_A_ × 56100 100_A_
_F_ = ------------ = ------- (3).
561 × _K_ _K_
It is evident that it is not necessary to calculate the acid value
(_A_) of the sample and the saponification value (_K_) of the free fat
acids, the ratio _A_/_K_ alone being required. It will be sufficient
therefore to substitute for _A_ and _K_ the number of cubic centimeters
of alkali solutions required for one gram of the fat and one gram of
the fat acids, respectively. If _a_ and _b_ represent these numbers the
formula may be written
100_a_
_F_ = ------- (4);
_b_
100_a_
and _N_ = 100 - _F_ = 100 - ------ (5).
_b_
To simplify the determinations, it may be assumed that the free fat
acids have the same molecular weight as those still in combination with
the glycerol in any given sample. On this assumption, the process may
be carried on by determining the acid value _A_ and the saponification
value _K_ for the total fat acids. The mean molecular weight _M_, the
percentage of free fat acids _F_, and the proportion of neutral fat
_N_, may then be calculated from the formulas (2), (3), (4), and (5).
Further, let _G_ = the quantity of glycerol and _L_ that of fat acids
obtainable from one gram of neutral fat, that is, ¹/₁₀₀ of _H_ the
percentage of total fat acids.
The molecular weight of the neutral fat in each case is 3_M_ + 38.
Therefore, 3_M_ + 38 parts of neutral fat yield 3_M_ parts of fat acids
and ninety-two parts of glycerol (C₃H₈O₃ = 92).
_H_ 3_M_
Then _L_ = ----- = ---------- (6);
100 3_M_ + 38
92
and _G_ = --------- (7).
3_M_ + 38
_N_ per cent of neutral fat yields, therefore, on saponification, the
following theoretical quantities of fat acids _F_, and glycerol _G_
expressed as parts per hundred.
3_M_
_F_ = _N_ × -------- (8);
3_M_ + 38
92
and _G_ = _N_ × --------- (9).
3_M_ + 38
Formula (9) expresses also the total yield of glycerol from any given
sample. For a further discussion of this part of the subject a work of
a more technical character may be consulted.[323]
=353. Determination of a Free Fat Acid in a Fat.=—The principle of
the method rests upon the comparative accuracy with which a free fat
acid can be titrated with a set alkali solution when phenolphthalien
is used as an indicator. Among the many methods of manipulation which
the analyst has at his command there is probably none more simple and
accurate than that depending on the solution of the sample in alcohol,
ether, chloroform, or carbon tetrachlorid. Any acidity of the solvent
is determined by separate titration and the proper correction made.
Either an aqueous or alcoholic solution of the alkali may be used,
preferably the latter. The alkaline solution may be approximately or
exactly decinormal, but it is easier to make it approximately so and
to determine its real value before each operation by titration against
a standard decinormal solution of acid. About ten grams of the sample
and fifty cubic centimeters of the solvent will be found convenient
quantities.
_Example._—Ten grams of rancid olive oil dissolved in alcohol ether
require three and eight-tenths cubic centimeters of a solution of
alcoholic potash to saturate the free acid present. When titrated with
decinormal acid the potash solution is found to contain 25.7 milligrams
of potassium hydroxid in each cubic centimeter. The specific gravity
of the oil is 0.917 and the weight used therefore 9.17 grams. Then the
total quantity of potassium hydroxid required for the neutralization of
the acid is 25.7 × 3.8 = 97.7 milligrams.
The acid value _A_ is therefore:
3.8 × 25.7
_A_ = ----------- = 10.6
9.17
It is customary to regard free acid as oleic, molecular weight 282. On
this assumption the percentage of free acids in the above case is found
by the formula
3.8 × 25.7 × 282
_A_ (per cent) = ----------------- = 5.35
561 × 9.17
=354. Identification of Oils and Fats.=—Properly, the methods of
identifying and isolating the different oils and fats should be
looked for in works on food adulteration. There are, however, many
characteristics of these glycerids which can be advantageously
discussed in a work of this kind. Many cases arise in which the analyst
is called upon to determine the nature of a fat and discover whether
it be admixed with other glycerids. It is important often to know in
a given case whether an oil be of animal or vegetable origin. Many of
the methods of analysis already described are found useful in such
discriminations. For instance, a large amount of soluble or volatile
acids in the sample under examination, would indicate the presence
of a fat derived from milk while the form of the crystals in a solid
fat would give a clue to whether it were the product of the ox or the
swine. In the succeeding paragraphs will be briefly outlined some of
the more important additional methods of determining the nature and
origin of fats and oils of which the history is unknown.
The data obtained by means of the methods which have been described,
both physical and chemical, are all useful in judging the character and
nature of a glycerid of unknown origin. The colorations produced by
oxidizing agents, in the manner already set forth will be found useful,
especially when joined to those obtained with cottonseed and sesame
oils yet to be described. For instance, the red coloration produced by
nitric acid of 1.37 specific gravity is regarded by some authorities
as characteristic of cottonseed oil as well as the reduction by it of
silver nitrate. The coloration tests with silver nitrate (paragraph
=320=) and with phosphomolydic acid (paragraph =318=) are also helpful
in classifying oils in respect of their animal or vegetable origin.
The careful consideration of these tests, together with a study of the
numbers obtained by treating the samples with iodin, and the heat of
bromination and sulfuric saponification, is commended to all who are
interested in classifying oils. In addition to these reactions a few
specific tests are added for more detailed work.
=355. Consistence.=—It has already been said that oils are mostly of
vegetable origin and the solid fats of animal derivation. In the animal
economy it would be a source of disturbance to have in the tissues a
large body of fat which would remain in a liquid state at the normal
temperature of the body. Nearly all the animal fats are found to have
a higher melting point than the body containing them. An exception is
found in the case of butter fat, but it should be remembered that this
fat is an excretion and not intended for tissue building until it has
undergone subsequent digestion. Fish oils are another notable exception
to the rule, but in this case these oils can hardly be regarded as true
glycerids in the ordinary sense of that term.
In general, it may be said that a sample of a glycerid, which in its
natural state remains liquid at usual room temperatures, is probably
an oil of vegetable origin. Fish oils have also an odor and taste
which prevent them from being confounded with vegetable oils. In oils
which are manufactured from animal glycerids such as lard oil, the
discrimination is more difficult but peculiarities of taste and color
are generally perceptible.
=356. Nature of the Fat Acid.=—When it is not possible to discriminate
between samples by the sensible physical properties just described,
much light can be thrown on their origin by the determination of their
other physical properties, such as specific gravity, refractive index,
melting point, etc., in the manner already fully described. Further
light may be furnished by saponification and separation of the fat
acids. The relative quantities of oleic, stearic, palmitic, and other
acids will help to a correct judgment in respect of the nature of the
sample. The vegetable oils and lard oils, for instance, consist chiefly
of olein; lard and tallow contain a large proportion of stearin; palm
oil and butter fat contain considerable portions of palmitin, and
the latter is distinguished moreover by the presence of soluble and
volatile acids combined as butyrin and its associated glycerids.
Oleic acid can be rather readily separated from stearic and palmitic
by reason of the solubility of its lead salts in ether. One method of
accomplishing this separation has already been described (paragraph
=339=).
=357. Separation with Lime.=—A quicker, though perhaps not as accurate
a separation of the oleic from the palmitic and stearic acids, is
accomplished by means of lime according to the method developed by
Bondzyuski and Rufi.[324] This process is used chiefly, however, to
separate the free fat acids (palmitic, stearic) from the neutral fat
and the free oleic acid. It probably has no point of superiority over
the lead process.
=358. Separation of the Glycerids.=—The fact that olein is liquid at
temperatures allowing palmitin and stearin to remain solid, permits of
a rough separation of these two classes of bodies by mechanical means.
The mixed fats are first melted and allowed to cool very slowly. In
these conditions the stearin and palmitin separate from the olein in
a crystalline form and the olein is removed by pressure through bags.
In this way lard is separated into lard oil, consisting chiefly of
olein, and lard stearin, consisting largely of stearin. Beef (caul) fat
is in a similar manner separated into a liquid (oleo-oil) and a solid
(oleo-stearin) portion. It is evident that these separations are only
approximate, but by repeated fractionations a moderately pure olein or
stearin may be obtained.
=359. Separation as Lead Salts.=—Muter’s process, with a special
piece of apparatus, has already been described (=339=). For general
analytical work the special tube may be omitted. In a mixture of
insoluble free fat acids all are precipitated by lead acetate, and the
resulting soap may be extracted with ether, either with successive
shakings or in a continuous extraction apparatus. In this latter case
a little of the lead stearate or palmitate may pass into solution in
the hot ether and afterwards separate on cooling. When the operation
is conducted on from two to three grams of the dry mixed acids, the
percentage proportions of the soluble and insoluble acids (in ether)
can be determined. The lead salt which passes into solution can be
decomposed and the oleic acid removed, dried and weighed. Dilute
hydrochloric acid is a suitable reagent for decomposing the lead soap.
The difference between the weight of the oleic acid and that of the
mixed acids before conversion into lead soap furnishes the basis for
the calculation. For further details in respect of the fat acids the
reader may consult special analytical works.[325]
=360. Separation of Arachidic Acid.=—Peanut oil is easily distinguished
from other vegetable glycerids by the presence of arachidic acid.
The method used in this laboratory for separating arachidic acid is
a modification of the usual methods based on the process as carried
out by Milliau.[326] About twenty grams of the oil are saponified with
alcoholic soda, using twenty cubic centimeters of 36° baumé soda
solution diluted with 100 cubic centimeters of ninety per cent alcohol.
When the saponification is complete, the soda is converted into the
lead soap by treatment with a slight excess of a saturated alcoholic
solution of lead acetate. Good results are also obtained by using
dilute alcohol, _viz._, fifty per cent, instead of ninety per cent, in
preparing the lead acetate solution.
While still warm the supernatant liquid is decanted, the precipitate
washed by decantation with warm ninety per cent alcohol and triturated
with ether in a mortar four times, decanting the ethereal solution in
each instance. By this treatment all of the lead oleate and hypogaeate
are removed and are found in the ethereal solution, from which they can
be recovered and the acids set free by hydrochloric acid and determined
in the usual way.
The residue is transferred to a large dish containing two or three
liters of pure water and decomposed by the addition of about fifty
cubic centimeters of strong hydrochloric acid. The lead chlorid formed
is soluble in the large quantity of water present, which should be
warm enough to keep the free acids in a liquid state in which form
they float as a clear oily liquid on the surface. The free acids are
decanted and washed with warm water to remove the last traces of lead
chlorid and hydrochloric acid. The last traces of water are removed
by drying in a thin layer in vacuo. Practically all of the acids,
originally present in the sample except oleic and hypogaeic, are thus
obtained in a free state and their weight is determined.
The arachidic acid may be separated almost quantitively by dissolving
the mixed acids in forty cubic centimeters of ninety per cent alcohol,
adding a drop of hydrochloric acid, cooling to 16° and allowing to
stand until the arachidic acid has crystallized. The crystals are
purified by washing twice with twenty cubic centimeters of ninety
per cent and three times with the same quantity of seventy per cent
alcohol. The residual impure arachidic acid is dissolved in boiling
absolute alcohol, poured through a filter and washed with pure hot
alcohol. The filtrate is evaporated to dryness and heated to 100° until
a constant weight is obtained. From the above data, the percentages of
oleic, hypogaeic, arachidic and other acids in the sample examined are
calculated.
In the above process, owing to the pasty state of the lead soaps, the
trituration in a mortar with ether is found troublesome. The extraction
of the lead oleate and hypogaeate is facilitated by throwing the pasty
ethereal mass on a filter and washing it thoroughly with successive
portions of about fifty cubic centimeters of ether. By this variation,
it was found by Krug in this laboratory, that less ether was required
and a more complete removal of the lead oleate effected. The solution
of the lead oleate is completed by about half a dozen washings with
ether as above described. The extraction may also be secured by placing
the lead soaps in a large extracting apparatus and proceeding as
directed in paragraph =40=. The residue is washed from the filter paper
into a large porcelain dish and decomposed as already described with
hydrochloric acid. After the separation is complete, the mixture is
cooled until the acids are solid. The solid acids are then transferred
to a smaller dish, freed of water and dissolved in ether. The ethereal
solution is washed with water to remove any traces of lead salt or of
hydrochloric acid. After the removal of the ether, the arachidic acid
is separated as has already been described.
The melting point of pure arachidic acid varies from 73° to 75°.
=361. Detection of Arachis= (=Peanut=) =Oil.=—Kreis has modified
the usual process of Renard for the detection of arachis oil, by
precipitating the solution of the fat acid with an alcoholic instead
of an aqueous solution of lead acetate, in a manner quite similar to
that described above.[327] The fat acids are obtained in the usual
manner, washed with hot water and the acids from twenty grams of the
oil dissolved in 100 cubic centimeters of ninety per cent alcohol.
The solution is cooled in ice-water and the fat acids precipitated by
the addition of fifteen grams of lead acetate dissolved in 150 cubic
centimeters of ninety per cent alcohol. The precipitate, after standing
for two hours, is separated by filtration through cotton wool and is
extracted for six hours with ether. The residue is boiled with 250
cubic centimeters of five per cent hydrochloric acid until the fat
acids appear as a clear oily layer upon the surface. The acids thus
obtained are washed with hot water to remove lead chlorid, dried by
pressing between blotting paper, dissolved in 100 cubic centimeters of
ninety per cent alcohol, cooled to 15° and allowed to stand for several
hours, after which time any arachidic acid present is separated by
crystallization and identified in the usual manner.
When it is not important to obtain all of the acid present, the process
may be simplified in the following manner:
The fat acids obtained from twenty grams of oil are dissolved in 300
cubic centimeters of ether and treated at the temperature of ice-water
with a quantity of the alcoholic lead acetate solution mentioned above.
Lead oleate remains in solution and the precipitate which forms after
a few hours consists almost wholly of the lead salts of the solid fat
acids. The precipitate is collected, washed with ether and identified
in the usual manner.
=362. Cottonseed Oil, Bechi’s Test.=—Crude, fresh cottonseed oil, when
not too highly colored, and generally the refined article, may be
distinguished from other oils by the property of reducing silver salts
in certain conditions. The reaction was first noticed by Bechi and has
been the subject of extensive discussions.[328]
The process as proposed by Bechi has been modified in many ways but
apparently without improving it. It is conducted as follows: One gram
of silver nitrate is dissolved in 200 cubic centimeters of ninety-eight
per cent alcohol and forty cubic centimeters of ether and one drop of
nitric acid added to the mixture. Ten cubic centimeters of the oil
are shaken in a test tube with one cubic centimeter of this reagent,
and then with ten cubic centimeters of a mixture containing 100
cubic centimeters of amyl alcohol and ten of colza oil. The mixture
is divided into two portions, one of which is put aside for future
comparison and the other plunged into boiling water for fifteen
minutes. A deep brown or black color, due to the reduction of silver,
reveals the presence of cottonseed oil.
In this laboratory the heating is accomplished in a small porcelain
dish on which is often deposited a brilliant mirror of metallic silver.
The white color of the porcelain also serves as a background for the
observation of the coloration produced. In most instances a green color
has been noticed after the reduction of the silver is practically
complete. Unless cottonseed oil has been boiled or refined in some
unusual way, the test, as applied above, is rarely negative. The
reduction of the silver is doubtless due to some aldehydic principle,
present in extremely minute quantities, and which may be removed by
some methods of technical treatment. The silver nitrate test therefore
is reliable when the reduction takes place, but the absence of a
distinct reaction may not in all cases prove the absence of cottonseed
oil.
=363. Milliau’s Process.=—Milliau has proposed the application of
the silver salt directly to the free fat acids of the oil instead of
to the oil itself.[329] About fifteen cubic centimeters of the oil
are saponified with alcoholic potash in the usual manner, 150 cubic
centimeters of water added to the dish and the mixture boiled until
the alcohol is evaporated. The fat acids are freed by the addition of
decinormal sulfuric acid and as they rise to the surface in a pasty
condition are removed with a spoon. The free acids are washed with
distilled water. The water is drained off and the free acids dissolved
in fifteen cubic centimeters of ninety-two per cent alcohol and two
cubic centimeters of a three per cent solution of silver nitrate.
The test tube containing the mixture is well shaken and placed in a
water-bath, out of contact with light, and left until about one-third
of the alcohol is evaporated. Ten cubic centimeters of water are
added, the heating continued for a few minutes and the color of the
supernatant fat acids observed. The presence of cottonseed oil is
revealed by the production of a lustrous precipitate which colors the
fat acids black. In some cases the process of Milliau gives better
results than the original method of Bechi, but this is not always the
case. It does away with the use of amyl alcohol and colza oil, but
its manipulation is more difficult. In all doubtful cases the analyst
should apply both methods.
=364. Detection of Sesame Oil.=—Milliau has pointed out a
characteristic reaction of this oil which may be used with advantage
in cases of doubtful identity.[330] The identification is based on the
fact that the free acids of sesame oil, or some concomitant thereof,
give a rose-red color when brought in contact with a solution of sugar
in hydrochloric acid.
The analytical process is conducted as follows: About fifteen grams
of the oil are saponified with alcoholic soda and when the reaction
is complete treated with 200 cubic centimeters of hot water and
boiled until the alcohol is removed. The fat acids are set free with
decinormal sulfuric acid and removed with a spoon as they rise to the
surface in a pasty state, in which condition they are washed by shaking
with water in a large test tube. When washed, the acids are placed in
an oven at 105° until the greater part of the water is evaporated and
the acids begin to become fluid. At this point they are treated with
half their volume of hydrochloric acid saturated with finely ground
sugar. On shaking the mixture, a rose color is developed which is
characteristic of the sesame oil. Other oils give either no coloration
or at most a yellow tint.
=365. The Sulfur Chlorid Reaction.=—Some vegetable oils, when treated
with sulfur chlorid, give a hard product similar to elaidin, while lard
does not. This reaction is therefore helpful in discriminating between
some vegetable and animal glycerids. The process which is described by
Warren has been used with some satisfaction in this laboratory.[331]
Five grams of the oil or fat are placed in a tared porcelain dish and
treated with two cubic centimeters of carbon bisulfid and the same
quantity of sulfur chlorid. The dish is placed on a steam-bath and its
contents stirred until the reaction is well under way. The heating is
continued until all volatile products are evaporated, the hard mass
being well rubbed up to facilitate the escape of imprisoned vapors.
The powdered or pasty mass is transferred to a filter and washed with
carbon bisulfid to remove all unaltered oil. The washing with carbon
bisulfid is hastened by pressure and about 200 cubic centimeters of the
solvent should be used. After drying, the weight of insoluble matter is
obtained and deducted from the total weight of the sample used.
The color and tenacity of the hard, insoluble portion are
characteristic. The quantitive part of the operation appears to have
but little value, but applied qualitively in this laboratory it
produces hard, leathery masses with cotton, olive and peanut oils,
and but little change in lard and beef fats. Qualitively applied,
the process is conducted as described above but without making the
weighings. In this instance it is as easy of application as the process
of Bechi and is deserving of greater attention than has been given it
by analysts.
In the combination which takes place between the sulfur and the fat it
is probable that only addition products are formed, since the quantity
of alkali required for saponification is not diminished by previously
treating the fat with sulfur chlorid.[332] The reactions which take
place are probably well represented by the following equations, in
which oleic acid is treated with sulfur chlorid:
C₁₈H₃₄O₂ + S = C₁₈H₃₄S.O₂.
C₁₈H₃₄S.O₂ + NaOH = C₁₈H₃₄SO₂Na + H₂O.
=366. Detection of Cholesterin and Phytosterin in
Glycerids.=—Cholesterin is often found in animal glycerids and a
corresponding body, phytosterin, is sometimes found in oils of a
vegetable origin.[333] When one of these two bodies is present it may
be useful in distinguishing between animal and vegetable glycerids.
They are detected as follows: Fifty grams of the glycerids in each
case are saponified with alcoholic alkali, preferably potash, in order
to have a soft soap. After saponification is complete, the alcohol is
evaporated and the residual soap dissolved in two liters of water.
The mixture is shaken with ether and the ethereal solution evaporated
to a small bulk. The residue, which may contain a small quantity of
unsaponified fat, is again treated with alcoholic potash and subjected
a second time to the action of ether, as indicated above, with the
addition of a few drops of water and of alcohol if the emulsion
separate slowly. The ethereal extract finally secured is allowed to
evaporate slowly and the cholesterin (phytosterin) is obtained in a
crystalline form. The melting point of the cholesterin crystals is 146°
and that of the phytosterin 132°.
Cholesterin crystallizes in thin rhombic tables while phytosterin
separates in stellar aggregates or in bundles of long needles.
When dissolved in chloroform the two products show different color
reactions with sulfuric acid, cholesterin giving a cherry and
phytosterin a blue-red tint. In a mixture of animal and vegetable
glycerids the two products are obtained together and the melting point
of the mixture may afford some idea of the relative quantities of each
present. It is evident, however, that no reliable judgment can be
formed from these data of the relative proportions of the two kinds of
glycerids in the original sample.
=367. Cholesterin and Paraffin in Ether Extracts.=—In ethereal extracts
of some bodies, especially of flowers of the chrysanthemum, paraffin
is found combined with cholesterin. The two bodies may be separated as
follows:[334]
The ether extract is treated with aqueous then with alcoholic potash
several times; the residue soluble in ether is a solid body melting at
from 70° to 100°.
If the ethereal solution be cooled in a mixture of snow and salt, a
crystalline deposit is formed. This substance, purified by repeated
precipitations, is obtained colorless in fine crystalline scales
melting at 64°. It is very soluble in ether, benzene and chloroform,
almost insoluble in cold alcohol, and somewhat soluble in hot.
Its percentage composition is:
Per cent.
Carbon 85.00
Hydrogen 14.95
It is therefore a paraffin.
The ethereal solution, freed by the above process from paraffin, leaves
on evaporation a crystalline mass which is cholesterin, retaining
still a small quantity of fat matters. In treating the crystals with
alcoholic potash these fat bodies are saponified and the residue is
taken up with ether. The cholesterin is obtained in fine needles
melting at from 170° to 176°. It presents all the reactions of
cholesterin, especially the characteristic reaction with chloroform and
sulfuric acid.
=368. Absorption of Oxygen.=—Among oils a distinction is made between
those which oxidize readily and those which are of a more stable
composition. Linseed oil, for instance, in presence of certain metallic
oxids, absorbs oxygen readily and is a type of the drying oils, while
olive oil represents the opposite type.
The method of determining the quantity of oxygen absorbed is due to
Livache and is carried out as follows:[335]
Precipitated metallic lead (by zinc) is mixed in a flat dish, with
the oil to be tested, in the proportions of one gram of lead to
three-quarters of a gram of oil, and exposed to the air and light of
the workroom. The dish is weighed from time to time until there is no
longer any increase in weight.
Instead of lead, finely divided copper has been used by Krug in this
laboratory, but the percentage of absorption of oxygen is not so
high with copper as with lead. Krug found the quantities of oxygen
absorbed, after nine days, by the samples treated with copper and lead
respectively to be the following:
Copper, per cent Lead, per
oxygen absorbed. cent oxygen
absorbed.
Olive oil 1.69 2.03
Cottonseed oil 4.25 5.30
Peanut oil 2.74 3.87
Linseed oil 5.55 7.32
Livache found that linseed oil absorbed about twice as much oxygen as
indicated by the data just given.
=369. Elaidin Reactions.=—In discriminating between oils and fats
having a preponderance of olein and others with a smaller proportion of
that glycerid, the conversion of the olein into its isomer elaidin is
of diagnostic value. The following will be found a convenient method of
applying this test:[336]
About ten cubic centimeters of the oil are placed in a test tube
together with half that quantity of nitric acid and one gram of
mercury. The mixture is shaken until the mercury dissolves when the
mass is allowed to remain at rest for twenty minutes. At the end of
this time it is again shaken and placed aside. In from one to three
hours the reaction is complete. Olive, peanut and lard oils give
very hard elaidins. The depth to which a plunger of given weight and
dimensions sinks into an elaidin mixture at a given temperature, has
been used as a measure of the percentage of olein contained in the
sample of oil, but it is evident that such a determination is only
roughly approximate. Copper may be used instead of mercury for the
generation of the oxids of nitrogen, but it is not so effective. The
vapors of nitric oxids may also be conducted directly into the oil
from a convenient generator. The reaction may also be accomplished
by shaking the oil with nitric acid and adding, a drop at a time, a
solution of potassium nitrite.