=259. The Copper Tartrate Method.=—The lactose in milk is readily
estimated by the gravimetric copper method described in paragraph
=143=. Before the application of the process the casein and fat of
the milk should be removed by an appropriate precipitant, and an
aliquot part of the filtrate, diluted to contain about one per cent
of milk sugar, used for the determination. The clarification is very
conveniently secured by copper sulfate or acetic acid, as described
in the next paragraph. A proper correction should be made for the
volume occupied by the precipitate and, for general purposes, with
whole milk of fair quality this volume may be assumed to be five per
cent. One hundred grams of milk will give a precipitate occupying
approximately five cubic centimeters. In the analytical process, to
twenty cubic centimeters of milk, diluted with water to eighty, is
added a ten per cent solution of acetic acid, until a clear whey is
shown after standing a few minutes, when the volume is completed to 100
cubic centimeters with water, and the whole, after thorough shaking,
thrown on a filter. An aliquot part of the filtrate is neutralized with
sodium carbonate and used for the lactose determination. This solution
contains approximately one per cent of lactose. In a convenient part
of it the lactose is determined and the quantity calculated for the
whole. This quantity represents the total lactose in the twenty
cubic centimeters of milk used. The weight of the milk is found by
multiplying twenty by its specific gravity. From this number the
percentage of lactose is easily found. In this process the milk is
clarified by the removal of its casein and fat. Other albuminoids
remain in solution and while these doubtless disturb the subsequent
determination of lactose, any attempt at their removal would be equally
as disadvantageous. The volume of the precipitate formed by good, whole
milk when the process is conducted as above described, is about one
cubic centimeter, for which a corresponding correction is readily made.
=260. The Official Method.=—The alkaline copper method of determining
lactose, adopted by the Association of Official Agricultural Chemists,
is essentially the procedure proposed by Soxhlet.[215]
Dilute twenty-five cubic centimeters of the milk, held in a half
liter flask, with 400 cubic centimeters of water and add ten cubic
centimeters of a solution of copper sulfate of the strength given for
Soxhlet’s modification of Fehling’s solution, page 129; add about seven
and a half cubic centimeters of a solution of potassium hydroxid of
such strength that one volume of it is just sufficient to completely
precipitate the copper as hydroxid from one volume of the solution of
copper sulfate. In place of a solution of potassium hydroxid of this
strength eight and a half cubic centimeters of a half normal solution
of sodium hydroxid may be used. After the addition of the alkali
solution the mixture must still have an acid reaction and contain
copper in solution. Fill the flask to the mark, shake and filter
through a dry filter.
Place fifty cubic centimeters of the mixed copper reagent in a beaker
and heat to the boiling point. While boiling briskly, add 100 cubic
centimeters of the lactose solution, prepared as directed above, and
boil for six minutes. Filter immediately and determine the amount of
copper reduced by one of the methods already given, pages 149-155.
Obtain the weight of lactose equivalent to the weight of copper found
from the table on page 163.
=261. The Copper Cyanid Process.=—It has been found by Blyth that the
copper cyanid process of Gerrard gives practically the same results
in the determination of sugar in milk as are obtained by optical
methods.[216] The milk for this purpose is clarified, by precipitating
the casein with acetic acid in the following manner:
Twenty-five cubic centimeters of milk are diluted with an equal
volume of distilled water, and strong acetic acid added until the
casein begins to separate. The liquid is heated to boiling and, while
hot, centrifugated in any convenient machine. The supernatant liquid
obtained is separated by filtration and the solid matter thrown upon
the filter and well washed with hot water. The filtrate and washings
are cooled and completed to a volume of 100 cubic centimeters. This
liquid is of about the proper dilution for use with the copper cyanid
reagent. The percentage of sugar determined by this reagent agrees well
with that obtained by the optical method, when no other sugars than
lactose are present. If there be a notable difference in the results
of the two methods other sugars must be looked for. The presence
of dextrin may be determined by testing a few drops of the clear
liquor with iodin, which, in the presence of dextrin, gives a reddish
color. Other sugars are determined by obtaining their osazones. For
this purpose the filtrate obtained as above should be concentrated
until the volume is about thirty cubic centimeters. Any solid matter
which separates during the evaporation is removed by filtration. The
osazones are precipitated in the manner described in paragraph =147=.
On cooling, the almost solid crystalline mass obtained is placed on
a filter, washed with a little cold water, the crystals then pressed
between blotting paper and dried at a temperature of 100°. The dry
osazones obtained are dissolved in boiling absolute alcohol, of which
just sufficient is used to obtain complete solution. The alcoholic
solution is set aside for twelve hours and the separation of a
crystalline product after that time shows that dextrose or invert sugar
is present. Milk sugar alone gives no precipitate but only a slight
amorphous deposit. The lactosazone is precipitated by adding a little
water to the hot alcoholic solution and the crystals thus obtained
should be dissolved in boiling absolute alcohol and reprecipitated by
the addition of water at least three times in order to secure them
pure. The osazones are identified by their melting points, paragraph
=172=. The first part of this method does not appear to have any
advantage over the optical process by double dilution (p. 278), and
requires more time.
=262. Sugars in Evaporated Milks.=—In addition to the lactose normally
present in evaporated milks the analyst will, in most cases, find large
quantities of sucrose. The latter sugar is added as a preservative and
condiment. By reason of the ease with which sucrose is hydrolyzed,
evaporated milk containing it may have also some invert sugar among
its contents. A method of examination is desirable, therefore, which
will secure the determination of lactose, sucrose and invert sugar
in mixtures. The probability of the development of galactose and
dextrose during the evaporation and conservation of the sample, is not
great. The best method of conducting this work is the one developed by
Bigelow and McElroy.[217] The principle on which the method is based
rests on the fact that in certain conditions, easily supplied, the
sucrose and invert sugar present in a sample may be entirely removed
by fermentation and the residual lactose secured in an unchanged
condition. The lactose is finally estimated by one of the methods
already described.
The details of the process follow:
On opening a package of evaporated milk, its entire contents are
transferred to a dish and well mixed. Several portions of about
twenty-five grams each are placed in flasks marked at 100 cubic
centimeters. To each of the flasks enough water is added to bring
all the sugars into solution and normal rotation is made certain by
boiling. After cooling, the contents of the flasks are clarified by
mercuric iodid in acetic acid solution. The clarifying reagent is
prepared by dissolving fifty-three grams of potassium iodid, twenty-two
grams of mercuric chlorid, and thirty-two cubic centimeters of
strongest acetic acid in water, mixing the solutions and completing the
volume to one liter. The clarification is aided by the use of alumina
cream (=84=). The flask is filled to the mark, and the contents well
shaken and poured on a filter. After rejecting the first portion of
the filtrate the residue is polarized in the usual manner. Two or more
separate portions of the sample are dissolved in water in flasks of the
size mentioned, heated to 55°, half a cake of compressed yeast added
to each and the temperature kept at 55° for five hours. The residue in
each flask is treated as above described, the mercuric solution being
added before cooling to prevent the fermentative action of the yeast,
and the polarization noted.
By this treatment the sucrose is completely inverted, while the
lactose is not affected. The percentage of sucrose is calculated by
the formulas given in paragraph =94=, using the factor 142.6. At the
temperature noted the yeast exercises no fermentative, but only a
diastatic action.
In each case the volume of the precipitated milk solids is determined
by the double dilution method, and the proper correction made (p. 278).
The lactose remaining is determined by chemical or optical methods,
but it is necessary, in all cases where invert sugar is supposed to be
present, to determine the total reducing sugars in the original sample
as lactose. If the quantity thus determined and the amount of sucrose
found as above are sufficient to produce the rotation observed in the
first polarization, it is evident that no invert sugar is present. When
the polarization observed is less than is equivalent to the quantity
of sugar found, invert sugar is present, which tends to diminish the
rotation produced by the other sugars. In this case it is necessary to
remove both the sucrose and invert sugar by a process of fermentation,
which will leave the lactose unchanged.
This is accomplished by conducting the fermentation in the presence
of potassium fluorid, which prevents the development of the lactic
ferments. For this purpose 350 grams of the evaporated milk are
dissolved in water and the solution boiled to secure the normal
rotation of the lactose. After cooling to 80°, the casein is thrown
down by adding a solution containing about four grams of glacial
phosphoric acid and keeping the temperature at 80° for about fifteen
minutes. After cooling to room temperature, the volume is completed
to one liter with water, well shaken and poured onto a filter. An
aliquot part of the filtrate is nearly neutralized with a noted volume
of potassium hydroxid. Enough water is added to make up, with the
volume of potassium hydroxid used, the total space occupied by the
precipitated solid, corresponding to that part of the filtrate, and if
necessary, refilter. The volume occupied by the precipitated solids is
easily determined by polarization and double dilution. The filtrate,
obtained from the process described above, is placed in portions of
100 cubic centimeters each in 200 cubic centimeter flasks with about
twenty milligrams of potassium fluorid in solution, and half a cake of
compressed yeast. The yeast is broken up and evenly distributed, and
the fermentation is allowed to proceed for ten days at a temperature
of from 25° to 30°. At the end of this time experience has shown that
all of the sucrose and invert sugar has disappeared, but the lactose
remains intact. The flasks are filled to the mark with water and the
lactose determined by chemical or optical methods. By comparing the
data obtained from the estimation of the total reducing sugars before
fermentation or inversion and the estimation of the lactose after
fermentation, the quantity of invert sugar is easily calculated. The
experience of this laboratory shows that invert sugar is rarely present
in evaporated milks, which is an indication that the sucrose added
thereto does not generally suffer hydrolysis. The mean percentage of
added sucrose found in evaporated milks is about forty.