and orthophosphoric acid

There are compounds that to not fit into either of the groups discussed above, e.g., nitro compounds, amides, negatively substituted amines, nitriles, azo compounds, hydrazo compounds, sulphones, thiols and thioethers All of these compounds contain nitrogen or sulphur atoms

Heating and combustion tests The most simple means of ensuring that a

sample is an organic substance is to heat it with concentrated sulphuric acid

or chromic acid [4] Organic substances turn black under the influence of

concentrated sulphuric acid, or turn the yellow colour of chromic acid mixture green, owing to the reduction of chromium(VI) ions

The thermal decomposition of several organic compounds yields pounds with lower molecular weight and characteristic chemical properties These may be inorganic (hydrogen sulphide, hydrogen cyanide, etc.) or organic compounds (formaldehyde, acetaldehyde, methanol, acetic acid, etc.) The same compound may give different decomposition products when heated under oxidizing or reducing conditions For example, sulphur-containing compounds may release hydrogen sulphide or sulphur dioxide when heated under reducing or oxidizing conditions, respectively Certain compounds undergo decomposition with the formation of volatile aldehydes or acids, and these decomposition products can be detected in the vapour by means of simple reactions Detectable decomposition products of some compounds are shown in Table 4

com-In the heating test the sample is placed in a narrow test-tube, the open end

of which is covered with paper impregnated with a suitable reagent, then heating is cautiously started Reducing conditions are ensured by the carbon

-and hydrogen contents of the substance Air can be expelled from the test-tube with an inert gas prior to starting the test

The ignition test is carried out as follows About 30-50 mg of sample are placed on the end of a narrow porcelain plate, then this end is moved slowly towards the small, colourless flame of a micro burner Under the influence of heat, certain substances undergo sublimation or vaporization, and the vapours are ignited in the flame In this way, compounds with high carbon and low oxygen contents can be clearly recognized, as their vapours make the flame strongly luminous and sooty Almost all aromatic compounds show this behaviour Compounds with low carbon content, relatively rich in oxygen (mainly aliphatic compounds except for high-molecular-weight hydrocarbons) produce a slightly luminous or colourless flame Some substances swell on heating, then undergo melting and boiling; they usually contain water of crystallization Others exhibit explosion-like phenomena during burning, such as nitro compounds Polyhalogenated compounds burn only slightly or not at all The odour of combustion products can be very characteristic; sugars have a caramel smell, and proteins give off a smell of burning hair However, these smells are not given by all carbohydrates and amino compounds, and all such sensory observations can be very misleading without a thorough knowledge of the materials involved

Preliminary tests with mixtures With liquid or powder mixtures (e.g., drug

formulations) the preliminary tests reviewed above are less promising than with homogeneous substances but, in favourable situations when only two-

or three-component mixtures are involved, they can still be applied With liquid mixtures, when not starting directly with fractional distillation, qualitative evaporation tests can be carried out A few drops of the liquid are heated on a watch-glass and, if the boiling points of the components are significantly different, this fact can be observed If a solid residue is obtained, the sample was, in fact, a solution

Solubility tests with powder mixtures can be accomplished in a conical test-tube with 0.1 c m3 divisions When two or more components are present

in similar amounts, the decrease in the amount of sedimenting non-dissolved material can be noted The test is repeated with the residue using another solvent, and the solubilities of two or more components in various solvents can be established

With powder mixtures, heating tests can provide useful information when the decomposition products of the constituents are different The reagent paper covering the open end of the test-tube is replaced with another while increasing the temperature further

Differences in the behaviour of gases evolved from components that have different decomposition temperatures can also be observed during the

ignition test As in this instance a uniform rate of heating cannot be ensured, subsequent phenomena characteristic of the individual components can be observed only when the decomposition temperatures are greatly different When these tests do not yield unambiguous information, it is better to turn

to simple separation methods on the micro- or semimicro-scale, such as distillation, extraction and sublimation Preliminary studies on the com-ponents separated qualitatively are recommended prior to starting systematic analysis

The information obtainable from preliminary tests may provide valuable help, but it should be stressed that simple separation methods are rarely complete and the products should be regarded as contaminated even in the preliminary tests

Thermo-micro technique When discussing the preliminary tests, it was

mentioned that organic compounds often undergo characteristic changes during heating In the course of the heating and ignition tests, the thermal decomposition products are identified by sensing or simple chemical reactions These methods are regarded as preliminary tests, as they are rather uncertain and the same phenomenon or reaction may be obtained with several different compounds Several tenths of a gram of sample are required for these examinations

With the development of Koflers' hot-stage microscope a new testing technique called the "thermomicro method" become available This is not only suitable for the determination of certain physical constants (melting point, molecular weight, refractivity, etc.), but also several important conclusions can be drawn from the behaviour of the crystals of organic compounds during heating to the melting point, which also facilitates or supports identification For this purpose, only a few micrograms of sample are required

According to the above description, thermomicro methods are those in which the behaviour of milligram amounts of a sample are studied as a function of temperature in order to obtain information regarding the nature

of the substance They are suitable for the determination of physical constants also, but this aspect will be discussed in detail in Chapter 4

Changes in modification It is well known that substances with identical

chemical properties can take different crystal forms For example, elemental sulphur can exist in three modifications at different temperatures The conversion into the opposite direction during cooling is slow, and the crystal forms stable at higher temperatures can exist for long periods (sometimes several years) in the metastable state This phenomenon of polymorphism and its temperature dependence also occur with organic compounds, in fact more frequently than with elements and inorganic compounds

( a ) ( b ) ( c ) Fig 1 Changes in the crystal form of diethylbarbituric acid with increasing temperature

(a) 175°C; (b) 183°C; (c) 184°C

The temperature below the melting point of the substance at which the

crystal shape changes is called the transition point; such data are given in the

literature for many compounds Thus, for example, plate-shaped crystals of

diethylbarbituric acid turn into needles at a surprisingly high rate between

175°C and 183°C, as shown in Fig 1

The transition is caused by an increase in the internal energy content of the

crystal under the influence of heat; crystal shapes stable at elevated

temperature but unstable at lower temperature are formed The endothermic

or exothermic nature of the conversion can be determined

thermoanalyti-cally; however, the change in heat content during cooling becomes apparent

only when the crystal form actually changes, that is, not at the theoretical

transition point but at the end of the supercooling period

The physical properties of the individual crystal modifications (e.g.,

melting point, heat of melting, density, refractivity, sometimes solubility) are

different For example, the less stable modification is more volatile and more

soluble In this way, a distinction can be made between two modifications at a

given temperature

Polymorphous substances that suddenly turn into an unstable

modification when melted then cooled below the freezing point are called

enantiomorphous These obey the Ostwald law, which states that in physical

changes the less stable modification is formed first Substances that show

unidirectional changes during melting are called monotropic

Sublimation Several organic compounds undergo sublimation before

melting, and the melting point can be determined only in closed systems The

tension of sublimation of these substances in the solid state reaches unity at a

temperature lower than the melting point This can be utilized for the transfer

of a substance from a warmer to a cooler location, and this process is called

sublimation Such substances can be melted only at pressures higher than 105

Pa The temperature of sublimation of organic compounds can be determined, but not with high accuracy Earlier literature data on the temperatures of sublimation of various substances are uncertain and unreliable, as the temperature of sublimation is strongly dependent on the experimental conditions such as pressure, surface area and the distance to be covered by the substance to reach the cooler location

The temperature of sublimation can conveniently be determined with a hot-stage microscope, under controlled conditions A glass ring is placed on a microscope slide lying on the hot stage of the microscope and about 1 mg of sample is placed on the slide in the middle of the ring The glass ring is covered with a glass cover and the microscope is focused on the cover Heating is started at a slow rate until the first crystals of the sublimate appear on the cover The temperature difference between the microscope slide and the glass cover depends on the height of the glass ring

Sublimation can be carried out at lower temperatures under reduced pressure

Almost all substances sublimate at the melting point, and in some instances this may take place to a significant extent (2-3°C) before the melting point is reached; this indicates that this phase transformation is being approached The sublimate often has a crystal shape entirely different from that of the original sample The sublimate may be drops or crystals, or both forms may

be present simultaneously Sublimate drops are usually obtained when the temperature of sublimation is near to the melting point Crystalline sublimates may consist of small crystal grains, needles or plates It often occurs that the individual crystals are surrounded by drops and, as drops have a tension (vapour-pressure) higher than that of the crystals, the surrounding drops will evaporate and be deposited on the crystals, which will thus grow When the substance is very volatile, the crystals appearing on the upper cover will grow into each other and show a textile-like pattern For example, caffeine exhibits this phenomenon, whereas with camphor a dendritic crystal structure develops

There is a correlation between the temperature of sublimation and the melting point The difference between the two values increases with increasing melting point For instance, the sublimation temperature of substances with melting points of 50°C and 200°C are about 40°C and 150°C, respectively The microsublimation procedure carried out on the hot-stage of a microscope is not only a valuable preliminary testing method, but also provides a possibility for purification and separation on the microscale, as the sublimate crystals are usually very pure and are obtained in amounts sufficient for the determination of the melting point or for use in some

microchemical reactions Sublimation can often be applied to the separation

of components of mixtures when only one of them shows this phenomenon or the sublimation temperatures are sufficiently different

In preliminary testing, loss of material during sublimation is usually negligible When, however, only a small sample is available, sublimation should be carried out at a temperature about 10°C lower than the melting point, where the process takes place relatively rapidly, without loss, and the product is sufficiently pure

In the knowledge of the optimal temperature of sublimation, a metal (platinum) cover can be used on the top of the glass ring, and this cover can be cooled If the sublimate is obtained on a relatively cool surface, unstable crystal modifications often occur owing to rapid cooling When sublimation is accomplished slowly (that is, at a relatively low temperature and by the use of

a high ring), it is interesting and informative to observe the growth of the crystals of the sublimate

Decomposition processes Several organic substances undergo

position before the melting point is reached The temperature of position can sometimes found in the literature, but it is often only stated as a fact (e.g "decomposed, caramelized, exploded") instead of giving the melting point in concrete data C The process of partial or complete decomposition

decom-on heating can be recognized from the discoloratidecom-on and degradatidecom-on of crystals Partial decomposition is indicated by the fact that, after the melting point has been reached, the melt solidifies at a temperature far below the freezing point, and this solid has a much lower melting point than the original sample, owing to the depression of the melting point caused by the presence of decomposition products The refractivity is often altered by the decom-position when measured in the melt

If the sample contains water or alcohol of crystallization, these are liberated before the melting point is reached In the literature, the temperature given for the release of water of crystallization is usually 100-110°C but, according to experience, this process may start at 60-70°C in several instances Removal of water of crystallization is indicated by the fact that the originally transparent crystals become opaque or, occasionally, dark coloured Changes in crystal structure often accompany this process The crystals may be suspended in a drop of paraffin oil on a microscope slide, then heated In this way, liberation of gases or vapours in the form of bubbles can be observed if gaseous decomposition products or water vapour (alcohol of crystallization) are released By placing a layer of a suitable reagent on the paraffin phase in a short glass tube with a small inner diameter the gas evolved can even be identified A similar procedure is to cover the glass ring with a glass cover holding a drop of reagent on its lower (inner) side

Monitoring of thermal decomposition processes of organic compounds by, e.g., differential thermogravimetry, recording the weight loss as a function of temperature, is still an unsolved problem Several attempts have been made in this field, but organic substances, except in a few instances such as the decomposition of alkaline earth metal oxalates, show a continuous weight loss even when a very slow heating rate is applied, and no steps appear on the thermogram This is explained by the fact that in the course of thermal decomposition of organic compounds several simultaneous processes take place, and recombination reactions are also possible, and thus the occasional separate steps overlap Therefore, characteristic steps (indicating removal of water of crystallization, decarboxylation under the influence of heat, etc.) can

be observed with the simplest organic compounds by this technique Again, the differential thermoanalytical technique can be applied only with difficulty to the detection of relatively small changes in internal heat such as those due to changes in crystal structure Melting and solidification are accompanied by larger changes in enthalpy, which can be utilized in the determination of melting or freezing points This will be treated in detail in Chapter IV

Other possibilities provided by thermomicro-analysis, such as monitoring changes in crystal structure, examination of the structure of isomorphous crystals, and the behaviour of liquid crystals, and also several other theoretical and practical problems, were discussed in Ref [5]

The technique of thermomicro-analysis not only can be employed in the examination and identification of homogeneous pure organic compounds but also, although to a much more limited extent, it is suitable for studies on binary or multicomponent mixtures For instance, drug powder mixtures of given composition can be recognized from their thermal behaviour

In the main, however, the thermal behaviour of binary or multicomponent mixtures of entirely unknown composition is not characteristic, except when individual crystals of the components can be distinguished (e.g., on the basis

of their colour or shape), under a microscope A mixed melting point is observed during heating, but this provides no basis for recognition Finally, the whole mixture melts, then solidifies on cooling, but the crystal pattern developed is not characteristic of the components either It may happen that during heating, before the mixed melting point is reached, the individual components exhibit different behaviour, for example, undergo sublimation, and thus some of them can be recognized This is essentially a separation process

The other separation technique also applicable to small samples is the called absorption procedure, which is carried out as follows First, the starting (eutectic) melting point of the sample is determined A microscope slide,

so-together with a hardened filter-paper ring is placed on the hot-stage microscope, and a thin layer of the sample to be purified is placed on it A second slide is placed across the first on the filter-paper, they are pressed together, then the hot stage of the microscope is heated to the eutectic temperature determined earlier The eutectic melt is absorbed by the filter-paper and a transparent spot appears Crystals of non-melted material with a melting point higher than the eutectic temperature adhere in a pure state to the glass cover and can be removed from the hot stage Then the filter-paper is replaced with another, the temperature is increased by a few degrees and the procedure is repeated By repeating this absorption process four or five times,

as required, sufficient amounts of satisfactorily pure substance are obtained This can be subjected to identification tests (melting point, refractivity, etc.) The procedure takes 10-15 min

References to Chapters 1 and 2

1 Feigl, F.: Tiipfelanalyse Organischer Teil, Akademie Verlagsgesellschaft, Frankfurt, 1960

2 McGookin, A.: Organic Qualitative Analysis In: Wilson, C L., Wilson, D W (Eds.):

Comprehensive Analytical Chemistry, Elsevier, Amsterdam, 1959 Vol IA, Chapter V

3 Fulton, C G.: Modern Microcrystal Tests for Drugs Wiley, New York, 1969

4 Evans, D L.: Studies in Qualitative Inorganic Analysis Part XXVI Preliminary Tests for Organic Matter, Mikrochimica Acta, 385 (1967)

5 Kofler, L., Kofler, A., Brandstatter, M.: Thermo-Mikro-Methoden Universitatsverlag,

Innsbruck, 1954

of natural origin Phosphorus may be present in certain weed-killers Compounds that contain silicon are treated as a separate branch of chemistry (organo-silicon compounds), and the importance of organometallic com-pounds is increasing

The presence or absence of hetero elements is of crucial importance with respect to further investigations, as preliminary tests for functional groups are based on elemental analysis data

Qualitative elemental analysis can also provide valuable information for quantitative analysis by confirming the presence of the element to be determined and the order of magnitude of the amount present, which may be helpful in deciding the size of sample to be taken or the choice of the methods

to be applied

In the course of qualitative elemental analysis, the covalent bonds between the elements are eliminated and the substance is converted into ions or compounds that can be detected by simple and sensitive reactions It rarely happens that the element can be detected directly in a solution of the organic sample, for example, oxygen can be detected by the Ferrox test, and loosely

bound halogen or sulphur atoms can be detected in certain compounds In most instances, the organic substance must be decomposed, mineralized, and the detection reactions carried out in the solution of the decomposition product, usually by applying methods of inorganic qualitative analysis Mineralization should not necessarily be quantitative, that is, losses may occur in the whole sample or in one or another of the elements or simple compounds to be detected Losses of material, of course, reduce the sensitivity

of detection

In qualitative analysis, an important requirement is to apply sensitive reactions, that is, small samples should be sufficient Therefore, mineralization should be accomplished with relatively small amounts of active agents in vessels of small capacity, so that the residue can be dissolved in a few drops (maximum 1-2 c m3) of water For this purpose, metal micro-bombs, micro-test-tubes or capillary tubes can be used It is important to use reagents that neither interfere with the detection nor reduce its sensitivity The volatility of the sample should also be taken into account When it is volatile or readily decomposes by heat, a mineralizing agent that reacts with the substance in the cold should be used, or the mineralization step should be carried out in a closed vessel

Liberation of gases often takes place during mineralization, and the components can be detected from these gases In such instances the simple apparatus shown in Fig 2 (actual size) may be useful These were developed

Fig 2 Apparatus for qualitative microanalysis suggested by Feigl

(a) Detection in a d r o p h a n g i n g on the b u l b ; / — d r o p ; 2—solution of a n a l y t e ; (b) detection on a strip of filter-paper h a n g i n g on the h o o k ; /—filter-pa per; 2—solution of a n a l y t e ; (c) detection of gaseous reaction p r o d u c t s o n a strip of filter-paper (1) a d h e r i n g

to the stopper; 2—solution of a n a l y t e ; ( d ) d e t e c t i o n of gases in the reagent solution placed in the s t o p p e r ; / — r e a g e n t p a p e r ; 2— solution of a n a l y t e ; (e) detection on a strip of reagent p a p e r covering t h e m o u t h of the flask, when the b u l b of the flask has t o be

heated; / — watch glass covering of the filter-paper strip; 2 — s t r i p of filter-paper i m p r e g n a t e d with the reagent s o l u t i o n ; 3—

reagent solution

(a) (b) (c) (d) (e)

by Feigl, and detection is carried out simultaneously with mineralization by means of spot-tests on filter-paper in a nearly closed system

Mineralization can be achieved by the use of oxidizing or reducing agents Neutral substances an^l mixtures can also be applied, which react with the volatile thermal decomposition products then release it on the addition of acids, bases or other agents for detection purposes

Of the strong oxidizing agents, concentrated nitric acid is used mainly in closed systems Very vigorous oxidizing action is exerted by a mixture of concentrated sulphuric acid and chromic acid on slight heating When the sample is not volatile, the mixture can be used in open test-tubes

Of solid oxidizing agents, sodium peroxide, mixtures of sodium carbonate and sodium nitrate, potassium permanganate, manganese(II) oxide, cobalt

(II, III) oxide (C03O4), copper(II) oxide, etc., are employed most frequently

The oxidizing power decreases in approximately the above order

For reducing purposes, alkali metals are favoured Their melting points are lower than 100°C and, when suspended in an organic solvent, they are suitable for the direct reduction of organic substances soluble in the given solvent Even the most resistant organic fluorine compounds can be mineralized by heating with alkali metals in a closed metal bomb at high temperature (600-800°C)

Special mineralization procedures will be discussed in connection with the detection of the individual elements

Organic compounds undergo decomposition when irradiated with strong light (xenon lamp) [1] This takes place most readily with iodine and bromine compounds and, most slowly with fluorine compounds

Detection of several elements together or individually by means of subsequent reactions can be carried out after mineralization with metallic potassium The classical method is the Lassaigne decomposition procedure, which is carried out as follows

In Fig 3, a thin-walled glass fusion tube with a bulb is shown (diameter 4-5 mm, length of the stem 40-50 mm) The sample (0.5-2 mg) is placed

in the bulb, then the end of the tube is pushed into a slice of potassium metal (2-3 mm thick) The small plug of potassium is pushed into the bulb with a thin glass rod Another piece of potassium is cut, but pushed down only to just above the bulb This plug will prevent the rapid egress of vapours of the pyrolysis products The tube is held with tongs and heated slowly and carefully in the small flame (1 cm high) of a micro burner until the reaction between the melted metal and the sample starts The bulb is heated for a further 20-30 s, then placed rapidly, while red-hot, into 3-4 drops of water in a cavity on a spot-test slide The glass bulb breaks when it comes into contact with the water and the melt is dissolved The red-hot bulb can also be dropped

into a micro-test-tube containing about 1 c m3 of water The reaction of residual potassium with water is usually not too vigorous, but the use of eye-protecting glasses is advisable Nitro compounds, when heated with potassium metal, may produce reactions with explosion-like phenomena In the aqueous solution obtained, the elemental constituents are in an ionic

Fig 3 The Lassaigne method of decomposition of organic material

/ S a m p l e ; 2- pieces of metallic potassium

state The solution may be black owing to the presence of carbon particles, which are filtered off, e.g., by covering the cavity of the spot-test slide with four

or five layers of filter-paper rings cut to a size larger than that of the cavity itself When they have absorbed the solution, the filter-paper rings are transferred to other cavities on the spot-test slide, where detection of ions is accomplished, with acidification with a drop of sulphuric acid when necessary

The solution obtained from the mineralization procedure with potassium can also be examined by the ring-oven technique [2] In this way, nitrogen, sulphur, chlorine, bromine and iodine can be detected in 1-2 mg of sample by means of suitable reactions

Luis et al [3] employed nascent hydrogen generated in a capillary for the detection of sulphur, antimony and arsenic Luis and Sa [4] elaborated an ultramicro technique for the detection of gases evolved from organic matter

using a microscope; 1 ng of substance could be detected in 1-100 x 10" 9 d m3

of solution

Specific gas chromatographic detectors are capable of detecting fractions

of a microgram of halogens and phosphorus This technique will be dealt with

at length in connection with the detection of these elements

Mass spectrometry also provides the possibility for the elemental analysis

of extremely small amounts of organic matter

2 Detection of carbon

In most instances, organic compounds burning with a luminous flame during the combustion test are assumed to contain carbon The detection of carbon is of importance mainly with compounds in which the carbon atoms are strongly substituted by other atoms, or when organic contaminants are to

be detected in inorganic matter

An old macro and semimicro method is to mix the sample with a two- or three-fold amount of finely powdered copper(II) oxide and to place the

mixture in a thin, long (about 5 x 150 mm) heat-resistant tube The

test-tube is closed with a rubber stopper carrying a thin glass test-tube bent at 90° The test-tube is mounted in slanting position and the outlet tube is immersed in a solution of calcium or barium hydroxide The bottom of the test-tube is heated strongly, the carbon dioxide formed by the reaction with copper(II) oxide leaves and the reagent solution becomes turbid At least 20-50 mg of sample are required for this test When a sufficient amount of hydrogen is also present in the sample, the water formed will condense on the cool upper walls

of the test-tube Small amounts of water vapour may be carried away, however, by the gas evolved, and thus condensed drops cannot be observed

A more sensitive method of detection is based on the reducing action of carbon (carbon-containing organic material)

Yellow molybdenum(VI) oxide is reduced by carbon to blue molybdenum(V) oxide:

4 M o 0 3 + C = 2 M o 2 0 5 + C 0 2

A positive reaction is given by substances that do not contain carbon but have

a reducing character, and also by ammonium salts:

6 M o 0 3 + 2 N H 3 = 3 H 2 0 + N 2 + 3 M o 2 0 5

The sample is placed in a heat-resistant test-tube (7 x 75 mm), which is then

half filled with finely powdered molybdenum(IV) oxide Air is sucked out of the test-tube (or expelled from it with an inert gas), then it is mounted in a slanting position and heated from the top downwards A blue ring is formed at the bottom of the test-tube, its thickness being nearly proportional to the carbon content of the sample In this way, 1-5 |xg of carbon can be detected

When silver arsenate ( A g3A s 04) is heated with a carbon-containing substance, the arsenate is converted first into the arsenite:

2 A g 3 A s 0 4 + C = 2 A g 3 A s 0 3 + C 0 2

which then undergoes disproportionation with the formation of coloured silver metal:

black-A g 3 A s 0 3 = 2 Ag + A g A s 0 3

The test is carried out as with molybdenum(VI) oxide

The reaction can be made more sensitive if the contents of the test-tube are moistened with a solution of molybdenate in hydrochloric acid after cool-ing Silver chloride and blue-coloured molybdenum blue are formed This is suitable for the detection of about 5 /ig of carbon

Carbon contents of organic matter will reduce potassium iodate to iodide

at 300-400°C The melt is dissolved and acidified, then elemental iodine is liberated and this can be detected very sensitively with starch indicator solution [5] Detection limit of this reaction is about 0.5 jig of carbon All the above reactions may be regarded as evidence only when the blank test is negative

Each reaction can be utilized for the detection of low carbon (organic matter) contents of inorganic substances having no reducing action in themselves

Organically bound carbon can be detected with mercury(II) amido chloride (HgNH2Cl), or with mercury(II) oxide and ammonium chloride In this reaction, hydrogen cyanide is formed, this can be detected with benzidine [6] An even better reagent is a solution of copper(II) acetate and benzidine acetate [7]

Luis et al [8] developed an apparatus where the atmosphere is entirely free

of carbon dioxide The organic sample is decomposed in it by a dry or wet procedure, and the carbon dioxide formed is allowed to react in a capillary tube with a reagent that contains lead acetate The reagent solution becomes turbid According to Luis et al 1 ng of carbon can be detected in this way, that

is, this procedure is about 1000 times more sensitive than any of the mentioned procedures

above-3 Detection of hydrogen

Sensitive methods for the detection of hydrogen are based on the fact that hydrogen reacts with the oxygen in organic compounds on heating to yield water Water liberates an easily detectable gas from the reagent used For

example, sodium sulphite releases hydrogen sulphide on reacting with water (which is formed from the hydrogen content of the organic matter):

N a 2 S 0 3 + 3 C = 3 C O + N a 2 S

N a 2 S + H 2 0 = N a 2 0 + H 2 S

Hydrogen sulphide is detected with a filter-paper moistened with lead acetate

or sodium pentacyanonitrosylferrate(III) (sodium nitroprusside) solution in

the atmosphere of the test-tube Device e in Fig 2 is very suitable for this

purpose

The sample is mixed with a five-fold amount of anhydrous sodium sulphite and placed into a flask with a long neck A filter-paper ring impregnated with the reagent solution is placed on the stopper of the flask, then the flask is heated until red hot Under the influence of hydrogen sulphide, the paper becomes black-coloured or red when lead acetate or sodium pentacyano-nitrosylferrate(III) solution is used, respectively

Potassium thiocyanate, when heated to about 400°C after melting, will decompose with the formation of very reactive elemental sulphur:

KSCN?±KCN + S

which combines with the hydrogen content of the sample, yielding hydrogen sulphide The other possible reaction path is the reaction of potassium thiocyanate with water formed from the hydrogen and oxygen contents of organic matter:

4 Detection of oxygen

When applying a less sensitive old method, oxygen is detected by mixing the sample with carbon, heating in nitrogen atmosphere, then using, e.g., palladium chloride for the detection of the carbon monoxide formed [10,11]

A more sensitive reaction was reported first by Davidson [12], based on the observation that iodine turns brown when dissolved in oxygen-containing solvents, but becomes violet when the solvent does not contain oxygen

Davidson dealt only with practical aspects of the test, without discussing the theoretical background His "Ferrox test" is carried out as follows

Aqueous solutions of iron(III) thiocyanate, with a dark red colour, can be extracted with diethyl ether or amyl alcohol, whereas oxygen-free solvents (benzene, carbon tetrachloride, chloroform) do not dissolve it In this way, oxygen-containing (polar) and oxygen-free (apolar) solvents can be distin-guished When larger amounts of a polar (oxygen-containing), compound are dissolved in an apolar solvent, discoloration of the solvent is observed

In a simple, but not too sensitive, procedure, a piece of filter-paper is impregnated with a solution of iron (III) thiocyanate in diethyl ether or methanol, dried, then dipped into the solvent or the solution of the sample in

an apolar solvent Another variation is to place a drop of the solution onto the paper In positive reaction, the "Ferrox paper" becomes red The Ferrox paper should always be freshly prepared

A more sensitive and more readily preserved reagent is obtained by combining separate 20 c m3 solutions of potassium thiocyanate (5 g) and iron(III) chloride ( F e C l3 6 H20 ) (4 g) and extracting the mixture 2-3 times with 5 c m3 portions of ether This ethereal reagent solution can be stored in a dark bottle for several weeks When the reagent is to be used, a glass rod is immersed in it repeatedly and the diethyl ether is allowed to evaporate between the immersions In this way, a sufficient amount of solid reagent will adhere to the rod, which is used for stirring the solution of the sample in a cavity on the spot-test slide Two drops of an apolar solvent are used to dissolve the sample The test is positive if the drop becomes red, and negative

if only particles of the reagent falling from the glass rod can be seen in the colourless drop The reaction can also be carried out in melts when the sample

is insoluble

Feigl [13] explained the reaction by the formation of stable, coloured solvates between the oxygen-containing compounds and iron(III) thio-cyanate complex Oxygen-free compounds are incapable of forming these solvates

There are several difficulties in the application of the Ferrox test For example, coloured samples, as well as solid, poorly soluble substances (oxygen-containing compounds are usually poorly soluble in apolar solvents), and those which decompose when melted, cannot be tested by this method [13] Further some sulphur- and nitrogen-containing compounds free from oxygen show positive reactions, as under certain conditions, sulphur and nitrogen atoms can form the same stable solvates as the oxygen atom with the reagent O n the other hand, negative reaction may be observed if the formation of the solvate is prevented for steric reasons (e.g., salicylic acid gives

a negative reaction, whereas benzoic acid gives a positive reaction) Other

reasons, such as the lack of a free electron pair on the oxygen atom (in the furan ring), may also be responsible for a negative reaction

For these reasons, iron(III) thiocyanate was replaced [14] with the complex potassium cobalt(II) tetrathiocyanate / K [ ( S C N )4C o ] ~ prepared from potassium thiocyanate and cobalt(II) chloride mixed in stoichiometric amounts This reagent can be stored, and is used in the solid state or as a melt

as follows

About 10 mg of reagent powder is added to 0.5-1 c m3 of sample (solvent or solution) in a micro-test-tube, which is then closed and shaken vigorously If a positive reaction occurs, the solvent becomes a vivid blue

If insoluble samples are stable up to 200-300°C, about 5 mg of sample are placed on a microscope slide, and 3-5 mg of reagent are added Heat is applied slowly until the mixture melts and becomes vivid blue if a positive reaction occurs Of course, much smaller samples are sufficient when the hot-stage microscope is used

This reaction is more reliable than the Ferrox test, as sulphur compounds (allyl sulphide, ethylvinyl sulphide, carbon disulphide) do not give positive reactions

Of oxygen-free compounds, only nitriles produce a blue colour in this test Certain primary amines give a colour other than blue owing to the presence of the amine ligand in the complex (aniline, butyl amine) The colour produced

by certain secondary and tertiary amines is pale green

A negative reaction is given by some oxygen-containing compounds, such

as furan and thymol, whereas tetrahydrofuran gives a positive reaction

In the original paper on this topic [14] about 500 compounds were listed, about 300 of which showed positive and about 200 negative reactions

5 Detection of nitrogen

Methods designed for the detection of nitrogen in organic compounds can

be divided into groups according to the nature of the simple containing compound applied in the final identification reaction

nitrogen-Nitrogen can be detected most simply, but with low sensitivity, in the form

of ammonia, which has a characteristic smell and is alkaline; it can be detected very sensitively as ammonium ions by the Nessler reaction

Nitrogen can be detected as cyanide ions obtained during reductive decomposition, formed from the nitrogen and carbon contents of the substance

With certain compounds, gaseous nitrogen can easily be liberated, and the only problem here is the identification of nitrogen gas

When oxidative decomposition, that is, combustion in an oxygen atmosphere, is applied, the nitrogen content is converted into nitrogen oxides, which can be detected very sensitively by means of the nitrite reaction Decomposition is usually carried out according to the Lassaigne method When mineralization is accomplished on the micro-scale, potassium cyanide

is formed in the reaction of potassium, carbon and nitrogen A small crystal,

or half a drop of a concentrated solution of iron(II) sulphate is placed in the middle of one of the filter-paper rings Iron (II) sulphate always contains some iron(III) ions, also Iron(II) ions react with potassium cyanide to yield potassium hexacyanoferrate(II):

6 KCN + F e S 0 4 = K 4 [ F e ( C N ) 6 ] + K 2 S 0 4

Hexacyanoferrate(II) ions and iron(III) ions produce a Prussian blue colour, and sometimes a precipitate is also obtained:

3[Fe(CN ) 6 ] 4 - + 4 F e 3 + = F e 4 [ F e ( C N ) 6 ] 3

The detection limit of this reaction is about 15 \ig of nitrogen

On the semimicro scale, the following procedure has been suggested for decomposition and detection [15]

About 1-10 mg of sample is placed in a heat-resistant test-tube (about 10

x 120 mm) and a piece of sodium is placed on it The sample and sodium are

thoroughly pressed together with a glass rod After a short period the tube is mounted in a vertical position and, the bottom is heated slowly until the piece of sodium melts and the reaction is completed Then a few milligrams of sample are added to the micro-test-tube, which is heated further for 30 sec After cooling, about 0.1 c m3 of methanol is added in order to decompose the excess of sodium, the residue is dissolved in about 2 c m3 of water and the black carbon residue is removed by centrifugation The clear supernatant is removed and diluted to 4-5 c m3, then 0.5-1 c m3 aliquots are used for the detection of nitrogen by the following procedure

test-Alkalinity of the solution is reduced by the addition of 1 drop of 1 N

hydrochloric acid, then 10-15 mg of powdered iron(II) sulphate and one drop

of 30% potassium fluoride solution are added and the mixture is heated to

boiling After cooling, one drop of iron(III) chloride solution and sufficient 6 N

sulphuric acid to dissolve the iron hydroxide precipitate are added dropwise

A blue colour appears at a maximum of 2-3 min later

If sulphur is present in the sample, sulphide ions interfere with the detection In order to prevent this interference, one drop of 10% lead acetate solution is added to the solution obtained in the decomposition procedure