(c
LIBRARY
E ID r
Hemoglobin 1:100.
hemoglobin 1:100.
Carboxyhcemoglobin 1 .100.
M ethcsmoglobin : neutral.
Hfsmatin in acid alcohol.
Hcsmochromogen in alkaline solution, 1:100.
Hcematoporphyrin in 1%
Hesmatoporphyrin in 5%
Urobilin.
Uroerythrin.
Chlorophyll in living nettle leaf.
a — Chlorophyll in ether.
b — Chlorophyll in ether.
Carotin in alcohol.
Xanthophyll in alcohol.
SPECTRA.
PRACTICAL ORGANIC AND BIO-CHEMISTRY
R. H. A. PLIMMER, D.Sc.
HEAD OF THE BIO-CHEMICAL DEPARTMENT OF THE ROWETT RESEARCH INSTITUTE IN ANIMAL NUTRITION, UNIVERSITY OF ABERDEEN AND NORTH OF SCOTLAND COLLEGE OF AGRICULTURE, AND LECTURER ON APPLIED BIO-CHEMISTRY, UNIVERSITY OF ABERDEEN. FORMERLY, READER IN PHYSIOLOGICAL CHEMISTRY, UNIVERSITY OF LONDON, UNIVERSITY COLLEGE
WITH COLOURED PLATE AND OTHER ILLUSTRATIONS IN THE TEXT
NEW IMPRESSION
LONGMANS, GREEN AND CO.
39 PATERNOSTER ROW, LONDON
FOURTH AVENUE & 30TH STREET. NEW YORK
BOMBAY, CALCUTTA, AND MADRAS
1920
LIBRARY
Made in (treat Hritain
PREFACE TO REVISED EDITION.
FOR this edition the text has been thoroughly revised. Several sections have been rewritten and some new methods of preparation and analysis have been incorporated.
R. H. A. P.
November, 1917.
PREFACE.
IN this edition the same method is adopted as was followed in the former edition, entitled " Practical Physiological Chemistry," but further experience in teaching has led me to believe that the book would be improved and made more useful if its scope were extended. New sections upon organic chemistry and organic substances found in plants, together with methods used in more advanced work, are therefore in- cluded.
As the basis of the book is Organic Chemistry this term is used in its title, and since the subject is also treated from the botanical side, the term Bio-Chemistry is substituted for Physiological Chemistry, a term too often restricted to the aspect of the animal side of the subject.
The book is still intended mainly for medical students, but it contains the essentials for all students of Biology. The medical student will gain rather than lose by having additional matter, though it may not be of immediate use to him. A survey of the sections upon plant compounds should give him a wider outlook and a deeper insight into the wonderful sub- stances connected with the phenomena of Life, and withal, they may be useful for reference.
In order to help the student in using the book the essentials of the subject are printed in large type, while the botanical and more advanced portions are printed in small type. The more important practical experiments suitable for a preliminary course are indicated by asterisks and correspond to the small type of the former edition.
I am much indebted to Dr. C. Lovatt Evans for his assistance in the sections on " The Function of Haemoglobin " and "The Analysis of Blood Gases". Mr. W. W. Reeve, B.Sc., has kindly assisted me with the proofs.
R. H. A. P.
PREFACE TO FORMER EDITION.
THIS book was originally compiled as a handbook for prac- tical work in Physiological Chemistry at University College, London, since no single text or class book covered the complete course, or treated Physiological Chemistry as part of the subject of organic Chemistry, or even as an in- dependent subject.
The present book must still be regarded mainly as a compilation. It represents an attempt to give to the worker a nearly complete statement of the whole subject. Each section has a short explanatory summary of the essential points, so as to connect the various sections together. The essential points are illustrated by the practical experiments, which are printed in different type.
The illustrations are also compiled from various sources. These are mentioned underneath each figure. The illustra- tions of apparatus not so mentioned have been drawn from my own sketches. For those of the osazone crystals, haemin, and tyrosine, I am indebted to Miss V. G. Sheffield, who has also kindly helped in reading the proof sheets.
In most physiological chemistry laboratories the strengths of the reagents employed are very various, e.g. dilute acetic acid may be i per cent., or 2 per cent, or 5 per cent., or even 10 per cent. In order that all workers may employ a reagent of standard concentration, a list of reagents has been carefully drawn up and is appended.
vii
CONTENTS.
PAGE
DEFINITION i
RECOGNITION OF AN ORGANIC COMPOUND f . . 2
ISOLATION AND PREPARATION OF PURE ORGANIC COMPOUNDS. CRITERIA
OF THEIR PURITY 3
I. Purification of a Liquid by Distillation. Determination of the Boiling- point, 4; II. Separation of Liquids, 8 ; Evaporation of Liquids, 13; III. Separation of Solid and Liquid. Filtration, 15; IV. Purification of Solids by Crystallisation, 17 ; Determination of the Melting-point, 23 ; V. Separation of Solids, 25 j Isolation of Solids from Solution, 26.
COMPOSITION OF ORGANIC COMPOUNDS 28
A. Elementary Composition. Detection of the Elements, 28 ; B. Quantita- tive Composition. Estimation of the Elements, 31 ; C. Calculation of Results, 41 ; D. Determination of Molecular Weight, 42.
IDENTIFICATION OF AN ORGANIC COMPOUND 46
HYDROCARBONS 49
A. Saturated, 49 ; B. Unsaturated (a) Olefines, 54, (b) Acetylenes, 56.
HALOGEN DERIVATIVES OF THE HYDROCARBONS 57
Monohalogen Derivatives. Alkyl Halides, 57 ; Dihalogen Derivatives, 58 ; Trihalogen Derivatives. Chloroform, 59. lodoform, 62.
ALCOHOLS 63
Methyl Alcohol, 63 ; Ethyl Alcohol, 64 ; Propyl Alcohols, 68 ; Butyl Alcohols, 68 ; Amyl Alcohols, 69 ; Higher Alcohols, 70.
ESTERS 71
Esters of Inorganic Acids, 71 ; Esters of Organic Acids, 72 ; Hydrolysis of Esters, 74.
ETHERS. ETHYL ETHER 76
MERCAPTANS AND SULPHIDES • . . .78
ALDEHYDES 80
Formaldehyde, 80; Acetaldehyde, 80; Estimation, 85; Chloral, 86; , Chloral Hydrate, 86.
KETONES. ACETONE 88
THE FATTY ACIDS . . ........ 93
Formic Acid, 94 ; Acetic Acid, 96 ; the Higher Fatty Acids, 99. HALOGEN SUBSTITUTION DERIVATIVES OF THE FATTY ACIDS . . .100
ACID OR ACYL CHLORIDES 101
ACID ANHYDRIDES ............ 102
be
CONTENTS
UNSATURATED ALCOHOLS, ALDEHYDES AND FATTY ACIDS « . .103
HYDROXY-, KETO- AND DIBASIC ACIDS ....... 106
AMINES ............. 124
AMIDES ............. 129
THE AMINO ACIDS ........... 137
BETAINES ............. 150
CYANOGEN COMPOUNDS .......... 152
GUANIDINE AND ITS DERIVATIVES ........ 164
Dl-, TRI- AND POLYHYDRIC ALCOHOLS ....... 173
FATS AND OILS. WAXES. LECITHINS ....... 175
THE CARBOHYDRATES . . ........ 183
THE MONOSACCHARIDES ......... 184
PROPERTIES AND REACTIONS OF THE MONOSACCHARIDES . .190 A. Glucose, 190 ; B. Fructose, 194 ; C. Galactose, 195 ; D. Mannose, 195 ; E. Pentoses, 195 ; F. Glycuronic Acid, 196.
THE DISACCHARIDES : CANE SUGAR; LACTOSE; MALTOSE . .197
TRISACCHARIDES AND TETRASACCHARIDES ..... 202
CHITIN AND CHONDROITIN ........ 203
THE POLYSACCHARIDES : STARCH ; DEXTRINS ; GLYCOGEN ; CELLULOSE 205
GLUCOSIDES ........... 214
ESTIMATION OF CARBOHYDRATES ........ 216
A. Estimation by Means of the Polarimeter, 216; B. Estimation by Reduction of Copper Salts, 221 ; C. Estimation by Fermentation, 233 ; Estimation of Pentoses, 234 ; Estimation of Disaccharides, 235 ; Estimation of Polysaccharides, 236.
CARBOCYCLIC COMPOUNDS .......... 237
AROMATIC COMPOUNDS .......... 238
Benzene and its Monosubstitution Derivatives, 240 ; Disubstitution Deriva- tives of Benzene, 260 ; Trisubstitution Derivatives of Benzene, 270 ; Tetrasubstitution Derivatives of Benzene, 273.
TANNINS ............. 274
HETEROCYCLIC COMPOUNDS ......... 276
UREIDES ........... . 277
A. Ureides of Monobasic Acids, 277 ; B. Ureides of Hydroxy and Alde- hyde Acids, 277 ; C. Ureides of Dibasic Acids, 279.
PYRIMIDINES ... ... . 282
GLYOX ALINE OR IM1NAZOLE DERIVATIVES ...... 284
PURINES ...... ...... 286
NUCLEIC ACIDS .... ..... 299
FURFURANE, OR FURANE, AND ITS DERIVATIVES ..... 303
THIOPHENE AND ITS DERIVATIVES . . ..... 304
PYRROLE AND ITS DERIVATIVES ........ 304
PYRIDINE AND ITS DERIVATIVES ........ 307
PAGE
HYDROAROMATIC COMPOUNDS 310
The Inositols 310
The Terpenes 312
The Cholesterols 319
Bile Acids 322
COMPLEX AROMATIC COMPOUNDS ........ 330
THE ANTHOXANTHINS 338
THE ANTHOCYANS 340
INDOLE AND ITS DERIVATIVES 342
QUINOLINE AND ISOQUINOLINE . . 3$O
THE ALKALOIDS ... 351
THE PROTEINS 361
The General Reactions of the Proteins, 365 ; A. Colour Reactions, 365 ; B. Coagulation Reactions, 368 ; C. Precipitation Reactions, 368. Derivatives of Proteins, 370 ; Metaproteins, 370 ; Proteoses and Peptones, 371 ; Separation of Proteoses and Peptones, 372 ; Peptone, 373.
APPENDIX TO PROTEINS. COLLOIDS AND COLLOIDAL SOLUTIONS . .374
ENZYMES. FERMENTATION 391
Localisation of the Enzymes and the Chemical Changes in the Organism, 396 ; Demonstration of the Action of Enzymes, 399 ; the Cataly- tic Action of Enzymes, 413 ; the Synthetical Action of Enzymes, 415 ; the Measurement of the Activity of Enzymes, 416. Appendix to Digestion —
I. The Acids in the Gastric Contents, 427 ; II. The Constituents of Bile, 429; III. Gall Stones, 431.
THE INDIVIDUAL GROUPS OF PROTEINS 432
Protamines 432
Histones 433
Coagulable Proteins. Albumins. Globulins 435
The Coagulable Proteins of Egg- White, 436 ; Blood, 438 ; Milk, 446 ; Muscle, 447 ; Other Animal Tissues, 449 ; Plants, 450.
Gliadins and Glutelins . . . .452
The Scleroproteins -454
Phosphoproteins 457
Milk, 457 ; Egg- Yolk, 463.
Nucleoproteins 465
Glucoproteins 468
Chromoproteins 472
Blood, 472. The Reactions of Haemoglobin in Defibrinated Blood. Spec- troscopic Examination of Haemoglobin and its Derivatives, 478. Estimation of Haemoglobin, 486. Crystals of Oxyhaemoglobin and Derivatives, 491. The Function of Haemoglobin as Carrier of Oxygen, 497. The Blood as Carrier of Carbon Dioxide, 510. THE CHEMICAL CONSTITUTION OF H^MIN AND H^MATOPORPHYRIN . 516 THE PIGMENTS OF LEAVES. THE CHLOROPHYLLS AND CAROTINOIDS . 519 METABOLISM. INTEGRATION OF THE CHEMICAL PROCESSES . . .533
COMPOSITION OF THE COMMONER TISSUES USED AS FOOD-STUFFS FOR
ANIMALS 537
xii CONTENTS
PAGE
ANALYSIS OF NORMAL URINE 539
Micro-Methods for the Analysis of the Nitrogenous Constituents of Urine 556
Appendix to Urine 564
I. The Pigments, 564 ; 2. Urinary Sediments, 565 ; 3. Urinary Calculi, 568 ; 4. Inborn Errors of Metabolism, 569 ; 5. Pathological Urines: (i) Diabetic, 570; (2) Protein, 571; (3) Blood, 572; (4) Bile, 572.
ANALYSIS OF TISSUES 573
A. The Inorganic Constituents 573
B. Proteins 575
C. Nitrogenous Extractives 577
D. Carbohydrates 585
E. Lactic Acid 59°
F. Aceto-acetic Acid 593
G. /3-Hydroxybutyric Acid 597
H. Fat 601
I. Cholesterol 603
TABLES 605
LIST OF REAGENTS 610
INDEX 618
DEFINITION.
THE substances composing the organic material connected with the phenomena of life, and the great majority of the products of vital activity, are mixtures of compounds of the element Carbon.
From these substances the chemist has isolated numerous pure carbon compounds and prepared others ; he has also synthesised carbon compounds from their elements.
About 1 50,000 carbon compounds are now known. The possibility of their existence is due to the unique property which the element carbon possesses of being able to combine with itself; compounds are known which contain in their molecules from one up to sixty atoms of carbon directly joined together.
Of these 150,000 carbon compounds only a small number are directly concerned in vital processes.
The chemistry of all the carbon compounds is termed organic chemistry.
The chemistry of those carbon compounds which are the con- stituents of living matter and are concerned in vital processes is physiological or biological chemistry ; the term physiological chemistry more frequently refers to the compounds and their functions in animals ; the term biological chemistry comprises the compounds and their functions in both plants and animals. The changes which they undergo and the functions which they fulfil in the living plant or animal form the subject of chemical physiology.
Though a distinction can be made between biological chemistry and chemical physiology, the two subjects are so closely interrelated that they are essentially only different aspects of the same subject. No biological change can be followed until a knowledge of the chemical properties of the substances involved has first been acquired. Chemi- cal physiology is thus dependent on biological chemistry, or Bio- chemistry. Bio-chemistry is the branch of organic chemistry which deals with the natural organic compounds and with the functions of these compounds in nature.
I
RECOGNITION OF AN ORGANIC COMPOUND.
Organic compounds are distinguished from inorganic compounds by being combustible: on heating they generally char, sometimes take fire, and on prolonged heating completely burn away leaving no ash. Inorganic compounds when heated do not char and they leave a residue. A mixture of an organic compound and an inorganic compound will also char and leave a residue. There are a few excep- tions to this general rule : oxalic acid and its salts amongst the organic compounds do not char on heating ; amongst the inorganic com- pounds the ammonium salts volatilise leaving no ash. An oxalate leaves a residue of the oxide of the metal with which it is combined.
The following experiments exemplify these statements : —
1. A small piece of paraffin wax heated upon platinum foil will melt, take fire, and will completely burn away leaving no residue.
2. A crystal of cane sugar heated in the same way will melt, char, and on further heating will disappear completely.
3. A few crystals of common salt heated on platinum foil will melt, and unless heated very strongly, e.g. with a blowpipe flame, will remain as a solid white mass when allowed* to cool.
4. A small piece of soap heated as above will char, the vapours evolved may take fire, and when the charred particles have all vanished a white or nearly white residue will R-rmin.
Note. — // is in this way that substances composed of organic and inorganic matter are recognised. The composition of the inorganic residue is found out by the usual m 'thods of inorganic analysis after the organic matter has been destroyed by heating.
5. No appreciabl- will be seen on heating a little oxalic acid or an o, e.g. calcium oxalate.
6. Ammonium chloride volatilises on heating and leaves no residue.
7. To prove the presence of carbon in oxalic acid or in an oxilate the sub-
!K a'u-tl in a smull glass tube and the gases evolved are passed into or hiryta water. A precipitate of calcium or barium carbonate indicates
1 carbon.
8. On h.-atin^ ammonium chloride as in 7, there is no formation of carbonate.
F CATION AND PREPARATION OF PURE ORGANIC COMPOUNDS.
CRITERIA OF THEIR PURITY.
The organic material which composes all animal and vegetable cells consists of a mixture of numerous compounds. In order to in- vestigate their evolution and their degradation in nature it is necessary to separate these compounds from one another and to prepare each of them in a state of purity. The pure substance can then be analysed and its chemical and physical properties ascertained. Knowledge of the pure compounds shows their chemical relationship to one another and an idea of their role in nature is obtained. This idea is proved or disproved by an investigation of the changes which the organism, as a whole or individual portions of it, can effect in these substances.
In the study of the chemical properties of the compounds, other compounds are formed by their interaction. These compounds also require isolation and purification. The principal operations in organic and biological chemistry will thus consist in the isolation and prepara- tion of pure compounds.
The methods of separating organic compounds are based upon differences in the properties of the substances under investigation. These differences are taken advantage of as much as possible ; some- times they are so gross that the separation is simple, sometimes they are so small that the separation is of extreme difficulty, and in these cases a separation can only be effected when sufficient material is available.
Solid organic compounds are more numerous than liquid ; gases are comparatively rare.
Solids are most usually separated and purified by solution in water or organic solvents. The methods for the manipulation of liquids are therefore most frequently used and are described first.
4 PRACTICAL ORGANIC AND BIO-CHEMISTRY
I. PURIFICATION OF A LIQUID BY DISTILLATION- DETERMINATION OF THE BOILING-POINT.
A liquid is purified by distillation. The criterion of the purity of a liquid is its boiling-point A pure liquid has a constant boiling- point
A. Drying and Cleaning of Apparatus.
Since organic liquids are very frequently not miscible with water all the apparatus which is used with them must be dry.
The apparatus may be dried in an oven for some time, but on removal to prevent deposition of aqueous vapour a current of air is blown through them whilst they are hot and during cooling. The current of air is most conveniently got from bellows, or by suction with a pump. The tubing, or if the glass vessel be narrow, a glass tube inserted in the tubing, is placed inside the vessel so that the farthest extremity is dried and cooled first.
More usually since the vessels are wet with water, they are rinsed with alcohol after draining as much water away as possible, then with ether after again draining. A current of air from the bellows or water- pump is drawn through to evaporate the ether. The vessel may be warmed in a luminous flame, when no more ether is present and the vessel is not quite dry, and air driven through as above. It is im- portant that all the ether be evaporated, since it may become ignited or form an explosive mixture inside the vessel.
Apparatus which contains charred matter may be cleaned by oxidising it away with potassium bichromate and sulphuric acid, or by heating in it a mixture of concentrated sulphuric and nitric acids, washing with water and proceeding as above to dry it.
B. Distillation of a Liquid and Determination of its Boiling- point.
The liquid is placed in a clean, dry fractionating or distilling flask — a round-bottom flask with a side tube in its neck 1 — of suitable size so that only about half or at most two-thirds of the space is filled. Some small pieces of unglazed porcelain, or porous earthenware, or pieces of platinum, are added to ensure steady boiling without bump- ing. The neck of the flask is closed with a well-fitting cork 2 which
1 With liquids of high boiling-point the flask should have the side tube low down so as to prevent decomposition by the high temperature.
Rubber corks are dissolved by many organic liquids and consequently are not used except in special cases.
PREPARATION OF PURE COMPOUNDS 5
is bored to carry a thermometer. The position of the thermometer is so adjusted that the bulb is just below or opposite the side tube and not touching the walls. The side tube is connected by a cork to a clean, dry condenser, which is supported by a clamp, and a slow stream of cold water is allowed to flow through the condenser. A receiver (flask) is placed at the other end of the condenser (Fig. i).
A water condenser is not used for liquids boiling above I2O° ; the vapours are condensed by being passed through a simple tube (the inner tube of the above condenser). The vapours of liquids boiling at very high temperatures are condensed in the side tube of the distilling flask. If the vapours condense to a solid on cooling, the solid is melted by a flame so that the liquid runs into the receiver and does not block up the side tube.
Fio. i.
Liquids boiling below 1 00° are heated on a water-bath, liquids boiling above 1 00° are heated directly with a flame, which is moved round and round under the bottom of the flask until boiling begins. When boiling commences it must be kept on continuously and vigorously and not interrupted by the removal of the flame or by draughts.
When the vapour from the boiling liquid reaches the thermometer, the temperature is seen to rise rapidly, and then becomes stationary at a definite temperature. This is the boiling-point of the liquid. Drops of condensed liquid are usually seen to fall from the end of the thermometer into the flask. The heating is continued until all the liquid boiling at this temperature has distilled over into the receiver. The portion remaining in the distilling flask contains the impurities.
6 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Towards the end of the distillation the thermometer may be seen to rise slowly and the last portion to distil 1° higher than the first por- tion. Most pure liquids boil over a range of -5° or 1° or sometimes more.
Chloroform and aniline may be used as examples.
Correction for Boiling-point.
A correction should be applied when the mercury in the thermometer passes outside the neck of the flask as this portion is ccoled below the tem- perature of the vapour of the liquid. The following amount is added to the
rved temperature T —
n(T — /) x "000154
where n is the number of scale divisions projecting, / the temperature of the air recorded by another thermometer held at a point about the centre of the projecting thread and screened from heat radiation from below by a sheet of cardboard.
A short thermometer registering only 50° and which can be completely inserted in the vapour is used for more accurate determinations. Sets of thermometers registering intervals of 50° between o° and 360° can be pur- chased.
Determination of the Boiling-point of Small Quantities.
When insufficient liquid is available for distillation its boiling-point can be determined by placing it in a test tube and heating it through an opening in a sheet of asbestos. The thermometer is held in the vapour.
If only a few drops of liquid are available the boiling-point can be ascer- tained by introducing it into a small test tube, attaching the test tube to a thermometer by a rubber band and heating the two together in a beaker con- taining sulphuric acid. In the liquid is placed a piece of capillary tubing, a melting-point tube (p. 24) which is sealed near the end immersed in the liquid. As the temperature of the bath rises bubbles escape from the capillary and ascend through the liquid. At the boiling-point they form a continuous
;m and the temperature of the bath is noted. Several determinations are made with fresh pieces of capillary and the mean will give the boiling-point of the liquid.
PREPARATION OF PURE COMPOUNDS
C. Distillation in Vacuo.
Liquids which boil at high temperatures and decompose on distillation under atmospheric pressure can frequently be distilled under reduced pressure.
The liquid is distilled from a fractionating flask, the side tube of which is inserted in another fractionating flask, or other stout vessel with a side tube, which acts as receiver. This is kept cold by allowing a stream of cold water to run over it ; the water is collected in a funnel, which serves as a support to the receiver and it runs thence to the waste. The vacuum is produced by connecting the side tube of the receiver with a water pump or a mechanical pump. A gauge is in connection between the apparatus and the pump by a T piece so that the pressure can be ascertained.
To ensure continuous ebullition without bumping a slow stream of air, or carbon dioxide from a Kipp apparatus if the liquid tends to oxidise or de- compose in the air, is passed through the liquid by inserting in the cork a tube with a long capillary which reaches almost to the bottom. The supply of air is regulated by means of a screw, pinch on a piece of pressure-tubing placed on the end of the glass tube. The apparatus is shown in Fig. 2.
TO PUMP
FIG. 2.
A distilling flask with a double neck as in Fig. 7 (p. n) is more convenient than a simple distilling flask ; the neck with the side tube carries the thermo- meter, the other neck the capillary.
The heating of the flask during distillation in vacua is best effected by means Of a bath ; overheating is prevented, and the flask and its contents can be completely immersed which ensures a uniform temperature.
Rubber corks are used in vacuum distillation as they more easily prevent leakage in of air. If they are attacked by the vapours of the liquid they may be protected by placing a piece of cork between their ends and the vapours.
PRACTICAL ORGANIC AND BIO-CHEMISTRY
another.
II. SEPARATION OF LIQUIDS. A. Mechanically.
(a) Two liquids, if they are not miscible, are easily separated from one They are placed in a tap funnel or separating funnel (Fig. 3), either cylindrical or pear-shaped according to the volume, the stopper is removed and the heavier liquid run out and collected. The (lighter liquid is poured out through the top so as to avoid contamina- tion by coming in contact with drops of the other liquid remainingiin the stem.
(b) Two liquids, if they are miscible, may not both be soluble in the same solvent ; such a solvent must not be miscible with one of the constituents. On shaking the mixture with the solvent in a separating funnel, the mixture will separate into two layers. In shaking up two liquids in a separating funnel, the stopper of the funnel is held in the palm of one hand, and as usually an increase of pressure occurs, especi- ally if ether and water be the liquids, the tap of the
Fio. j.
funnel is occasionally opened after allowing the liquids to settle at the other end. After thoroughly shaking, the liquids are allowed to separate, the stopper is removed and the heavier liquid is run out. The insoluble liquid is shaken up once or twice more with more solvent, and is puri- fied by drying, if necessary, and distillation. The solvent, if water be one of the liquids, is dried by shaking it, or allowing it to stand for 12-24 hours with solid calcium chloride, or sodium sulphate, or potas- sium carbonate. It is separated from the soluble constituent by frac- tional distillation, or treated as the case necessitates.
As an example, a mixture of equal parts of alcohol (50 c.c.) and chloroform may be made. On shaking up with two volumes of water in a separating funnel, the chloroform separates and sinks. It is re- moved and the other constituents are poured out. The chloroform is returned and shaken once more with water. It is run out and the moisture is removed by shaking or standing with some calcium chloride from which it is filtered and then distilled.
PREPARATION OF PURE COMPOUNDS
B. Fractional Distillation.
A mixture of two or more miscible liquids may be separated from one another by fractional distillation if their boiling-points differ by 20-30°.
The mixture is distilled as described previously (p. 4), preferably from a flask with its side tube high in the neck and the thermometer is carefully watched. The first portion which distils will consist mainly or entirely of the more volatile constituent which has a higher vapour pressure ; the last portion will consist of the less volatile constituent having a lower vapour pressure or higher boiling-point. Between these portions there may be a small intermediate fraction consisting of a mixture of the liquids. The three portions or fractions are collected in separate receivers.
Eg.-
A mixture of equal volumes of chloroform and aniline will give on fractional distillation a fraction boiling at 61° (almost pure chloroform), an intermediate fraction, and a final fraction boiling at 183° (almost pure aniline). During the distillation of such a mixture when the boiling-point of a constituent exceeds 120° the water should be run out of the condenser.
Redistillation of the first and last fractions will give each of them in a state of purity.
If a mixture of liquids contains constituents which have boiling-points fairly close to one another, the fractionation must be repeated several times until each fraction is found to have a constant boiling-point.
The separation of such a mixture is greatly facili- tated by the use of a fractionating column or still-head. This is simply a device to lengthen the neck of the distilling flask so that the higher boiling fractions are exposed to the air and condensed before they reach the condenser and run back into the flask. Numerous forms have been invented ; two efficient forms are those of Hempel and of Young (pear still-head, Fig. 4).
The former consists of a glass tube filled with glass beads and a side tube. The latter consists of a piece of glass tubing upon which are blown 2, 3, 4 or more bulbs of a pear shape, and a side tube. The liquid is placed in a round-bottom flask, the fractional column inserted and this in turn connected by iN side tube to the condenser. The mixture in the flask, to which several small pieces of
FIG. 4.
io PRACTICAL ORGANIC AND BIO-CHEMISTRY
porous earthenware have been added, is heated over a gauze at such a rate that the condensed liquid comes over drop by drop. Fractions are collected at 5° or 10° ranges of temperature.
The redistillation of each fraction is carried out as follows : In the apparatus which has been washed out and dried fraction I is put which boiled, say, at 90-95°, and distilled till the thermometer shows 95°, when the distillation is stopped and fraction II, 95-100°, is added to the remainder in the flask. On distilling, fractions below 95° are collected in the first receiver and the second fraction is distilled till the temperature reaches 100°. Distillation is stopped and the next fraction added. The process is continued until all the fractions have been redistilled. Finally the main fractions boiling at the extremes will be obtained.
A mixture of benzene, toluene and xylene, such as occurs in coal tar, may be taken in illustration.
Constant Boiling Mixtures.
It frequently happens that two liquids form a mixture which has a constant boiling-point and behaves like a single liquid. Such a mixture cannot be separated by fractional distillation, although more or less separation may be possible by distilling at a different pressure.
Such a mixture may have a boiling-point which is lower than either of its constituents, or higher.
Excess of either constituent in the mixture beyond that forming the constant boiling mixture can be separated by fractional distilla- tion and it will distil over either before or after the mixture according to the boiling-points. Such constant boiling mixtures are mixtures of ethyl alcohol and water, methyl alcohol and acetone, benzene and alcohol, pyridine and water, water and formic acid, chloroform and acetone. They can only be separated by chemical means.
Fractional Distillation in Vacuo.
The same apparatus as described above for distillation in vacua can be used for fractional distillation in vacuo, but as each fraction distils the apparatus must be disconnected so as to insert a new receiver. To avoid releasing the vacuum and disconnecting the apparatus, and to allow fractional distillation to proceed continuously several contrivances have been suggested. Usually the vapours of liquids distilled in vacuo condense without being cooled by water or ice, and an apparatus as in Fig. 5 may be used. The receivers are turned into position when necessary. A very simple form is shown in
PREPARATION OF PURE COMPOUNDS
ii
Fig. 6: the apparatus can be rotated on the cork connecting it with the distilling flask when a new fraction begins to distil over.
FIG. 5. FIG. 6.
An apparatus of the type in Fig. 7 is the most convenient. By means of the several taps the receiver can be shut off and its vacuum released, whilst distillation continues and the fraction collects in the bulb. The fresh
FIG.
receiver is exhausted whilst the taps to the bulb and distilling flasks are closed; no great decrease of vacuum occurs as the small receivers are rapidly exhausted.
If necessary the bulb and receiver can be cooled by a stream of cold water, or by immersing the receiver in ice or a freezing mixture.
12 PRACTICAL ORGANIC AND BIO-CHEMISTRY
C. Steam Distillation.
Very frequently a separation of a liquid or a solid from a mixture can be effected by the process of steam distillation. The liquid, or solid, has usually a higher boiling-point than water, but the vapours of the liquid and of the water do not interfere with each other. The effect of the steam is to reinforce the vapour pressure of the liquid, so that the liquid distils with water under atmospheric pressure at a lower temperature.
Steam is generated in a large flask, or tin can, which is provided with a cork carrying a long safety tube about 80 cm. long, reaching almost to the bottom, and with a delivery tube. The flask is half- filled with water. The delivery tube when the steam is ready is con- nected to the flask containing the mixture. This flask is placed in a
FIG. 8.
sloping position so as to prevent splashing of its contents and mechanical carrying over of substance into the condenser. The steam is passed into the bottom of the vessel by a tube which is bent so that its end lies in a vertical position, and to prevent condensation of the steam the flask is also heated. The steam and other vapour reach a long condenser by which water and substance are condensed and are col- lected in a receiver (Fig. 8).
The separation of substance and water is effected by filtration if solid, by simple separation if liquid, or by extraction with a solvent such as ether, chloroform, etc. The solvent is then dried, removed by distillation, and the substance or substances obtained by distillation or fractional distillation.
PREPARATION OF PURE COMPOUNDS 13
EVAPORATION OF LIQUIDS.
Only aqueous solutions can be evaporated over a flame, and the evaporation should be completed over a water-bath to prevent charring the substance as it becomes concentrated. Evaporation over a flame must be carefully watched to prevent charring and also to avoid spurting when the solution begins to concentrate.
Most organic liquids which are used as solvents are readily inflam- mable and must not be brought near a flame.
When a considerable amount of solvent is present it is removed by distillation and in cases where the solvent (ether, acetone, ligroin, etc.) is very readily inflammable the distillation must be carried out on a water-bath, heated by a flame specially protected by a gauze, or by steam, or electrically. If the bath be heated by a burner, the end ol the condenser should be placed as far away as possible and a sheet of cardboard or asbestos interposed between the burner and receiver.
Not only is evaporation by distillation absolutely necessary with inflammable liquids, but also it is economical. The solvent is recovered and after purification can be used again.
When only small quantities of liquids up to 25 c.c. require evapora- tion they are set aside, away from flames, and allowed to evaporate spontaneously, or they may.be put upon a warm water-bath, with the flame extinguished.
Another common procedure is to evaporate small quantities by placing them in a vacuum desiccator and exhausting. The evaporation is greatly accelerated if the liquid be previously warmed on a water-bath and whilst warm put into the desiccator.
Evaporation in l/acuo.
Evaporation in vacua is the most rapid method of concentrating solutions, especially if the temperature can be kept at an elevated point.
Very frequently evaporation of solutions must be carried out at a low tem- perature (35-45°) to prevent decomposition. This is carried out in the same way as distillation in vacua, the distilling flask being kept in water at 35-45°- With large flasks of 2-3 litres capacity and a good vacuum of about 15 mm., a litre of water can be distilled off in 2-2^ hours at 35-45°. Instead of a distil- ling flask an ordinary flask fitted with capillary and bent tube may be used and a similar one as receiver.
14 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Frothing During Evaporation in l/acuo.
Aqueous solutions of extracts of plant or animal matter have a great tendency to froth during concentration in vacua.
The froth can be broken by allowing drops of alcohol to fall upon it from a tap funnel inserted into the distilling flask. The alcohol vapour helps in the evaporation when the vacuum has been established. • \
The liquid to be evaporated can be allowed to flow into the exhausted vessel through a spiral tube at such a rate that no large volume is ever present in the flask.
A contrivance to catch the froth and return it to the flask has been devised by Davis and Daish. It has been very useful for evaporating plant extracts. It is shown in Fig. 9.
To Water Pump
FIG. 9. — From the Journal of Agricultural Science, Vol. V, p. 435 (Cambridge Uni- versity Press).
. Any froth which is formed is broken by the piece of copper gauze in B and the liquid runs back into the flask A. The vapours are condensed by the condenser D and collected in G. The glass piece E is introduced so as to allow of the emptying of G : the tap is turned and the screw clamp on the pressure-tubing at S closed ; liquid collects in E whilst G is removed, emptied, replaced and evacuated. The tube M leads to a manometer, the bottles P and H and the valve J serve to regulate inequalities of pressure.
PREPARATION OF PURE COMPOUNDS
III. SEPARATION OF SOLID AND LIQUID. FILTRATION.
The mixture of solid and liquid may consist of suspended particles consisting of residues of filter paper, etc., which do not require ex- amination, or it may consist of matter requiring examination, obtained either naturally or by evaporation of the liquid, or it may consist ot solid in a crystalline form purified by crystallisation.
Obviously the procedure to adopt is filtration. Filtration is effected in several ways depending on the material.
(a) Fluted or Pleated Filter Paper.
Suspended matter is removed by filtration through a filter paper folded in the ordinary way to fit a funnel, or better and more rapidly through a fluted or pleated filter paper which exposes as large a sur- face as possible to the liquid. A filter paper is folded into quarters in the ordin- ary way. Each quarter is then bisected by folding towards the hollow of the central fold, and each of these divisions is bisected again in such a way that the hollows and ridges alternate (Fig. 10). More pleats are obtained in the same way by bisecting the divisions and folding alternately. The paper is thoroughly pressed to make the pleats permanent.
Such a filter paper is used for rapidly filtering off a small number of particles and also for filtering off solid matter which is not crystalline, such as the residues re- maining after extracting animal and plant
tissues with solvents, residues which are not easily filtered by the other methods.
(b) Filter Plate. Buchner Funnel.
The filtration of crystalline compounds is best effected by means of a perforated porcelain plate placed in a funnel or a complete funnel of porcelain of this pattern (Buchner or Hirsch funnel). The perforations are covered over with a filter paper of the right size and to prevent breaking of the paper two thicknesses may be used, or better hardened filter paper. The paper is wetted with the liquid and sucked down by a vacuum produced by a filter pump (Fig. 1 1).
The substance is placed upon the paper and the liquid drained off as
i6
PRACTICAL ORGANIC AND BIO-CHEMISTRY
completely as possible, the solid being pressed down with a spatula or flat piece of glass. The solid is washed two or three times with solvent which is added in small portions. The whole of the solid must be wetted and the solvent drained off completely before more is added.
Fio. n.
(f) By Filter Presses.
Large quantities of solid matter with small amounts of liquid are separated by means of a filter press. The material is pressed either between layers ol cloth or in a sheet of cloth which can be folded to make a sort of bag. Squeezing by hand in the latter method will remove some of the liquid and the greater pressure from a press will remove nearly all. The dry residue can be taken out, stirred up with solvent and again pressed out.
The greatest pressure usually attainable is from a Buchner press or a hy- draulic press. * The solid matter must be in a fairly dry condition and, if moist, is mixed with some absorbent, generally siliceous earth or " Kieselguhr ". Liquid is squeezed out of this mass by the high pressure.
Subsequent filtration through a fluted paper is usually necessary after fil- tration by a filter press as particles come through the pores in the cloth.
(d) Through a Layer of Neutral Material.
Solutions containing colloidal particles are generally most difficult to filter as the particles either come through fluted filter paper or soon clog the pores.
Osborne has used filter paper pulp as a medium for filtering solutions of pro- teins. A sufficient quantity of pulp is nv'xed with the liquid and this is poured up >n the paper on a Buchner funnel, where it forms a layer exposing a large sur- face and prevents the underneath paper from breaking and becoming clogged.
A layer of siliceous earth upon a Uuchner funnel has al.^o been found very efleci ufficient quantity of the siliceous earth is mixed in a small mor-
tar with the liquid and poured upon the funnel so as to forma layer from 1-2 cm. thick, and th.e liquid is then filtered through this.
The first poitions of filtrate may be cloudy, and must be returned to the filter and filtered again.
PREPARATION OF PURK COMPOUNDS 17
IV. PURIFICATION OF SOLIDS BY CRYSTALLISATION.
The majority of solid organic compounds are crystalline, but many of the complex solids, such as starch, glycogen, various pro- teins, which belong to the class of substances termed colloids, have not yet been prepared in a crystalline form and are only known in an amorphous state. Others, such as the fats, though obtainable in a crystalline form, are mixtures of closely related substances and it is extremely difficult, or almost impossible, to separate them into individual compounds.
The separation of a solid in a crystalline form is essential to its preparation in a pure condition. Its recrystallisation, when once obtained in a crystalline form, will lead to its preparation in a state of purity.
The criterion of the purity of a crystalline solid compound is its melting-point. A pure solid organic compound melts sharply at a definite temperature. An impure organic compound does not melt sharply and it melts at too low a temperature. The knowledge of the melting-point of a compound helps in its identification.
(a) Choice of Solvent for Crystallisation.
The purification of a solid by crystallisation depends very largely upon the choice of a suitable solvent. The best condition for puri- fication is very slight solubility in the cold solvent and ready solubility in the boiling solvent. A hot saturated solution of the solid will de- posit the greater part of the solid in a crystalline condition on cooling, and if the solution be allowed to cool slowly the crystals which are deposited will be more regular than if the solution be cooled rapidly.
In order to ascertain the solubility of a substance in a solvent the substance must be in a fine state of division. A small quantity of the substance is finely powdered in a watch glass with a glass rod, or better in a small agate mortar with a pestle.
A few milligrams of the substance are then placed in a small test tube, a few drops of solvent are added and the solid well stirred or shaken with it. If the solid is apparently not soluble in this amount of solvent more is gradually added, and so it can be determined whether the substance is easily soluble, moderately soluble, or in- soluble in the cold liquid.
Substances which are readily soluble in cold solvent are not usually recrystallised from this medium since there will be no great difference in solubility in the hot and cold liquid, and a large quantity of solid will remain in solution even when crystals are obtained,
1 8 PRACTICAL ORGANIC AND BIO-CHEMISTRY
The solvent in those cases where the substance is slightly soluble or insoluble is now heated ; if the solid dissolves easily more is added until the solution is saturated ; if not, more solvent is added so as to bring, if possible, the solid into solution. The solution is cooled by holding under running water and it is noticed how much of the solid crystallises out. If a considerable quantity separates out, the solvent will probably be suitable for recrystallising larger quantities.
Sometimes crystallisation does not occur spontaneously on cooling, but it may be started by scratching the sides of the test tube or by adding a crystal of the solid
The following solvents are most frequently used : —
(1) water
(2) alcohol
(3) acetone
(4) benzene
(5) chloroform
(6) ligroin
(7) glacial acetic acid
(8) methyl alcohol
The following substances — cane sugar, oxalic acid, benzoic acid, urea and succinic acid — may be taken as examples for observing the difference in solubility in water and alcohol, and for the choice of solvent for crystallisation.
(b) Recrystallisation.
If the suitable solvent has been found to be water, or glacial
acetic acid or a liquid which is not inflammable and boils at a fairly high temperature, the recrystallisa- tion may be carried out in a beaker heated over a gauze.
If the suitable solvent has been found to be alcohol, acetone, ligroin, benzene — liquids which are volatile and inflammable — the re- crystallisation must be carried out in a flask to which is attached a reflux or inverted condenser, as in Fig. 12.
Solvents boiling below 90° are heated on the water-bath, above 100° over a flame, .through a wire gauze and with an air condenser (inner tube of condenser or a tube about 80 cm. long by "5-1 cm. in diameter) as reflux.
FlO. 12.
PREPARATION OF PURE COMPOUNDS 19
(i) Solution.
The substance to be recrystallised is powdered finely and placed in a flask or beaker with a small quantity of solvent. Excess of solvent must be avoided as the object is to prepare a hot saturated solution. The solvent is boiled. If, after boiling for some time, a consider- able amount of solid remains, more solvent is cautiously added (through the condenser) and the boiling continued ; solvent is added until the whole of the solid, except insoluble matter, is dissolved. It should be noted that thuj last portions of a solid often only dissolve with difficulty and fresh solvent should not be added too soon.
(ii) Filtration.
Particles of insoluble impurity are now filtered off by rapidly filter- ing the hot solution through a pleated filter paper. Very frequently the solution is very concentrated and begins to crystallise immediately filtration is commenced. To avoid this a funnel with a very short stem or without stem is used, and it is previously heated in an oven or by passing through a flame ; the solution is filtered whilst it is still hot.
When the tendency to crystallise immediately is very pronounced the filtra- tion must be carried out through a funnel heated by steam. The funnel may be surrounded by coils of metallic piping through which steam from a generator is passed, or it may be enclosed in a larger metal funnel with two walls between which there is water and from the outer of which there is a projection for heating the contents by a flame. Care must be taken that inflammable liquids do not become ignited if this form of hot-water funnel be used. The water can be raised to boiling and the flame removed.
If crystallisation of solid should commence dur-ngthe filtration, the funnel and paper are placed over the flask, the paper pierced and the particles washed with a little solvent into the flask and the solution again boiled up.
The filtrate is collected in a beaker of such a size that it is not filled more than two-thirds.
Some liquids " creep," and if the vessel be filled too full or if shallow dishes be used the solution will creep over the edge and de- posit crusts of impure crystals over the edges.
If crystals begin to separate before the filtration is completed, it is best to heat the filtrate until solution is again effected. To exclude dust and prevent evaporation, the beaker is covered with a clock glass with its convex side uppermost. Condensed drops of solvent will then run towards the side and not drop into the liquid and disturb the formation of the crystals. The solution is set aside in a cool place to crystallise.
Crystallisation may be complete as soon as the solution is cold, or it may take several hours, or days.
20 PRACTICAL ORGANIC AND BIO-CHEMISTRY
(iii) Collecting of Pure Crystals.
Any crusts of crystals which may have formed on the sides and edges of the vessel by evaporation must be removed before the re- mainder of the crystals are collected, as they are impure. They are carefully scraped off with a spatula and collected and returned to the mother liquor after filtration.
The pure crystals are collected on a filter plate or on a Buchner funnel (p. 16). The vessel is rinsed out with a little solvent which is used also for washing the crystals, and washing is done two or three times.
(iv) Drying of Pure Crystals.
The crystals are left to drain as completely as possible in the funnel and are then transferred to either
(1) several thicknesses of filter paper and the liquid pressed
out ;
(2) a piece of unglazed porous plate, carefully dusted before
use;
(3) a watch or clock glass and dried in the air.
To keep out dust they are covered over by a clock glass or funnel which is raised up about 2 cm. on supports so that an air space is left for the solvent to evaporate ; this may take 24 hours ; or they are dried by placing them in a vacuum desiccator over sulphuric acid, soda lime, etc.
Sometimes the crystals, if placed on a watch glass and when they are nearly free from solvent, are dried by putting the glass containing them on a boiling water-bath. The crystals in this case should not melt below 1 00° or contain sufficient solvent that they dissolve in it on warming. They are cooled by placing in a desiccator.
Some substances, e.g. carbohydrates, are very difficult to dry completely, but can be obtained anhydrous by keeping them in vacua at 100° or 130° in presence of phosphorus pentoxide for a few hours. A convenient form of ap- paratus for drying such compounds is shown in Fig. 13. The substance is put into a tube or boat and placed in the central vacuum vessel. This is connected by a ground joint to a bulb containing P.2O5 in the neck of which there should be some glass wool to prevent 1 './)., from entering the vacuum tube. It is ex- hausted and heated by inserting in a jacket tube, which is kept hot by the vapour of a liquid boiled in a flask below and condensed above.
PREPARATION OF PURE COMPOUNDS
.CONDENSER
21
VAPOUR JAGt
c
VACUUM
A TOB = 8 INCHES
E TO 0=54 •• B TO F= 3 »
TO FLASK
FIG. 13.
(v) Mother Liquor.
The mother liquor generally contains some dissolved solid, which should be recovered. The impure crusts, if any, are added to the liquid and the liquid is concentrated by distilling or evaporating (p. 13) until crystals begin to separate. The solution is poured or filtered into a beaker and allowed to cool ; a second crop crystallises out and is treated as above. A third and more crops may be obtained on further concentration. These are not so pure as the original crop, but may be recrystallised and obtained pure.
(vi) Decolorising Solutions.
Substances containing tarry or resinous impurities or colouring matter cannot sometimes be freed from them by simple recrystallisation. During recrystallisation and while the solid is in solution (especially aqueous or alcoholic), the solution is boiled for 2-5 minutes or longer with a small quantity of blood charcoal which is removed by filtration. The first portions of filtrate generally require filtering again through the same paper as the finely divided charcoal passes through at first. To remove colouring matter from a solution which should be colour- less, prolonged boiling with several quantities of charcoal is sometimes necessary.
The purification by crystallisation of about 5 gm. benzoic acid, oxalic acid and succinic acid from water and of urea from alcohol serve as simple examples of the method of recrystallisation.
22 PRACTICAL ORGANIC AND BIO-CHEMISTRY
(f) Crystallisation from a Mixture of Solvents.
A solid may be too readily soluble in some solvents, but insoluble in other solvents, and consequently its recrystallisation from a single solvent may be difficult. Crystallisation may be then effected by using a mixture of solvents. A concentrated solution is made in the hot solvent and the other solvent is added whilst the first is still hot until the mixture becomes turbid, when it is allowed to cool. Sometimes it is better to add the second solvent to a strong solution until it is turbid, heating until the turbidity has gone, and adding, if necessary, more of the first solvent. On cooling crystals may appear.
Alcohol and water, acetone and water, benzene and ligroin, chloroform and ligroin, alcohol and ether are mixtures of solvents frequently used.
Trials should be made to ascertain the best mixture of solvents.
(d) Crystallisation by Evaporation.
Another possibility is that the substance is easily soluble in all solvents. Crystallisation is then effected by partial evaporation. The filtered solution is placed in a shallow dish, i.e. a crystallising dish ; it is covered with a funnel or clock glass which is raised up so as to leave an air space and the dish is pre- ferably placed on a clock glass in case the solution creeps, so that the crystals can be scraped up without contamination from the bench. Crystals gradually form and they are removed before complete evaporation has occurred. Crusts are removed and treated as described above ; the crystals are filtered off, washed with very little solvent and dried. The mother liquor in this case will contain a large amount of solid.
The evaporation need not be conducted in the air ; it may be hastened by placing the solution in a desiccator over sulphuric acid, etc., and under reduced pressure, or by evaporation by distilling the solution ; if aqueous, evaporation over a flame or on a water-bath.
(e) Solids Soluble with Difficulty in all Solvents.
If a solid is almost insoluble in all solvents recrystallisation is effected, after preliminary testing, by boiling the solid for some time with the best solvent, filteiing off the insoluble portion, and evaporating the filtrate to a small volume. The crystals which separate are treated in the way described above.
Operations with Small Quantities.
Filters of small sizes are obtainable to retain the solid. Small filter flasks to contain the mother liquor are obtainable in the shape of test ttfbes, or a test tube may be placed inside the filter flask so that the mother liquor collects in it.
When there is too little solid and mother liquor for filtration, the crystals and mother liquor are placed upon a piece of porous earthenware or between sheets of filter paper, so that the liquid is absorbed.
Recrystallisation of the solid may be effected in a small test tube, and the mother liquor may be dissolved out of the filter paper or earthenware, if required.
PREPARATION OF PURE COMPOUNDS
DETERMINATION OF THE MELTING-POINT.
A small quantity of the finely powdered substance is introduced into a melting-point tube; this is attached! to a thermometer and the two are heated together in a bath until the substance is seen to melt. The first determination of an unknown substance is usually only approximate: it is repeated, heating rapidly to within 10° and then more slowly.
A small beaker containing water is used as a bath if the melting- point is below 1 00°, and the thermometer in a cork is held in the centre of it by a clamp. The beaker is heated over a gauze by a small flame and the liquid is stirred with a circular glass stirrer.
A flask of about 50 ex. capacity with a long neck (10-20 cm.) filled about two-thirds with strong sulphuric acid is more generally used. The thermometer is secured in a cork into which a notch is cut to
FIG. 14.
allow hot air to escape when the flask is heated and to see the gradua- tions if the mercury reaches this level. The flask is held by a clamp and heated with a flame directly, the burner being inclined at an angle and held by the hand so that it is heated round and round and not directly in the centre (Fig. 1 4). After frequent use the acid becomes dark in colour, but it will become clear again if a tiny crystal of potassium nitrate be added.
Paraffin wax is most generally used when the melting-point of a substance is above the boiling-point of sulphuric acid (290°). The solid substance is introduced and melted until it fills two-thirds of the space. It becomes brown after being used several times and must be renewed.
24 PRACTICAL ORGANIC AND BIO-CHEMISTRY
A melting-point tube consists of a capillary of thin glass about I mm. in diameter, $-6 cm. long and closed at one end. It is made by heating near one end a dry piece of glass tubing of about I cm. bore in a blow-pipe flame until it is red-hot and soft, removing it from the flame and pulling it out carefully just when the glass begins to harden. A long capillary tube is thus made. Several more such lengths can be made from the glass tube if the capillary so made be broken off about 2-3 inches from the remainder of the glass tube. The long capillaries are cut into short lengths of about 5-6 cm. by scratching at these distances with a file and breaking by bending. Short lengths and lengths of too small bore must be rejected. One end of each capillary is sealed by holding it in a small flame. The tubes so made are preserved in a corked dry test tube.
The powdered substance is introduced by scooping up solid with the open end and making it fall to the other end by gently tapping the closed end on the bench. This process is repeated until sufficient of the substance to occupy a lengh of 2-5 mm. in the capillary has been introduced and shaken down, or pushed down with a fine wire, so as to form a compact and continuous layer.
The filled melting-point tube is attached to the thermometer so that the substance is on a level with the bulb. If a bath of water or paraffin wax be used the attachment is made with a strip of rubber cut from a length of rubber tubing. If a bath of sulphuric acid be used the attachment is made by adhesion. The thermometer is wetted with acid and if held horizontally the acid runs along it. The melting-point tube is wetted with acid by drawing it along the thermometer and it will adhere when the surfaces of contact are wet and will not fall off on putting it carefully into the bath.
The pure specimens of benzoic acid, succinic acid and urea may be used for determination of the melting-point.
Corrected Melting-point Determinations.
Just as in the case of boiling-point determinations a correction should be made for the thread of mercury outside the bath. The small thermometers of 50° range may be used and attached by platinum wire to the ordinary ther- mometer. A corrected thermometer is generally used which has been cali- brated against the small ones.
PREPARATION OF PURE COMPOUNDS 25
V. SEPARATION OF SOLIDS.
It is almost impossible to formulate a general scheme for the separation of a mixture of solids. The separation depends to a very large extent upon whether all the constituents, or whether only one constituent, or whether one or more groups of compounds in the mixture, is to be investigated ; it depends also upon the nature of the constituents in the mixture and of those requiring separation.
A scheme of extraction with various solvents, such as the following, may be adopted for the separation of the constituents in a natural product.
Extraction with Solvents.
The solvents most commonly used in separating one or a group of constituents from plant or animal material are : —
(1) Ether, chloroform, petroleum ether, acetone, methyl alcohol.
(2) Dilute mineral acid (HC1).
(3) Dilute alkali — sodium carbonate and sodium hydroxide.
(4) Water or glycerol.
When solvents such as ether, petroleum ether, etc., which are not miscible with water are used as the first solvent, the material is gener- ally dried preparatory to their use.
The extraction of the material can be carried out in several ways : —
(1) If ether, etc., be used, the extraction is effected in a Soxhlet apparatus (p. 178), or, more simply, by hanging up the material in a paper or linen bag in a wide tube so that condensed liquid falls upon the material and from the material back to the extracting solvent
(2) In a percolator.
(3) Or simply in a glass or metal vessel. The mass is frequently stirred with the solvent and filtered off and the residue pressed out.
Drying of Material.
Plant and animal tissues are composed to a very large extent of water, which varies from 10-75 Per cent- m amount, and in order to ensure proper contact of solvent, which is immiscible with water, with the material it is necessary to dry it before extraction.
Plant tissues are generally more easily dried than animal tissues ; the material is brought into a fine state of division by chopping, or grinding, or some other process, by hand or by machinery, and it is exposed to temperatures ranging from 40-100°, depending on the constituent required.
Animal tissues which consist mainly of protein are more troublesome to obtain in a dry state. They are more difficult to bring into a fine state of
$6* PRACTICAL ORGANIC AND BIO-CHEMISTRY
division and on simple drying by exposure the surface forms an impenetrable skin, preventing evaporation of water. Mincing, chopping, grinding with sand and other processes are used to prepare them for drying.
It is essential that drying be carried out rapidly to prevent metabolic changes going on during the process of drying.
(a) Exposure to Air or Indifferent Gas.
The finely ground material is spread over a large surface on glass plates, or if vegetable on a metal mesh. It can be left exposed to air at the ordinary temperature or in a room or box at 40° or higher, or better by blowing a current of clean air, heated to 40°, over the surface. To prevent oxidation of the constituents during drying, the material is placed in a vacuum desiccator or suitable chamber through which a current of carbon dioxide or nitrogen can be blown instead of air.
After exposure to air or indifferent gas, more water can generally be removed by placing the material in a vacuum desiccator over dehydrating agents.
(b) Treatment with Alcohol, or Acetone.
Since alcohol and acetone are miscible with water in all proportions, they not only help to remove water, but also they disturb the conditions in the tissue, which are necessary for metabolic changes, by precipitating proteins, etc., and by disturbing the peculiar solubility of fatty and protein material. The material is mixed with about an equal bulk of solvent or sufficient to cover it completely and the mass is allowed to stand for 12-24 hours. The material may be put into boiling solvent and heated for about an hour.
The solvent is filteied or strained off and the tissue treated again with the same solvent, or with ether, etc. Substances are soluble in the mixture of alcohol or acetone and water separated from the tissue and this extract requires examination.
(c) Admixture with Neutral Dehydrating Agent.
Animal tissues after grinding are sometimes mixed with anhydrous sodium sulphate, or calcium sulphate, by grinding together equal parts in a mortar. The mixture sets after some time to a cake which can be finely ground and extracted with ether, acetone, etc. By boiling with alcohol proteins are coagulated and the other constituents may be extracted with water.
ISOLATION OF SOLIDS FROM SOLUTION.
The extracts obtained above, either directly or after concentration by evaporation, evaporation in vacuo, distillation, etc. (if acid or alkaline after neutralisation), or solutions of solid compounds whether natural or obtained by chemical reactions between compounds, may be treated in several ways in order to separate the solid compound.
A. Precipitation.
(i) By adding another solvent.
(ii) By acidifying. Aqueous or alkaline aqueous extracts may contain acids. On acidifying with mineral acid, the acid or acids, if they are insoluble or soluble with difficulty in water, will be pre- cipitated.
PREPARATION OF PURE COMPOUNDS V)
(iii) By making alkaline. Aqueous and acid extracts may contain bases ; when made alkaline with sodium carbonate or sodium hydrox- ide, the base or bases may be precipitated.
(iv) If no precipitate has been obtained in (ii.) or (iii.) the solution may be submitted to steam distillation. Volatile acids and bases distil over.
(v) Acids may be precipitated from the exactly neutral solution as insoluble salts of heavy and other metals by adding lead acetate, mercurous nitrate, calcium chloride, etc.
(vi) Bases may be precipitated by adding to the previously acidi- fied solution phosphotungstic acid, or other reagents used for precipitat- ing alkaloids from solutions.
(vii) Bases may be precipitated (especially from alcoholic solution) as double salts with mercuric chloride, gold chloride, platinum chloride.
B. Extraction.
The acid or alkaline extract may be extracted with an immiscible solvent by repeated shaking or by means of an extractor (p. 600).
C. Fractional Crystallisation.
The solution on evaporation may deposit crystals. On filtering and evaporating further, another crop of crystals may be obtained.
D. Salting Out.
Proteins, polysaccharides, soaps and other complex compounds separate out when their aqueous solutions are saturated with sodium chloride, magnesium, zinc and ammonium sulphates.
E. Dialysis.
Mixtures of colloids and crystalloids are separated by dialysis.
F. By Preparing Suitable Chemical Derivatives.
Some knowledge of the chemical nature of the compound is essential before a derivative can be prepared. The principal reactions of the various groups o,f componnds are given under the separate headings.
Two other procedures are occasionally employed in separating solids : — (i) Sublimation. On carefully heating a mixture in a basin, one or
more solids may be volatilised. Tiie vapours are condensed on a cool clock
glass or funnel and the substance is thus obtained.
(ii) Sedimentation. A mixture of solids of different specific gravity
may be separated by shaking with a liquid of suitable density.
Separation may not be effected by applying any one of the above methods. Usually only an incomplete separation is made and further separation is carried out by using another method.
COMPOSITION OF ORGANIC COMPOUNDS.
A pure carbon compound which has been prepared is analysed, i.e. the elements besides carbon contained in it and their amounts are determined.
Carbon compounds may contain the elements hydrogen, oxygen, nitrogen, halogens, sulphur, phosphorus, etc., either singly or collectively. Usually all the possible elements are not present, but the proteins contain carbon, hydrogen, oxygen, nitrogen and sulphur ; some con- tain also phosphorus and a few contain halogens. Proteins belong to the most complex of the organic compounds. The method of analysis of the compound is varied according to the elements which are present. The elementary composition, or detection of the elements, precedes the quantitative composition. Since all organic compounds contain carbon and most of them contain hydrogen it is not absolutely essential that the presence of these elements should be ascertained. Their quantita- tive analysis is carried out simultaneously under the same conditions.
A ELEMENTARY COMPOSITION.
DETECTION OF THE ELEMENTS.
i. Carbon and Hydrogen.
(a) A small portion of the substance (e.g. cane sugar) is gently heated in a test tube or on platinum foil. It melts and chars. The charring denotes the presence of carbon. There is a condensation of water on the sides of the tube where it is cool ; this denotes the presence of hydrogen.
(b) About 5 grms. of finely powdered cupric oxide are dried thoroughly by heating in a small ^crucible. Whilst still warm1 it is mixed with a little of the substance (e.g. oxalic acid) and the mixture is introduced into a hard glass tube. The end is closed with a cork through which passes a glass tube, bent at right angles. This end is dipped into a little baryta water contained in a small beaker or test tube. On heating the mixture in the hard glass tube, water will condense on the cooler parts of the tube — presence of hydrogen ; and the baryta water will become turbid owing to the formation of barium carbonate — presence of carbon.
1 As cupric oxide takes up water on cooling it must be used warm, otherwise it must be allowed to cool in a desiccator over sulphuric acid.
28
COMPOSITION OF ORGANIC COMPOUNDS 29
2. Nitrogen.
(a) As Ammonia. — A portion of the substance (e.g. caseinogen) is ground up with soda-lime and heated in a dry test tube. Ammonia is given off as shown by the smell, by litmus paper and by the production of white fumes when a glass rod dipped in hydrochloric acid is held over the mouth of the tube (Will and Varrentrapp's method).
The peculiar smell of burning flesh, horn, etc., produced on heat- ing such substances alone, also indicates the presence of nitrogen.
(f) As Sodium Cyanide. — A small piece of metallic sodium is heated in a small dry test tube of hard glass until the metal begins to boil ; successive minute portions of the substance (dried egg albumin) are added. The heating is continued for a short time, the tube is cooled, and the lower end of it is broken in a. mortar, containing a few drops of alcohol ; water is added when effervescence has ceased. The solu- tion is transferred to a test tube, warmed and filtered. To the filtrate some ferrous sulphate solution (this must be freshly prepared by dis- solving a few small crystals in a little water) and caustic soda are added and it is boiled for a few minutes. It is cooled and a drop or two of ferric chloride and excess of dilute hydrochloric acid are added. A precipitate or coloration of Prussian blue indicates the presence of nitrogen (Lassaigne's method).
Note. — It is important that the substance be made to come into proper contact with the sodium.
Castellands Modification of this Test. — A small quantity of the sub- stance is intimately mixed with about ten times its quantity of equal parts of magnesium powder and dry sodium carbonate and gently heated until the magnesium burns ; it is then heated to redness as with the sodium (£). The remainder of process is carried out as described above, i.e. breaking the tube in a mortar, etc.
3. Halogens.
(a) Beilsteiris Test. — A piece of copper wire is heated in a Bunsen flame until the flame is no longer coloured green. A little of the sub- stance, e.g. chloroform, is placed on it and it is again heated. Copper chloride is formed which colours the flame green.
(ti) Halogen may also be detected by means of sodium employed just as in the nitrogen test. The filtered solution is acidified with nitric acid, boiled to remove any hydrocyanic acid which will be formed if the substance also contains nitrogen, and then treated with silver nitrate.
30 PRACTICAL ORGANIC AND BIO-CHEMISTRY
The nature of the halogen may be determined by treating a little of the acidified solution with chlorine water, and then testing with starch solution for iodine, or by extracting with carbon bisulphide or chloroform for bromine.
(c) Heating with Lime. — Halogens are best detected by heating with quicklime. The substance is finely powdered and mixed inti- mately with lime (if liquid, e.g. chloroform, the lime is moistened with the substance) and then heated strongly. When cool, water is added and the lime dissolved in nitric acid. On adding silver nitrate, a pre- cipitate of silver halide is obtained if halogen be present.
4 Sulphur.
(a) As sodium sulphide. A small portion of the substance is heated with metallic sodium as described under 2 (b). The hot tube is broken in a little water, the contents are filtered and tested for sodium sulphide with (i) lead acetate, (2) sodium nitroprusside.
(b) As sulphate. A small portion of the substance (dried fibrin) is fused in a crucible with three times its quantity of fusion mixture {2KNO3 + Na2CO3}. The mixture is heated cautiously at first round the edge and the heating is continued after the fusion until all charred particles have vanished. The mass, when cool, is extracted with hot water and the filtered solution is tested for sulphates with barium chloride in the presence of mineral acid (HC1 or HNO3).
5. Phosphorus.
(a) Some caseinogen is fused with fusion mixture as described for sulphur, the fused mass is extracted with hot water, and the solution is divided into two parts. To the one part is added excess of nitric acid and ammonium molybdate : a yellow precipitate on warming indicates "phosphoric acid; to the other part excess of ammonia is added and phosphates are precipitated with magnesia mixture.
(ft) A small quantity of caseinogen in a small flask is covered with 5-10 c.c. of concentrated sulphuric acid and an equal volume of con- centrated nitric acid is added. The mixture is heated gently over a small flame (in the draught crnmber) until the mixture becomes colourless. If it becomes brown, it is cooled, more nitric acid is added and it is heated again. When it is colourless, it is allowed to cool, water and a little ammonium nitrate solution are added and it is heated nearly to boiling; on adding ammonium molybdate solution, a yellow colour or precipitate indicates the presence of phosphoric acid (Neumann's method).
COMPOSITION OF ORGANIC COMPOUNDS 31
6. Other Elements.
Amongst natural compounds the most important other elements combined with carbon are iron, e.g. iron in haemoglobin, ferrocyanides, and magnesium, e.g. magnesium in chlorophyll. Copper is found in certain other animal pigments. Silicon is present in certain vegetables, e.g. in grasses. Organic silicon, arsenic, antimony and magnesium compounds have been prepared in the laboratory. These elements are best detected after the organic matter has been completely re- moved by burning either alone or in the presence of an oxidising agent (fusion mixture). Thus : —
Detection of Iron in Haemoglobin. — A small portion of haemo- globin is heated in a crucible with 3-4 times its quantity of fusion mixture until all the organic matter has been oxidised. The mass, when cold, is dissolved in dilute hydrochloric acid. The solution is filtered and the filtrate is tested for ferric salts with (a) ammonium thiocyanate and (b) potassium ferrocyanide.
B. QUANTITATIVE COMPOSITION.
ESTIMATION OF THE ELEMENTS.
i. Carbon and Hydrogen.
An organic compound on oxidation with copper oxide is con- verted into carbon dioxide and water. The amount of each element contained in the compound is determined by weighing the carbon dioxide and water produced from a known weight of the compound.
FIG. 15. — Combustion Furnace,
The analysis is carried out in a long tube of hard glass, a com- istion tube, about 80 cm. long, which is heated in a furnace (Fig. 15).
32 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Five-eighths of the length of the tube is filled with coarse coppec oxide which is kept in position by narrow plugs of oxidised copper gauze. Next to the copper oxide there is a small boat (copper or porcelain) of suitable size, containing a known weight of the substance, and the remaining space is filled by a roll of oxidised copper gauze (Fig. 1 6). This end of the tube is connected with a gasometer
FIG. 1 6.-
A = Oxidised Roll of Copper Gauze.
B = Boat.
C = Coarse Copper Oxide.
-From Price and Twiss' " Practical Organic Chemistry ",
containing air (or oxygen) and a current of air freed from carbon dioxide and water by passing through potash and sulphuric acid or calcium chloride, is passed through the combustion tube so as to drive out the products of the combustion and to help in the oxidation. To the other end of the combustion tube are attached absorption tubes (Fig. 1 7), of which there are various forms, to collect the carbon dioxide
Calcium Chloride Tube. Potash Absorption Bulbs.
FIG. 17. — From Price and Twiss' " Practical Organic Chemistry ".
and water. The first absorption tube, generally of U-shape, contains calcium chloride1 or pumice wetted with concentrated sulphuric acid; the second, generally complex in shape so as to give several surfaces, contains caustic potash of 33 per cent, strength; a small tube con- taining calcium chloride is attached so as to retain water vapour, which may be carried away during the passage of the gases. These tubes are weighed before and after the combustion..and their increase in weight gives the required data.
1 After filling the tube with calcium chloride dry carbon dioxide must be passed through it until its weight remains constant.
COMPOSITION OF ORGANIC COMPOUNDS 33
In practice, the combustion tube is filled, as described, with coarse copper oxide, which has been heated to redness in a copper basin and allowed to cool. The plugs and the roll are made of copper gauze which is rolled round a piece of copper wire and heated in a blowpipe flame to oxidise the metallic copper and burn away any organic matter. A space is left for the boat. The tube is heated in the furnace at a low red heat and a current of dry clean air or oxygen is passed through the tube from the gasometer. The carbon dioxide and water present in the tube and on the copper oxide are thus removed and the copper is completely oxidised to copper oxide. The heating of that portion of the combustion tube into which the boat is to be placed is discontinued so that this portion cools to room temperature whilst the rest of the tube is kept at a red heat. The absorption tubes are filled and weighed. About '2 gm. of substance is exactly weighed out into the boat which has been heated and cooled in a desiccator. When the end of the combustion tube is cool the absorption tubes are attached, that for water next to the tube. From the other end the roll is removed with a hooked copper wire, the boat quickly introduced and the roll replaced. The tube is closed and the pure air or oxygen current passed through at a rate of about 3 bubbles every two seconds. The roll is heated commencing at the end farthest from the boat and the heating is gradually extended from this point towards the boat and the coarse copper oxide until the substance has burnt away and the whole tube is heated from end to end. The air or oxygen current is continued for about half an hour after the combustion is finished so as to drive out the water and carbon dioxide. Any water which condenses on the end of the com- bustion tube is driven into the absorption tubes by means of a small flame, or hot brick, held under the end of the combustion tube. When the oxida- tion is completed, the absorption tubes are removed, allowed to cool for -J to i hour and weighed.
This method of analysis requires some modification if elements other than hydrogen or oxygen are present in the substance.
(a) Halogens. — On combustion, the halogen in an organic compound is evolved as hydrogen halide, or as halogen. To prevent its entry into the ab- sorption tubes it is combined with silver as silver halide. This is effected by putting at the end of the combustion tube a roll of silver gauze.
(b) Nitrogen. — Oxides of nitrogen may be evolved when organic nitrogen- ous compounds are analysed. A roll of metallic copper gauze, prepared by heating a roll in a blowpipe flame and dropping it into a few c.c. of methyl alcohol contained in a test tube (held in a duster) and drying at 100°, is intro- duced at the end of the combustion tube. Any oxides of nitrogen are thus reduced to nitrogen and prevented from being absorbed by the potash.
(f) Sulphur and Phosphorus. — To prevent hydrogen sulphide or hydrogen phosphide being formed, the oxidation of the organic compound is effected with lead chromate instead of copper oxide and the substance is mixed with it instead of being placed in the boat. Lead chromate may replace the irse copper oxide entirely or about half of it.
Organic phosphorus compounds are very difficult to oxidise completely id frequently give results for carbon which are too low by about -5 to i per it.
34
PRACTICAL ORGANIC AND BIO-CHEMISTRY
2. Nitrogen.
(a) Dumas' Method. — On heating an organic compound contain- ing nitrogen with copper oxide, its nitrogen is given off as nitrogen. The gas given off from a known weight of substance is collected and its volume measured, from which value its weight can be calculated.
The analysis in practice is carried out in a similar way to that described for carbon and hydrogen, but the substance is mixed with finely powdered copper oxide and introduced into the tube; and a roll of reduced copper gauze is placed at the end of the combustion tube so as to reduce any oxides of nitrogen, which may be formed, to nitrogen. Instead of a current of air, a current of carbon dioxide is passed through the tube. The gas is collected in a Schiff s nitro- meter over caustic potash which absorbs the carbon dioxide leaving the nitrogen (Fig. 1 8).
CO* r*r-
FIG. 18.
There are several ways of passing the carbon dioxide through the combustion tube ; it may be evolved from a Kipp apparatus or it may be evolved by heating magnesite, either contained in a special tube or in the combustion tube, which in this case is sealed at the end. It is obvious that before carrying out the analysis all the air must be ex- pelled from the apparatus.
COMPOSITION OF ORGANIC COMPOUNDS 35
(ff) Kjeldahl's Method.— The principle of this method consists in oxidising the substance with concentrated sulphuric acid ; the nitrogen is converted into ammonia. The solution is made alkaline with caustic soda and distilled. The ammonia is evolved and is collected in excess of standard acid ; on subsequent titration with standard alkali the amount evolved is given by difference.
This method is much simpler to carry out than the Dumas1 method, but it cannot be employed for all nitrogen- containing compounds.
Dakin and Dudley l have found that pyrrole and its derivatives, piperidine and some of its derivatives give satisfactory results if the heating of the sub- stance— about -15 gm. — with sulphuric acid be continued for at least four hours after the solution becomes clear. Pyridine, quinoline, pyrazole and their derivatives do not give satisfactory results. \
Of other groups of compounds, nitro-, nitroso-, azo-, diazo-, hydrazo-, aminoazo- compounds, also compounds of nitric and nitrous acid, i.e. those compounds containing nitrogen joined to oxygen or another nitrogen atom, give satisfactory results if they are previously reduced with tin. Osazones do not give satisfactory results.
On account of its simplicity this method has found extensive use in biological chemistry. The large group of compounds — the pro- teins— all contain nitrogen ; the amount of protein in a solution is estimated by determining the nitrogen content (see below) ; and the amount of nitrogen in urine is a factor of importance in studying metabolism.
In most laboratories there is an apparatus in which six determina- tions can be carried out at the same time as in Fig. 20, p. 37.
Example: Estimation of Nitrogen in Egg-white Solution.
A known volume, say 5 c.c., of the egg-white solution is placed with a pipette into a clean round-bottom Jena glass flask of 700 cc. capacity. I o or 20 c.c. of pure concentrated sulphuric acid and a crystal of copper sulphate, about the size of a pea and weighing about 0^25 gm., which helps in the oxidation, are added. (l gm. of potassium sulphate is also sometimes added for this purpose, as it raises the temperature.) The flask is heated in a fume-cupboard until the liquid, which at first becomes brown from charring, becomes quite or nearly colourless, a pro- cess which takes about an hour. The flask is allowed to cool and is half-filled with distilled water. By means of a special distillation tube it is connected to a condenser set up in a vertical position as in Fig. 19, No. I.
*J. Biol. Chem., 1914, 17, 275.
36 PRACTICAL ORGANIC AND BIO-CHEMISTRY
With a pipette a quantity of standard sulphuric acid (5 c.c. of N or 50 c.c. -iN H2SO4) are measured out into a clean beaker or flask (preferably conical) of about 600 c.c. capacity, and this is placed below the condenser so that the end of the condenser just reaches the surface of the liquid It is preferable to add a few drops of indicator, methyl orange or alizarin red,1 before the distillation is commenced in case more acid is required than that originally taken ; the change in colour of the indicator gives notice of this fact.
Sometimes the distillation is carried out without using a condenser (Fig. 19, No. 2) ; the end of the special distilling tube is then dipped into the standard acid. There is in this case usually greater danger of the liquid being sucked back into the flask which is being distilled, and further the glass is attacked by the ammonia with liberation of alkali, which causes inaccuracies in the determination.
No. i. No. 2.
FIG. 19.
The round-bottomed flask is removed, a piece of porous earthenware is added and excess of caustic soda solution (50 c.c. of 40 per cent, for every 10 c.c. concentrated H2SO4 used) is run in under the dilute acid without mixing. The flask is connected again to the condenser seeing that all corks fit tightly. The soda and acid are mixed and the ammonia is distilled off into the acid. In about half an hour the ammonia will have completely passed over into the standard acid To
1 Cochineal, congo red and other indicators may also be used, or the titration may be effected using sodium iodate and potassium iodide and standard thiosulphate solution (P- 563).
COMPOSITION OF ORGANIC COMPOUNDS 37
No. i.
No. 2.
FIG. 20. — Apparatus for estimation of nitrogen by Kjeldahl's method.
38 PRACTICAL ORGANIC AND BIO-CHEMISTRY
test if the distillation is finished the flask is lowered and the condensed water is allowed to wash out the inside of the condenser tube for about two minutes : after this time the distillate is tested with litmus paper to ascertain if any more ammonia is being evolved. When finished, the outside of the condenser tube is washed with distilled water and the contents of the flask are titrated with -I N alkali.
The difference between the amount of standard alkali and standard acid gives the amount of ammonia evolved, from which the amount of nitrogen can be calculated : —
5 c.c.- N H2SO4 = 50 c.c. -iN HaSO4.
37-4 c.c. -iN NaOH used.
12*6 c.c. *iN difference.
12-6 c.c. -iN H2SO4 = 12-6 c.c. -iN NH3
= r.2'6 c.c. *iN Nitrogen = 12*6 x 0-0014 gm. Nitrogen. .'. 5 c.c. solution contain o!oi764 „ „
.-. 100 c.c. „ „ 0-3528 „ „
The amount of protein to which this amount of nitrogen corresponds is ascertained as follows : — •
Proteins contain from 1 5 to 1 6 per cent, of nitrogen. Taking 1 5 -5 as
T OO
the average value, — — or 6*45 times the amount of nitrogen gives the amount of protein. The factor 6-25 is, however, generally used ico c.c. solution thus contain 6-25 x 03528 gm. protein = 2-2 gm.
COMPOSITION OF ORGANIC COMPOUNDS 39
3. Halogens.
After completely oxidising all the organic matter to carbon dioxide and water, the halogens are present as inorganic compounds and are estimated in the usual way.
The commonest method is to oxidise a known weight of the sub- stance in a sealed tube with fuming nitric acid at 200° for several hours a few crystals of silver nitrate being at the same time placed in the tube : silver halide is formed and this is washed out of the tube and weighed (Carius).
In practice, about -2 gm. of the substance is weighed out into a narrow test tube about 2 in. long. Some silver nitrate crystals are placed in a thick- walled combustion tube sealed at one end and covered with 10-20 c.c. of concentrated nitric acid, care being taken not to wet the sides of the tube with acid. This is done by introducing the acid through a tube with a long capillary. The test tube containing the substance is put in avoiding contact of the sub- stance with the acid. The open end of the tube is now sealed in the following way : a glass rod for a handle is fastened by heating to the side of the tube at the open end. The tube is heated near this end in a blow-pipe flame in such a way that the walls collapse together ; when it is nearly closed the end is drawn out so as to form a capillary tube with even and thick walls. The capillary is sealed by pulling off the end with the handle attached. The tube is wrapped in asbestos paper and carefully placed, capillary outwards, in an iron tube which can be closed with a screw cap. The iron jacket and tube are placed in a specially constructed furnace in such a position that the capillary point faces a wall. Should the tube burst, the contents are then not blown into the room. The tube is heated to about 200-220° for 4 or 5 hours. The furnace and contents are allowed to cool. The cap of the iron tube is re- moved and the capillary point allowed to project a little. The point is heated in a flame. When the glass is soft the pressure inside the tube forces an opening. Owing to the high pressure inside the tube it is unsafe to open the tube in any other way. The capillary is cut off and the contents washed out into a beaker. The silver halide is filtered off and weighed by the usual method employed in inorganic chemistry.
Less frequently, halogens are estimated by heating the substance in a combustion tube with quicklime.
A thin layer of quicklime is placed at the closed end of a combustion tube. Next to this is put a mixture of the substance (about -2 gm.) with quicklime and then another layer of quicklime. The tube is heated in a furnace, as in the estimation of carbon and hydrogen, the layers of quicklime being heated to redness before the mixture of substance and lime.
The contents of the tube, after the oxidation, are dissolved in nitric acid and the halogen precipitated with silver nitrate. The silver halide is filtered off, washed, dried and weighed.
A convenient method of estimating chlorine is that described by Neumann. The substance is oxidised with a mixture of nitric acid and sulphuric acid. Hydrochloric acid is evolved, and this is collected in a solution of silver nitrate. After boiling the solution for about half an hour to remove hydrogen cyanide, which is also formed if nitrogen be present in the substance, the silver chloride is filtered off, washed, dried and weighed.1 1 See J. Physiol., vol. 31, p. 65.
40 PRACTICAL ORGANIC AND BIO-CHEMISTRY
4- Sulphur.
This element is most generally estimated by the same method as the halogens (Carius) ; sulphuric acid is formed and precipitated as barium sulphate.
It is more convenient to oxidise the substance in a nickel crucible with sodium or barium peroxide ; the contents are acidified with hydrochloric acid and the barium sulphate formed is weighed. Still more convenient is the oxidation mixture used for estimating total sulphur in urine (see p. 542).
5. Phosphorus.
Phosphorus is usually estimated in the same way as sulphur by the Carius method, the phosphoric acid formed being precipitated as ammonium magnesium phosphate.
The most rapid and 'convenient method is that of Neumann. The substance is oxidised in an open flask with a mixture of nitric and sulphuric acids. The phosphoric acid formed is precipitated as ammonium phosphomolybdate and this is then estimated by solu- tion in excess of ^N caustic soda and subsequent titration with '$N sulphuric acid. The difference between -$N NaOH and *5N H2SO4 multiplied by I -268 gives the number of milligrams of P2O5 in the given weight of substance taken.1 This method is described on p. 545.
Micro-Analyses.
Minute quantities of substance can be analysed by the methods devised by Pregl. These methods are difficult to perform and require much practice. The full details are given by Pregl in Abderhalden's " Handbuch der Biochemischen Arbeitsmethoden," vol. v., part 2, p.
Folin has also described a method for estimating nitrogen with special reference to its estimation in urine and blood. Its technique is comparatively simple and is given on p. 558.
i See J. Physiol., 1906, 33, 439.
COMPOSITION OF ORGANIC COMPOUNDS 41
C. CALCULATION OF RESULTS.
With the exception of oxygen all the elements present in an organic compound are thus estimated. The amount of oxygen is found by difference.
From the figures obtained the percentage composition is calculated, i.e. the amount given by 100 grams of substance, thus: —
0*2009 gm. substance gave o 2987 gm. CO3 and 0*1092 gm. H2O. 0-1887 gm- » M I5'2 c.c. moist N at 16-5° and 767 mm.
Now, 0-2987 gm. CO2 = 0-2987 x H gm. C = 0-2987 x -1- gm. C = 0-0815 gm. C.
44 II
0*1092 gm. H2O = 0-1092 x 2- gm. H = 0-1092 x - gm. H = 0-01213 gm. H. io 9
15-2 c.c. moist N at 767 mm. and 16-5° C. = *5 2 x 753 x 2?3 c.c. at o° and 760 mm.
760 x 289-5
s= 14-2 C.C.
28
= 14-2 x gm.
22,400 = 0-01775 gm- N.
/.percentage of C = °'o8l5XIO° = 40*56. 0-2009
H - °'01213 x I0° - 6-04.
0*2009 N== 0-01775x100^
0-1887
O by difference = 44-00. Total = 100-00
The formula of the compound is obtained by dividing the percent- ages by the atomic weights of the elements ; the ratio of the number of atoms to each other is then obtained by dividing by the lowest value : —
C 12^ = 3-38*0*67 = 5. H ^£f = 6*o4^o-67 = 9. N O? = 0-67 -f 0*67 = i. O li^ = 2-75 -r 0*67 = 4-1.
The formula of the compound is therefore C5H9NO4.
In any estimation only a difference of o*2-O'3 per cent, is allowed between the values found and those calculated from the formula. The calculated values are
C = 40*81. diff. = - 0*25. H = 6*12. = + 0-08.
N = 9*52. = - 0*12.
The analysis was therefore sufficiently accurate.
* Vapour pressure of water at 16*5° C. = 14*0 mm. .*. pressure on gas = 767 - 14 = 753
42 PRACTICAL ORGANIC AND BIO-CHEMISTRY
D. DETERMINATION OF THE MOLECULAR WEIGHT.
As will be seen later, several organic compounds can have the same empirical formula, thus, for instance, lactic acid C3H6O3 and glucose C6H12O6 have the same empirical formula, namely CH2O.
In order to ascertain which of these formulae is the correct one, a molecular weight determination is carried out, i.e. the weight of the molecule of the substance compared with that of an atom of hydrogen (Avogadro's law).
The methods employed to determine the molecular weight are of two kinds : (a) physical, (fr) chemical.
(a) Physical Methods.
I. Victor Meyer's Method.— Of the physical methods, that by Victor Meyer is the most frequently used when the substance can be vaporised without decomposition. A known weight of the substance is converted into vapour at a temperature 40-50° above its boiling- point in a special apparatus. The air previously contained in the apparatus is displaced by the vapour, collected in a graduated cylinder and its volume measured ; this volume, after making corrections for
temperature and pressure, corresponds to that
occupied by the substance.
Thus, if v c.c. are given by w grammes substance,
, w x 22,400 .*. 22,400 c.c. are given by
«= mol. wt.
The apparatus employed is shown in the accompanying Fig. 21. A liquid, boiling 40-50° above the temperature at which the substance is volatilised, is boiled in the round bulb of the outer vessel. As soon as the temperature is constant and no more air escapes from the inner vessel by the side tube, the inverted graduated cylinder, filled with water, is placed over the end of the side tube, the cork is removed and a known weight of substance, contained in a small glass vessel, is dropped through the open- ing into the inner vessel and the cork is quickly FIG. 21. replaced The substance is rapidly vaporised
and the vapour displaces an equal volume of air, which is driven out and collected and measured in the graduated cylinder.
COMPOSITION OF ORGANIC COMPOUNDS
43
2. Raoult-Beckmann Method. — Substances dissolved in a liquid lower its freezing-point. It was shown by Raoult that the freezing- point was lowered the same number of degrees when weights of different substances proportional to their molecular weights were dis- solved in the same volume of liquid. Each liquid was found to have a definite freezing-point. By employing this value as a constant, the molecular weight of an unknown substance can be found. It is given by the formula
M =
IPO x C x w
where C is the constant, w the weight of the substance, W the weight of the solvent, and d the depression of the freezing-point.
The constants are: — water 19 benzene 49 acetic acid 39 phenol 76
Conversely by determining the lowering of the freezing-point, the amount of salt in a solution can be ascertained, e.g. in serum, urine.
The apparatus (Fig. 22) devised by Beckmann con- sists of the freezing-point tube C with side opening D. It is closed by a cork through which a Beckmann ther- mometer T and a stirrer S (through a glass tube) pass. The Beckmann thermometer is a large thermometer graduated usually in -^^ parts of a degree and having a range of only 5-6 degrees.1 The tube C is placed in a wider tube B which serves as a jacket and prevents too rapid cooling. This is fixed in position in a freezing mixture of salt and ice in the vessel A by a cork which fits the opening in the brass lid L. The brass lid has also openings for the passage of a stirrer E and a thermometer. In carrying out a determination a known weight of solvent is placed in C and its freezing-point is taken. The tube is then removed and the solid allowed to melt. A known weight of substance is then intro- duced through D, dissolved in the liquid and the freez- ing-point again determined.
Several determinations of the freezing-point of the solvent and the solvent containing the substance should be taken. Whilst the freezing-point is being taken the FIG liquid becomes super-cooled. To prevent very great gjcyj super-cooling it is vigorously stirred with the stirrer. At the freezing-point the temperature rises and the highest point reached taken as the freezing-point.
Similarly, a rise in the boiling-point of a solvent, when substances are dissolved in it, will give the molecular weight of the substance.
Micro-Molecular Weight Determinations.
Micro-molecular weight determinations may be made by Barger's method.2
1 It is so constructed that mercury can be removed from the thread or introduced into the thread from a small bulb at the top. It can thus be used for any liquid. a Trans. Chem, Soc., 1904, 85, 286.
2.— (From
Find- :
44 PRACTICAL ORGANIC AND BIO-CHEMISTRY
(b) Chemical Methods.
When the substance is an acid or a base the molecular weight can be determined by chemical methods, (i) In the case of an acid : —
The molecular weight can be calculated from the amount of standard alkali required to neutralise, using phenolphthalein as indicator, a known weight of the acid, according to the equation
H acid + NaOH = H2O + Na acid. e.g. x c.c. *iN NaOH = y gm. of acid.
.-. 40 gm. NaOH = 4° x •?
x x 0-004 ass mol. wt. of acid.
If the acid be dibasic or tribasic, two or three molecules of sodium hy- droxide will be required. The presence of such an acid will be indicated by titrating the acid using methyl orange, or alizarin red, and phenolphthalein as indicators. The acid salt will be neutral to methyl orange or alizarin red, the neutral salt to phenolphthalein. The basicity of the acid is definitely ascer- tained by the analysis of the salt and the free acid.
It is most usual to employ the silver salt of an acid. A quantity of the salt is prepared by adding silver nitrate to the neutral solu- tion of the acid, filtering off the silver salt, washing and drying it. A known weight is heated in a crucible and the metallic silver obtained is weighed.
a gm. of silver salt gave b gm. of silver. If the acid be monobasic it will contain i atom of silver,
107-9 gin- of silver will be contained in IO7 9 * a gm> Qf sjlver salt>
0 Since the silver replaces i atom of hydrogen
.-. I07'9 x a _ IOj.Q + r is the mol. wt. of the acid.
o If the acid be dibasic it will contain 2 atoms of silver,
107*9 x 2 gm. of silver will be contained in I07 9 x 2 x a ^ Qf sijver gajtf
0 Since the silver replaces 2 atoms of hydrogen
.». i107'9 x 2 x ° _ (I07-9 x 2) + 2 is the mol. wt. of the acid. 0
The zinc salt or barium salt is also sometimes employed ; a known weight of salt is heated with a drop of concentrated sulphuric acid in a crucible ; zinc or barium sulphate is obtained from which the amount of barium or zinc is calculated.
COMPOSITION OF ORGANIC COMPOUNDS 45
(2) In the case of a base : —
Organic bases form double salts with metallic salts, such as platinum chloride, mercuric chloride. On heating the double salt in a crucible, a residue of the metal is left. The estimation of the amount of metal in a known weight of the compound gives the molecular weight.
Ammonia and platinum chloride give the compound ammonio- platinum chloride,
(NH3 . HC1)2 . PtCl4.
The organic bases form analogous compounds, the base replacing the ammonia. Their general formula is therefore (B . HC1)2 . PtCl4 or B2 . H2PtCl6.
The molecular weight of 2 molecules of base is thus
B2 . H2PtCl6 - H2PtCl6. e.g. x gm. of salt ga.vey gm. of platinum. .'. 194-8 gm. platinum will be given by X-^ — ^Uf gm. salt B2 .
Now 194-8 gm. platinum are contained in 409-8 gm. H2PtCl6. ... ^g4>8 xj* _ 40g.g is the molecular weight of B2.
I94>8 X * - 409-8
Hence molecular weight of i molecule of base is
The gold salts have the general formula,
B . HC1 . AuCl3 or BHAuCl4, from which the molecular weight of the base is calculated in a similar
way.
46 PRACTICAL ORGANIC AND BIO-CHEMISTRY
IDENTIFICATION OF AN ORGANIC COMPOUND.
Knowing the formula of a pure organic compound from its analysis and molecular weight, it ha^ to be identified. The compound may be a known or an unknown one.
To find out if the compound is known reference is made to Richter's " Lexicon of Carbon Compounds " 1 in which the melting- points and other constants of the various compounds are given. Cor- responding properties identify the substance.
If the compound be unknown, further analysis is necessary ; it must be ascertained to what group of carbon compounds the unknown body belongs, whether it is an alcohol, an ester, an acid, a carbo- hydrate, an amide, an amine, a protein, etc. With the complex natural substances this is a matter of great difficulty, and it may take many years before a question is settled ; e.g. tyrosine was discovered in 1846 and its constitution only definitely proved in 1882.
The identification of an unknown substance, or the rapid identifica- tion of a known substance, is greatly facilitated by a few preliminary tests. If in solution a portion of it should be evaporated to see if there is a residue and whether it is solid or liquid. The residue can be tested for the elements present, especially nitrogen. If there is no residue the solution must be distilled and the boiling-point observed.
1. Colour. Vegetable colouring matters : if blue, they are changed to red by acid and the blue colour is restored, or changed to a green, by ammonia ; if yellow, they are changed to brown by alkali and the colour is restored by acid.
Ferric salts and copper salts are reddish-brown and blue or green respectively.
Many coloured compounds show absorption spectra, such as haemoglobin and its derivatives.
2. Taste. Tasting must be done carefully on account of the extremely poisonous nature of some organic compounds. A drop of a weak solution in water or alcohol may be used. Acids have a sour and astringent taste. Alkaloids and glucosides are bitter. Sugars and glycerol are sweet.
1 The most recent compounds are given in the yearly volumes of the Journals of the English and Foreign Chemical Societies.
IDENTIFICATION OF AN ORGANIC COMPOUND 47
3. Odour. The odour is sometimes characteristic.
4. Appearance. The appearance under the microscope gives evi- dence of homogeneity or impurity. The microscopical appearance is very Useful in identifying the different kinds of starch. Many substances have a characteristic crystalline structure, e.g. cholesterol, cystine, osazones of carbohydrates, etc.
5. Effect of Heat. By heating the substance firstly on platinum, secondly in a small dry tube many valuable details can be ascertained. The odour may be peculiar, the substance may melt, char, decompose, sublime, or boil. The melting-point and boiling-point of solids and liquids can be observed directly after such a preliminary examination.
6. Detection of the Elements. By ascertaining whether the substance does or does not contain nitrogen, it may be placed in either of the following groups :
Non-nitrogenous. Nitrogenous.
Hydrocarbons. Cyanogen compounds.
Alcohols, phenols Amides.
Esters, ethers. Amines.
Aldehydes. Amino acids.
Ketones. Guanidine compounds.
Acids and Salts. Purines.
Fats and cholesterol. Proteins.
Carbohydrates. I
7. Solubility.
(a) Alkali salts and salts of bases, ^ o> ^
The lower alcohols, aldehydes, acids, ketones, amides, amines
The polyhydric alcohols and carbohydrates
Phenols and hydroxy acids
In general, compounds containing several OH groups
(£) Aromatic acids are insoluble or very slightly soluble, but dis- solve in boiling water. Starch is insoluble and gives an opalescent solution with hot
water. Tyrosine, cystine and uric acid are soluble with difficulty in
water.
Fats, higher fatty acids and cholesterol are insoluble in water, but soluble in ether.
82 ; 8
rt
48 PRACTICAL ORGANIC AND BIO-CHEMISTRY
8. Behaviour towards Reagents.
(a) Reaction of aqueous solution to litmus.
A marked acid reaction indicates an acid or a phenol ; if there is an odour, it may be a volatile fatty acid or a phenol.
A neutral reaction indicates a salt of an acid or a base ; an al- cohol, aldehyde, ketone (note smell), carbohydrate. An ester in alcoholic or ethereal solution has also a neutral reaction.
An alkaline reaction indicates a base, or an acid dissolved in excess of alkali.
(b) Sodium carbonate: acids insoluble in water, e.g. uric acid, also
cystine, tyrosine dissolve ; bases insoluble in water do not dissolve or if in solution are precipitated.
(c) Sodium hydroxide: ammonium salts are decomposed with
evolution of ammonia and bases are liberated from their salts. On boiling, amides are decomposed, and esters are hydrolysed.
(d) Hydrochloric acid: acids insoluble in water do not dissolve or
if in solution are precipitated ; bases insoluble in water dis- solve, e.g. tyrosine, cystine, aniline.
(e) Sulphuric acid. (/) Nitric acid.
(g) Bromine water. \K) Permanganate.
(i) Effect of heating with soda lime. The exactly neutral solution may be tested with (/) Schiffs reagent — for aldehydes.
(k) Ammoniacal silver nitrate — for aldehydes, reducing carbo- hydrates, etc.
(/) Fehlings solution — for aldehydes, reducing carbohydrates, etc. (m) Ferric chloride — for phenols (violet or green colour), for acetoacetic acid (claret colour), for formates, acetates (reddish-brown colour,
precipitate on boiling), for lactates, oxalates (yellow colour). (n) Calcium chloride — for oxalates, urates, etc. (insoluble calcium
salts are precipitated).
(o) Sodium nitroprusside and sodium hydroxide : — Acetone gives a red colour, changing to purple with acetic acid. Creatinine ,, a red ,, „ ,, yellow ,, ,, „
Indole ,, a blue-violet „ ,, ,, blue ,, ,, „
Confirmatory tests must be made after an indication of the nature of the substance has been obtained, according to the reactions given under the various groups of compounds.
HYDROCARBONS.
A. SATURATED.
The simplest organic compounds are the hydrocarbons, which con- sist of carbon united with hydrogen.
Carbon is a tetravalent element, but, unlike other elements, the carbon atom can combine with itself many times, thus 2, 3, 4, 5, 6, etc., carbon atoms can be combined together.
_U_i_U_L i i i . i i i
The hydrocarbons are the compounds in which the remaining valencies of the carbon atoms so joined together are satisfied by hydrogen, as for example in
H H H H H H
H— C-H H-C— C— H H— C— C— C— H
H H H H H H
Methane. Ethane. Propane.
They form a homologous series of compounds in which the member containing i carbon atom more than the preceding one also contains 2 hydrogen atoms more, i.e. the members differ from one another by CH2. They possess the general formula CnH2« + 2-
If we continue the process of adding CH2 to propane, two ways are possible : it may be added to one of the end carbon atoms or to the middle carbon atom. The two compounds
H
H-C— H H H H H H | H CH3
H— C— C— C— C— H and H— C C C— H or CH,— C— CH3
I I I I III I
HHHH HHH H
Butane. Isobutane or trimethyl-methane.
are thus obtainable. Continuing the process we obtain
49
50 PRACTICAL ORGANIC AND BIO-CHEMISTRY
H
H— C— H HHHHH HH I H CH3
H— C— C— C— C— C— H H— C— C C C— H or C3HB— C— CH,
Pentane. Dimethyl-ethyl-methane.
H
H— C— H H I H H
H— C C C— C— H H
4 A
H— C— H
i
Dimethyl-ethly-methane. Tetramethyl-methane.
Two of these compounds are identical in structure, so that only three compounds can be derived from butane and isobutane.
Two or more compounds which have the same empirical composi- tion (C4H10 or C5Hi2), but a different structure as represented by the graphic formulas, are therefore possible. Such compounds are known as isomers.
The compounds with a straight chain of carbon atoms are termed normal compounds. Those with a branched chain of carbon atoms are regarded as derivatives of methane, the radicles CH3, C2H6, C3H7, etc., being termed methyl, ethyl, propyl, etc., which shows their origin from the parent hydrocarbon.
In accordance with this theory an enormous number of hydro- carbons are possible ; those containing from I up to 60 atoms of carbon in their molecule are actually known. The theory was advanced and developed to account for their large number.
The saturated hydrocarbons are the basis of the nomenclature and classification of all the carbon compounds. The carbon compounds are classified according as to whether they contain I, ?, 3, or 4, etc., carbon atoms in their molecule, i.e. whether they are derived from methane, ethane, propane, butane, etc.
The saturated hydrocarbons are thus distinguished by the suffix am ; the prefix meth means I carbon atom ; eth means 2. carbon atoms ; prop means 3 carbon atoms, and so on.
The majority of the saturated hydrocarbons are natural substances. The lower members of the series of the hydrocarbons (up to 4 carbon atoms, which are gases) are formed by the dry distillation of diverse organic substances and are contained in coal gas. Methane occurs in
HYDROCARBONS 51
coal seams, but was originally called marsh gas, because it was found to escape from the water of ponds, where it is now known to be formed by the decomposition of cellulose. By a similar process of bacterial action it may be produced in the intestines of animals. The middle members, containing 5-16 atoms of carbon, are liquids, and are con- tained in petroleum, which consists of a mixture of saturated hydro- carbons. The higher members are solids.
Two theories have been advanced to account for their formation. According to the first, they are the products of the dry distillation of animal remains ; according to the second, they are formed by the action of water upon the metallic carbides, of which the interior of the earth is supposed to consist. If the former supposition be the correct one, as the most recent work tends to show, they become of still greater interest in biological chemistry.
Several fractions are separated by the fractional distillation of the natural mineral oil. The following are the principal fractions from American petroleum : —
1. Cymogene, B.P. o°\ gases which are liquefied by pressure and used for producing
2. Rhigolene, B.P. i8°J cold by evaporation.
3. Petroleum ether or naphtha, B.P. 5o°-6o°, contains chiefly pentane and hexane.
4. Benzoline or Benzine, B.P. 7o°-go°, contains chiefly hexane and heptane.
*"<* heptane and octane.
7. Paraffin oil or Kerosene, B.P. i5o°-3oo°, contains chiefly octane to hexadecane.
8. Vaseline, B.P. above 300°, contains chiefly heptadecane to heneicosane (CZIHU).
The fractions may be purified by shaking with concentrated sulphuric acid and caustic soda to remove unsaturated hydrocarbons.
The portions of higher boiling-point are decomposed by overheat- ing or by distilling under pressure (cracking process) and yield fractions of lower boiling-point.
The other natural mineral oils, found in Russia, Roumania, etc., are also distilled fractionally. They contain generally less of the lower boil- ing fractions and a greater quantity of naphthene hydrocarbons (p. 237).
Liquid hydrocarbons are also prepared by the distillation of bitu- minous shale.
The higher members, which are solid, remain as distillation residues, and are also found in nature, e.g. ozokerit.
The distillation residues are converted into oil and paraffin wax by freezing and pressing ; the liquid portion forms lubricating oil and the solid portion paraffin wax. The residues and fractions may be purified by treatment with sulphuric acid and caustic soda.
52 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Examination of a Commercial Specimen of Hydrocarbons by Fractional Distillation.
On distilling 50-100 c.c. of ligroin or kerosene from a small dis- tilling flask attached to a condenser and observing the temperature indicated by the thermometer, it will be seen that the temperature never remains constant for any length of time. The substance is a mixture. Several fractions which boil within 10° or 20° ranges of temperature can be collected in separate receivers. By redistilling these fractions and using a fractionating column (cf. p. 9) a pure product with a constant boiling-point can eventually be obtained.
Properties.
The saturated hydrocarbons have a peculiar odour. They are in- soluble in water, but are soluble in alcohol, ether and other organic liquids.
Inflammability.
Marsh gas and the other gases burn with a non-luminous flame and form explosive mixtures when mixed with a certain proportion of oxygen or air.
'f he lower members amongst the liquids are also inflammable and burn with a more or less luminous flame. E.g. if about 3 c.c. of ligroin be placed in a watch glass and a lighted match applied it will burn.
The higher liquid members do not burn until they have been warmed. E.g. on applying a lighted match to about 3 c.c. of kerosene contained in a watch glass, the flame is extinguished, but if the kerosene be warmed on the water-bath to about 40° and a lighted match again applied, the vapours of the kerosene will be ignited.
In a lamp the kerosene rises to the surface of the wick by capil- larity, and on applying a light the oil becomes hot and turns into inflammable vapour.
Inertness towards Chemical Reagents.
The saturated hydrocarbons are very inert substances ; they are not acted upon by concentrated acids or alkalies except under special conditions, and on account of their stability they are known as the paraffins horn flarum affinis, little affinity.
E.g. on shaking about I c.c. of ligroin or kerosene with
(a) concentrated sulphuric acid,
(f) concentrated nitric acid,
(r) potassium permanganate solution, d bromine dissolved in chloroform,
HYDROCARBONS 53
there is no reaction, unless the commercial mixture of hydrocarbons contain hydrocarbons belonging to the unsaturated series.
They are acted upon by the halogens forming substitution products (P. -57).
Synthetical Preparation.
1. The lower members of the series can be prepared by the action of water on certain metallic carbides, e.g. : —
Marsh gas is evolved if about 2 gm. of aluminium carbide in a test tube be covered with water. The gas may be collected in an inverted test tube and shown to be inflammable.
2. Saturated hydrocarbons can be prepared by the dry distillation of the dry sodium salt (i part) of a fatty acid with soda lime (3 parts).
E.g. Methaneiis given off when fused sodium acetate and soda lime in the above proportions are heated together in a test tube. The evolved gas may be ignited at the mouth of the tube. CH3COONa + NaOH = CH4 + Na2CO3.
3. Saturated hydrocarbons are prepared from the correspond- ing halogen derivative by reduction with hydriodic acid, zinc-copper couple, zinc and hydrochloric acid, etc.
C2H5I + HI = C2H6 + I8
4. The higher members of the series are prepared from the lower members by treating the dry halogen derivative (alkyl halide) with zinc or with sodium : —
CHJ + aNa + CH3I = CH3 . CH3 + 2NaI 2CH8I+ Zn = CH8.CH8 + Znla.
54 PRACTICAL ORGANIC AND BIO-CHEMISTRY
B. UNSATU RATED.
In addition to the series of saturated hydrocarbons there are other series which contain less hydrogen in the molecule and are represented by the general formulae CnHZn (olefines) and CWH2^_2 (acetylenes). The two compounds ethylene or ethene, C2H4, and acetylene or ethine, C2H2, are the first and typical examples. They are represented by
the constitutional formulae : —
CH2 CH
I! HI
CH2 CH
Ethylene. Acetylene.
The four valencies of the carbon atoms are not completely satisfied by hydrogen atoms and they are therefore termed the unsaturated hydro- carbons. The unsaturated hydrocarbons are given the suffix ene and ine respectively. The higher members are derived from the corre- sponding saturated hydrocarbons by the loss of two or four hydrogen atoms and the insertion of a double or triple bond. Isomers exist amongst the higher members, and further, compounds are known which contain two or more double bonds in their molecule, e.g.
CH
>C— CH=CH2 CH/
Isoprene.
It should be noted that the double and the triple bonds do not indi- cate greater, but on the contrary lesser, stability.
(a) OLEFINES.
Preparation.
1. The olefines are most usually prepared by abstracting the ele- ments of water from alcohols by means of dehydrating agents — zinc chloride, sulphuric acid, phosphoric acid : —
C2H6OH = CH2=CH2 + H20.
The preparation of ethylene by this method is described on p. 58.
2. Olefines are prepared by the action of alcoholic potash upon
the alkyl halide (p. 57).
C2H5I + KOH = CH2=CH2 + H2O + KI. Ethylene may be prepared as follows : —
50 c.c. of a 20 per cent, solution of caustic potash in alcohol is placed in a 250 c.c. distilling flask in the neck of which a tap funnel is fastened with a cork. The distilling flask is fixed on a stand at an angle so that its neck may be attached to an inverted condenser (or its neck bent at an angle). A glass tube suitably bent leads from the condenser to a water trough. The potash solution is warmed and about 15 c.c. of ethyl iodide are slowly dropped in. Ethylene is evolved and potassium iodide is precipitated. When all the air has been displaced from the apparatus the gas may be collected in a gas cylinder over water.
. — Ether is formed in the reaction according to the equation : — KOC2H0 + C2H5I = KI + C2H6 . O . C3H8.
HYDROCARBONS 55
Properties.
The lower members with 2, 3 and 4 atoms of carbon are gases. The higher members are liquids and solids. They are lighter than water in which they are only slightly soluble. They are soluble in alcohol, ether and other organic liquids. They are inflammable and burn with a luminous, smoky flame.
Addition Reactions.
(1) Hydrogen. When mixed with hydrogen and passed through a hot tube over platinum black, or finely divided nickel, they are con- verted into saturated hydrocarbons : —
L/XT-o— — - v-/Alo -J- ^~*o ^™ JtiQ"1"" \-/Xjl««
The catalyst can be suspended in an inert solvent and a mixture of ethylene and hydrogen bubbled through the liquid.
(2) Halogens. The olefines combine with the halogens, chlorine and bromine, but less readily with iodine, to form halogen compounds containing two atoms of halogen (see p. 57): —
CH2=rCH2 + Br.. = CH2Br . CH2Br.
(3) Halogen Acids. The following reaction occurs : —
CH2=: CH2 + HI = CH3 . CH2I.
(4) Sulphuric Acid. The alkyl hydrogen sulphate (p. 71) is formed by addition : —
CH2=CH2 + H2SO4 = CH3 . CH2 . HSO4.
This reaction serves for the separation of saturated and unsaturated hydrocarbons.
(5) Hypochlorous Acid. Chlorhydrins are formed : —
CH2=CH2 + HOCl = CH2OH . CH2C1
Ethylene chlorhydrin.
(6) Potassium Permanganate. The olefines are oxidised by dilute permanganate : —
CH2=CH2 + H20 + O = CH2OH . CH2OH Ethylene glycol.
This reaction may be used for detecting unsaturated compounds in a mixture of hydrocarbons.
56 PRACTICAL ORGANIC AND BIO-CHEMISTRY
(b) ACETYLENES.
Preparation.
Acetylene is formed by the incomplete combustion of other hydro- carbons, but is most usually prepared by the action of water upon cal- cium carbide : —
CaC2 + H20 = C2H2 + CaO.
The hydrocarbons of this group are prepared by the action of alco- holic potash upon halogen compounds in the same way as ethylene : —
CH2Br CHBr
+ KOH = || + KBr + H2O
CH2Br CH2
Vinyl bromide. CHBr CH
|| + KOH = I)) + KBr + H2O.
CH2 CH
Properties.
The lower members are gases, the higher members are liquids. Acetylene is soluble in water (i : i) and other organic liquids. Acetone dissolves thirty-one times its own volume of the gas at N.T.P. Acety- lene burns with a smoky, intensely hot flame which is very luminous ; it is consequently employed for illuminating purposes, the burners, generally of clay, being designed so that complete cpmbustion is effected.
Addition Reactions.
Acetylene and its homologues behave like the defines, but react with two molecules : —
(1) Hydrogen.
CaH2 + H2 = C2H4 (ethylene) C2H4 + Ha = C2H6 (ethane).
(2) Halogen Acid.
CH2 C2H, + HC1 = || (vinyl chloride)
CHC1
CH2 CH.
|| ' +HC1= | (ethylidene chloride).
CHC1 CHC12
(3) Halogens.
CH CHBr
(I) + Br2 = || (acetylene dibromide)
CH CHBr
CHBr CHBr2
|| + Bra «= I (ethane tetrabromide).
CHBr CHBr2
Acetylene and the other members of the series form characteristic compounds with copper, silver and other heavy metals.
Cuprous acetylide, C2Cu2,and silver acetylide, C2 Ag2, are precipitated as amorphous compounds when acetylene is passed through ammoni- acal solutions of cuprous chloride or silver nitrate. In the dry state these compounds are very explosive ; they are decomposed on treat- ment with hydrochloric acid or potassium cyanide yielding acetylene. Acetylene may be separated from other hydrocarbons by this property.
HALOGEN DERIVATIVES OF THE HYDROCARBONS.
The only chemical property of the saturated hydrocarbons is that they are attacked by the halogens yielding halogen substitution deriva- tives, one atom of hydrogen being progressively replaced by an atom of halogen ; thus, from methane by the action of chlorine, we can obtain
CH3C1 CHaCl2 CHC1S CC14.
A mixture of the compounds results and the reaction is slow, so that, in practice, these compounds are not prepared from the hydrocarbon, but from other compounds.
The unsaturated hydrocarbons differ from the saturated hydrocar- bons in their behaviour to the halogens. They react by addition, thus, e.g. ethylene combines with two atoms of bromine, forming the saturated compound, ethylene dibromide : —
CH2 CH2Br
CH2 CH2Br.
Dihalogen compounds of this type are generally prepared by this reaction.
MONOHALOGEN DERIVATIVES. ALKYL HALIDES.
Preparation.
The alkyl halides are prepared from the corresponding alcohol by the action of the halogen acid, or by the action of the phosphorus halide :—
CH8OH + HBr = CH3Br + H2O 3CH3OH + PL, = CHJ + H3PO. CH3OH + PC18 = CH3C1 + POC13 + HC1.
Preparation of Methyl Iodide.
1 8 gm. of methyl alcohol and 5 gm. of red phosphorus are placed in a small flask (250 c.c.) and a reflux condenser is attached to it. 50 gm. of iodine are slowly added by detaching the flask from the condenser and rapidly refixing. Heat is evolved in the reaction and loss of alcohol and iodide is prevented by the condenser. The apparatus and mixture is allowed to stand for 12 to 24 hours so that the reaction completes itself. The contents of the flask are distilled from a water-bath. The distillate is shaken in a separating funnel with dilute caustic soda to remove iodine and hydriodic acid, and if sufficient has been used the lower layer of methyl iodide will be colourless. The lower layer of methyl iodide is separated, allowed to stand with a little calcium chloride till it is clear and distilled from a water-bath (b.p. 44°. About 45 gm. or 75 per cent, of the theoretical yield should be obtained.
57
$8 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Preparation of Ethyl Bromide.
A distilling flask of about i litre capacity is closed by a cork and its neck attached to a condenser. To the end of the condenser is attached an adapter (a bent tube wide at one end and narrow at the other, p. 1 2) which dips under water contained in a conical flask of about 250 c.c. capacity cooled by standing in ice. 54 c.c. (100 gm.) of sulphuric acid are mixed in the flask with 75 c.c. (60 gm.) of absolute alcohol and cooled to the tem- perature of the air. 100 gm. of coarsely powdered potassium bromide are added to the contents of the flask and the mixture is heated on a sand bath or carefully on a gauze. The contents boil and froth up and heavy oily drops of ethyl bromide collect under the water in the receiver. If the frothing is too great the flask is removed from the source of heat for a minute. The heating is continued so long as oily drops distil over. The contents of the receiver are placed in a separating funnel and the lower layer collected. It is purified by returning to the separating funnel and shaking with a dilute solution of sodium carbonate. The ethyl bromide is then shaken with water to remove alkali and it is placed in a clean dry distilling flask and left in con- tact with calcium chloride till it is clear. The flask is furnished with a ther- mometer, attached to a condenser and the ethyl bromide (b.p. 35-40°) distilled over from a water- bath. About 75 to 80 gm. should be obtained.
DIHALOGEN DERIVATIVES.
Methylene chloride, CH2C12, is generally prepared by reducing chloroform in alcoholic solution with zinc and hydrochloric acid.
Methylene iodide, CH2I2, is prepared by reducing iodoform with hydriodic acid.
Methylene bromide, CH2Bra, is prepared by treating methylene iodide with bromine: —
CH2I2 + 2Br2 = CHaBr2 + 2BrI.
The numerous isomers of the halogen derivatives of the higher hydrocarbons are prepared by various methods, e.g. : —
(a) by addition of halogen to unsaturated hydrocarbons ;
(V) by the action of phosphorus pentachloride upon the aldehydes and ketones.
Preparation of Ethylene Dibromide.
Ethylene is prepared by dropping a mixture of 30 c.c. of absolute alcohol and 80 c.c. sulphuric acid from a tap funnel upon a mixture of 124 c.c. of alcohol and 1 08 c.c. of concentrated sulphuric acid contained in a 2 litre flask and mixed with sand to prevent frothing, the mixture being gently heated until a steady stream of gas is evolved. The evolved gas is passed through two wash bottles l with safety tubes containing caustic soda solution to remove sulphur dioxide into two ordinary wash bottles containing 50 c.c. bromine and i c.c. of water and 15 c.c. of bromine and i c.c. of water respectively. These two bottles are placed in a basin of water to which ice may be added to prevent the contents becoming warm during the reaction. The outlet tube is connected to a tower containing soda lime so that bromine vapour does not escape into the room. The bromine in the bottles is gradually decolorised
1 It may be necessary to change these bottles for fresh ones during the preparation.
HALOGEN DERIVATIVES OF HYDROCARBONS 59
and changes into ethylene bromide which may have a straw-yellow colour. The heavy liquid product is shaken in a separating funnel with dilute caustic soda solution and then with water. It is dried with calcium chloride and purified by distillation (b.p. 130-132°). About 60 gm. should be obtained.
. Properties of the Monohalogen and Dihalogen Derivatives.
The monohalogen derivatives are liquids heavier than water in which they are insoluble or only slightly soluble. They have a peculiar smell and do not burn readily. Their properties are in general like those of chloroform (p. 60).
Chemically the monohalogen derivatives or alkylhalides are very reactive substances and readily exchange the atom of halogen with other atoms or groups. They are thus largely used for introducing alkyl radicles into other compounds, thus : —
1. CH3I + 2H = CH4 + HI (p. 53).
2. CH3I + Zn + CH3I = C2H6 + ZnI2 (p. 53).
3. CH3I + 2Zn + CH3I = CH3 . Zn . CH3 + ZnL.
4. CH3I + KOH = CH3OH + KI (p. 63).
aqueous
5. C2H5I + KOH = C2H4 +KI + H20 (p. 54).
alcoholic
6. C0H5I + NH3 = C2H5NH2 + HI (p. 124).
7. C;H5I + KCN = C9H5CN + KI (p. 158).
8. C2H5I + KN02 = C2H5N02 + KI.
9. C2H5I + KHS = C2H5HS + KI (p. 78).
The dihalogen derivatives are very similar to the monohalogen de- rivatives in both their physical and chemical properties. Both the halogen atoms can be replaced by OH, NH2, etc.
TRIHALOGEN DERIVATIVES. CHLOROFORM.
Preparation.
100 gm. of fresh bleaching powder are rubbed up in a mortar with 200 c.c. of water so as to form a paste, the paste is rinsed into a large flask of about 1000 c.c. capacity with another 200 c.c. of water ; 25 c.c. of acetone or alcohol are added and the mixture shaken up thor- oughly. The flask is connected by means of a bent tube to a con- denser and receiver and gently heated through a wire gauze. As soon as the reaction commences, which is shown by the frothing, the flame is removed. When the frothing has subsided and the reaction has moderated, the contents of the flask are boiled until no more chloro- form distils over with the water. The chloroform consists of heavy oily drops which sink in water, and it forms the lower layer of the distillate.
The distillate is transferred to a separating funnel and shaken with a little dilute caustic soda solution ; the lower layer of chloroform
60 PRACTICAL ORGANIC AND BIO-CHEMISTRY
form is drawn off into a clean, dry flask and dried by adding anhydrous calcium chloride, either by shaking for 5-10 minutes or allowing to stand from 12-24 hours, until it is clear. The chloroform is filtered into a clean, dry distilling flask and distilled.
The mechanism of the reaction by which the chloroform is formed is probably : —
1. The oxidation of the alcohol to aldehyde (p. 80),
CH8 . CHaOH + O «= CH, . CHO + H2O.
2. The chlorinatiori of the aldehyde to chloral,
CH, . CHO + 3C12 = CC13 . CHO + 3HC1.
3. The decomposition of the chloral to chloroform and calcium formate by the calcium hydroxide (p. 87),
2CC13 . CHO + Ca(OH)2 = 2CHC13 + (HCOO)2Ca.
Purification of Commercial Chloroform.
Chloroform prepared from alcohol, methylated spirit (methylated chloroform) or acetone may contain chlorine, hypochlorous acid or hydrochloric acid, aldehyde, etc.
The specimen is shaken several times with water, the chloroform is separated, dried with (i) calcium chloride, (2) phosphorus pentoxide and distilled.
The last traces of alcohol may also be removed by adding slices of metallic sodium, allowing to stand for 12-24 hours and then distilling.
Properties.
Chloroform is a volatile colourless liquid with a distinct and sharp odour and sweetish taste. It boils at 61° and has a sp. gr. of I '483-1 '487.
Its vapour does not burn, but when mixed with alcohol the com- bined vapours burn with a smoky flame edged with green.
It is soluble in about 200 volumes of cold water (-44 gm. in 100 c.c.) to which it gives a sweet taste.
It mixes in all proportions with absolute alcohol, ether, benzene, petroleum ether. It is slightly soluble in dilute alcohol and readily dissolves fats, resins, india-rubber, camphor, iodine, bromine.
Many specimens of commercial chloroform undergo change on keeping, especially in the light, and are liable to contain chlorine, hypochloro'us acid or hydrochloric acid. This decomposition is hin- dered by the addition of I per cent, of alcohol. The bottle should be kept in the dark. I c.c. of chloroform on evaporation should leave no residue and if allowed to evaporate on clean filter paper should leave no disagreeable odour.
Chloroform is decomposed by boiling with aqueous alkali, more rapidly in alcoholic solution, into alkali formate and chloride : — CHC18 + 4NaOH = HCOONa + sNaCl + 2HaO.
HALOGEN DERIVATIVES OF HYDROCARBONS 61
A few drops of chloroform are heated with dilute caustic soda. The presence of chloride is tested for in a small portion of the solu- tion, the remainder is neutralised exactly, if it be still alkaline, and heated with mercuric chloride solution. A deposit of mercurous chloride and mercury shows the presence of formate.
Tests for Impurities in Chloroform.
A quantity of the specimen is shaken up with two volumes of water. The water is separated and silver nitrate is added. Pure chloroform gives no reaction, but a precipitate of silver chloride indicates the presence of chlorides. If, on heating, the precipitate blackens the presence of aldehyde or formic acid is indicated. The water should not react with blue litmus.
Chloroform is not soluble in concentrated sulphuric acid. Any darkening which occurs on shaking them together is due to the presence of aldehyde, methyl alcohol, etc. The presence of alcohol in chloroform may be detected by shaking some of the specimen with five volumes of water, filtering through a wet paper, and testing for alcohol in the filtrate by the iodoform reaction (p. 67).
Tests for Chloroform.
(1) A red or yellow precipitate of cuprous oxide is formed on add- ing some solution of chloroform in water to Fehling's solution (p. 84) and heating.
(2) Carbylamine Reaction. — To the dilute solution of chloroform in water is added some alcoholic sodium hydroxide and a drop of aniline and the mixture heated. Phenyl isonitrile or carbylamine is formed, which has a disgusting smell : —
CHC13 + 3KOH + C6H5NH2 = C6H6NC + aKCl + 3H2O. This reaction is extraordinarily sensitive and will detect one part iof chloroform in 5000 parts of alcohol. It is also given by bromoform, odoform, chloral, trichloracetic acid and substances which yield chloro- form when treated with alkali.
From liquids, such as blood, it is better to remove the chloroform as described under estimation and to test the liquid in the receiver.
Estimation of Chloroform.
Hydrochloric acid is formed when chloroform vapour mixed with hydrogen is passed through a red hot tube.
Hydrogen is slowly passed into a flask containing the solution of chloroform and the flask is gently heated. The mixed vapours are passed through a short, heated combustion tube containing platinum wire gauze or loose asbestos and into a receiver containing water. The contents of the receiver are titrated with standard alkali or precipitated with silver nitrate. As acetylene and hydrogen cyanide may also be present the contents ot receiver should be boiled before titrating or precipitating.
This procedure may be used for detecting and estimating chlorofc blood and other liquids which do not contain other chlorinated compounds.
62 PRACTICAL ORGANIC AND BIO-CHEMISTRY
IODOFORM.
Preparation.
4 gm. of crystallised sodium carbonate are dissolved in 20 c.c. of water. 2 c.c. of absolute alcohol and 2 gm. of iodine are added, and the solution* warmed to about 70° on the water-bath until it is decolorised. lodoform separates as a lemon-yellow powder. It is filtered off, washed with cold water and dried on an unglazed plate.
The melting-point of the preparation serves to prove its identity.
Properties.
lodoform is a light yellow, shining crystalline solid with a per- sistent unpleasant odour. It has a characteristic microscopic appear- ance— hexagonal plates, stars, or rosettes, and melts at 119°. On gently heating it sublimes without change, but on heating strongly it is decomposed : violet vapours of iodine are formed and a deposit of carbon is left.
lodoform is nearly insoluble in water (i part in 10,000) and in dilute acids and alkalies. It is slightly soluble in alcohol (i part in 50) but more easily soluble in absolute alcohol (i part in 23). It is easily soluble in ether, chloroform and carbon disulphide, but very slightly soluble in glycerol, benzene and petroleum ether. In its chemical properties iodoform closely resembles chloroform.
Tests for Impurities in lodoform.
1. No residue should remain when it is heated in the air.
2. It should be completely soluble in boiling alcohol, but insoluble in brine.
3. On shaking up with water and filtering, the filtrate should give no re- action with barium chloride or silver nitrate.
4. If picric acid be present as adulterant, it may be detected (a) By testing the aqueous extract with potassium cyanide when a reddish-brown coloration is produced, (b) By treating with caustic soda solution and shaking this solution with chloroform. Picric acid remains in the aqueous solution. (c) By extracting the acid with dilute sodium carbonate solution, neutralising exactly with acetic acid and adding potassium nitrate ; potassium picrate is precipitated.
ALCOHOLS.
Alcohols are hydrocarbons in which a hydrogen atom (or more in the case of the higher members, e.g. glycerol) has been substituted by a hydroxyl or OH group. This relationship is shown : —
1. By the action of water and aqueous alkalies upon the halogen mono-substituted hydrocarbons : —
CH3 . Cl + HOH = HCl + CH3 . OH.
2. By the action of phosphorus pentachloride upon the alcohol : —
CH3 . OH + PC15 = CH3 . Cl + POC13 + HCl.
All alcohols are designated by the suffix -ol, e.g. methyl alcohol or methanol, ethyl alcohol or ethanol.
Most of the alcohols are natural substances and serve as the start- ing point for the preparation of other compounds.
METHYL ALCOHOL. CH3 . OH. Commercial Methyl Alcohol.
Preparation.
Methyl alcohol, together with acetone, acetic acid, methyl acetate and other substances is formed in the dry distillation of wood. The acid. aqueous distillate is known as pyroligneous acid; on standing wood tar separates out The acid liquid contains 1-2 per cent, of methyl alcohol, '!-'$ per cent, of acetone and about 10 per cent, of acetic acid. It is distilled until the distillate has a specific gravity of •9-1. The crude wood spirit so obtained is a greenish-yellow liquid with disagreeable odour. It is mixed with about 2 per cent, of lime and again distilled. This retains the acetic acid as calcium acetate, the neutral substances— methyl alcohol, acetone, acetaldehyde, methyl acetate passing ovef. This distillate is wood spirit and contains about 93 per cent, of methyl alcohol. It is diluted with water to precipitate oily impurities and is again treated with lime and distilled. Basic impurities are removed by distilling it with -I -'2 per cent, of sul- phuric acid and the methyl alcohol boiling at 64-66° is collected.
Methyl alcohol is also prepared by dry distillation from vinasses- the mass remaining after fermentation of the residues from the pn
paration of beet sugar.
63
64 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Pure Methyl Alcohol.
Commercial methyl alcohol contains acetone. By dissolving anhydrous oxalic acid (prepared by heating oxalic acid at 100°) in the boiling spirit, methyl oxalate is formed ; it separates in crystals on cool- ing. The crystals are filtered off, washed free from acetone with water, and then decomposed into oxalic acid and methyl alcohol by boiling with water or ammonia. Methyl alcohol is obtained on distillation and is dehydrated by distilling over quicklime (see under ethyl alcohol).
Pure methyl alcohol may also be obtained by boiling commercial methyl alcohol with anhydrous calcium chloride. Calcium chloride crystallises out in combination with methyl alcohol as CaCl2 +4CH3OH from the saturated solution on cooling. The crystals are drained from the mother liquor and are decomposed by heating ; methyl alcohol is evolved and is collected.
The acetone may also be removed by passing chlorine into it form- ing trichloracetone. The methyl alcohol is separated by fractional distillation.
Properties.
Methyl alcohol is a colourless liquid which boils at 66° and has a sp. gr. of 797 at 15°. It closely resembles ethyl alcohol in its properties, but it does not give the iodoform reaction.
ETHYL ALCOHOL.
Preparation.
Ethyl alcohol is obtained by the fermentation of sugar by yeast and occurs in all fermented liquids such as wine and beer. It is made chiefly from potatoes and cereals, the starch being first converted into the sugar, glucose, which is fermented by the yeast and changed into alcohol and carbon dioxide.
1. Rectified Spirit.
The alcohol produced by fermentation is separated from the fer- mented liquor by distillation. The distillate is then fractionally re- distilled, or rectified, so as to separate as much water as possible and the greater part of the higher alcohols. The product is rectified spirit. It contains about 84 per cent, by weight of ethyl alcohol and has a sp. gr. of 0-838 at 15°.
2. Methylated Spirit.
The rectified spirit is denatured and rendered unfit for drinking purposes by the addition to it of one-ninth of its volume of wood spirit and three-eighths of I per cent of mineral naphtha or paraffin oil.
Since 1905 methylated spirit has been obtainable in approved scientific institutions free of duty and free from mineral naphtha.
ALCOHOLS 65
3. Absolute Alcohol.
Rectified spirit is filtered through charcoal and fractionally distilled, the first portions which contain aldehyde and the last portions which contain fusel oil being rejected. The middle fraction is distilled over quicklime and commercial absolute alcohol is obtained. This contains about -5 per cent, of water. Pure alcohol is prepared from this by adding the requisite quantity of metallic sodium or calcium and again distilling.
4. Absolute Alcohol from Methylated Spirit.
Methylated spirit (i litre) is boiled upon a water-bath under a reflux or inverted condenser (p. 18) with about 30 gm. of caustic soda for one hour in a 2-litre flask. Acetone, aldehyde and other impurities are destroyed and the spirit turns brown. The contents of the flask are distilled and the distillate collected in another flask of the same capacity containing 400-500 gm. of quicklime. The flask is connected with a reflux condenser and either allowed to stand for twenty- four hours or heated for one hour on a water-bath. The liquid is distilled again without pouring off from the flask. The yield of absolute alcohol is about 80 per cent., 'and it contains 2-3 per cent, of water. By treating it again with half the previous quantity of quicklime the amount of water may be reduced to less than I per cent. The boiling- point (76-78°) may be determined by distilling 50 c.c. in a small apparatus.
Properties.
(1) Ethyl alcohol is a colourless, pleasant-smelling liquid with a hot taste. It boils at 78° and has a sp. gr. of 79384 at 15-5° or 60° F.
(2) It mixes with water in all proportions. Absolute alcohol is very hygroscopic and readily absorbs water on exposure to the air.
On mixing alcohol with water there is an evolution of heat and a contraction in bulk.
The addition of water to methylated spirit produces a cloudiness due to the precipitation of the mineral naphtha.
(3) Alcohol burns with a faint blue non-luminous flame even when mixed with considerable amounts of water.
On mixing 10 c.c. of alcohol with 10 c.c. of water in a measuring cylinder the evolution of heat will be noticed, and when the mixture is cold the diminution in volume can be measured. By placing the mixture in a small basin and applying a light it will be seen whether it is or is not inflammable.
66 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Detection of Water in Absolute Alcohol.
(a) If the alcohol contains a considerable quantity of water its presence will be shown by adding some anhydrous copper sulphate which turns blue.1
(ff) 0-5 per cent, of water may be detected by adding a crystal of potassium permanganate ; the liquid will assume a pink colour.
(r) Traces of water in absolute alcohol according to Yvon may be detected by means of calcium carbide. If water be present, bubbles of acetylene are given off and the liquid becomes milky, due to the formation of calcium hydroxide.
Reactions.
1. Action of Sodium.
On adding about I gm. of sodium to 20 c.c. of absolute alcohol in a small flask there is an evolution of hydrogen just as with water, but the reaction is by no means so violent. The gas which is evolved may be collected in an inverted test tube and shown to be hydrogen by burning.
When the sodium has dissolved the solution is evaporated to dry- ness on the water-bath. A white solid — sodium ethoxide — remains, which is very hygroscopic and is decomposed by water, yielding alcohol which can be recognised by its smell and by the iodoform test :—
C2H5OH + Na = C2H5ONa + H C2H5ONa + H2O = C2H5OH + NaOH.
This reaction shows that one of the hydrogen atoms in alcohol is replaceable by sodium.
2. Action of Phosphorus Pentachloride.
On adding a little phosphorus pentachloride to a small quantity of alcohol, a vigorous action occurs and hydrochloric acid fumes are evolved. Ethyl chloride and phosphorus oxychloride are the other products. The smell of ethyl chloride will be noticed when the hydrochloric acid fumes cease to be given off. This reaction shows the presence of an hydroxyl or OH group : —
C2HBOH + PC15 = C2H5C1 + POC13 + HC1.
1 Prepared by gently heating a crystal of copper sulphate in a crucible until it falls to powder.
ALCOHOLS 67
Tests.
(1) Smell. — Even in dilute solutions alcohol may be detected by its smell.
(2) Oxidation to Acetaldehyde. — On warming a little dilute alcohol in a test tube with a few drops of potassium dichromate and some dilute sulphuric acid the pungent characteristic odour of aldehyde will be observed and the solution turns green : —
C2H5OH + O = CH3 . CHO + H2O.
(3) Formation of Ethyl Acetate. — The fruity odour of ethyl acetate is produced when some of the dilute solution is heated with concentrated sulphuric acid and a little solid sodium acetate.
(4) lodoform Reaction (Lieben). — About an equal volume of iodine in potassium iodide is added to a very dilute solution of alcohol — I or 2 drops in half a test tube full of water — and then, drop by drop, caustic soda till the mixture is decolorised. On gently warming the mixture, iodoform is formed and may be recognised by its characteristic smell. A yellow crystalline precipitate will separate if the solution of alcohol is not too weak.
Note. — This very sensitive reaction is not characteristic of alcohol as it may be given by aldehyde, acetone, acetic ester and other sub- stances which contain the grouping CH3 — C joined to oxygen.
Alcohol gives the reaction on warming, acetone gives the reaction in the cold.
Estimation of Alcohol in Beer, Wines, Spirits.
The amount of alcohol in these liquids is ascertained by distilling off the alcohol and determining the specific gravity of the distillate.
(a) 100 c.c. beer are distilled and 80 c.c. distillate are collected.
(b) 100 c.c. wine + 80 c.c. water and a little tannin are distilled and nearly 100 c.c. distillate are collected.
(c) 50 c.c. spirit + 100 c.c. water, or 25 c.c. spirit + 150 c.c. water, are distilled and nearly 100 c.c. distillate are collected.
The distillate is made up to 100 c.c. with water, the liquids are mixed, and the sp. gr. at 15-5° or 60° F. is determined by weighing in a sp. gr. bottle. The amount is given by referring to an alcohol specific gravity table for the percentage by weight. The amount in the sample is ascertained from the formula : —
sp. gr. of distillate x amount of distillate in c.c. x per cent, of alcohol from table
sp. gr. of sample x amount of sample taken = percentage of abs. ale. by weight in the sample. If the specific gravity of the sample be unknown, it may be calculated from
wt. of distillate x per cent, of alcohol from table
wt. of sample taken = percentage of abs. ale. by weight in the sample.
68 PRACTICAL ORGANIC AND BIO-CHEMISTRY
PROPYL ALCOHOLS. C3H7OH.
Normal Propyl Alcohol. CH3 . CH2 . CH2OH.
Normal propyl alcohol is formed in the process of alcoholic fermen- tation and is contained in fusel oil, from which it is obtained by frac- tional distillation. It is present to the extent of about 3 per cent, in the fusel oil obtained from potato spirit.
It is a liquid resembling ethyl alcohol but with a less pleasant smell, and burns with a luminous flame. It boils at 97° and has a sp. gr. of -807 at 15°.
3
Isopropyl Alcohol. \CHOH.
CH/
Isopropyl alcohol is prepared either by the reduction of acetone with sodium amalgam or from isopropyl iodide by boiling it with lead hydroxide and water. Isopropyl iodide is prepared by the action of phosphorus and iodine upon dilute glycerol.
It is a liquid resembling normal propyl alcohol, but it boils at 82°" and has a sp. gr. of 792 at 15°.
CH2
BUTYL ALCOHOLS. C4H0OH.
Four isomers are possible in the case of the butyl alcohols : — CH2OH CH2OH CH3 CH3
CH CHOH COH
CH3 CH3 CH2 CH3 CH3
Normal Primary Normal Tertiary
primary isobutyl secondary butyl
butyl alcohol. butyl alcohol,
alcohol. alcohol.
The chief of these is primary isobutyl alcohol which is formed in alcoholic fermentation and is separated by fractional distillation from the fusel oil.
Normal primary butyl alcohol has once been found in fusel oil. It is formed by the action of the Schizomycetes upon glyceroL Bacillus butylicus (contained in the excrement of cows) produces 6-& per cent, from glycerol and 10 per cent, from mannitol. Normal secondary butyl alcohol and tertiary butyl alcohol are prepared by synthetical methods. The butyl alcohols are liquids with the following boiling-points and specific gravities at 20° : —
B.P. Sp. Gr. B.P. Sp. Gr.
Normal primary 117° '810 Primary isobutyl 107° -806
Normal secondary 100° '8c8 Tertiary 83° 786
They are not miscible with water in all proportions ; normal primary butyl alcohol requires 12 parts of water to dissolve it.
ALCOHOLS 69
AMYL ALCOHOLS. C3HnOH.
Eight isomers are possible, all of which are known :—
B.P. Sp. Gr. at 20°.
1. Normal primary, CH3 . CH2 . CH2 . CH2 . CH2OH 138° -817
CH3x
2. Isobutyl carbinol, ^CH . CH2 . CH2OH 130° -810
CH3/
3. Secondary butyl carbinol, /CH . CH2OH 128° -816
CH3CH/
-4. Tertiary butyl carbinol.
(primary) CH3— C . CH2OH u3«
CHg^
5. Methyl propyl carbinol, CH3
(secondary) >CHOH 119-
CH3 . CH2 . CH./
112° -819
CH3.CH2
7. Diethyl carbinol, \CHOH
(secondary) CH3 . CH2/
5. Dimethyl ethyl carbinol, CH3 — C . OH 102'
(tertiary) CH3 . CH2/
Secondary butyl carbinol contains an asymmetric carbon atom (see lactic acid) and consequently exists in a dextro- and a laevo-form. Together with primary isobutyl alcohol and propyl alcohol the two amyl alcohols, isobutyl carbinol and laevo-secondary butyl carbinol, are the principal constituents of fusel oil — and they together constitute fermentation amyl alcohol.
The fusel oil from potatoes or cereals contains chiefly isobutyl carbinol, secondary butyl carbinol being present only to 13-22 per cent. The fusel oil from beet molasses contains 48-58 per cent, of secondary butyl carbinol.
Fermentation amyl alcohol is a strongly refractive liquid which boils at 130-131° and is very slightly soluble in water — 3-3 volumes in loo volumes of water at 22°. Its vapours, on being inhaled, produce a peculiar sensation in the head, causing headache, etc. The two alcohols cannot be separated by fractional distillation, but only by chemical means. The mixture generally referred to as amyl alcohol is frequently used as a solvent.
70 PRACTICAL ORGANIC AND BIO-CHEMISTRY
HIGHER ALCOHOLS.
A hexyl alcohol has been isolated from the fusel oil obtained from grape skins. Two primary hexyl alcohols occur as esters : n-primary hexyl alcohol in the oil from the seeds of the parsnip, Heracleum gigantettm, and 3-methyl- pentanot in Roman camomile oil.
Normal primary heptyl alcohol is prepared by the reduction of oenan- thylic aldehyde which is obtained by distilling castor oil.
Normal primary octyl alcohol occurs in the oil from the fruits of the parsnips, Heracleum sphondylium^ Heracleum giganteum and Pastinaca sativa.
Normal nonyl alcohol is prepared by reducing with sodium and alcohol methyl heptyl ketone which is contained up to 5 per cent, in oil of rue.
Normal secondary undecylic alcohol is prepared by reducing methyl nonyl ketone, which occurs in camomile in large quantities.
n-dodecyl alcohol occurs as ester in oil of Cascara sagrada.
Normal hexadecyl alcohol, or cetyl alcohol, C16H3SOH, the most important of foe higher alcohols, is easily prepared from spermaceti, in which it is present as ester, by hydrolising the ester with alcoholic soda, diluting with water, filtering off and recrystallising the cetyl alcohol from alcohol. Cetyl alcohol has also been described as being present in the fat from an ovarian dermoid cyst. Cetyl alcohol is a white solid which melts at
5°°.
Ceryl alcohol, C27H55OH, or more probably C^H^OH, is prepared from Chinese wax, and melts at 76-79°.
Myricyl alcohol, C30H6iOH, is best prepared from carnauba wax. Beeswax contains this alcohol or the alcohol C3iH63OH.
Psylla-stearyl alcohol, CssHggO, has been obtained from the fat of the leaf louse (Psylla alni).
ESTERS.
Alcohols are like the bases NaOH, KOH in containing an OH group. The bases combine with acids to form salts. Alcohols com* bine with acids to form esters.
An enormous number of esters is possible since any alcohol can be combined with any acid, inorganic or organic. The organic acids may be grouped into three classes : (a) those insoluble or very insoluble in water ; (£) those soluble in water, but volatile with steam ; (c) those soluble in water, but not volatile with steam.
Preparation.
There are several methods of preparing esters :—
(1) By the action of the acid upon the- alcohol in the presence of a dehydrating agent, or catalyst.
(2) By the action of concentrated sulphuric acid upon the sodium salt of the acid and the alcohol.
(3) By the action of the acid chloride, or anhydride, upon the alcohol.
(4) By the action of the alkyl halide upon the silver salt.
ESTERS OF INORGANIC ACIDS.
Halogen Acids.
These compounds are the same as the monosubstituted halogen derivatives of the hydrocarbons (p. 57): —
C2H5OH + HC1 = C2H5C1 + H20.
Amyl Nitrite.
The calculated quantity of concentrated sulphuric acid is allowed to drop slowly upon the calculated quantity of sodium nitrite mixed with the calcu- lated quantity of amyl alcohol contained in a flask cooled by a freezing mixture. Amyl nitrite floats to the surface as an oil. It is separated, washed with water, dried with calcium chloride and distilled.
Ethyl Sulphuric Acid. Barium and Potassium Ethyl Sulphate.
10 c.c. of concentrated sulphuric acid are poured carefully into and mixed with 20 c.c. of ethyl alcohol ; the mixture becomes hot. It is heated on a water-bath under a reflux condenser for |-i hour. On cooling it is poured into about 200 c.c. of cold water. The acid solution is neutralised to litmus by stirring it up with calcium or
71
72 PRACTICAL ORGANIC AND BIO-CHEMISTRY
barium carbonate. Carbon dioxide is evolved and the excess of sulphuric acid is precipitated as insoluble sulphate ; this is filtered off after heating on a water-bath for -J-i hour. The solution contains calcium or barium ethyl sulphate (as shown below under hydrolysis).
UO\ C2H5O\
C2H5OH + \S02 = \S02 + H20
HCT HCT
C2H60\ 2 \S02
HCT
The clear filtrate is heated on the water-bath and treated with a strong solution of potassium carbonate (10 gm.) or potassium oxalate until no further precipitate is formed :^
/O— SO2OC2HS C2H5O\
Ba/ + K2C03 = BaC03 + 2 \SOr
\0-S02OC2H5 KCT
The barium carbonate is filtered off and the filtrate evaporated to a small volume (a drop withdrawn on a glass rod should crystallise on cooling). The crystals which form after standing for several hours are filtered off, washed with dilute alcohol and dried between sheets of filter paper. The mother liquor yields more crystals on further evaporation. The salt is dis- solved in boiling alcohol under a reflux condenser, filtered, using a hot- water funnel, and allowed to crystallise out.
ESTERS OF ORGANIC ACID.
Ethyl Acetate.
Molecular proportions of glacial acetic acid (50 c.c) and absolute alcohol (50 c.c.) are mixed in a distilling flask, and I per cent, by volume of concentrated sulphuric acid (i c.c.) is added. The distilling flask is connected to a condenser and receiver and the mixture is dis- tilled. A yield of 86*5 per cent, of ester is obtained (Senderens, method). Ethyl sulphuric acid appears to be the catalyst : —
C2H5HS04 + C2H5OH = (C2H5)2S04 + H2O (C2H5)2SO4 + CH3COOH = C2H5HSO4 + CHaCOOC2H5.
The distillate which contains water, alcohol and acetic acid is purified by shaking it in a separating funnel with strong sodium carbonate solution which is added in small quantities until the aqueous por- tion shows an alkaline reaction. The aqueous portion is withdrawn and the ester shaken with saturated salt or strong calcium chloride solution to remove alcohol ; the ester layer is separated and dried by contact with solid calcium chloride. It is then distilled from a dry flask, the portion passing over when the thermometer reads 74-78° being collected.
ESTERS
73
Ethyl Benzoate.
20 gm. of benzoic acid are dissolved in 75 c.c. of absolute alcohol and I c.c. of concentrated sulphuric acid is added. The flask containing the mixture is connected to a reflux condenser and gently heated for 1-2 hours over a gauze, a piece of porcelain being added to prevent bumping. The esterification is complete when, on testing by pouring a few drops into water, only oil drops and no crystalline benzoic acid is seen. The whole is then poured into about 400 c.c. of water and the oil is allowed to settle. The water is decanted off and the remainder is shaken up with ether in a separating funnel. The aqueous layer is withdrawn, the ethereal layer shaken with sodium carbonate solution and then with water. It is dried with calcium chloride and the ether distilled off over a water-bath. The ester is distilled over a flame and the fraction boiling from 210-215° is collected. Ethyl benzoate boils at 2 1 3°.
Properties.
Esters are usually liquids having a sweet and fragnant odour ; a few are solid.
Neutral esters of monobasic, dibasic, etc., acids are insoluble in water, or only slightly soluble, e.g. ethyl formate and acetate.
Acid esters of dibasic, etc., acids are soluble in water, e.g. ethyl sulphuric acid, ethyl oxalic acid, etc.
Neutral esters are soluble in alcohol and ether, acid esters may be soluble in alcohol, but are insoluble in ether.
Esters are comparatively inert substances and are unaffected by cold dilute sodium carbonate, sodium hydroxide, hydrochloric acid, sul- phuric acid, but there are exceptions, e.g. methyl oxalate, which is decom- posed by cold dilute caustic soda. They are acted upon by sodium more- or less readily (cf. ethers). All esters are hydrolysed by boiling with water, acids, or alkalies. The last method of hydrolysis is knowri as saponification. They are thus converted into their constituents, namely acid and alcohol. The recognition of these identifies the ester. Esters which are of frequent occurrence in animals and plants are identified in this way. The hydrolysis is effected by boiling under a reflux condenser with aqueous sodium hydroxide, or 80 per cent, sulphuric acid. If only the acid is to be identified hydrolysis is effected by boiling with alcoholic sodium hydroxide. The alcohol is isolated by distilling the alkaline liquid if the alcohol be volatile, by extracting the alkaline solution with ether if not volatile, and it is identi- fied by the reactions for alcohols. If the alcoholic portion of the ester be a phenol or an aromatic alcohol it does not distil and is not extracted
74 PRACTICAL ORGANIC AND BIO-CHEMISTRY
from an alkaline solution. The solution must be firstly acidified to liberate the phenol (see under phenols). The acid, which is formed by hydrolysis with alkali, is liberated by acidifying the cold solution with mineral acid — sulphuric acid. If insoluble it is filtered off; if soluble and volatile with steam, distilled ; if soluble and not volatile, it is extracted with ether or precipitated as insoluble calcium or other salt.
HYDROLYSIS OF ESTERS.
Ethyl Sulphuric Acid.
Ethyl sulphuric acid is not readily hydrolysed by alkali, but it is decomposed by boiling with acid.
The solution of calcium or barium ethyl sulphate obtained above contains one or other of these bases as shown by adding dilute sulphuric acid, the insoluble sulphate being precipitated.
If a portion of the solution be heated with dilute hydrochloric acid f°r 3~5 minutes, the insoluble sulphate is again precipitated : — C3H60 . S02 . Ov
/Ba + 2H2O = 2C0H5OH + BaSO4 + H2SO4. C2H50 . S02 . O/
Ethyl Acetate.
About 10 c.c. of ethyl acetate are placed in a flask with about 80 cc of sodium hydroxide, and the mixture is boiled over a gauze under a reflux condenser for 20-30 minutes until no more oily drops are visible and until the smell of ethyl acetate has disappeared. A piece of unglazed porcelain is added with advantage to prevent bumping of the liquid during the heating : —
CH3COOC2H5 + NaOH = CH3COONa + HOC2H0.
The flask is connected with a condenser and about a quarter of the liquid is distilled over.
This liquid contains the alcohol. It may be identified by the tests for ethyl alcohol.
The alcohol is separated and identified by saturating the solution with solid potassium carbonate, collecting the alcohol in a pipette, determining its boiling-point and performing other reactions for the alcohol.
The liquid remaining in the flask is acidified with dilute sulphuric acid and again distilled as long as the distillate reacts acid to litmus.
The distillate is neutralised and evaporated down and the acetic acid prepared and identified (p. 97).
ESTERS 75
Ethyl Benzoate.
The hydrolysis is effected as described above for ethyl acetate.
The insoluble acid is more readily prepared by hydrolysing with alcoholic soda and then identified : —
5-10 gm. of the ester are placed in a flask and boiled under a reflux condenser with excess of caustic soda (1-2 gm.), dissolved in 10 c.c. water and 100 c.c. of alcohol, for 10-15 minutes. The saponification is continued until a few drops poured into water show no oily drops of unchanged ester.
If any insoluble sodium benzoate separates out, it is dissolved by adding a little water through the condenser.
The solution is poured into an evaporating basin, water added and the alcohol evaporated off on the water-bath. On cooling and after adding about 25-50 c.c. of water the solution is acidified with dilute mineral acid. Benzoic acid is precipitated. It is washed with water, recrystallised from hot water, and identified (p. 256).
Ethyl Oxalate.
10-20 gm. of ethyl oxalate are hydrolysed with caustic soda solution containing sufficient alkali (6-12 gm.) as described undei -ethyl acetate and the alcohol is distill *d off.
The acid contained in the solution on acidifying with mineral acid is not precipitated nor is it volatile with steam. The acid solution may be extracted several times with ether, the ethereal solution dis- tilled to remove the ether, and the acid which is left identified.
Oxalic acid is more easily separated as its calcium salt. The acid liquid is carefully neutralised with soda and calcium oxalate is precipi- tated by adding calcium chloride. The acid is obtained as described under oxalic acid (p. 108).
76 PRACTICAL ORGANIC AND BIO-CHEMISTRY.
ETHERS.
Preparation.
Ethers are prepared either by distilling alcohols with concentrated sulphuric acid or by the action of sodium ethoxide upon an alkyl halide : —
/ C2H5OH + H2SO4 = C2H5HSO4 + H2O \C,HSOH + C2H6HS04 = C2H5OC2H5 + H2S04 CH3ONa + CH3I = CH3OCH3 + Nal.
If in the first preparation a different alcohol be used in the second reaction, and if in the second preparation the alkoxideand halide con- tain different radicles, mixed ethers are formed, e.g.
CH8.O.C2H6.
ETHYL ETHER.
Preparation.
Ethyl ether is generally prepared by distilling ethyl alcohol with sulphuric acid — hence its name of sulphuric ether. According to the equation the sulphuric acid is combined and again liberated so that it should be possible to convert an unlimited quantity of alcohol into- ether, but bye-products are formed which interfere with the reaction. The process is known as the continuous process.
A distilling flask of about 500 c.c. capacity is fitted with a tap funnel and a thermometer, the bulb of which reaches nearly to the bottom of the flask. The neck of the flask is connected to a long condenser and the receiver is cooled by standing in ice water. A mixture of no c.c. of absolute alcohol and 80 c.c. of concentrated sulphuric acid is placed in the flask and heated to 140-145°. At this temperature ether is formed and absolute alcohol is dropped in from the tap funnel at the same rate as the liquid distils. The preparation is continued until about twice the volume of alcohol originally mixed with the sulphuric acid has been added. The distillate consists of ether, alcohol, water and sulphurous acid. It is put into a separating funnel and shaken with dilute caustic soda. The alkaline layer is withdrawn and the upper layer of ether shaken with saturated salt solution, which is also withdrawn. The ether is put into a distilling flask, which is loosely corked, and dried by being allowed to stand in contact with calcium chloride for 12- 24 hours. The flask is connected with a condenser and the ether distilled off from a water-bath (b p. 35°).
Purification of Ethyl Ether.
The ether obtained above contains traces of alcohol and water. These can only be removed by treatment with metallic sodium. The ether is placed in a flask, provided with a calcium chloride tube to prevent access of moisture and to allow the escape of hydrogen, and several slices of sodium are added. When no further effervescence is observed the ether is decanted into a dis- tilling flask and distilled from a water-bath. Pure ether of constant boiling- point 35° is collected.
ETHERS 77
Purification of Commercial Methylated Ether.
This ether is made by the continuous process from methylated spirit and •contains water, alcohol and other impurities. The ether may be washed with water to remove most of the alcohol. By distilling it over solid caustic potash, aldehydic impurities are destroyed. It is dried by standing over calcium chloride and then treated with metallic sodium.
Sometimes, 'after treatment with sodium, ether is left in contact with phosphorus pentoxide and then distilled from the solid dehydrating agent.
Distillation of Ether. Precautions.
As ether is very inflammable and exceedingly volatile no flame should be in the neighbourhood. Ether should never be distilled over a free flame and the most convenient way, if steam or electric heaters are not available, is to heat a water-bath, extinguish the flame, -and immerse the distilling flask containing the ether in the hot water.
Large quantities of ether should not be distilled from a large flask, but a small flask provided with a tap funnel should be employed. As the ether distils a fresh quantity can be added without interrupting the distillation. The ether should be collected in small receivers and transferred to a larger reservoir.
Properties.
The first member of the series, dimethyl ether, is a gas.
Ethyl ether, or simply ether, the chief representative of the group is a very volatile, colourless liquid with a pleasant characteristic smell. It boils at 35° and has a sp. gr. of 7195 at 15°. It is sparingly soluble in water, less soluble in glycerol. It mixes in all proportions with alcohol, chloroform, benzene, ligroin, and is largely used as a sol- vent for fats, resins, etc. The lower members of ethers of the aliphatic series are also volatile liquids, like ethyl ether, which boil at a lower temperature than the alcohol from which they are derived. The highest members are odourless solids.
The ethers are inert compounds and are not acted upon by phosphorus pentachloride and sodium (distinction from alcohols), or by .aqueous or alcoholic potash (distinction from halogen compounds and ^esters).
The lower members— especially those containing methyl and ethyl radicles — are decomposed by heating with hydriodic acid fDrming alkyl iodides (distinction from hydrocarbons). This reaction is used in estimating methoxy— CH3O— and ethoxy C2H5O groups in organic compounds (Zeisel's method).
78 PRACTICAL ORGANIC AND BIO-CHEMISTRY MERCAPTANS AND SULPHIDES.
The sulphur compounds corresponding to the alcohols, i.e. thio-alcohols,. are known as mercaptans ; the sulphur compounds corresponding to the ethers, i.e. thio-ethers, are known as sulphides, or alkyl sulphides. Bisulphides are also known.
CH3 . SH C2H5 . S . C2H5 C2H5 . S— S . C2H6
Methyl mercaptan. Ethyl sulphide. Ethyl disulphide.
Mercaptans.
Methyl mercaptan is a product of the putrefaction of proteins. It occurs in the urine after a diet of asparagus and gives it the peculiar unpleasant odour.
Preparation.
Mercaptans are prepared : —
(1) By heating the alcohol with phosphorus pentasulphide : —
5CH3OH + P2S5 = 5CH3SH + P205.
(2) By heating the alkyl halide or alkyl potassium sulphate with potas-
sium hydrosulphide : —
CH3I + KSH = CH3SH + KI C2H5O . SO2 . ONa + KSH = C2H6SH + NaKSO4.
About 2-5 c.c. of a saturated solution of sodium ethyl sulphate are made alkaline with sodium hydroxide and an equal volume of sodium hydrosulphide (33 Per cent.) is added. On warming ethyl mercaptan is formed which is- recognised by its garlic-like unpleasant odour.
Properties.
Methyl mercaptan is a gas, ethyl mercaptan is a colourless liquid boiling at 36°. The other mercaptans are also liquids which are insoluble in water and have a disgusting smell.
Like the alcohols they react with sodium with evolution of hydrogen : —
2CH3SH + Na2 = 2CH3SNa + H2.
The mercaptans react with mercuric oxide forming crystalline com- pounds : —
2C2H6SH + HgO = (C2H8 . S)2Hg + H2O.
These compounds are termed mercaptides, the name of the group being; derived from the mercury compounds. On oxidation with nitric acid the mercaptans yield sulphonic acids : —
CH3SH + 30 = CH3 . SO3H.
The sulphonic acids are isomeric with alkyl hydrogen sulphites. The latter compounds are esters and are hydrolysed by alkali ; the sulphonic acids are stable. In the sulphonic acids the sulphur atom is joined to carbon, in the sulphites it is joined to oxygen : —
o CH-°
\OH
Methyl sulphonic acid. Methyl hydrogen sulphite.
MERCAPTANS AND SULPHIDES 79
Alkyl Sulphides.
Ethyl sulphide, C2H5 . S . C2H5, is another product of the putrefaction of proteins, being derived from cystine (p. 143).
Preparation.
Sulphides are obtained : —
(1) By the action of phosphorus pentasulphide upon ethers : —
5(C2H5)20 + P2S5 = 5(C2H5)2S + P205.
(2) By the action of potassium sulphide on an alkyl halide or alkyl
potassium sulphate : —
2C0H5I + K2S = 2KI + (C0H5)2S 2C2H5KSO4 + K2S = 2K2SO4 + (C2H5)aS.
Properties.
The sulphides are colourless, neutral liquids with very unpleasant smell ; ethyl sulphide boils at 91°.
They resemble the ethers in being comparatively stable compounds. On oxidation with nitric acid, they are converted into sulphones which are stable crystalline compounds : —
(C2H5)2S + 02 = (C2H6)2S02.
Alkyi Bisulphides.
Bisulphides are formed when mercaptans are exposed to the air : —
2C2H5SH + O = H20 + C2H5 . S— S . C2H5, or by the action of iodine upon sodium mercaptides :—
2C2H5S . Na + I2 = 2NaI + C2H5 . S— S . C2H5.
ALDEHYDES.
Aldehydes are the first products of oxidation of primary alcohols, e.g. :—
CH3 CH3 CH.
I -* I /OH -» I
CHoOH CH( CHO
\OH
Ethyl alcohol. Hypothetical. Acetaldehyde.
The hypothetical intermediate compound does not exist; it at once loses a molecule of water and is converted into aldehyde. Two OH groups cannot exist attached to one carbon atom ; aldehyde is formed by loss of water. There are a few exceptions, such as chloral hydrate,
CC13
\ /OH
CH^
\OH.
Formaldehyde is formed from methyl alcohol, propyl aldehyde from primary propyl alcohol, etc.
/H
The group — CHO or — C<f is characteristic of aldehydes.
%0
Preparation.
When the alcohol is available the aldehyde is usually prepared by oxidation ; otherwise it may be prepared by the dry distillation of molecular proportions of calcium formate and the calcium salt of the corresponding acid (compare ketones).
FORMALDEHYDE.
Preparation.
Formaldehyde is prepared by passing the vapour of methyl alcohol mixed with air over heated platinum or copper, or other substances. The formaldehyde formed by oxidation is passed into water.
Formol, or formalin, is a commercial aqueous solution containing 40 per cent, of formaldehyde.
ACETALDEHYDE.
Preparation.
25 gm. of coarsely powdered potassium bichromate and 100 c.c. of water are placed in a distilling flask of 250 c.c. capacity. The flask is connected with a condenser and a strong current of cold water is made to flow through it. Through a tap funnel, secured in the neck of the
80
ALDEHYDES 81
flask by a well-fitting cork, a mixture of 25 gm. (30 c.c. of absolute alcohol and 35 gm. (20 c.c.) of concentrated sulphuric acid is slowly added to the contents of the flask which have been gently warmed and the flame removed. During the addition the contents of the flask, which darken in colour, are occasionally shaken. A mixture of alde- hyde, alcohol and water distils over. When the mixture has been added the flask is heated until all the aldehyde (recognised by smell) has distilled over.
The tests for acetaldehyde can be carried out with this distillate
(P. 84).
Purification.
The solution is redistilled through an inverted condenser filled with water at 30-35°. Water and alcohol are condensed, but the aldehyde passes on. The aldehyde vapour is passed through a 100 c.c. pipette into about 30 c.c. of pure dry ether contained in a bottle standing in ice.
Pure ammonia, prepared by gently heating concentrated ammonia solution with a small flame and dried by passage through a tower containing quick- lime, is passed into the ether until it is saturated. Aldehyde ammonia crystallises out. After standing for one hour the ether is decanted off, the crystals are drained on a Buchner filter and washed with ether.
The crystals of aldehyde ammonia are dissolved in an equal weight of water, and the solution is distilled with a mixture of 1*5 parts of sulphuric acid and 2 parts of water from a water-bath, which is gradually raised to boiling. The receiver is cooled in ice. The distillate is dried with calcium chloride from which the aldehyde is distilled in a bath at 20° and collected in a receiver in ice. The aldehyde must be preserved in a well-stoppered bottle.
Properties of Aldehydes.
Formaldehyde is a gas at the ordinary temperature easily soluble in water and alcohol and with a peculiar pungent smell.
Acetaldehyde is a colourless liquid having a fruity pungent smell and boils at 21°. It is easily soluble in water, alcohol and ether.
The next members of the series of aldehydes are also liquids and resemble acetaldehyde very closely in their properties.
Polymerisation.
Paraformaldehyde or Paraform.
On evaporating formalin (about I c.c.) in a watch-glass on a water-bath a solid mass of paraformaldehyde is left.
If a portion of the solid be heated in a test tube dissociation occurs at about 100°, the mass melts between 153 and 172°, a white sublimate is formed and formaldehyde is evolved.
82 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Paracetaldehyde or Paraldehyde.
On adding a drop of concentrated sulphuric acid to about I c.c. of acetaldehyde, a violent reaction occurs and the liquid becomes hot. Par- aldehyde (CH3 . CHO)3 separates out as an oil on diluting with water. Acetaldehyde is re-formed if the acid aqueous liquid be heated.
Paraldehyde is a colourless liquid which boils at 124°.
Metaldehyde.
The polymer, metaldehyde, is formed from acetaldehyde when it is treated with hydrochloric acid gas or dilute sulphuric acid at a low temperature.
Aldol Condensation.
Solutions of formaldehyde and acetaldehyde when kept with dilute solutions of lime or potassium carbonate undergo aldol condensation.
Formaldehyde gives a sweet syrup which contains monosaccharides,. especially dl-fructose : —
HCHO + HCHO = HCHOH . CHO
HCHO + HCHO + HCHO = HCHOH . CHOH . CHO,
etc.
Acetaldehyde gives aldol : —
CH3 . CHO + CH3 . CHO = CH3 . CHOH . CH2 . CHO.
These reactions of aldehyde probably take place in nature. In plants, under the "action of light and chlorophyll, carbon dioxide is reduced to formaldehyde which undergoes aldol condensation into sugars. The higher fatty acids are probably formed from acetaldehyde jn this way in both animals and plants.
Action of Ammonia.
Hexamethylene Tetramine.
Formaldehyde behaves differently to the other aldehydes.
On adding ammonia gradually to formalin (i c.c. in 5 c.c. water) it is absorbed. On now adding excess of ammonia and evaporating on the water-bath hexamethylene tetramine, or urotropin, remains as a white solid : —
6CH2O + 4NH3 = (CH2)6 N4 + 6H2O.
Hexamethylene tetramine consists of colourless crystals soluble in about 1-5 parts of hot or cold water and 10 parts of alcohol. It is volatilised on heating and it is converted into ammonium sulphate and formaldehyde on treatment with strong sulphuric acid.
Aldehyde Ammonia.
On passing dry ammonia gas into a dry ethereal solution of acetaldehyde, acetaldehyde ammonia is formed : —
/OH CH3 . CHO + NH3 = CH3 . CH/
^NH2.
Acetaldehyde ammonia is a white crystalline compound easily
ALDEHYDES 83
soluble in water and alcohol, and easily decomposed by acids and alkalies.
On dissolving a little aldehyde ammonia and heating with dilute sulphuric acid, aldehyde is given off. Ammonia is also evolved on heating with dilute caustic soda.
Aldehyde Sodium Bisulphite.
On adding 1-2 c.c. of a cold saturated solution of sodium bisulphite to 5-10 drops of aldehyde and shaking vigorously, aldehyde sodium bisulphite crystallises out : —
,OH
CH3 . CHO + NaHS03 = CH3 . CH\
XS03NA.
Aldehyde Cyanhydrin.
Hydrogen cyanide combines with aldehydes forming cyano-
hydrins : —
,OH
CH, . CHO + HCN = CH3 . CH\
XCN.
In this way another carbon atom can be added to organic com- pounds. Compounds containing the CN group are hydrolysed by acids or alkalies and converted into the corresponding acid (see cyano- gen compounds) : —
,OH OH
CH-.CHx + 2H20 = CH3.CH< + NH8.
\CN \COOH
Aldehyde Hydrazone.
Aldehydes combine with hydrazine and substituted hydrazines,. especially phenylhydrazine, forming hydrazones.
The calculated quantities of aldehyde (-5 c.c.), phenylhydrazine hydrochloride (-2 gm.) and cryst. sodium acetate (-5 gm.)-are dissolved in about 10 c.c. of water and warmed ; an oil (acetaldehyde phenyl- hydrazone) is formed : —
CH3 . CHO + H2N . NH . C6H5 = CH3 . CH : N . NH . C«H5 + H2O.
Aldoxime.
Aldehydes combine with hydroxylamine forming oximes : —
CH3 . CHO + H2NOH = CH3 . CH : NOH + H2O
(acet)aldoxime.
The calculated quantity of hydroxylamine hydrochloride is dissolved in water, the equivalent quantity of caustic soda required to liberate the hydroxylamine is added and then the calculated quantity of the aldehyde. The mixture is shaken and allowed to stand until it no longer reduces Fehling's solution. The oxime is extracted with ether, most of the ether dis- tilled off, and the concentrated solution poured into a basin. The crystals which separate are drained on a porous plate and recrystalhsed from hgrom.
S4 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Tests.
Aldehydes are easily further oxidised into the corresponding fatty -acids containing the same number of carbon atoms and they conse- quently behave as reducing agents.
Reduction of Metallic Oxides in Alkaline Solution.
(a) Stiver.
An ammoniacal solution of silver hydroxide is prepared by adding dilute ammonia to silver nitrate until the precipitate first formed just re-dissolves. Some dilute aldehyde solution is added and the mixture is placed in a cold water-bath and heated to the boiling- point. A mirror of metallic silver forms on the glass.
A very sensitive reagent may be prepared by mixing equal volumes of 10 per cent, silver nitrate and sodium hydroxide and then adding ammonia •drop by drop till the silver hydroxide dissolves.
A mirror is formed immediately if the solution contains i per cent, of acetaldehyde, in 30 seconds if i per thousand ; a yellow-brown mirror forms in 5 minutes if i per 10,000 be present.
(£) Copper.
Dilute aldehyde solution reduces Periling' s solution x on warming with the formation of cuprous oxide.
Action of Sodium Hydroxide.
Except with formaldehyde, benzaldehyde and a few other alde- hydes, caustic soda solution decomposes dilute aldehyde solutions on warming. Yellow to brownish-red resins which rise to the surface — aldehyde resin — are formed. The liquid has usually a peculiar smell. Aldehyde resin is insoluble in water, but soluble in alcohol and ether.
Formaldehyde is converted into methyl alcohol and formic acid.
Oxidation.
Aldehydes are converted into the corresponding acid on warm- ing their solutions with potassium bichromate and dilute sulphuric .acid, the solution becoming green.
The aldehyde may be identified by preparing the acid by oxidation.
Schiffs Test.
A solution of magenta, or fuchsin, is decolorised by bubbling sulphur dioxide through it. On adding the dilute aldehyde solution the purple-red colour returns.
Numerous other sensitive tests have been described for aldehydes, •especially formaldehyde. The following one has been used more particularly in testing for formaldehyde in distillates from plant leaves, etc.
1 Fehling's solution consists of copper sulphate, caustic soda and Rochelle salt (sodium potassium tartrate). On adding caustic soda to copper sulphate a blue precipitate of cupric hydrate Cu(OH)2 is formed, which turns black on boiling. The presence of the Rochelle salt keeps the Cu(OH)2 in solution forming a deep blue solution. This solution does not keep, so that it must be freshly made for each experiment. For this purpose two solutions are therefore kept. The one contains the copper sulphate, the other the Rochelle salt and caustic soda. When required for use, equal parts of each are mixed together, and this forms the reagent.
ALDEHYDES 85
Rimini's Test.
A small quantity (2 drops) of phenylhydrazine is added to the solution, then a drop of dilute freshly prepared sodium nitroprusside solution and a few drops of sodium hydroxide solution. A deep blue colour forms if form- aldehyde be present ; the colour changes through green and brown to red.
Schryver has modified this test and made it more sensitive : 2 c.c. of a freshly prepared and filtered i per cent, solution of phenylhydrazine hydrochloride are added to 10 c.c. of the solution of formaldehyde, then i c.c. of a 5 per cent, solution of sodium ferricyanide and 5 c.c. of hydrochloric acid ; a magenta colour is formed. This test will show the presence of i part oi formaldehyde in 100,000 to 1,000,000 parts of solution. No colour is given by acetaldehyde.
ESTIMATION. Formaldehyde.
(a) By Converting into Hexamethylenetetramine.
25 c.c. of normal ammonium hydroxide solution are placed in a 100 c.c. strong bottle provided with a rubber stopper. A measured volume of the solution (not containing above -5 gm. of formaldehyde) is added. The cork is securely fastened by tying and the bottle is submerged in a cold water-bath which is then heated to boiling for i hour, the bottle being kept under water the whole time. The bottle is cooled, opened and the contents titrated with standard acid until the methyl orange which is used as indicator first becomes red.
A series of bottles should be taken containing different amounts, or none,. of the aldehyde solution. Allowing for the blank each c.c. of normal am- monium hydroxide used corresponds to *o6oi gm. of formaldehyde.
The estimation should be carried out in water. The formaldehyde is therefore distilled from its original solution, e.g. milk, plant extracts, and the distillate is used.
• (b) By Titrating with Iodine and Sodium Thiosulphate.
A known volume of the solution (10 c.c.) is mixed with 25 c.c. of -iN iodine solution, and sodium hydroxide is added drop by drop till the liquid becomes clear yellow. The flask is closed for 10 minutes, dilute hydrochloric acid is added, and the free iodine is titrated with -iN thiosulphate.
2 atoms of iodine = i molecule of formaldehyde.
Good results are not given by this method for aldehydes other than form- aldehyde.
Acetaldehyde.
By Combination with Sulphite.
The solution of sulphite is prepared by dissolving 12*6 gm. of sodium sulphite in 400 c.c. of water, adding 100 c.c. of -iN sulphuric acid and diluting to 1000 c.c. with the purest ethyl alcohol of 95 per cent.
The volume of aldehyde solution, not containing more than 2 per cent, of aldehyde, is placed in a 100 c.c, measuring flask. A known volume of the sulphite solution is added and the mixture diluted to 100 c.c. with the purest 50 per cent, alcohol. A blank with the reagents is carried out simul- taneously.
The flasks are kept at 50° for 4 hours, cooled and titrated with standard iodine solution, using starch as indicator.
Each c.c. of -iN iodine solution corresponds to -0032 gm. of SO2 or -0022 gm. of acetaldehyde.
86 PRACTICAL ORGANIC AND BIO-CHEMISTRY
CC13 CHLORAL. |
CHO.
Preparation.
Chloral is prepared by the prolonged action (about 10 days) of dry chlorine upon absolute alcohol. The gas is passed into the cold alcohol until it is saturated and acquires a sp. gr. of i "400 and the temperature is gradually raised to 1 00°. Chloral alcoholate is formed. An equal weight of concentrated sulphuric acid is added and the mixture is distilled. The fraction passing over between 94 and 100° is collected, neutralised with calcium carbonate and again distilled.
Properties.
Chloral is a colourless oily liquid with a peculiar penetrating smell, having a sp. gr. of 1*502 at 18°. It boils at 97° and is soluble in ether and chloroform.
Metachloral.
On keeping, or on leaving in contact with moderately concentrated sulphuric acid, chloral polymerises to metachloral, a solid which is sparingly soluble in boiling water, but insoluble in cold water, alcohol and ether. The polymerisation does not occur with pure chloral and may be hindered by adding chloroform. On heating to 180° meta- chloral is decomposed and chloral distils over.
Chloral Alcoholate.
If chloral be mixed with an equivalent quantity of absolute alcohol, chloral alcoholate is formed.
It consists of white crystals which melt at 46° and boil at 1 13*5° and are readily soluble in chloroform (distinction from chloral hydrate).
/OH CHLORAL HYDRATE. CC13 . CH<
XOH.
Preparation.
* Equivalent parts of chloral (6 c.c.) and water (i c.c.) are mixed together. The mixture becomes hot and solidifies to a mass of crystals of chloral hydrate.
Properties.
Chloral hydrate is a white crystalline solid, which melts at 50-51°. It is soluble in I -5 times its weight of water, also in alcohol, ether, petroleum ether and carbon disulphide. It is soluble with difficulty in cold chloroform.
Pure chloral hydrate is completely volatile on heating and com- •mences to boil rapidly at 97-98°.
ALDEHYDES 87
Reconversion into Chloral.
About 2 gm. of chloral hydrate are placed in a dry test tube and covered with concentrated sulphuric acid and the mixture is warmed gently. Chloral is formed and floats to the surface.
An aqueous solution heated with zinc to 50° and gradually treated with dilute acid yields aldehyde and paraldehyde which may be distilled off.
Tests for Chloral and Chloral Hydrate.
Aqueous solutions in the cold give no reaction with silver nitrate. On adding a few drops of ammonia and boiling, metallic silver is deposited.
Aqueous solutions reduce Fehling's solution on heating. Traces of chloral may be detected by the carbylamine reaction for chloro- form (p. 61).
Decomposition of Chloral by Alkali.
Chloral or chloral hydrate is rapidly decomposed by caustic alkali with the formation of chloroform and alkali formate : — CC1SCH(OH)2 + NaOH = CHC13 + HCOONa + H2O.
The odour of chloroform is noticed at once on gently warming an aqueous solution of chloral with caustic soda. Estimation.
1. By measuring the volume of chloroform.
2 5 gm. of chloral hydrate or chloral are placed in a graduated cylinder and excess of sodium hydroxide solution (80-100 c.c.) are carefully added. The tube is kept well cooled at first on account of the violence of the reaction. Afterwards the cylinder is closed and shaken. On standing the liquid be- •comes clear and separates into two layers. When cold (at 17°) the volume of the lower layer of chloroform is measured. The volume in c.c. multiplied by i -84 gives the number of grams of chloral, or by 2-064 of chloral hydrate, in the sample.
2. By titrating the acid.
i-2. gm. are dissolved in water and shaken with barium carbonate to remove any acid. The carbonate is filtered off and washed, Excess of normal caustic soda (100-150 c.c.) is added and the solution titrated with normal acid, using litmus as indicator.
Each c.c. of alkali neutralised = '1475 gm- chloral or '1655 gm. chloral hydrate.
Butyric Chloral Hydrate.
This compound is formed when chlorine is passed into paraldehyde or acetaldehyde. It is a white crystalline substance with peculiar fruity flavour -and melts at 78°.
88 PRACTICAL ORGANIC AND BIO-CHEMISTRY
KETONES.
Ketones are the first products of the oxidation of secondary alcohols, e.g. : —
CH3 CH3 CH3
I I /OH I
CHOH ->C< -> CO
I XOH I
CH3 CH3 CH3
Isopropyl alcohol. Hypothetical. Acetone.
The same statements apply here as in the case of the formation of aldehydes.
The group >CO is characteristic of ketones.
Acetone is the first member of the homologous series of ketones and the chief representative.
ACETONE.
Preparation.
Acetone is formed in the dry distillation of wood and is separated from methyl alcohol by fractional distillation (p. 63).
Acetone is also prepared by the dry distillation of calcium or barium acetate : —
CH3 . COO. CH3v
>Ca = )CO + CaC03.
CH3 . COO/ CH3/
50-100 gm. of dry calcium acetate are placed in a retort or dis- tilling flask and at first heated gently, afterwards more strongly, and the vapours are passed through a condenser. A brownish liquid col- lects in the receiver. It contains acetone, aldehyde and higher ketones. The acetone is separated by fractional distillation.
KETONES 89
Purification.
The proper quantity of crude acetone (100 gm. or 125 c.c.) is added to the calculated quantity of sodium bisulphite (70 gm.) in saturated solution (this should smell of sulphur dioxide, if not, SO2 is passed into it until it smells strongly of the gas), and the mixture is shaken vigorously in a closed vessel. Heat is evolved and a mass of crystals, C3H6O . NaHSO3, separates out. After standing, the crystals are filtered off on a Buchner funnel and well drained. They are placed in a distilling flask and decomposed by adding a solution of sodium carbonate (40 gm.). The solution is distilled, preferably using a frac- tionating column, until the thermometer reaches 60°.
The distillate is dried with calcium chloride and the acetone distilled off.
Properties.
Ketones closely resemble aldehydes in most of their properties, but there are several differences.
Acetone is a colourless, pleasant smelling liquid which boils at 56° and has a sp. gr. of 797 at 15°. It is very volatile and in- flammable. It mixes with water, alcohol and ether in all proportions. Like alcohol it can be separated from water by saturating the solution with potassium carbonate.
Polymerisation and Condensation.
Acetone does not polymerise like aldehyde, but when distilled with moderately concentrated sulphuric acid it is converted into mesitylene (sym. trimethylbenzene).
Action of Ammonia.
Acetone does not form simple condensation products with ammonia like aldehyde does, but it reacts forming diacetonamine, C6H13ON, and triaceton- amine, C9H17ON.
Acetone Sodium Bisulphite.
On shaking together about I c.c. of acetone and 5 c.c. of a cold saturated solution of sodium bisulphite, acetone sodium bisulphite crystallises out : —
CH3\ CH3\ /OH
>CO + NaHSO3 = )C\
CH/ CH3/ >S03Na.
Acetone Cyanhydrin.
Acetone combines with hydrogen cyanide forming the addition
compound, acetone cyanhydrin : —
CH3V CH3X /OH
\CO + HCN = >C
CH/
90 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Acetone Phenylhydrazone.
Acetone combines with hydrazine and substituted hydrazines forming hydrazones : —
* Acetone phenylhydrazone is formed as an oil when acetone is mixed with phenylhydrazine hydrochloride and sodium acetate :—
CH3x CH3\
pCO + H2N . NHC6H5 = \C : N . NHC6H5 + H2O.
Acetoxime.
Combination occurs between acetone and hydroxylamine when the calculated quantities are allowed to react together as described under aldehyde (p. 83) :—
3v 3\
>CO + H2NOH = >C : NOH + H0O.
CH/ C&/
* Tests for Acetone.
Acetone is more stable than aldehyde and does not behave as a reducing agent.
* Acetone reduces ammoniacal silver nitrate solution on prolonged boiling.
* Acetone does not reduce Fehling's solution.
* Acetone does not give a resin when heated with sodium hy- droxide.
* Acetone does not give SchifT's test.
These four reactions are characteristic only for aldehydes. Oxidation.
* Acetone is oxidised on heating with potassium bichromate and sulphuric acid and yields acetic and formic acids : —
CH3\
)CO + 30 = CHXOOH + HCOOH. CH3/
The constitution of a ketone is determined by identifying the acids it yields on oxidation. *
From 2-5 gm. of the ketone are mixed in a flask attached to a reflux condenser with 30-50 c.c. water and the calculated quantity of sulphuric acid is added. The calculated quantity of finely powdered potassium bichromate is added in portions of '5-1 gm. If the oxidation is very energetic, the contents should be cooled and kept at 50-60°. The flask is finally heated on the water- bath for 15 minutes. The acids are then distilled and collected in the receiver (see under acids).
v
KETONES 91
lodoform Reaction (Lieberi}.
Acetone gives iodoform in the cold ; 3-5 drops of sodium hydrox- ide are added to about 2 c.c. of the solution and then, drop by drop iodine solution until the liquid is faintly yellow. lodoform separates at once.
If ammonia be used in place of sodium hydroxide and iodine solution be added drop by drop, a small black precipitate of nitrogen iodide is formed. On standing, or on warming, this disappears and iodoform is produced; this reaction may serve to distinguish acetone and alcohol.
Sodium Nitroprusside Test (Legal}.
On adding about 5 drops of freshly prepared sodium nitroprusside solution to about 5 c.c. of the dilute acetone and about I c.c. of sodium hydroxide, a ruby-red colour is produced. This fades to yellow oa- standing.
If the reaction be repeated and the solution acidified at once with*, acetic acid, a purple-red colour is produced.
Rothera suggests that the reaction be carried out by adding 3 drops of 5 per cent, sodium nitroprusside and 1-2 c.c. of ammonia to the dilute acetone and a small quantity of solid ammonium sulphate. A permanganate colour slowly develops, reaches a maximum in about 30 minutes and then fades away.
Creatinine does not react under these conditions ; a brownish-red^ colour is given by aldehydes.
Salicylic Aldehyde Test.
i gm. of solid potassium hydroxide is added to 10 c.c. of the acetone- solution, and before it dissolves 10 drops of salicylic aldehyde are added. On warming to 70° a purple-red contact ring appears. If the potash has dis- solved before adding the salicylic aldehyde .the liquid becomes yellow, red, and finally purple-red.
JVta?.— The iodoform, nitroprusside and salicylic aldehyde reactions are carried out preferably incolourless solutions. The acetone should be separated by distillation and the distillate tested.
92 PRACTICAL ORGANIC AND BIO-CHEMISTRY
Estimation of Acetone.
1. Acetone is most usually estimated by converting it into iodoform with -excess of iodine and caustic soda and titrating the excess of iodine with thio- sulphate (Messinger's method).
In the case of wood spirit *5 c.c. are added to 25 c.c. 'of N sodium hydroxide contained in a stoppered bottle of 200 c.c. capacity ; the mixture is well shaken and allowed to stand 5-10 minutes. 'aN iodine solution is slowly run in from a burette drop by drop, shaking thoroughly till the upper portion of the solution on standing for a minute becomes quite clear. A few more c.c. of the iodine solution are run in so as to have an excess of about 25 per cent, and the solution is allowed to stand 10-15 minutes. 25 c.c. of N sulphuric acid are added and the iodine which is liberated is titrated with •iN sodium thiosulphate solution, using starch as indicator.
i c.c. *iN iodine solution = '00967 gm. acetone.
A blank experiment should be made as sodium hydroxide may contain nitrite.
Aldehydes and other compounds which react with iodine are included in this estimation, if present.
2. Jolles has suggested the estimation of acetone by conversion into ace- tone sodium bisulphite with excess of sodium hydrogen sulphite and titration of the excess of sulphite with standard iodine solution.
3-4 times the excess of the bisulphite solution of known strength is added to the acetone solution ; after standing for 30 hours the excess is titrated with *iN iodine solution.
i mol. of NaHSO3 = 2 atoms of I = i mol. of acetone.
3. Deniges makes use of an insoluble compound of acetone with mercuric •sulphate for the estimation of acetone. The reagent is prepared by dissolving 5 gm. of mercuric oxide in 100 c.c. of water to which 20 c.c. of sulphuric acid have been added.
The acetone content of the solution must be not greater than '2 per cent. so that strong solutions must be diluted. 25 c.c. of the reagent are added to 25 c.c. of the solution and the mixture heated on the water-bath for 10 minutes. The precipitate is filtered off on a weighed filter, washed with not more than 100 c.c. of cold water, dried at 100° and weighed. The amount of acetone in the precipitate, 3Hg5S2O11 . 4C3HGO, is obtained by multiplying by the factor -0609.
This reaction can be carried out volumetrically by estimating the excess or mercury. The mercuric sulphate solution must therefore be of known strength. The filtrate and washings from the precipitate are collected and made up to 100 c.c. To 20 c.c. of this solution 15 c.c. of ammonia, 50 c.c. of water and 10 c.c. potassium cyanide solution (13 gm. per litre) are added. The excess of cyanide is estimated by titration with *iN silver nitrate solution, using potassium iodide as indicator, until there is a slight permanent precipitate.
Since acetone is a decomposition product of aceto-acetic acid and the two compounds are usually associated in tissues and extracts of organs, the estimation of acetone in urine, etc., is combined with the .estimation of aceto-acetic acid (p. 593).
THE FATTY ACIDS.
The fatty acids are the second products of oxidation of the primary alcohols, the aldehydes being the intermediate products. Secondary alcohols and ketones also give rise to fatty acids on oxidation, but the number of carbon atoms in the molecules of the fatty acids so formed is less than in the original secondary alcohol. Conversely, on reduction fatty acids give aldehydes and primary alcohols, thus : —
CH3 . CH2OH 2 CH3 . CHO ^ CH8 . COOH.
The fatty acids are characterised by the presence of the carboxyl or —COOH group.
They occur widely distributed in nature, both in the free state and in combination with glycerol as the fats.
Only those acids containing an even number of carbon atoms occur in combination as fats, and as far as is known they all have a straight chain of carbon atoms. Acids with an uneven number of carbon atoms and with branched chains of carbon atoms are also found in nature.
The lower members of the series of the fatty acids up to capric acid with lo carbon atoms are volatile with steam and hence are termed the volatile fatty acids. They are separated in this way from the higher members which are not volatile with steam. They thus form two groups.
In the following list are given the names of the homologous series of hydrocarbons,. primary alcohols, aldehydes and fatty acids : —
|
Number of |
Saturated |
Primary |
||
|
Carbon Atoms. |
Hydrocarbon. |
Alcohol -CH2OH. |
AlrlphvHo Ffltty Acid -CHO -CC-OH. |
|
|
I |
Methane |
Methyl |
Formaldehyde Formic |
|
|
2 3 4 5 |
Ethane Propane Butane Pentane |
Ethyl Propyl Butyl Amyl |
Acetaldehyde Propionic aide Butyric Valeric |
Acetic hyde Propionic Butyric Valerianic |
|
6 I g |
Hexane Heptane Octane Nonane |
Hexyl a? Nonyl |
Caproic Oenanthic Caprylic Pelargonic |
Caproic Oenanthic Caprylic Pelargonic |
|
10 ii |
Decane Undecane |
Decyl Undecyl |
Capric Undecylic |
Capric Undecylic |
|
12 13 14 15 16 17 18 |
Dodecane Tridecane Tetradecane Pentadecane Hexadecane Heptadecane Octadecane |
Dodecyl Tetradecyl Cetyl Octadecyl |
Laurie Tridecyiic Myristic Palmitic Margaric Stearic |
Laurie Tridecyiic Myristic Pentadecylic Palmitic Margaric Stearic |
|
19 |
Nonadecane |
— |
— |
|
|
20 |
Eicosane |
— |
— Aracnic |
|
|
21 22 |
Heneicosane Docosane |
— |
— Behenic |
|
|
23 24 |
Tricosane Tetracosane |
|
Lignoceric |
|
|
25 |
— |
— |
||
|
26 |
— |
Ceryl |
— Cerotic |
|
|
11 |
Heptacosane |
|
— — |
|
|
29 3° |
~~ |
Myricyl |
Melissic |
93
94 PRACTICAL ORGANIC AND BIO-CHEMISTRY
FORMIC ACID. H . COOH.
Preparation.
Formic acid was first prepared by distilling crushed ants with water — hence its name. The stings of some insects and plants also probably contain it. It occurs together with acetic and other lower fatty acids in urine. It can be obtained by oxidising methyl alcohol with potassium permanganate. It is formed in the decom- position of chloroform by alkali (p. 60), by the action of water upon hydrogen cyanide '(p. 154), and its alkaline salts are obtained by the reaction of carbon monoxide with alkalies : —
CO + KOH = HCOOK.
It is manufactured by heating glycerol with oxalic acid. It has been shown by Chattaway that in this reaction glyceryl acid oxalate is formed ; on raising the temperature carbon dioxide is evolved and glyceryl monoformin is produced On hydrolysing this ester with a further quantity of oxalic acid, formic acid is produced and the acid .oxalate again formed. There is thus a continuous reaction : —
CH2OH CH2O— OC . COOH
HOOC |
CH.H + I = CHOH + HoO
HOOC I CH2OH CH2OH.
CH2O— OC . COOH CH2O— OC . H
CHOH = CO2 + CHOH
CH2OH CH./)H.
CH,O— OC . H
COOH CHOH + | = HCOOH + CHOH
COOH CH2OH CH2OH.
Properties.
Formic acid is a colourless volatile liquid with pungent odour. It has a sp. gr. of 1-221 at 20°, freezes at 8-3°, and boils at 100°. It is a very strong acid, about 12 times as strong as acetic acid, and produces blisters on the skin and intense irritation.
It dissolves in water, alcohol and ether, and in general properties resembles acetic acid.
The formates crystallise well and are prepared in the same way as acetates (p. 97). The lead and magnesium salts are insoluble in alcohol ; the corresponding acetates are soluble. The acids may therefore be separated by treating a concentrated solution of these salts with alcohol; the formate is then precipitated. Potassium formate is almost insoluble in alcohol and may thus also be separated from the acetate, .which is soluble.
THE FATTY ACIDS 95
Reactions and Detection.
A solution of formic acid must be exactly neutralised with soda or ammonia before the tests can be carried out. Solid formates are obtained by evaporating their solutions to dryness.
(1) On boiling a solution of a formate with dilute sulphuric acid, formic acid is evolved. Its pungent odour is only perceptible with strong solutions.
(2) On heating a solid formate with concentrated sulphuric acid, carbon monoxide is evolved and it may be ignited at the mouth of the test tube.
(3) Ethyl formate is formed when solid formates are heated with alcohol and concentrated sulphuric acid.
(4) A red solution containing ferric formate is obtained when ferric chloride or ferric nitrate is added to a solution of a formate. On heating a reddish-brown precipitate of basic ferric formate is produced.
Formic acid differs from acetic acid in its reducing properties which are due to the presence of the aldehyde group CHO in its molecule.
(5) In concentrated solution it forms with silver nitrate a white crystalline precipitate of silver formate. This precipitate darkens on standing owing to reduction to metallic silver. A precipitate is not formed in dilute solution, but the solution is reduced on heating with separation of metallic silver. The reduction is retarded in the pre- sence of ammonia.
(6) On adding mercuric chloride solution and heating a pre- cipitate of mercurous chloride is produced, which, on further heating, may be reduced to metallic mercury.
96 PRACTICAL ORGANIC AND BIO-CHEMISTRY
ACETIC ACID. CH3.COOH.
Preparation.
Acetic acid is one of the few products made commercially by biological methods, i.e. by the oxidation of dilute alcohol by means of the micro-organism Mycoderma aceti, or " mother of vinegar". Me- chanical contrivances are used in order to expose a large surface of the alcoholic liquid to the air so that the acetification is as rapid as possible.
Wine, red and white, cider, beer and malt, and sugar prepared from starch are the materials from which the vinegar is made. Be- sides acetic acid vinegar contains other organic acids, sugar, dextrin and colouring matters which were present in the original material. The amount of acetic acid in the solution varies from about 3-12 per cent. , the average quantity being about 5 per cent.
A large quantity of acetic acid is produced by the dry