Chapter VIII: Analysis of Raw Materials and Finished Products

General Methods of Analysis of Fats and Oils—Special
Tests for Individual Oils—Analysis of Butter,
Margarine, Lard, Cheese, Chocolate.

The adulteration of fats and oils becomes year by year more scientific, the latest developments of chemical research being prostituted to the purpose of enabling sophisticated articles to elude the vigilance of the public analyst, whose duty it is to detect such adulteration. A striking instance of this is furnished by the methods of adulterating butter described in Chapter IV.

As was there mentioned, a method for the examination of pure butter, formulated by Reichert in 1879 and subsequently slightly modified by Meissl and Wollny, was based on the presence of volatile fatty acids, such as butyric acid, in butter. Butter substitutes, as usually made, do not contain these volatile fatty acids, at any rate in appreciable quantity, except when cocoanut oil has been added, and this, therefore, afforded a means of detecting the presence of artificial in genuine butter. It has been found, however, that some manufacturers of butter substitutes, especially on the Continent, have actually added these volatile fatty acids, ostensibly to improve their flavour, but there can be little doubt with the real object of enabling the artificial product to be sold as genuine butter.

It is unfortunately the case that the unscrupulous manufacturer is generally slightly in advance of the examining authority, and as fast as the latter detects one form of adulteration, and devises means for its ready determination, some new form of fraud is invented to take its place.

As a result of this competition between expert adulterator and public analyst, the methods for the examination of edible fats are constantly being improved and increased, and some extremely fantastic and far-fetched tests have been suggested. In the following pages, however, only those most usually adopted in a works’ or commercial laboratory are described.

GENERAL METHODS OF EXAMINATION OF FATS AND OILS

=Raw Materials.=—The appearance, colour, and odour of the sample should be observed, and any characteristic feature recorded. The taste is also frequently of value in judging the purity of a fat or oil, but deductions from this can only be made after considerable experience.

The following physical and chemical data may be determined:—

=Specific Gravity.=—In the case of oils liquid at ordinary temperatures, this is usually taken at 15° C., the weight of a given volume of the oil being compared with that of the same volume of water, at 15° C., the symbol used to denote this being

15°
“_d_ ———— ”.
15°

If a sufficient quantity of the oil is available a Westphal balance or a hydrometer may be employed for the purpose; if only a small quantity can be obtained, a specific gravity bottle or Sprengel or Nicol pycnometer should be used. This latter method is to be preferred where great accuracy is desirable. The specific gravity bottle or pycnometer must be first calibrated by filling it up to the mark with distilled water at 15° C., and weighing it.

The specific gravity of solid fats is taken at some higher temperature at which they are fluid, preferably at the temperature of a boiling water-bath, which will generally be found to be about 99° C. This is compared with the weight of a similar volume of water at 15° C., and is represented by

99°
“_d_ ———— ”.
15°

The specific gravity of oils and fats is liable to increase with age, and also varies with the method of treatment during refining.

The specific gravity of butter fat is best taken at a temperature of 35° C., for reasons which are dealt with under the heading of “Butter,” in Chapter IV.

=Free Fatty Acids.=—These are determined by warming on the water-bath for a few minutes a weighed quantity (2 to 10 grms. according to the degree of acidity of the fat) of the fat or oil, with 25 c.c. of purified methylated alcohol, which has been neutralised immediately prior to use, with N/10 potassium hydroxide solution, and after adding phenol-phthalein solution, slowly running in from a burette N/2 or N/10 alcoholic potassium hydroxide solution until a faint permanent pink colour is produced. Each c.c. of N/2 or N/10 alkali corresponds to 0·141 or 0·0282 grm. respectively of free fatty acids, expressed as oleic acid, and from this the percentage of acidity is calculated.

The free acidity is frequently expressed as the _acid value_, which represents the number of milligrams of potassium hydroxide, KOH, required to neutralise the acidity in 1 grm. of oil or fat.

_Example._—If 6·656 grms. fat required 1·5 c.c. N/10 alcoholic potassium hydroxide solution to neutralise it, then

1·5 × 0·0282 × 100
—————————————————— = 0·63 per cent.
6·656

free fatty acids, expressed as oleic acid.

The _acid value_ in this case would be

1·5 × 0·00561 x 1000
———————————————————— = 1·26.
6·656

The maximum permissible limit for free acidity depends on the nature of the fat or oil. For edible tallows it should not exceed 3 per cent., for lard 0·5 per cent., and for cocoanut oil 2 per cent.

=Saponification Value.=—It has been shown in Chapter I, how the various glycerides contained in fats and oils are split up or saponified by the action of caustic alkalies, one molecule of a triglyceride such as stearin requiring three molecules of potassium hydroxide for its saponification (cf. p. 4).

The composition of the various fats and oils being fairly constant, the amount of alkali required for the saponification of any given weight is also nearly constant. Koettstorfer first utilised this fact in 1879 for the analysis of butter fat, and it has now become the basis of one of the most important factors in the analysis of nearly all fats and oils, the amount of any fat which is saponified by 1 grm.-molecule or 56·1 grms. of caustic potash being termed its _saponification equivalent_.

The more usual method of expressing the same thing is the number of milligrams of potassium hydroxide required to saponify 1 grm. of fat, which is called the _saponification value_. The difference between the saponification and acid values is spoken of as the _ester value_.

To convert saponification equivalent into saponification value, it is merely necessary to divide 56·1 by the saponification equivalent, and multiply the quotient by 100.

To determine the saponification value, about 2 grms. of the fat or oil are weighed out into a conical flask of about 200 c.c. capacity, 25 c.c. of _neutral_ methylated spirit added, and 25 c.c. of an approximately N/2 alcoholic solution of potassium hydroxide run in from a burette, similar quantities of methylated spirit and alcoholic potassium hydroxide solution being also placed in another flask to serve as a blank test. The two flasks are now fitted with reflux condensers (which may be simply glass tubes, about four feet long and half an inch in diameter, inserted through a cork), and are placed on a steam or water-bath. The contents are then boiled until saponification of the fat is complete, which may take from thirty to sixty minutes, and is known to be accomplished when all globules of oil disappear. A few drops of phenol-phthalein solution are now added to each flask, and N/2 hydrochloric or sulphuric acid carefully run in from a burette until the pink colour is discharged. The difference in the amount of acid required by the two flasks indicates the quantity of potassium hydroxide required to saponify the weight of fat or oil taken.

_Example._—2·1314 grms. fat required 10·7 c.c. N/2 hydrochloric acid to neutralise unabsorbed alkali.

In the blank test 25 c.c. of approximately N/2 alcoholic potassium hydroxide solution required 25·6 c.c. of N/2 hydrochloric acid to neutralise it.

N
25·6 - 10·7 = 14·9 c.c. ————
2

alcoholic potassium hydroxide solution required to saponify the fat, and

2·1314 × 1000 × 2
————————————————— = 286·1
14·9

saponification equivalent, and

56·1 × 100
———————————— = 196·1,
286·1

the saponification value.

If the quantity of fat or oil employed for the estimation of acidity is only about 2 grms., the saponification value may also be determined on the same quantity. After proceeding as described above for the acidity estimation, 25 c.c. of approximately N/2 alcoholic potassium hydroxide solution are added to this, and the process continued as described above.

=Iodine Absorption.=—This test, devised by Hübl in 1884, and subsequently modified by Wijs, by Hanus, and by Waller, is based upon the capacity of unsaturated fatty compounds to absorb iodine, with formation of addition compounds. The amount of iodine absorbed is therefore a measure of the unsaturated compounds present in a fat, and is very fairly constant for any given fat in the fresh condition. The action of Hübl’s solution is attributed by Ephraim to the presence of iodine monochloride, but by Wijs to that of hypoiodous acid. The percentage of iodine absorbed is usually recorded as the _iodine number_ or _iodine value_.

In Hübl’s method, two solutions are required—(1) containing 25 grms. iodine in 500 c.c. of absolute alcohol, and (2) containing 30 grms. mercuric chloride in 500 c.c. of absolute alcohol. These solutions should be kept separate, and only mixed about twelve to twenty-four hours before use.

The process is carried out as follows:—Into a tightly fitting stoppered bottle is introduced 0·2 to 0·6 grm. of the fat, 10 c.c. chloroform added, and 25 c.c. of the mixed Hübl solution run in from a burette. The bottle is then firmly stoppered, and allowed to stand in a dark place for four hours, a similar bottle containing the same quantities of chloroform and Hübl solution being placed by its side as a blank experiment.

At the end of four hours, 20 c.c. of a freshly prepared 10 per cent. potassium iodide solution and 150 c.c. of water are added to each bottle, and the excess of iodine titrated with recently standardised N/10 sodium thiosulphate solution, the bottles being vigorously shaken during the titration, and fresh starch solution used for determining the final point. The difference in the number of c.c. of N/10 sodium thiosulphate solution required by the contents of the two bottles is a measure of the iodine absorbed by the fat. This figure, multiplied by the iodine equivalent of the sodium thiosulphate solution (found by titrating it with a known weight of pure resublimed iodine), and by 100, and divided by the weight of fat taken, gives the iodine value of the fat.

_Example._—0·539 grm. oil taken. Blank bottle, with iodine solution and chloroform only, required 61·4 c.c. N/10 thiosulphate; bottle containing oil required 25 c.c. N/10 thiosulphate. The iodine equivalent of the thiosulphate solution was found to be 1 c.c. = 0·0126 grm. I.

61·4 - 25·0 × ·0126 × 100
Then ——————————————————————————— = 85.1 iodine value.
·539

_Wijs’ Method._—The Hübl method has now to a very large extent been displaced by the Wijs process, in which the iodine is absorbed by the fat much more quickly, only about thirty minutes’ contact being required. The Wijs iodine reagent consists of a solution of iodine monochloride in glacial acetic acid, and may be prepared by either weighing out 7·9 grms. of iodine trichloride (which must be done in a weighing bottle), and 8·7 grms. of iodine, dissolving these separately in glacial acetic acid, mixing and making up to a litre with glacial acetic acid, or by dissolving 13 grms. of iodine in a litre of glacial acetic acid, and passing chlorine into the solution until the iodine is all converted into the iodine monochloride—a point which may be determined by the gain in weight, or, with a little practice, by the change in colour of the solution. The details of the process are exactly similar to those in the Hübl method, except that it is preferable to dissolve the fat in carbon tetrachloride instead of in chloroform.

=Bromine Absorption.=—This is similar in principle to the iodine absorption, and though numerous processes, both gravimetric and volumetric, have been proposed for its determination, it has now been almost entirely superseded by determination of the iodine absorption. A process devised by McIlhiney (_J. Amer. Chem. Soc._, 1894, 295, and 1899, 1084), however, deserves attention, for it estimates both the added and the substituted bromine.

The weighed quantity, say 0.5 grms. of the oil or fat, is weighed out, and dissolved in 10 c.c. of chloroform, and 20 c.c. N/3 solution of bromine in chloroform added. After two or three minutes 20 to 30 c.c. of a 10 per cent. potassium iodide solution are added, and the liberated iodine titrated with standard N/10 thiosulphate, the result giving the bromine forming addition compounds. After this, 5 c.c. of neutral 2 per cent. potassium iodate solution are introduced, and the liberated iodine titrated, the result in this case corresponding to the hydrobromic acid formed by the bromine in producing substituted bodies, or, in other words, giving the _bromine substitution value_.

=Titre, or Solidifying Point of the Fatty Acids.=—Many methods for determining the melting and solidifying points of fatty matters have been proposed, but the _titre test_, due to Dalican, is that which is now most generally adopted. This consists in determining the solidifying point of the fatty acids separated from a fat or oil, a figure which is an important characteristic of most fats, and in the case of tallows is largely employed as the basis for their commercial valuation. It is, of course, essential that in the preparation of artificial butters from a standard formula the firmness of the fats used should be as nearly as possible constant, and this is best determined by the titre test.

The test is carried out by first saponifying the fat with alcoholic sodium hydroxide solution, decomposing the resulting soap with dilute sulphuric acid, and after washing and drying the liberated fatty acids, determining their solidifying point. One ounce of the sample is melted in a shallow porcelain basin on a water-bath, and 30 c.c. of a 25 per cent. solution of sodium hydroxide added, together with 50 c.c. of redistilled methylated spirit. The contents of the basin are now evaporated on the water-bath, with constant stirring, until a pasty mass of soap is formed, and this is redissolved in a further 50 c.c. of redistilled methylated spirit, and again evaporated to dryness on the water-bath. The solid soap thus obtained is dissolved in water, sufficient dilute sulphuric acid added to decompose it, and the whole warmed until the fatty acids melt to a clear oily liquid on the surface. The water underneath is now siphoned off, more distilled water added to wash out any remaining trace of mineral acid, and again siphoned off, this treatment being repeated until the washings are no longer acid to litmus paper. The melted fatty acids are next poured on to a dry filter paper, which is inserted in a funnel resting on a beaker, and the latter is placed either in the water-bath or in an air-oven at about 100° C. until the clear fatty acids have filtered through it.

From 10 to 15 grms. of these dry fatty acids are transferred to a wide test tube, about six inches long and one inch in diameter, which is inserted through a cork into a flask or wide-mouthed bottle, to protect the tube from draught. The tube is closed with a loosely fitting perforated cork, through which passes a short range thermometer (0° to 60°), accurately graduated in fifths of a degree centigrade, and having its bulb just immersed in the fatty acids as near the centre as possible.

The temperature is now raised to a few degrees above the melting-point of the fatty acids, and allowed to cool down without stirring. As soon as the fatty acids just begin slowly to solidify, they are stirred round gently with the thermometer, the temperature on which will gradually fall till a minimum point is reached. Stirring is now discontinued, and the rise in temperature, which is usually produced by the heat given out by the acids in crystallising, is observed. The maximum temperature attained by the fatty acids during this rise is the “titre” of the sample.

=Refractive Index.=—The determination of the refractive power of a fat or oil, or of its fatty acids, is frequently very useful in judging the purity of a sample, or in drawing conclusions as to the composition of a mixture of fats.

The refractive index itself may be either directly determined by means of an Abbé total reflection refractometer, or an Amagat and Jean oleo-refractometer (_Analyst_, 1890, 87), or, as is more usual in the case of butter, the refractive power may be read off on an arbitrary scale by means of a Zeiss butyro-refractometer.

One of the great advantages of this test is the ease and rapidity with which a number of samples may be examined, while a further advantage of the Abbé and Zeiss instruments is that only a very small quantity of the sample—5 or 6 drops—is necessary.

The different forms of apparatus are fully illustrated and described in the catalogues of most firms supplying chemical apparatus, and it is therefore unnecessary to give a description here.

Different observers employ various temperatures for determining refractive indices, but that most usual for oils and fats, with the exception of butter, is 60° C. The best temperature of observation for butter is 40° C.

=Unsaponifiable Matter.=—The unsaponifiable matter present in the ordinary animal and vegetable fats is very small in amount, and the addition of any paraffin or other hydrocarbon is therefore readily detected by estimating the unsaponifiable matter. The usual method is to saponify about 5 grms. of the fat, dissolve the soap in water, and extract the unsaponified fatty matter with ether. The saponification is effected by boiling the fat with 50 c.c. of approximately N/2 alcoholic potassium hydroxide solution under a reflux condenser, with frequent agitation, for about an hour. The soapy solution is then evaporated to dryness in a porcelain basin on a steam or water-bath, and the soap obtained is dissolved in about 200 c.c. of hot water and transferred, as soon as sufficiently cool, to a 10 oz. separating funnel. To this is now added 50 c.c. ether, and the whole well shaken, and allowed to separate. The aqueous soap solution at the bottom is now run into another similar separator, and the ethereal extract washed with water to remove any soap dissolved therein. The washings are added to the aqueous soap solution, which is again extracted with a second 50 c.c. of ether, separated, the ethereal extract washed with water, and the extraction repeated a third time. The three washed ethereal extracts are then transferred to a tared flask, the ether distilled off in a water-bath, and the residue dried in the oven at 100° C. till constant in weight. This residue is the unsaponifiable matter in the weight of fat taken, whence the percentage may be calculated.

Difficulty often occurs during this process through the formation of an emulsion between the ethereal and the aqueous solutions, which prevents a sharp separation of the two layers. To overcome this, various expedients are recommended, such as the addition of a few c.c. of alcohol or glycerin or of more ether or water, careful warming, or gentle rotation.

The unsaponifiable matter may consist of cholesterol, a constituent of many animal fats, of phytosterol, a substance similar to cholesterol found in vegetable fats, of solid alcohols, such as cetyl and ceryl alcohols, present in spermaceti and Chinese wax, or of hydrocarbons, which do not occur naturally in either animal or vegetable fats, but are occasionally added as adulterants.

To examine the unsaponifiable matter for cholesterol and phytosterol a small quantity is dissolved in acetic anhydride, and one drop of the solution added to one drop of 50 per cent. sulphuric acid on a white porcelain tile, when, if either is present, a blood red to violet coloration is produced. They may be distinguished from each other by their crystalline form, cholesterol crystallising in laminæ, phytosterol in needle-shaped tufts; or by the melting-points of their acetates, cholesteryl acetate melting at 114°·3-114°·8, and phytosteryl acetate at 125°·6-137° C.

The fact that the unsaponifiable matter of animal fats contains cholesterol, while that of vegetable oils and fats contains phytosterol, has been made the basis of a test for detecting the presence of vegetable oils and fats in butter or lard. This test, which was first proposed by Bömer (_Zeit. Untersuch. Nahr. Genussm._, 1898, 81), consists in saponifying the fat with alcoholic potash, and extracting the unsaponifiable matter with ether, which is then distilled off, and the residue recrystallised from alcohol. The process has been subsequently improved by converting the cholesterol or phytosterol into the acetic esters by heating it with acetic anhydride, and determining the melting-point of the resulting ester. As mentioned above, that of cholesterol melts at about 114° C., that of phytosterol at 125°·6-137° C., and, according to Bömer, an acetate melting at 117°-118° C. corresponds to an addition of 1 to 2 per cent. of vegetable oil; at 120°-121° C., to an addition of 2 to 3 per cent. of vegetable oil; and at 123°-125° C., to an addition of 3 to 4 per cent. of vegetable oil.

The following method for carrying out this test, which is known as the _phytosteryl acetate test_, is described by Revis and Bolton (Allen’s _Commercial Organic Analysis_, ii. p. 301).

Fifty grms. of the clear fat are boiled with 75 c.c. of 95 per cent, alcohol, cooled, and the alcohol poured off, a second extraction being made with a further 75 c.c. of alcohol. These combined extracts, which will contain the greater part of the cholesterol and phytosterol and some fat, are transferred to a porcelain basin, and an excess of solid sodium hydroxide added, the mixture being then evaporated, with gentle stirring. After most of the alcohol has evaporated, more than sufficient sodium bicarbonate is added to convert the excess of sodium hydroxide into sodium carbonate, then some sand, and the whole evaporated to dryness, ground up in the dish, and extracted with light petroleum spirit. The residue from the ether is heated with 5 c.c. of (approximately) N/2 alcoholic sodium hydroxide solution, and again evaporated to dryness, with sand. A fresh extraction with petroleum spirit is made, followed by evaporation, and the residue is taken up with the smallest possible quantity of absolute alcohol. If necessary, the solution is boiled with animal charcoal and some 95 per cent. alcohol, filtered and evaporated to dryness. The crystals obtained are examined microscopically, then converted into their acetate by boiling with acetic anhydride in a covered watch-glass, evaporating off the excess of acetic anhydride on the water-bath, and recrystallising them from absolute alcohol.

Further tests for cholesterol have also been published by Lifschutz (_Ber. Deut. Chem. Ges._, 1908, 252-5) and Golodetz (_Chem. Zeit._, 1908, 160).

The method of the former depends on the oxidation of cholesterol to oxycholesterol ester and oxycholesterol. A few mgrms. of the unsaponifiable matter are dissolved in 2 to 3 c.c. of glacial acetic acid, a little benzoyl peroxide added, and the solution boiled, after which four drops of strong sulphuric acid are added. If cholesterol is present, a violet-blue or green colour is produced, the violet colour being due to oxycholesterol ester, the green to oxycholesterol.

Golodetz has devised two tests:—(1) the addition to a small quantity of the unsaponifiable matter of one or two drops of a mixture of 5 parts of concentrated sulphuric acid with 3 parts of formaldehyde solution, this reagent turning cholesterol a blackish-brown colour; and (2) the addition of one drop of 30 per cent. formaldehyde solution to a solution of the unsaponifiable matter in trichloracetic acid, when, if cholesterol is present, an intense blue coloration is produced.

Of the less frequently used methods of examination the following may be mentioned:—

=Valenta’s Acetic Acid Test=, which depends on the solubility of most oils and fats in hot glacial acetic acid of 1·0562 sp. gr., the temperature at which a warmed mixture of 3 c.c. melted fat and 3 c.c. acetic acid becomes turbid on cooling being noted.

The test has been slightly modified by Pearmain and Moor, who use a short stoppered tube, into which is weighed 2·75 grms. of the fat or oil, followed by 3 c.c. of acetic acid. The tube is then stoppered and heated in a water-bath, the temperature being raised until the contents of the tube become clear on shaking, after which the source of heat is removed, and the tube allowed to cool down gradually in the centre of the water-bath until the contents again become slightly turbid. The temperature at which this takes place is recorded, and is a fairly definite figure for any given oil. This test is of some value in the examination of butter for margarine, as is pointed out in Chapter IV. p. 48; the temperature at which the solution of the latter in acetic acid becomes turbid being very much higher than that of the former.

The following are the figures obtained by Pearmain and Moor by the above method:—

° C.
Butter fat 23-38
Margarine 94-97
Lard 97-99
Tallow 96-99
Cotton-seed oil 71-89
Sesame oil 90-97
Olive oil 83-91

The drawback of this test is that slight variations in the strength of the acetic acid, such as inevitably result from opening the bottle, cause considerable variations in the temperatures of turbidity. To obviate this it is advisable to compare the results with those given by specimens of butter fat of known purity.

=Maumené’s Test=, first proposed in 1852, consists in observing the rise of temperature which takes place when the fat or oil is mixed with concentrated sulphuric acid. Various methods of applying the test have been proposed by different authorities, that suggested by Archbutt (Allen’s _Commercial Organic Analysis_) being as follows:—Fifty grms. of oil are weighed into a 200 c.c. beaker, and the latter immersed in a capacious vessel of water, together with the bottle of strong sulphuric acid, until they are both at the same temperature, which should not be far from 20° C. The beaker containing the oil is then wiped, and placed in a cotton-wool nest previously made for it in a cardboard drum, or a wider beaker. The immersed thermometer is then observed, and the temperature recorded. Ten c.c. of the concentrated sulphuric acid should then be withdrawn from the bottle with a pipette, and allowed to run into the oil. During the addition of the acid, which should occupy about one minute, the mixture must be constantly stirred with the thermometer, and the agitation continued till no further rise of temperature ensues. This point is readily observed, as the mercury remains constant for a minute or two, and then begins to fall. Very different results are obtained according to the strength of acid employed, the best strength being 97 per cent.

Thomson and Ballantyne (_Journ. Soc. Chem. Ind._, 1891, x. 233) proposed to determine the “_specific temperature reaction_” of the oil, this being obtained by noting (1) the rise of temperature produced when 50 grms. of water are mixed with 10 c.c. of strong sulphuric acid in the same vessel and under the same conditions as those to be used for mixing the acid and oil; (2) mixing the oil and acid as described above, and then multiplying the rise in temperature produced by the oil-acid mixture by 100, and dividing by the rise in temperature given by the water-acid mixture. The following figures are given by Thomson and Ballantyne:—

Temperature.
Oil. Water = 100° C.
Olive 89-95°
Arachis 105-137°
Cotton-seed 163-170°

It has been shown by one of the authors (M.) (_Analyst_, 1901, xxvi. 169), that if the oil be dissolved in an inert solvent, such as carbon tetrachloride, the rise in temperature on adding sulphuric acid is usually proportional to the iodine value, i.e. the degree of unsaturation of the oil.

=Bromine Thermal Value.=—The heat reaction with bromine has been recommended as a rapid means of ascertaining the degree of unsaturation of oils and fats by Hehner and Mitchell (_Analyst_, 1895, xx. 146), a weighed quantity of the sample being dissolved in chloroform or acetic acid, and the rise in temperature on addition of bromine noted. The oil, chloroform, and bromine having first been brought to the same temperature, 1 grm. of oil is dissolved in 10 c.c. chloroform in a Dewar’s vacuum-jacketed test tube, and 1 c.c. of bromine added from a special pipette. This consists of a 1 c.c. pipette with a narrow tube, bent twice at right angles, connected to its top, the horizontal portion of the tube containing caustic lime kept in position by asbestos plugs. The mixture is immediately stirred, and the rise in temperature measured with a thermometer graduated in fifths of a degree. In the case of most ordinary oils and fats a relationship is shown to exist between the rise in temperature and the iodine value, so that this ratio having once been ascertained for the apparatus employed, the iodine value may be readily calculated from the rise in temperature observed on adding the bromine.

The preceding methods of examination are more or less generally applicable to all edible fats and oils, but there are numerous special tests applicable only to individual oils, of which the following are the most important. The application of these is also discussed more generally in the sections dealing with the special oils.

SESAME OIL

_Baudouin’s Test._—This is carried out by dissolving 0·1 grm. of cane sugar in 10 c.c. hydrochloric acid of sp. gr. 1·2, and adding this to 20 c.c. of the oil under examination, shaking the mixture thoroughly and allowing it to stand. The presence of as little as 2 per cent. of sesame oil imparts a crimson red colour to the aqueous liquid. This reaction is given by even the most rancid oils, though the colour produced is less intense.

A modification of this, consisting in adding 0·1 c.c. of a 2 per cent. alcoholic solution of furfural to the hydrochloric acid instead of the 0·1 grm. sugar, was devised by Villavecchia and Fabris, who found the reaction to depend upon the formation of furfural; while Wauters suggested that instead of mixing the reagent with the oil, the latter should be poured upon the reagent, under which conditions less than 1 per cent. will impart a crimson colour to the surface of contact.

Sprinkmeyer and Wagner (_Zeit. Nahr. Genussm._ 1905, x. 347-353) have still further increased the delicacy of the test, so that as little as 0·1 per cent. of sesame oil may be detected. In their process about 100 grms. of the filtered fat are twice extracted with 20 to 30 c.c. of glacial acetic acid at 60° C. The acid extracts are separated and evaporated, and the residue tested as described above. In the case of butter containing colouring matter, which may interfere with the reaction, the latter may be removed by evaporating the acetic acid residue with 10 c.c. of alcohol and 5 c.c. of saturated barium hydroxide solution, and extracting the residue several times with light petroleum spirit, which is then evaporated, and the final residue treated with the furfural solution.

_Tocher’s Test._—A freshly made solution of 1 grm. of pyrogallol in 15 c.c. of concentrated hydrochloric acid is shaken up in a separating funnel with 15 c.c. of the oil, and allowed to separate. The aqueous liquid is then drawn off, filtered, and boiled for about five minutes, when, if sesame oil is present, it appears red by transmitted and blue by reflected light.

OLIVE OIL

An important test for the purity of this oil is based on the elaidin reaction (p. 11), the degree of hardness of the product obtained by treatment of the oil with nitrous acid and the time required for its solidification being observed.

The best method of applying the test is to make use of the action of nitric acid on mercury, a reagent being prepared by dissolving 1 c.c. of mercury in 12 c.c. of cold nitric acid of 142 sp. gr. When this is shaken with the oil in a wide-mouthed stoppered bottle in the proportion of 2 c.c. of reagent to 50 c.c. of oil, the shaking being repeated at intervals of ten minutes for two hours, and the temperature being kept constant at not less than 25° C., a bright lemon-yellow coloured solid mass is obtained with olive oil. The products of the reaction with almond, lard, sperm, and arachis oils are also solid, but those yielded by rape, sesame, cotton-seed, sunflower, cod-liver, and porpoise oils have a consistency resembling that of butter, while those from linseed and other drying oils are liquid.

COTTON-SEED OIL

There are two well-known tests for this oil, those of Bechi and Halphen, both of which have undergone various modifications.

Bechi’s, or the silver nitrate test, requires the preparation of two solutions: (1) containing 1 grm. of silver nitrate dissolved in 200 c.c. or alcohol (98 per cent. by volume), to which is added 40 c.c. of ether and 0.1 grm. of nitric acid; and (2) a mixture of 15 c.c. of rape oil with 100 c.c. of amyl alcohol. Ten c.c. of the oil to be tested are mixed in a test tube with 1 c.c. of solution (1), and then shaken with 10 c.c. of solution (2). The mixture is now divided into two equal parts, and one-half immersed in boiling water for fifteen minutes, after which it is withdrawn and compared with the unheated portion. In the presence of cotton-seed oil a reddish-brown coloration is developed.

_Halphen’s Test._—Equal parts of the oil or fat (or its fatty acids), amyl alcohol, and a 1 per cent. solution of sulphur in carbon bisulphide, are heated together in a test tube placed in a boiling water bath until effervescence ceases, and then transferred to a boiling brine-bath for about an hour, when, if cotton-seed oil is present, a pink coloration is produced. The reaction may be rendered much more rapid, according to Rupp (_Zeit. Untersuch. Nahr. Genussm._, 1907, xiii., 74), by heating the mixture in a stoppered flask.

The production of a coloration with Bechi’s or Halphen’s reagent does not invariably prove the presence of cotton-seed oil, as the pure fat of animals fed with cotton-seed cake, even some long time previously, has been found to give the reaction. On the other hand, failure to obtain a reaction does not prove the absence of cotton-seed oil, since heating the oil to 250° C. causes it to give negative results in these tests.

ARACHIS OIL

The great similarity in the properties of this oil and olive oil renders some means of distinguishing one from the other necessary, and a process for the purpose has been based on the different chemical composition of the fatty acids, the arachis oil containing a considerable proportion of arachidic and lignoceric acid.

This arachidic acid (the term also being used to include the lignoceric acid) may be determined by the following process due to Renard and modified by Lewkowitsch:—About 10 grms. of the oil are saponified with alkali, as described under the “titre test,” the soap dissolved in water, excess of alkali neutralised with acetic acid, and the lead salts of the fatty acids precipitated by addition of a solution of lead acetate, filtered off, and extracted with ether, all but the palmitate and arachidate being dissolved. These latter are decomposed with hydrochloric acid, the fatty acids separated from lead chloride, and dissolved in 50 c.c. of hot 90 per cent. alcohol.

On cooling this solution, arachidic acid will crystallise out if arachis oil is present, and the amount of arachidic acid may be estimated, if desired, by filtering it off, and washing it twice with 10 c.c. of 90 per cent. alcohol, and once with alcohol of 0.890 sp. gr. The residue on the filter is now extracted with boiling absolute alcohol in which arachidic acid is soluble, the solution evaporated to dryness, and the arachidic acid weighed.

This amount has to be corrected by the addition of 0·0025 grm. for each 10 c.c. of 90 per cent. alcohol used in the crystallisation and washing if the treatment has been carried out at 15° C., or 0·0045 for 10 c.c. if it was done at 20° C. Arachis oil contains about 5 per cent. of arachidic acid, so that twenty times the total amount of arachidic acid represents the quantity of arachis oil in the sample under examination. The melting-point of arachidic acid is 71°-72° C.

BUTTER

An analysis of butter for the purpose of the Food and Drugs Act should comprise determination of the water and examination of the fatty matter, together with tests for colouring matter and preservatives. In addition, estimations of the casein, ash, and salt are also sometimes made.

The effect of heating a little of the sample in a spoon is a useful rough indication of its purity. Pure butter produces considerable foam, and turns brown, but butter substitutes do not foam appreciably, and unless specially prepared to do so (see Chapter IV. pp. 45 and 71), do not become brown.

_Water._—The determination of water may be made by weighing out about 5 grms. of the butter in a platinum dish and placing it in a hot-air oven at a temperature of 100°-105° C., the drying being continued until the weight is constant.

_Examination of the Fat._—A quantity of the butter fat is prepared by heating about 50 grms. of the sample on the top of a water-bath until it has completely melted, and the water and casein have separated to the bottom, when the clear fat is decanted off and filtered through a dry, warm filter paper. The filtered fat should be clear and bright, and is then ready for examination.

Many of the general methods already described for the examination of oils and fats are applicable, and furnish useful information as to the purity of butter. Such, for example, are the specific gravity, the saponification value, the refractive power, and Valenta’s acetic acid test, the variations in which are discussed on p. 102.

_Refractive Power._—The instrument most usually employed is the Zeiss butyro-refractometer, which consists of an Abbé double prism, which opens for the reception of a few drops of the melted and, preferably, filtered fat. A telescope is attached for reading the refraction on an arbitrary scale graduated from 5°-105°. The prism should be maintained at 40° C. while the observation is being made. At this temperature the reading for pure butter is usually about 35°-38°. If the temperature is above 40° subtract 0·55° for each degree above, and if below 40° C. add 0·55° for each degree below.

_Valenta’s Acetic Acid Test._—Reference has already been made to the value of this in the examination of butter fat (p. 48). The mixture of 2·75 grms. butter fat and 3 c.c. glacial acetic acid is warmed to 40° C., when, if the butter fat is pure, the mixture should become clear. If this is the case, the liquid is now allowed to slowly cool down with constant stirring with a thermometer until it just becomes turbid, which should take place at about 30°-40° C.

With the exception of the determination of the refractive power these methods of examination have now very largely given place to processes based on the presence of volatile fatty acids in butter, and on their differentiation from the volatile fatty acids found in cocoanut oil (see pp. 50 _et seq._).

The modification of the Reichert process suggested by Wollny was, in its essential details, that adopted for the determination of butter in margarine by a Committee of the Society of Public Analysts appointed in 1900 to confer with the Principal of the Government Laboratory. The details of this process, which was formerly, until the large increase in adulteration of butter with cocoanut oil, or margarine containing cocoanut oil, the chief method for the examination of butter, are as follows (_Analyst_, 1900, 311):—

Five grms. of the melted and filtered fat are weighed into a 300 c.c. flask, of the form and size shown in the figure, and 2 c.c. of a solution prepared by dissolving 98 per cent. sodium hydroxide in an equal weight of water, taking care to prevent absorption of atmospheric carbon dioxide, and 10 c.c. alcohol (92 per cent.) are added. The mixture is heated under a reflux condenser connected with the flask by a T-piece for fifteen minutes in a bath containing boiling water. The alcohol is distilled off by heating the flask on the water-bath for about thirty minutes, or until the soap is dry. One hundred c.c. of hot water, which has been kept boiling for at least ten minutes, are added, and the flask heated until the soap is dissolved. Forty c.c. of N/1 sulphuric acid, and three or four fragments of pumice or broken pipe-stem, are added, and the flask is at once connected with a condenser by means of a glass tube 7 mm. wide and 15 cm. from the top of the cork to the bend. At a distance of 5 cm. above the cork is a bulb 5 cm. in diameter. The flask is supported on a circular piece of asbestos 12 cm. in diameter, having a hole in the centre 5 cm. in diameter, and is first heated by a very small flame to melt the insoluble fatty acids, though the heat must not be sufficient to cause the liquid to boil. The heat is increased, and, when fusion is complete, 110 c.c. of the liquid are distilled off into a graduated flask, the distillation lasting about thirty minutes (twenty-eight to thirty-two minutes). The distillate is shaken, 110 c.c. filtered, transferred to a beaker, 0·5 c.c. of an alcoholic phenol-phthalein solution (1 per cent.) added, and the filtrate titrated with N/10 sodium hydroxide or barium hydroxide until pink. A blank experiment is carried out in precisely the same way, using the same reagents, and omitting the fat, and the amount of N/10 alkali required to neutralise this distillate should not exceed 0·3 c.c. The difference between the volumes of alkali required with the butter fat and in the blank determination, multiplied by 1·1, gives the Reichert-Meissl-Wollny value.

The improvements in the methods of refining cocoanut oil during recent years have led to the addition of considerable quantities of cocoanut oil to edible fats, and thus has somewhat reduced the value of this process for distinguishing pure from adulterated butter, owing to the fact already mentioned, that cocoanut oil contains a distinct quantity of volatile fatty acids. A very large number of methods have been proposed for detecting cocoanut oil in butter, and such terms as “silver value,” “caprylic acid value,” “oxygen equivalent,” have appeared in the literature of the subject.

Probably the most valuable of all these tests is one devised by Polenské (_Zeit. Untersuch. Nahr. Genussm._, 1904, vii. 273-280), which is based upon the determination of the volatile insoluble fatty acids that distil over in the Reichert-Wollny process. This method, which is known as the Reichert-Wollny-Polenské process, is now very generally employed for the analysis of butter, but to obtain concordant results it must be carried out strictly under the specified conditions and in an apparatus of the form and dimensions shown in the figure (Fig. 2). The details of the process are as follows:—

Five grms. of the clear butter fat are weighed out into a 300 c.c. flask, and saponified as described on p. 111, with 20 grms. glycerin and 2 c.c. of a 50 per cent. aqueous solution of sodium hydroxide, the heating being effected with a Bunsen burner. When saponification is complete and the mixture perfectly clear, it is allowed to cool down below 100° C., and the soap dissolved in 90 c.c. of water. To this solution, which should be clear and almost colourless, are now added 50 c.c. of dilute sulphuric acid containing 2·5 per cent. sulphuric acid and some fragments of pumice, and the flask attached to the condenser. The distillation is now proceeded with as usual, the heat being so regulated that 100 c.c. of distillate passes over in nineteen to twenty minutes, and the temperature of the condensing water is between 18° and 20° C. As soon as 110 c.c. of distillate have been collected the receiver is changed, and a 25 c.c. cylinder put in its place. The receiver is now transferred (care being taken to mix its contents as little as possible) to a water-bath at a temperature of 10° C., where it is kept for ten minutes, the surface of the water being just above the 110 c.c. mark. The insoluble fatty acids rise into the neck of the flask, and, in the case of butter, are in the form of solid, opaque granules, and with pure cocoanut oil in the form of clear, oily drops. The latter are also obtained in the case of mixtures containing more than 10 per cent. of cocoanut oil. The liquid is mixed and filtered, the Reichert-Wollny value being determined on the filtrate by titration with N/10 alkali.

The condenser, cylinder, and receiver are now washed with 18 c.c. of water, the washings being passed through the filter paper, and the insoluble fatty acids remaining on the filter dissolved in alcohol. The solution obtained is titrated with N/10 barium hydroxide solution, using phenol-phthalein as indicator, the number of c.c. required being termed the “new butter value” of the fat.

This value was stated by Polenské to range, for pure butters having Reichert-Meissl values of 23·3-30·1, from 1·5-3·0, and for cocoanut oils having Reichert-Meissl values of 0·8-7·7, from 16·8-17·8. Mixtures with Reichert-Meissl values between 23 and 27 gave “new butter values” of from 1·6-1·9, a rise of 1·0 in the Reichert-Meissl figure corresponding with an increase of 0·1 in the “new butter value,” and each per cent. of cocoanut oil increasing the new butter value by 0·1 above that given by a genuine butter possessing the same Reichert-Meissl value. By this method it is possible to detect the presence in butter of 10 per cent. and upwards of cocoanut oil.

A process similar in principle, but differing in method has been proposed by Muentz and Coudon (_Ann. d. l’Inst. Agron._, 1904, iii.,