=450. Form of Fat in Milk.=—The fat in milk occurs in the form of
globules suspended in the liquid, in other words in the form of an
emulsion. Many authorities have asserted that each globule of fat is
contained in a haptogenic membrane composed presumably of nitrogenous
matter, but there is no convincing evidence of the truth of this
opinion. The weight of experimental evidence is in the opposite
direction. The supposed action of the membrane and the phenomena
produced thereby are more easily explained by the surface tension
existing between the fat globules and the menstruum in which they are
suspended.
Babcock affirms that the spontaneous coagulation of the fibrin present
in milk tends to draw the fat globules into clusters, and this tendency
can be arrested by adding a little soda or potash lye to the milk as
soon as it is drawn.[432]
The diameter of the fat globules is extremely variable, extending in
some cases from two to twenty micromillimeters. In cow milk, the usual
diameters are from three to five micromillimeters.
=451. Number of Fat Globules in Milk.=—The number of fat globules in
milk depends on their size and the percentage of fat. It is evident
that no definite statement of the number can be made. There is a
tendency, on the part of the globules, to diminish in size and increase
in number as the period of lactation is prolonged. To avoid large
numbers, it is convenient to give the number of globules in 0.0001
cubic millimeter. This number may be found within wide limits depending
on the individual, race, food and other local conditions to which the
animal or herd is subjected. In general, in whole milk this number will
be found between 140 and 250.
=452. Method of Counting Globules.=—The number of globules in milk is
computed with the aid of the microscope. The most convenient method
is the one devised by Babcock.[433] In carrying out this computation,
capillary tubes, from two to three centimeters long and about one-tenth
millimeter in internal diameter, are provided. The exact diameter of
each tube, in at least three points, is determined by the micrometer
attachment of the microscope, and from these measurements the mean
diameter of the tube is calculated. This known, its cubic content
for any given length is easily computed. Ten cubic centimeters of
the milk are diluted with distilled water to half a liter and one
end of a capillary tube dipped therein. The tube is quickly filled
with diluted milk and each end is closed with a little wax to prevent
evaporation. Several of these tubes being thus prepared, they are
placed in a horizontal position on the stage of the microscope and
covered with glycerol and a cover glass. The tubes are left at rest
for some time until all the fat globules have attached themselves
to the upper surfaces, in which position they are easily counted.
The micrometer is so placed as to lie parallel with the tubes, and
the number of globules, corresponding to each division of its scale,
counted. The mean number of globules corresponding to each division of
the micrometer scale is thus determined.
To compare the data obtained with each tube they are reduced to a
common basis of the number of globules found in a length of fifty
divisions of the micrometer scale in a tube having a diameter of 100
divisions, using the formula
10000_n_
_N_ = ---------,
_d_²
in which _n_ = the number of globules found in the standard length of
tube measured and _d_ = the diameter of the tube. It is not difficult
to actually count all the globules in a length of fifty divisions of
the scale, but the computation may also be made from the mean numbers
found in a few divisions. The usual number of globules found in a
length of 0.1 millimeter in a tube 0.1 millimeter in diameter, varies
from fifty to one hundred.
_Example._—The length of one division of the micrometer scale is 0.002
millimeter, and the internal diameter of the tube 0.1 millimeter. The
content of a tube, of a length of 0.002 × 50 = 0.1 millimeter, is
therefore 0.0007854 cubic millimeter.
The cubic content of a tube 100 scale divisions in diameter and fifty
in length is 0.0031416 cubic millimeter. The number of globules found
in fifty divisions of the tubes used is 40. Then the number which would
be contained in a tube of a diameter of 100 divisions of the micrometer
scale and a length of fifty divisions thereof is
10000_n_ 400000
_N_ = -------- = ------ = 160.
_d_² 2500
Since the milk is diluted fifty times, the actual number of globules
corresponding to the volume given is 8000. It is convenient to reduce
the observations to some definite volume, _exempla gratia_, 0.0001
cubic centimeter. The equation for this in the above instance is
0.0031416: 0.0001 = 8000: _x_, whence _x_ = 223, = number of fat
globules in 0.0001 cubic millimeter.
In one cubic millimeter of milk there are therefore 2,230,000 fat
globules, and in one cubic centimeter 2,230,000,000 globules. In a
single drop of milk there are from one to two hundred million fat
globules.[434]
=453. Classification of Methods of Determining Fat in Milk.=—The fat,
being the most valuable of the constituents of milk, is the subject
of a number of analytical processes. An effort will be made here to
classify these various methods and to illustrate each class with one
or more typical processes. In general the methods may be divided into
analytical and commercial, those of the first class being used for
scientific and of the other for trade purposes. For normal milk, some
of the trade methods have proved to be quite as accurate as the more
chronokleptic analytical processes to which, in disputed cases, a final
appeal must be taken. When the analyst is called upon to determine the
fat in a large number of samples of milk some one of the trade methods
may often be adopted with great advantage.
=454. Dry Extraction Methods.=—Among the oldest and most reliable
methods of determining fat in milk, are included those processes based
on the principle of drying the milk and extracting the fat from the
residue by an appropriate solvent. The solvents generally employed are
ether and petroleum spirit of low boiling point. The methods of drying
are legion.
In extracting with ether, it must not be forgotten that other bodies
than fat may pass into solution on the one hand and on the other any
substituted glycerid, such as lecithin or nuclein, which may be present
may escape solution, at least in part. Perhaps petroleum spirit,
boiling at from 45° to 60°, is the best solvent for fat, but it is
almost the universal custom in this and other countries to use ether.
=455. The Official Methods.=—In the methods adopted by the Association
of Official Agricultural Chemists two processes are recommended.
(1) _The Asbestos Process_: In this process it is directed to extract
the residue from the determination of water by the asbestos method
(=447=) with anhydrous pure ether until the fat is removed, evaporate
the ether, dry the fat at 100° and weigh. The fat may also be
determined by difference, after drying the extracted cylinders at 100°.
(2) _Paper Coil Method_: This is essentially the method proposed by
Adams as modified by the author.[435] Coils made of thick filter paper
are cut into strips 6.25 by 62.5 centimeters, thoroughly extracted with
ether and alcohol, or the weight of the extract corrected by a constant
obtained for the paper. If this latter method be used, a small amount
of anhydrous sodium carbonate should be added. Paper free of matters
soluble in ether is also to be had for this purpose. From a weighing
bottle about five grams of milk are transferred to the coil by a
pipette, taking care to keep dry the lower end of the coil. The coil,
dry end down, is placed on a piece of glass, and dried at a temperature
of boiling water for one hour, or better, dried in hydrogen at a
temperature of boiling water, transferred to an extraction apparatus
and extracted with absolute ether or petroleum spirit boiling at about
45°. The extracted fat is dried in hydrogen and weighed. Experience
has shown that drying in hydrogen is not necessary. The fat may be
conveniently dried in partial vacuo.
=456. Variations of Extraction Method.=—The method of preparing the
milk for fat extraction is capable of many variations. Some of the most
important follow:
(1) _Evaporation on Sand_: The sand should be pure, dry and of uniform
size of grain. It may be held in a dish or tube. The dish may be made
of tin foil, so that it can be introduced with its contents into the
extraction apparatus after the desiccation is complete. For this
purpose, it is cut into fragments of convenient size after its contents
have been poured into the extractor. The scissors used are washed with
the solvent.
(2) _Evaporation on Kieselguhr_: Dry kieselguhr (infusorial earth,
tripoli) may take the place of the sand as above noted. The
manipulation is the same as with sand.
(3) _Evaporation on Plaster of Paris_,[436] (_Soxhlet Method_),[437] (4)
_On Pumice Stone_, (5) _On Powdered Glass_, (6) _On Chrysolite_:[438]
The manipulation in these cases is conducted as with sand and no
detailed description is required.
(7) _Evaporation on Organic Substances_: These variations would fall
under the general heading of drying on paper. The following materials
have been used; _viz._, sponge,[439] lint,[440] and wood pulp.[441] In
these variations the principal precautions to be observed are to secure
the organic material in a dry state and free of any matter soluble in
the solvent used.
(8) _Dehydration with Anhydrous Copper Sulfate_: In this process the
water of the sample is absorbed by powdered anhydrous copper sulfate,
the residual mass extracted and the butter fat obtained determined by
saponification and titration.[442] In the manipulation about twenty
grams of the anhydrous copper sulfate are placed in a mortar, a
depression made therein in such a manner that ten cubic centimeters of
milk can be poured into it without wetting it through to the mortar.
The water is soon absorbed when the mass is ground with a little dry
sand and transferred to the extractor.
Petroleum spirit of low boiling point is used as a solvent, successive
portions of about fifteen cubic centimeters each being forced through
the powdered mass under pressure. Two or three treatments with the
petroleum are required. The residual butter fat, after the evaporation
of the petroleum, is saponified with a measured portion, about
twenty-five cubic centimeters, of seminormal alcoholic potash lye. The
residual alkali is determined by titration with seminormal hydrochloric
acid in the usual manner. From the data obtained is calculated the
quantity of alkali employed in the saponification. The weight of butter
fat extracted is then calculated on the assumption that 230 milligrams
of potash are required to saponify one gram of the fat.
=457. Gypsum Method for Sour Milk.=—In sour milk, extraction of the
dry residue with ether is attended with danger of securing a part of
the free lactic acid in the extract. This may be avoided, at least
in part, by making the milk neutral or slightly alkaline before
desiccation. This method is illustrated by a variation of Soxhlet’s
method of drying on gypsum proposed by Kühn.[443] The curdled milk is
treated with potash lye of forty per cent strength until the reaction
is slightly alkaline. For absorbing the sample before drying, a mixture
is employed consisting of twenty-five grams of plaster of paris, four
of precipitated carbonate of lime and two of acid potassium sulfate. To
this mixture ten grams of the milk, rendered alkaline as above noted,
are added in a desiccating dish, the excess of moisture evaporated at
100°, the residual mass finely ground and extracted with ether for
four hours. A little gypsum may be found in the solution, but in such
small quantities as not to interfere seriously with the accuracy of the
results obtained.
=458. Estimation of Fat in Altered Milk.=—In altered milk the lactose
has usually undergone a fermentation affording considerable quantities
of lactic acid. If such milks be treated by the extraction method for
fat, the results will always be too high, because of the solubility of
lactic acid in ether.
Vizern[444] has proposed to avoid this error by first warming the soured
milk for a few minutes to 40°, at which temperature the clabber is
easily divided by vigorous shaking. Of the milk thus prepared, thirty
grams are diluted with two or three volumes of water and poured onto a
smooth and moistened filter. The vessel and filter are washed several
times until the filtrate presents no further acid reaction. The filter
and its contents are next placed in a vessel containing some fine
washed sand. A small quantity of water is added, sufficient to form a
paste. With a stirring rod, the filter is entirely broken up and the
whole mass thoroughly mixed. Dried on the water bath the material is
subjected to extraction in the ordinary way. Several analyses made
on fresh milk and on milk kept for several months show that almost
identical results are obtained.
In respect of this process there would be danger, on long standing,
of the formation of free acids from butter glycerids, and these acids
would be removed by the process of washing prescribed. In this case the
quantity of fat obtained would be less than in the original sample.
=459. Comparison of Methods.=—An immense amount of work has been done
by analysts in comparing the various types of extraction methods
outlined above.[445]
The consensus of opinion is that good results are obtained by all the
methods when properly conducted, and preference is given to the two
methods finally adopted by the Association of Official Chemists. As
solvents, pure ether and petroleum spirit of low boiling point are
preferred. The direct extraction gravimetric processes are important,
since it is to these that all the other quicker and easier methods must
appeal for the proof of their accuracy.
=460. Wet Extraction Methods.=—It has been found quite impracticable to
extract the fat from milk by shaking it directly with the solvent. An
emulsion is produced whereby the solvent itself becomes incorporated
with the other constituents of the milk, and from which it is not
separated easily even with the aid of whirling. The disturbing element
which prevents the separation of the solvent is doubtless the colloid
casein, since, when this is previously rendered soluble, the separation
of the solvent holding the fat is easily accomplished.
The principle on which the methods of wet extraction are based is a
simple one; _viz._, to secure a complete or partial solution of the
casein and subsequently to extract the fat with a solvent immiscible
with water. The methods may be divided into three great classes;
_viz._, (1) those in which the solvent is evaporated from the whole of
the extracted fat and the residual matters weighed; (2) processes in
which an aliquot part of the fat solution is employed and the total fat
calculated from the data secured; (3) the density of the fat solution
is determined at a definite temperature and the percentage of fat
corresponding thereto determined from tables or otherwise. Methods (1)
and (2) are practically identical in principle and one or the other may
be applied according to convenience or to local considerations. The
methods may be further subdivided in respect of the reagents used to
secure complete or partial solution of the casein, as, for instance,
alkali or acid.[446]
=461. Solution in an Acid.=—A good type of these processes is the
method of Schmid.[447] In this process ten cubic centimeters of milk
are placed in a test tube of about five times that content, graduated
to measure small volumes. An equal quantity of hydrochloric acid is
added, the mixture shaken, boiled until it turns dark brown, and cooled
quickly. The fat is extracted by shaking with thirty cubic centimeters
of ether. After standing some time the ethereal solution separates and
its volume is noted. An aliquot part of the solution is removed, the
solvent evaporated, and the weight of fat in the whole determined by
calculation.
The schmid process has been improved by Stokes,[448] Hill,[449] and
Richmond.[450] The most important of these variations consists in
weighing instead of measuring the milk employed, thus insuring greater
accuracy. Dyer and Roberts affirm that the ether dissolves some of the
caramel products formed on boiling condensed milk with hydrochloric
acid, and that the data obtained in such cases by the process of Schmid
are too high.[451]
Since lactic acid is also slightly soluble in ether, sour milk should
not be extracted with that solvent. In these cases petroleum spirit,
or a mixture of petroleum and ether, as suggested by Pinette, may be
used.[452] Another variation consists in extracting the fat with several
portions of the solvent and evaporating all the extracts thus obtained
to get the total fat. This method is perhaps the best of those in which
the fat is extracted from the residual liquid after the decomposition
of the casein by an acid, and may be recommended as both reliable and
typical within the limitations mentioned above.
=462. Solution in an Alkali.=—The casein of milk is not so readily
dissolved in an alkali as in an acid, but the solution is sufficient
to permit the extraction of the fat. Soda and potash lyes and ammonia
are the alkaline bodies usually employed. To promote the separation
of the emulsions, alcohol is added with advantage. The principle of
the process rests on the observed power of an alkali to free the fat
globules sufficiently to allow them to dissolve in ether or some other
solvent. When the solvent has separated from the emulsion at first
formed, the whole or a part of it is used for the determination of fat
in a manner entirely analogous to that employed in the process with the
acid solutions described above. There are many methods based on this
principle, and some of the typical ones will be given below. Experience
has shown that extraction from an alkaline solution is more troublesome
and less perfect than from an acid and these alkaline methods are,
therefore, not so much practiced now as they were formerly.
=463. Method of Short.=—Instead of measuring the volume of the
separated fat, Short has proposed a method in which the casein is
dissolved in an alkali and the fat at the same time saponified. The
soap thus produced is decomposed by sulfuric acid and the volume of the
separated fat acids noted. This volume represents eighty-seven per cent
of the corresponding volume of fat.[453]
The solvent employed is a mixture of sodium and potassium hydroxids,
containing in one liter 125 and 150 grams, respectively, of these
alkalies. The sample of milk is mixed with half its volume of the
reagent and placed in boiling water for two hours. By this treatment
the casein is dissolved and the fat saponified. After cooling to about
60°, the soap is decomposed by the addition of equal parts of sulfuric
and acetic acids. The tubes containing the mixture are again placed in
boiling water for an hour and they are then filled with boiling water
to within one inch of the top. The tubes may either be furnished with a
graduation or the column of fat be measured by a scale.
=464. Method of Thörner.=—The process of Short is conducted by Thörner
as follows:[454]
Ten cubic centimeters of milk measured at 15° are saponified, in
tubes fitting a centrifugal, by the addition of one and a half cubic
centimeters of an alcoholic potash lye, containing 160 grams of
potassium hydroxid per liter, or one cubic centimeter of an aqueous
fifty per cent soda lye. The saponification is hastened by setting the
tubes in boiling water, where they remain for two minutes. The soap
formed is decomposed with a strong acid, sulfuric preferred, the tubes
placed in the centrifugal and whirled for four minutes, when the fat
acids will be formed in the narrow graduated part of the tube and the
volume occupied thereby is noted after immersion in boiling water.
Thörner’s process is not followed in this country, but is used to a
considerable extent in Germany.[455]
=465. Liebermann’s Method.=—In this method, fifty cubic centimeters
of milk, at ordinary temperatures, are placed in a glass cylinder
twenty-five centimeters high and about four and a half internal
diameter; five cubic centimeters of potash lye of 1.27 specific gravity
are added, the cylinder closed with a well fitting cork stopper and
thoroughly shaken.[456] After shaking, fifty cubic centimeters of
petroleum spirit, boiling point about 60°, are added. The cylinder is
again stoppered and vigorously shaken until an emulsion is formed.
To this emulsion fifty cubic centimeters of alcohol of ninety-five
per cent strength are added, and the whole again thoroughly shaken.
After four or five minutes the petroleum spirit, containing the fat,
separates. In order to insure an absolute separation of the fat,
however, the shaking may be repeated three or four times for about
one-quarter minute, waiting each time between the shakings until the
spirit separates.
Of the separated petroleum spirit twenty cubic centimeters are placed
in a small weighed flask. The use of the flask is recommended on
account of the ease with which the petroleum spirit can be evaporated
without danger of loss of fat. Instead of the flask a weighed beaker or
other weighed dish may be employed.
The petroleum spirit is carefully evaporated on a water-bath and the
residue dried at 110° to 120° for one hour. The weight found multiplied
by five gives the content of fat in 100 cubic centimeters of the
milk. The percentage by weight can then be calculated by taking into
consideration the specific gravity of the milk employed.
The results obtained by this method agree well with those obtained by
the paper coil method, when petroleum spirit instead of sulfuric ether
is used as the solvent for the fat. Sulfuric ether, however, gives
an apparently higher content of fat because of the solution of other
bodies not fat present in the milk.
=466. Densimetric Methods.=—Instead of evaporating the separated fat
solution and weighing the residue, its density may be determined and
the percentage of fat dissolved therein obtained by calculation, or
more conveniently from tables. The typical method of this kind is due
to Soxhlet, and until the introduction of modern rapid volumetric
processes, it was used perhaps more extensively than any other
proceeding for the determination of fat in milk.[457] The reagents
employed in the process are ether saturated with water and a potash lye
containing 400 grams of potash in a liter. The principle of the process
is based on the assumption that a milk made alkaline with potash will
give up all its fat when shaken with ether and the quantity of fat in
solution can be determined by ascertaining the specific gravity of the
ethereal solution.
[Illustration: FIG. 108. AREOMETRIC FAT APPARATUS.]
The apparatus is arranged as shown in Fig. 108, whereby it is easy to
drive the ethereal fat solution into the measuring vessel by means of
the bellows shown. In the bottle, seen at the right of the engraving
are placed 200 cubic centimeters of milk, ten of the potash lye and
sixty of the aqueous ether. The milk and potash are first added and
well shaken, the ether then added, and the contents of the bottle are
shaken until a homogeneous emulsion is formed. The bottle is then set
aside for the separation of the ethereal solution, which is promoted
by gently jarring it from time to time. When the chief part of the
solution has separated, a sufficient quantity of it is driven over
into the measuring apparatus, by means of the air bulbs, to float the
hydrometer contained in the inner cylinder. After a few moments the
scale of the oleometer is read and the percentage of fat calculated
from the table. All the measurements are made at a temperature of
17°.5. The temperature is preserved constant by filling the outer
cylinder of the apparatus with water. If the room be warmer than 17°.5,
the water added should be at a temperature slightly below that and
_vice versa_. The oleometer carries a thermometer which indicates the
moment when the reading is to be made.
The scale of the oleometer is graduated arbitrarily from 43 to 66,
corresponding to the specific gravities 0.743 and 0.766, respectively,
or to corresponding fat contents of 2.07 and 5.12 per cent, in the
milk, a range which covers most normal milks.
In the use of the table the per cents corresponding to parts of an
oleometer division can be easily calculated.
TABLE FOR CALCULATING PER CENT OF FAT IN MILK BY AREOMETRIC METHOD
OF SOXHLET.
Reading of Per cent fat Reading of Per cent fat
oleometer. in milk. oleometer. in milk.
43 2.07 55 3.49
44 2.18 56 3.63
45 2.30 57 3.75
46 2.40 58 3.90
47 2.52 59 4.03
48 2.64 60 4.18
49 2.76 61 4.32
50 2.88 62 4.47
51 3.00 63 4.63
52 3.12 64 4.79
53 3.25 65 4.95
54 3.37 66 5.12
=467. Application of the Areometric Method.=—Soxhlet’s method, as
outlined above, with many modifications, has been extensively used
in Europe and to a limited degree in this country, and the results
obtained are in general satisfactory, when the sample is a mixed one
from a large number of cows and of average composition.
The author has shown that the process is not applicable to abnormal
milk and often not to milk derived from one animal alone.[458]
The chief difficulty is found in securing a separation of the emulsion.
This trouble can usually be readily overcome by whirling. Any
centrifugal machine, which can receive the bottle in which the emulsion
is made, may be employed for that purpose.
Since the introduction of more modern and convenient methods of fat
determination, the areometric method has fallen into disuse and perhaps
is no longer practiced in this country. It is valuable now chiefly from
the fact that many of the recorded analyses of milk fat were made by
it, and also for its typical character in representing all methods of
analysis of fat in milk based on the density of ethereal solutions.
=468. The Lactobutyrometer.=—A typical instrument for measuring the
volume of fat in a milk is known as Marchand’s lactobutyrometer. It
is based on the observation that ether will dissolve the fat from
milk when the casein is wholly or partly dissolved by an alkali, and
further, that the fat in an impure form can be separated from its
ethereal solution by the action of alcohol. Experience has shown
that all the fat is not separated from the ethereal solution by this
process, and also that the part separated is a saturated solution in
ether. The method cannot be rigorously placed in the two classes given
above, but being volumetric demands consideration here chiefly because
of its historical interest.[459]
The instrument employed by Marchand is a tube about thirty centimeters
long and twelve in diameter, closed at one end and marked in three
portions of ten cubic centimeters each. The upper part is divided in
tenths of a cubic centimeter. The superior divisions are subdivided so
that the readings can be made to hundredths of a cubic centimeter.
The tube is filled with milk to the first mark and two or three drops
of a twenty-five per cent solution of soda lye added thereto. Ether is
poured in to the second mark, the tube closed and vigorously shaken.
Alcohol of about ninety per cent strength is added to the upper mark,
the tube closed, shaken and allowed to stand in a vertical position,
with occasional jolting, until the separation of the liquids is
complete. In order to promote the separation the tube is placed in a
cylinder containing water at 40°.
When the separation is complete the milk serum is found at the bottom,
the mixture of alcohol and ether in the middle and the fat at the top.
The mixture of ether and alcohol contains 0.126 gram of fat, and each
cubic centimeter of the separated ether fat 0.233 gram of fat. The
total volume of the separated fat, multiplied by 0.233, and the product
increased by 0.126, will give the weight of fat in the ten cubic
centimeters of milk employed.
_Example._—Milk used, ten cubic centimeters of 1.032 specific gravity
= 10.32 grams. The observed volume of the saturated ether fat solution
is two cubic centimeters. Then the weight of fat is 2 × 0.233 + 0.126 =
0.592 gram. The percentage of fat in the sample is 0.592 × 100 ÷ 10.32
= 5.74.
In the apparatus used in this laboratory the upper division of the
graduation is marked 12.6, because this represents the quantity of fat
which remains in the ether-alcohol mixture for one liter of milk. From
this point the graduation is extended downward to ninety-five, which,
for ten cubic centimeters of milk, represents 0.95 gram. After the fat
has separated, enough ninety-five per cent alcohol is added to bring
the upper surface exactly to the graduation 12.6. The number of grams
per liter of milk is then read directly from the scale.
In respect of applicability, the observation made regarding Soxhlet’s
areometric method may be repeated.
In practical work in this country the lactobutyrometer is no longer
used, but many of the recorded determinations of fat in milk have been
made by this method.
=469. Volumetric Methods.=—For practical purposes, the volumetric
methods of estimating fat in milk have entirely superseded all the
other processes. It has been found that the fat readily separates in a
pure state from the other constituents of milk whenever the casein is
rendered completely soluble; whereas no process has yet been devised
whereby the fat can be easily separated in a pure state from milk
which has not been treated with some reagent capable of effecting a
solution of the casein. The volumetric methods may be divided into
two classes; _viz._, (1) Those in which the fat is separated by the
simple action of gravity, and (2) those in which the natural action
of gravity is supplanted by centrifugal motion. Each of these classes
embraces a large number of variations and some of the typical ones will
be described in the following paragraphs. As solvents for the casein a
large number of reagents has been used, including alkalies and single
and mixed acids. In practice, preference is given to the least complex
and most easily prepared solvents.
=470. Method Of Patrick.=—A typical illustration of the method of
collecting the fat after solution of the casein, without the aid of
whirling, is found in the process devised by Patrick.[460]
The solvent employed is a mixture of acetic, sulfuric and hydrochloric
acids, saturated with sodium sulfate, in the respective volumetric
proportions of nine, five and two. The separation is accomplished in a
large test tube drawn out near the top into a constricted neck which is
graduated to measure the volume of the separated fat or to give direct
percentage results.
The tube should have a content of about twenty-five cubic centimeters
below the upper mark on the neck. In use 10.4 cubic centimeters of
milk and a sufficient quantity of the mixed acids to fill it nearly
to the upper mark are placed in the tube, together with a piece of
pumice stone, and the mixture boiled. On cooling below 100°, the fat
will separate and the volume thereof may be measured in the constricted
portion of the tube. The volume of the fat may be converted into weight
on multiplying by 0.88 at 60°, or more conveniently the percentage
of fat be taken from a table. In practice, the tube is filled with
the milk and acid mixture nearly up to the neck, its contents well
mixed and additional acid mixture added until the liquid is raised
in the tube above the neck. After mixing a second time, the contents
are boiled for five minutes and the fat allowed to collect in the
expanded part of the tube above the neck. When the fat has collected,
the mixture is boiled gently a second time for a few minutes. By this
treatment the fat is mixed with the upper portions of the acid liquid
and clarified. The clearing of the fat may be hastened by sprinkling
over it a little effloresced sodium sulfate. The fat is brought into
the graduated neck by opening a small orifice in the belly of the tube,
which is closed by means of a rubber band. When the temperature has
reached 60°, the space occupied by the fat is noted and the numbers
obtained express the percentage of fat in the sample.
This process is illustrative of the principle of analysis, but is no
longer used in analytical determinations.
=471. The Lactocrite.=—One of the earliest methods for fat estimation
in milk, depending on the solution of the casein and the collection
of the fat by means of whirling, is based on the use of a centrifugal
machine known as the lactocrite. This apparatus is modeled very like
the machine usually employed for creamery work,[461] and at one time
was extensively used, but it has now given place to less troublesome
and expensive machines. The acid mixture for freeing the fat of casein
is composed of glacial acetic acid carrying five per cent of sulfuric.
The samples of milk are heated with the acid mixture in test tubes
provided with stoppers and short glass tubes to return the condensation
products. The hot mixture is poured into a small metallic cylindrical
cup holding about three cubic centimeters. This cup fits by means of
an accurately ground shoulder on a metal casing, carrying inside a
heavy glass graduated tube of small internal diameter. The excess of
the milk mixture escapes through a small aperture in the metallic screw
cap of the metal holder. The metal holder is cut away on both sides in
order to expose the graduations on the glass tube. The glass tube is
held water-tight by means of perforated elastic washers. Thus prepared
the tubes are inserted in the radial holes of a revolving steel disk
previously heated to a temperature of 60°. The whirling is accomplished
in a few minutes by imparting to the steel disk a speed of about 6,000
revolutions per minute. At the end of this operation the fat is found
in a clear column in the small glass tube and the number of the
divisions it occupies in this tube is noted. Each division of the scale
represents one-tenth per cent of fat.
This apparatus is capable of giving accurate results when all its parts
are in good working order. In this laboratory the chief difficulty
which its use has presented is in keeping the joints in the glass metal
tube tight.
This description of the apparatus is given to secure an illustration of
the principle involved, a principle which has been worked out in later
times into some of the most rapid and practical processes of estimating
fat in milk.
=472. Modification of Lindström.=—Many modifications have been proposed
for conducting the determination of fat by means of the lactocrite,
but they do not involve any new principle and are of doubtful merit.
In the modification suggested by Lindström, which has attained quite
an extended practical application, the solvent mixture is composed of
lactic and sulfuric acids and the butyrometer tubes are so changed
as to permit the collection of the fat in the graduated neck after
whirling, by means of adding water. The apparatus is also adjusted
to secure the congelation of the fat column before its volume is
noted.[462] The analyst can read the fat volume at his leisure when it
is in the solid state and is not confused by changes of volume during
the observation. The best acid mixture has been found to be composed of
100 volumes of lactic, an equal amount of acetic and fifteen volumes of
sulfuric acids.[463]
[Illustration: FIG. 109. BABCOCK’S BUTYROMETER AND ACID MEASURE.]
=473. The Babcock Method.=—Among the many quick volumetric methods
which have been proposed for the determination of fat in milk, none has
secured so wide an application as that suggested by Babcock.[464]
The chief point of advantage in the use of this method is found in
effecting the solution of the casein by means of sulfuric acid of about
1.83 specific gravity. By this reagent the casein is dissolved in a few
moments without the aid of any other heat than that generated by mixing
the milk with the reagent. The bottle in which the separation is made
is shown in Fig. 109. The graduations on the neck are based on the use
of eighteen grams of milk. To avoid the trouble of weighing, the milk
is measured from a pipette graduated to deliver eighteen grams of milk
of the usual specific gravity. While it is true that normal milk may
vary somewhat in its density, it has been found that a pipette marked
at 17.6 cubic centimeters delivers a weight which can be safely assumed
to vary but slightly from the one desired. The graduated bottle holds
easily thirty-five cubic centimeters of liquid in its expanded portion
and the volume of milk just noted is mixed with an equal volume of
sulfuric acid, conveniently measured from the lip cylinder shown in the
figure. The complete mixture of the milk and acid is effected by gently
rotating the bottle until its contents are homogeneous. The final color
of the mixture varies from dark brown to black.
While still hot, the bottles are placed in a centrifugal machine and
whirled for at least five minutes. The most convenient machine, where
it is available, is the one driven by a jet of steam. The revolutions
of the centrifugal should be at least 700 per minute for a twenty inch
and 1,200 for a twelve inch wheel. After five minutes the bottles are
removed and filled to the upper mark or nearly so with hot water,
replaced in the machine and whirled for at least one minute. The fat
will then be found in a clearly defined column in the graduated neck of
the bottle. In reading the scale, the extreme limits between the lowest
point marked by the lower meniscus and the highest point marked by the
edge of the upper meniscus are to be regarded as the termini of the fat
column.
In testing cream by the babcock process, it may either be diluted
until the column of fat secured is contained in the graduated part of
the neck or specially graduated bottles may be used.
_Condensed Milk_: In applying the babcock test to condensed milk, it is
necessary to weigh the sample and to use only about eight grams.[465]
This quantity is placed in the bottle and dissolved in ten cubic
centimeters of water and the analysis completed as above. The reading
noted is multiplied by eighteen and divided by the weight of the sample
taken.
_Skim Milk_: In determining the fat in skim milk and whey, it is
desirable to use a bottle of double the usual capacity, but with the
same graduation on the neck. The percentage of fat noted is divided by
two.
_Cheese_: Five grams are a convenient quantity of cheese to employ.
To this quantity in the bottle are added fifteen cubic centimeters
of hot water and the flask gently shaken and warmed until the cheese
is softened. The treatment with acid and whirling are the same as
described above. The noted reading is multiplied by eighteen and
divided by five.
=474. Solution in Amyl Alcohol and Hydrochloric and Sulfuric
Acids.=—Leffmann and Beam have proposed to aid the solution of the
casein in sulfuric acid by the previous addition to the milk of a
mixture of equal volumes of amyl alcohol and hydrochloric acid.[466]
In this process the same graduated flasks may be used as in the
babcock process, or a special flask may be employed. In this case the
graduation of the neck is for fifteen cubic centimeters of milk, and
each one and a half cubic centimeters is divided into eighty-six parts.
The quantity of milk noted is placed in the flask, together with three
cubic centimeters of the mixture of amyl alcohol and hydrochloric acid,
and well shaken. To the mixture, sulfuric acid of 1.83 specific gravity
is added until the belly of the flask is nearly full and the contents
well mixed by shaking. When the casein is dissolved, the addition of
the sulfuric acid is continued until the flask is filled to the upper
mark and again the contents mixed. It is well to close the mouth of
the flask with a stopper while shaking. The bottle is placed in a
centrifugal and whirled for a few moments, when the fat is collected in
the graduated neck and its volume noted.
The process is also known in this country as the beimling method.[467]
The fat separated in the above process is probably mixed with a little
fusel oil, and therefore it is advisable to use the specially graduated
bottle instead of one marked in absolute volumes.[468]
The method, when conducted according to the details found in the
papers cited, gives accurate results, but is somewhat more complicated
than the babcock process and is not now used to any great extent in
analytical work.
=475. Method of Gerber.=—The method proposed by Gerber for estimating
fat in milk is based on the processes of Babcock, Beimling and Beam
already described. The tubes in which the decomposition of the milk and
the measurement of the fat are accomplished are of two kinds, one open
at only one end for milk and the other open at both ends for cheese.
They are closed during the separation by rubber stoppers.[469]
[Illustration: FIG. 110. GERBER’S BUTYROMETERS.]
The apparatus have been greatly improved and simplified since the first
description of them was published and have come into extensive use in
Europe and to a limited extent in this country.[470]
The butyrometer tubes are made of various sizes and shapes, but the
most convenient are those noted above as shown in Fig. 110.
Before adding the strong sulfuric acid, one cubic centimeter of amyl
alcohol is mixed with the milk in the butyrometer. This admixture
serves to clarify the fat and render the reading more easy.
The centrifugal is run by hand, and the required speed of rotation is
given it by means of a cord wrapped spirally about its axis, as shown
in Fig. 111. The cord in the new machines is replaced by a leather
strap working on a ratchet.
[Illustration: FIG. 111. GERBER’S CENTRIFUGAL.]
The process is more speedy than that of Babcock, and the results have
been shown by a large experience to be reliable and accurate.
The sulfuric acid employed is of 1.825 to 1.830 specific gravity. There
is no danger of loss by the formation of volatile ethers where the
quantity of amyl alcohol used does not exceed one cubic centimeter.
In a comparison of the respective merits of the methods of Babcock,
Thörner and Gerber, made by Hausamann, the first place is awarded to
the Gerber process.[471] In the figure 110, the butyrometers marked 2,
5 and 8 are for milk, and those numbered 1, 3 and 7 are for cream
and cheese. In conducting the analysis, ten cubic centimeters of the
sulfuric acid are placed in the butyrometer with one cubic centimeter
of the amyl alcohol. When mixed, eleven cubic centimeters of the
milk are added and the contents of the tube well mixed, the tube
stoppered and placed in the centrifugal. The larger tubes, open at both
ends, require double the quantities of the reagents mentioned. The
measurements are made at about 15°.
Minute directions for conducting the analyses with milk, skim milk,
buttermilk, cream, condensed milk, cheese and butter accompany each
apparatus.