=76. General Principles.=—Having described the instruments chiefly
employed in the optical examination of sugar solutions, the next step
is to apply them to the analytical work. A common set of directions for
use will be found applicable to all instruments with such modifications
only as are required by peculiarities of construction. With the
best made instruments it is always advisable to have some method of
controlling the accuracy of the observation. The simplest way of doing
this is to test the apparatus by standard quartz plates. These plates
are made from right-handed polarizing quartz crystal ground into plates
of definite thickness and accurately tested by standard instruments.
Theoretically such quartz plates deflect the plane of polarized light
in a degree proportionate to their thickness, but practically some
small deviations from the rule are found. With a source of light of the
same tint, and at a constant temperature, such plates become a safe
test for the accuracy of the graduation of polariscopes. They are more
convenient for use than pure sugar solutions of known strength which
are the final standards in all disputed cases. These quartz plates
are conveniently mounted in tubes of the same size as those holding
the sugar solution, and thus fit accurately into the trough of the
polariscope, the optical axis of which passes through their center.
The quartz plate when used for setting the scale of a polariscope
should be placed always in the same position. In some plates slight
differences of reading may be noticed on rotating the tubes holding
them. Theoretically, such differences should not exist, but in practice
they are sometimes found. The temperature of observation should also
be noted, and if not that at which the value of the plate was fixed a
proper correction should be made.
=77. Setting the Polariscope.=—While mention has been made of several
forms of apparatus in the preceding paragraphs, those in common use
are limited to a very small number. In this country quite a number of
color instruments may still be found, together with a few laurents,
and a constantly increasing number of shadow instruments for use with
lamp light. The following description of setting the polariscope is
especially adapted to the last named instrument, but the principles of
adjustment are equally applicable to all.
The scale of the instrument is first so adjusted by means of the
adjusting screws provided with each instrument, as to bring the zero
of the vernier and that of the scale exactly together. The telescope
or ocular is then adjusted until the sharp line separating the halves
of the field of vision is brought into focus. This being accomplished
an observation tube filled with pure water is placed in the apparatus
and the telescope again adjusted to bring the dividing line of the
field into focus. The beginner especially, should repeatedly study
this adjustment and be impressed with the fact that only in a sharply
defined field are practical observations of any worth. The importance
of having all the lenses perfect and all the cover glasses without a
flaw may be fully appreciated when it is remembered that the polarized
ray, already deprived of half its original luminous power, must pass
through several centimeters of crystallized calcium carbonate, and
half a dozen disks of glass and quartz, and as many lenses before
reaching the eye of the observer. Only with the greatest care and
neatness is it possible to secure the required degree of illumination.
The zero point having been well studied and accurately adjusted, the
scale of the instrument may be tried with a series of quartz plates of
known polarizing power at the temperature of the observation. In the
apparatus with double quartz wedge compensation, it will be noticed
that the marks on one scale are black and on the other red. The
black is the working and the red the control scale. To operate this
instrument, the red scale is placed exactly at the zero point. The
black scale is also placed at zero, and if the field of vision is not
neutral, it is made so by the micrometer screw with which the black
scale is provided. In a right-handed solution, the red scale is left
at zero and the black one moved to the right until neutrality in the
field of vision is reached and the reading is taken. The observation
tube containing the sugar solution is taken out and the red scale
moved until the field of vision is again neutral and the reading of
the red scale taken. The two readings should agree. Any failure in the
agreement shows some fault either in adjusting the apparatus or in its
construction, or some error in manipulation.
The double compensating shadow instruments are more readily tested
for accuracy in all parts of the scale than those of any other
construction. The two compensating wedges are cut with the greatest
care, one from a left-handed and the other from a right-handed
perfectly homogeneous quartz crystal. Since faults in these wedges are
due either to lack of parallelism of surface, or of perpendicularity
to the optical axis of the crystal, and since these faults of
crystallization or construction must be in a very limited degree common
they would not coincide once in many thousand times in the two wedges.
This is easily shown by the theory of probabilities. If, therefore, the
two readings made at any point, should not agree, it must be due either
to a fault in one of the wedges, or to a fault in reading or a lack of
adjustment, as has been mentioned. In such cases the readings should be
retaken and the errors are usually easily discovered.
=78. Control Observation Tube.=—Instead of using quartz plates of known
values for testing the accuracy of the scale, an observation tube may
be used, the length of which can be varied at the pleasure of the
observer.
The construction of a tube of this kind is shown in Fig. 40. The tube
B is movable telescopically in A by means of the ratchet wheel shown.
It is closed at D water-tight by a glass disk. The tube B fits as
accurately into A as is possible to permit of free movement, and any
liquid which may infilter between its outer surface and the inner
surface of A is prevented from gaining exit by the washer C, which
fits both tubes water-tight. The ratchet which moves B in A carries
a millimeter scale and vernier N whereby the exact thickness of the
liquid solution between the surfaces of the glass disks D and E can be
always determined.
[Illustration: FIGURE 41. CONTROL OBSERVATION TUBE.]
By this device the length of liquid under observation can be accurately
read to a tenth of a millimeter. The cover glass E is held in position
by any one of the devices in common use for this purpose in the case in
question, by a bayonet fastening. The funnel T, communicating directly
with the interior of A, serves to hold the solution, there being always
enough of it to fill the tube when D is removed to the maximum distance
from C, which is usually a little more than 200 millimeters.
Let the control tube be adjusted to 200 millimeters and filled with a
solution of pure sugar, which reads 100 per cent or degrees in a 200
millimeter tube. Since the degree of rotation is, other things being
equal, proportional to the length of the column of polarizing solution,
it follows that if the tube B be moved inward until the distance
between D and C is 100 millimeters, the scale should read 50° or per
cent. By adjusting the length of the distance between B and C it is
easily seen that every part of the scale can be accurately tested.
The tube should be filled by removing the funnel and closing the
orifice with a screw cap which comes with the apparatus. The cap E
is then removed and the tube filled in the ordinary manner. This
precaution is practiced to avoid carrying air bubbles into the tube
when filled directly through the funnel. With a little care, however,
this danger may be avoided, or should air bubbles enter they can be
easily removed by inclining the tube.
In case the solution used be not strictly pure it may still be employed
for testing the scale. Suppose, for instance, that a solution made up
in the usual way, has been made from a sample containing only 99.4 per
cent of sugar. Then in order to have this solution read 100° on the
scale the tube should be set at 201.2 millimeters, according to the
formula
200 × 100
--------- = 201.2.
99.4
By a similar calculation the position of the tube for reading any
desired degree on the scale can be determined. The importance of
controlling all parts of the scale in compensating instruments is
emphasized by the fact that a variation of only 0.016 millimeter in the
thickness of the compensating wedge will cause a change of one degree
in the reading of the instrument.
=79. Setting the Polariscope with Quartz Plates.=—Pure sugar is
not always at the command of the analyst, and it is more convenient
practically to adjust the instrument by means of quartz plates, the
sugar values of which have been previously tested for the character
of the light used. Assuming the homogeneity of a plate of quartz, the
degree of deflection which it imparts to a plane of polarized light
depends on the quality of the light, the thickness of the plate, and
the temperature.
In respect of the quality of light, red polarized rays are least, and
violet most deflected. The degree of rotation produced with any ray,
at a given temperature, is directly proportional to the thickness of
the plate. Temperature affects the rotating power of a quartz plate
in a degree highly significant from a scientific point of view and
not wholly negligible for practical purposes. The rotating power of a
quartz plate increases with the temperature and the variation may be
determined by the formula given below:[44]
The formula is applicable for temperatures between 0° and 100°. Its
values are expressed in degrees of angular measure which can be
converted into degrees of the sugar scale by appropriate factors:
_Formula.—a_ᵗ = _a_°(1 + 0.000146_t_);
in which _a_° = polarization in angular degrees at 0°, _t_ the
temperature of observation and _a_ᵗ the rotation desired.
_Example._—A quartz plate which has an angular rotation of 33° at 0°
will have a rotation at 20° of 33°.09834.
_a_ᵗ = 33(1 + 0.000146 × 20) = 33.09834.
Since in instruments using the ventzke scale one degree of the sugar
scale is equal to 0.3467 degree angular measure, the sugar value of the
quartz plate mentioned is equal to 95.47 percent; 33.09834 ÷ 0.3467 =
95.47.
The sugar value of this plate at 0° is 95.18 per cent; 33 ÷ 0.3467 =
95.18.
=80. Tables for Correcting Quartz Plates.=—Instead of calculating the
variation in quartz plates for each temperature of observation, it is
recommended by the Bureau of Internal Revenue of the Treasury, to use
control quartz plates the values of which at any given temperature, are
found on a card which accompanies each one.[45] The variations given,
are from temperatures between 10° and 35°. Three control plates are
provided with each instrument used by the Bureau, for polarimetric
work in the custom houses, or in ascertaining bounties to be paid on
the production of domestic sugars. For example, the case of a sugar
which polarizes 80°.5 may be cited. One of the control plates nearest
to this number, is found to have at the temperature of observation, a
polarization of 91°.4, the reading being made in each case at 25°. On
consulting the card which accompanies the control plate, it is seen
that its value at the temperature mentioned, is 91°.7. The reading of
the instrument is therefore too low by three-tenths of a degree, and
this quantity should be added to the observed polarization, making it
80°.8. In this method of correcting the reading for temperature, it is
assumed that the compensating wedges of the instrument, are free of
error at the points of observation. The plates used for the purpose
above, are all standardized in the office of weights and measures of
the Coast and Geodetic Survey, before delivery to the analysts.
=81. Applicability of Quartz Plates.=—Quartz plates which are correctly
set for one instrument or kind of light, should be equally accurate
for the sugar scales of all instruments, using the same sugar factor.
In other words a quartz plate which reads 99° on a scheibler color
polariscope, should give the same reading on the sugar scale of a
shadow compensating or a monochromatic direct reading apparatus using
26.048 grams of sugar.
The most useful quartz plates for sugar analysis, are those which give
the readings at points between 80° and 96°, which cover the limits of
ordinary commercial sugars. For molasses the plates should read from
45° to 55°. For sugar juices of the cane and beet, the most convenient
graduation would be from 10° to 20°, but plates of this value would be
too thin for practical work and are not in use. When quartz plates are
to be used for control purposes, they should be purchased from reliable
manufacturers, or better, tested directly against pure sugar solutions
by the observer.
In practice we have found quartz plates as a rule, true to their
markings.
=82. The Sugar Flask.=—Sugar solutions are prepared for polarization in
flasks graduated to hold fifty or one hundred cubic centimeters. For
scientific work a flask is marked to hold 100 grams of distilled water
at 4°. The weights are all to be reduced to a vacuum standard. One
flask having been marked in this way, others may be compared directly
therewith by means of pure mercury. For this purpose the flasks must be
perfectly dry and the mercury pure, leaving no stain on the sides of
the flask. The glass must also be strong enough to undergo no change in
shape from the weight of mercury used.
For sugar work the true 100 gram flask is not usually employed, but
one graduated by weighing at 17°.5. These flasks are graduated by
first weighing them perfectly dry, filling with distilled water and
again weighing fifty and fifty-five, or 100 and 110 grams of water at
the temperature named. Since the volume of water at 17°.5 is greater
than at 4° the sugar flask in ordinary use has a greater volume by
about 0.25 cubic centimeter than the true flask. The observer should
always secure a statement from the dealer in respect of the volume
of the flask used in testing the scale of the polariscope purchased.
In the graduation of a flask in true cubic centimeters, when brass
weights are used it will be necessary to correct the weight of each
gram of water by adding to it one milligram, which is almost exactly
the weight of the volume of air displaced by one gram of water in the
circumstances named. If the flask be first counterbalanced and it be
desired to mark it at 100 cubic centimeters the sum of the weights
placed in the opposite pan should be 100 - 0.100 = 99.900 grams. While
this is not a rigidly exact correction it will be sufficient for all
practical purposes. A liter of dry air weighs 1.29366 grams; and 100
cubic centimeters of water would therefore displace 0.129 gram of air.
But the brass weights also displace a volume of air which when deducted
reduces the correction to be made for the water to nearly the one
named. For convenience in inverting sugar solutions the flasks used in
practical work are graduated at fifty and fifty-five and 100 and 110
cubic centimeters respectively.
=83. Preparing Sugar Solutions for Polarization.=—If sugar samples
were always pure the percentage of sugar in a given solution could be
directly determined by immediate polarization. Such cases, however,
are rarely met in practice. In the majority of cases the sample is
not only to be brought into solution but is also to be decolorized
and rendered limpid by some one of the methods to be described. A
perfectly limpid liquid is of the highest importance to secure correct
observations. With a cloudy solution the field of vision is obscured,
the dividing line of the two halves, or the double line in the triple
field, becomes blurred or invisible and the intensity of illumination
is diminished. A colored liquid which is bright is far more easy to
polarize than a colorless liquid which is turbid. In fact, it is only
rarely in sugar work that samples will be found which require any
special decolorizing treatment other than that which is received in
applying the reagents which serve to make the solutions limpid. In the
following paragraphs the approved methods of clarifying sugar solutions
preparatory to observation in the polariscope will be described.
=84. Alumina Cream.=—The hydrate of alumina, commonly known as alumina
cream, is always to be preferred as a clarifying agent in all cases
where it can be successfully applied.[46] It is a substance that acts
wholly in a mechanical way and therefore leaves the sugars in solution
unchanged, carrying out only suspended matters. In the preparation
of this reagent a solution of alum is treated with ammonia in slight
excess, the aluminum hydroxid produced washed on a filter or by
decantation until neutral in reaction. The hydroxid is suspended
in pure water in proportions to produce a creamy liquid. Although
apparently very bulky, the actual space occupied by the amount of dry
hydroxid added in a few cubic centimeters is so small as to produce no
disturbing effect of importance on the volume of the sugar solution.
The cream thus prepared is shaken just before using and from one to
five cubic centimeters of it, according to the degree of turbidity of
the saccharine solution, are added before the volume in the flask is
completed to the mark. After filling the flask to the mark the ball of
the thumb is placed over the mouth and the contents well shaken and
allowed to stand for a few moments before filtering.
The alumina cream is well suited to use with solutions of commercial
sugars of not too low a grade and of most honeys and high grade
sirups. It is usually not powerful enough to clarify beet and cane
juices, molasses and massecuites.
=85. Basic Lead Acetate.=—A solution of basic lead acetate is an
invaluable aid to the sugar analyst in the preparation of samples for
polarimetric observation. It acts as a clarifying agent by throwing
out of solution certain organic compounds and by uniting with the
organic acids in solution forms an additional quantity of precipitate,
and these precipitates act also mechanically in removing suspended
matters from solution. The action of this reagent is therefore much
more vigorous than that of alumina cream. Coloring matters are often
precipitated and removed by treatment with lead acetate. It happens
therefore that there are few samples of saccharine bodies whose
solutions cannot be sufficiently clarified by lead acetate to permit of
polarimetric observation.
The reagent is most frequently employed of the following strength:[47]
Boil for half an hour in one and a half liters of water 464 grams of
lead acetate and 264 grams of litharge with frequent stirring. When
cool, dilute with water to two liters, allow to stand until clear, and
decant the solution. The specific gravity of this solution is about
1.267.
In a solution of basic lead acetate of unknown strength the percentage
of lead acetate may be determined from its specific gravity by the
following table:[48]
PERCENTAGE OF LEAD ACETATE CORRESPONDING TO
DIFFERENT SPECIFIC GRAVITIES AT 15°.
Percentage of Percentage of
Specific gravity. lead acetate. Specific gravity. lead acetate.
1.0127 2 1.2040 28
1.0255 4 1.2211 30
1.0386 6 1.2395 32
1.0520 8 1.2579 34
1.0654 10 1.2768 36
1.0796 12 1.2966 38
1.0939 14 1.3163 40
1.1084 16 1.3376 42
1.1234 18 1.3588 44
1.1384 20 1.3810 46
1.1544 22 1.4041 48
1.1704 24 1.4271 50
1.1869 26
=86. Errors Due to use of Lead Solutions.=—In the use of lead solutions
there is danger of errors intruding into the results of the work. These
errors are due to various sources. Lead subacetate solution, when
used with low grade products, or sugar juices, or sirups from beets
and canes, precipitates albuminous matters and also the organic acids
present. The bulk occupied by these combined precipitates is often of
considerable magnitude, so that on completing the volume in the flask
the actual sugar solution present is less than indicated. The resulting
condensation tends to give too high a polarimetric reading. With purer
samples this error is of no consequence, but especially with low grade
sirups and molasses it is a disturbing factor, which must be considered.
One of the best methods of correcting it has been proposed by
Scheibler.[49] To 100 cubic centimeters of a solution of the sample,
ten of lead solution are added, and after shaking and filtering
the polarimetric reading is taken. Another quantity of 100 cubic
centimeters of the solution with ten of lead is diluted to 220 cubic
centimeters, shaken, filtered, and polarized. Double the second
reading, subtract it from the first, multiply the difference by 2.2,
and deduct the product from the first reading. The remainder is the
correct polarization.
The process just described is for the usual work with beet juices and
sirups. For cane juices measured by the graduated pipette, hereafter to
be described, and for weighed samples of molasses and massecuites, the
following method of calculation is pursued.[50] To the sample dissolved
in water, add a measured portion of the lead subacetate solution, make
its volume 100 cubic centimeters and observe the polarimetric reading.
Prepare a second solution in the same way and make the volume double
that of the first and again take the polarimetric reading. Multiply the
second reading by two, subtract the product from the first reading and
multiply the remainder by two, and subtract the product from the first
reading.
_Example._—First polarization 30.0
Second polarization 14.9
Then 30 - (2 × 14.9 = 29.8) = 0.2
0.2 × 2 = 0.4
and 30 - 0.4 = 29.6
The corrected reading therefore shows that the sample contained 29.6
per cent of sugar.
=87. Error Due to Action of Lead Subacetate on Levulose.=—In the use of
lead subacetate solution not only is there danger of error due to the
causes just described, but also to a more serious one, arising from the
chemical interaction of the clarifying agent and levulose.[51]
Lead subacetate forms a chemical union with levulose and the resulting
compound has a different rotatory power from the left-handed sugar in
an uncombined state. By adding a sufficient quantity of subacetate
solution, the left-handed rotation of levulose may be greatly
diminished if not entirely destroyed. In this case the dextrose, which
with levulose forms inverted sugar, serves to increase the apparent
right rotation due to the sucrose in solution. The reading of the
scale is therefore higher than would be given by the sucrose alone.
If the lead subacetate could be added in just the proportion to make
the invert sugar neutral to polarized light, its use would render the
analysis more accurate; but such a case could only arise accidentally.
To correct the error, after clarification, the compound of levulose and
lead may be decomposed by the addition of acetic acid according to the
method of Spencer. In this case the true content of sucrose can only be
obtained by the method of inversion proposed by Clerget, which will be
described in another paragraph.
=88. Clarification with Mercuric Compounds.=—Where the disturbing
bodies in a solution are chiefly of an albuminoid nature, one of the
best methods of securing clarification is by the use of a solution
containing an acid mercuric compound.[52] In the case of milk this
method is to be preferred to all others. Albuminoid bodies themselves,
have the property of deflecting the plane of polarization, as a rule,
to the left, and therefore, should be completely removed from solutions
containing right-handed sugars such as lactose. For this purpose the
mercuric compound is more efficient than any other. It is prepared and
used as follows.[53] Dissolve mercury in double its weight of strong
nitric acid and dilute the solution with an equal volume of water. One
cubic centimeter of this solution is sufficient to clarify fifty times
its volume of milk.
=89. Decolorization by Means of Bone-Black.=—Where the means already
described fail to make a solution sufficiently colorless to permit of
the passage of a ray of polarized light, recourse should be had to a
decolorizing agent. The most efficient of these is bone-black. For
laboratory work it is finely ground and should be dry if added to an
already measured solution. When moist it should be added to the flask
before the volume is completed, and a correction made for the volume of
the dry char employed. Bone-black has the power of absorbing a certain
quantity of sugar, and for this reason as little of it should be
employed as is sufficient to secure the end in view. If not more than
one gram of the char be used for 100 cubic centimeters of solution,
the error is not important commercially. The error may be avoided by
placing the char on the filter and rejecting the first half of the
filtered solution. The char becomes saturated with the first portion
of the solution, and does not absorb any sugar from the second. This
method, however, does not secure so complete a decolorization as is
effected by adding the black directly to the solution and allowing to
stand for some time with frequent shaking.
=90. Remarks on Analytical Process.=—Since large weights of sugar are
taken for polarization, a balance which will weigh accurately to one
milligram may be used. In commercial work the weighing is made in a
counterpoised dish with a prominent lip, by means of which the sample
can be directed into the mouth of the flask after partial solution.
Where the air in the working room is still, an uncovered balance is
most convenient. With a little practice the analyst will be able to
dissolve and transfer the sample from the dish to the flask without
danger of loss. The source of light used in polarizing should be in
another room, and admitted by a circular opening in the partition. In a
close polarizing room, which results from the darkening of the windows,
the temperature will rapidly rise if a lamp be present, endangering
notably the accuracy of the work, and also interfering with the comfort
of the observer. The greatest neatness must be practiced in all stages
of the work, and especially the trough of the polariscope must be kept
from injury which may arise from the leaking of the observation tubes.
Dust and dirt of all kinds must be carefully excluded from the lenses,
prisms, wedges and plates of the instrument.
=91. Determination of Sucrose by Inversion.=—In the foregoing
paragraphs directions have been given for the estimation of sugar
(sucrose) by its optical properties. It has been assumed so far, that
no other disturbing bodies have been present, save those which could be
removed by the clarifying agents described. The case is different when
two or more sugars are present, each of which has a specific relation
to polarized light. In such cases some method must be used for the
optical determination of sucrose, which is independent of the influence
of the other polarizing bodies, or else recourse must be had to other
methods of analysis. The conversion of the sucrose present into invert
sugar by the action of an acid or a ferment, affords an opportunity for
the estimation of sucrose in mixed sugars, by purely optical methods.
This process rests upon the principle that by the action of a dilute
acid for a short time, or of a ferment for a long time, the sucrose
is completely changed, while other sugars present are not sensibly
affected. Neither of these assumptions is rigidly correct but each is
practically applicable.
The sucrose by this process of hydrolysis is converted into an equal
mixture of levulose and dextrose. The former, at room temperatures, has
the higher specific rotating power, and the deflection of the plane of
polarization in a solution of inverted sugar is therefore to the left.
The levorotatory power of invert sugar varies with the temperature,
and this arises from the optical properties of the levulose. The
influence of temperature on the rotating power of other sugars, is not
imperceptible in all cases, but in practice is negligible.
This method of analysis is invaluable in control work in factories,
in the customs and in agricultural laboratories. Since the rotating
power of levulose diminishes as the temperature rises, an accurate
thermometric observation must accompany each polarimetric reading. At
about 88° the rotatory powers of dextrose and levulose are equal, and a
solution of pure invert sugar examined at that degree, is found to be
neutral to polarized light.
=92. Clerget’s Method of Inversion.=—The classical method of Clerget
for the determination of cane sugar by double polarization before and
after inversion, was first described in a memoir presented to the
Society of Encouragement for National Industry on the 14th of October,
1846. The following description of the original method is taken from a
reprint of the proceedings of that Society, dated Nov. 1846:
Clerget points out first the observation of Mitscherlich regarding
the influence of temperature on the rotatory power of invert sugar,
and calls attention to the detailed experiments he has made which
resulted in the determination of the laws of the variation. From these
studies he was able to construct a table of corrections, applicable in
the analysis of all saccharine substances in which the cane sugar is
polarized before and after inversion. The basis of the law rests upon
the observation that a solution of pure sugar, polarizing 100° on the
sugar scale, before inversion, will polarize 44° to the left after
inversion at a temperature of zero. The quantity of sugar operated upon
by Clerget amounted to 16.471 grams in 100 cubic centimeters of liquid.
On the instrument employed by him this quantity of sugar in 100 cubic
centimeters gave a reading of 100° to the right on the sugar scale
when contained in a tube twenty centimeters in length. The process of
inversion carried on by Clerget is as follows:
The sugar solution is placed in a flask, marked on the neck at 100
and 110 cubic centimeters; or if smaller quantities are used, in a
flask marked on the neck at fifty and fifty-five cubic centimeters.
The flask is filled with the sugar solution to the first mark and
then a sufficient quantity of strong hydrochloric acid added to bring
the volume of the liquid to the second mark. The mouth of the flask
is then closed with the thumb and its contents thoroughly mixed by
shaking. A thermometer is placed in the flask which is set in a
water-bath in such a way that the water comes just above the level of
the liquid in the neck of the flask. The water is heated in such a
manner as to bring the temperature of the contents of the flask, as
determined by the thermometer, exactly to 68° and at such a rate as to
require fifteen minutes to reach this result. At the end of fifteen
minutes the temperature having reached 68° the flask is removed and
placed at once in another water-bath at the temperature of the room,
to which temperature the contents of the flask are cooled as rapidly
as possible. To make the polarimetric observation a tube twenty-two
centimeters in length is filled with the inverted sugar solution by
means of a tubulure in its center, which serves not only the purpose of
filling the tube but also afterwards to carry the thermometer, by means
of which the temperature of observation can be taken. If the sugar
solution be turbid, or contain any lead chlorid due to the previous use
of basic lead acetate in clarification, it should be filtered before
being introduced into the observation tube. This tube being one-tenth
longer than the original compensates for the dilution caused by the
addition of the hydrochloric acid in inversion.
When reading, the bulb of the thermometer should be withdrawn far
enough to permit the free passage of the ray of light and the exact
temperature of the solution noted.
The above outline of Clerget’s method of inversion is given in order
that the analyst may compare it with any of the variations which he may
find in other works. The chief points to which attention is called,
are, first, the fact that only a little over sixteen grams of sugar are
used for ten cubic centimeters of strong hydrochloric acid, and second,
that the time of heating is exactly fifteen minutes, during which time
the contents of the flask should be raised from room temperature to
exactly 68°.
From the above it is seen that the process of Clerget, as originally
described, can be applied directly to all instruments, using
approximately sixteen grams of sugar in 100 cubic centimeters.
Experience has also shown that even when larger quantities of sugar
are employed, as for instance, approximately twenty-six grams,
the inversion is effected with practical completeness in the same
circumstances. It is advised, therefore, that in all analytical
processes, in which cane sugar is to be determined by the process of
inversion with an acid, the original directions of Clerget be followed
as strictly as possible. Experience has shown that no one of the
variations proposed for Clerget’s original method has any practical
advantage and the analyst is especially cautioned against those
methods of inversion in which the temperature is continued at 68° for
fifteen minutes or in which it is allowed to go above that degree.
=93. Influence of Strength of Solution and Time of Heating on the
Inversion of Sucrose.=—As has been intimated, the strength of a sugar
solution and the time of heating with hydrochloric acid are factors
that must be considered in determining a formula for the calculation
of sucrose by inversion. The Clerget formula holds good only for the
conditions specified and these conditions must be rigidly adhered to
in order to secure the proper results. This matter has been thoroughly
studied by Bornträger, who also gives a nearly complete bibliography
of the subject.[54] As a result of his investigations it seems
well established that the original Clerget formula is practically
correct for the conditions indicated, Bornträger modifying it only
by substituting in the formula 143.66 for 144. This is so nearly the
same as the Clerget factor that it is not advisable to substitute it
therefor. If, however, the inverted sugar solution be diluted to double
its volume before polarization the factor proposed by Landolt, _viz._,
142.4, gives more nearly accurate results. If the hydrochloric acid be
neutralized before polarization by an alkaline body, the character of
the salt which is formed also influences, to a greater or less extent,
the specific rotatory power of the solution. Hydrochloric acid itself
also influences the rotation to a certain degree.[55]
=94. Calculation of Results.=—The percentage of sucrose in a solution
which has been polarized before and after inversion is calculated by an
appropriate formula from the data obtained or is taken directly from
tables. These tables are too long to insert here, and in point of fact
the calculation can be made from the formula almost as quickly as the
result can be taken from a table.
Two factors are commonly used in the calculations, one based on
the supposition that a sugar solution polarizing 100° to the right
will, after inversion, give a reading of 44° to the left, at zero
temperature. In the second formula in common use the polarization to
the left in the circumstances mentioned above is assumed to be 42.4, a
number reached by Landolt after a long series of experiments.[56] The
principle of the calculation of the percentage of sucrose is based upon
the original observation of Clerget to the effect that the algebraic
difference of the two readings, divided by 144, less half of the
temperature, will give the percentage of sucrose desired. The formula
by which this is obtained is
_a_ - _b_
_S_ = -----------.
_K_ - _t_
----
2
In this formula _a_ is the polarization on the sugar scale before
inversion, _b_ the polarization after inversion, _K_ the constant
representing the algebraic difference of the two polarizations of
pure sugar at 0° and _t_ the temperature of the observation. To _K_
may be assigned the values 144 or 142.4, the one in more common use.
In case the polarization, after inversion, is to the left, which is
more commonly the case, the sum of the two readings is taken for
_a_ - (-_b_) = _a_ + _b_; when both polarizations are to the right
or left the difference is taken. S is the percentage of sucrose desired.
_Example.—_Let the polarization before inversion be +95
and after inversion -26
and the temperature 20°
95 + 26
Then _S_= -------- = 121 ÷ 134 = 90.6.
144 - 10
Substituting the value 142.4 for _K_, the result of the calculation is
91.4.
In high grade sugars, therefore, the difference in the results secured
by taking the two values of _K_ amounts to about 1 per cent of sucrose.
For a further discussion of the theory and practice of inversion the
reader is referred to the articles of Herles, Herzfeld, and Wohl.[57]
=95. Method Of Lindet.=—Courtonne recommends the method of Lindet for
securing the inversion instead of the method of Clerget.[58] Modified
by Courtonne, the method is as follows:
Make two or three times the normal weight of sugar dissolved in water
to a volume of 200 or 300 cubic centimeters, as the case may be. After
thoroughly mixing proceed as follows:
_First, to Obtain the Polarization Direct._—Place fifty or 100 cubic
centimeters of the prepared solution in a flask marked at fifty and
fifty-five or at 100 and 110 cubic centimeters, add a sufficient
quantity of lead acetate to secure a complete clarification, make
the volume to fifty-five or 110 cubic centimeters, shake thoroughly,
filter, and polarize in a 220 millimeter tube.
_Second, to Obtain the Rotation after Inversion._—Place twenty cubic
centimeters of the original solution, in a flask marked at fifty cubic
centimeters, containing five grams of powdered zinc. The flask should
be placed in boiling water. Add, little by little so as to avoid a too
rapid evolution of hydrogen, ten cubic centimeters of hydrochloric
acid made of equal parts of the strongest acid and water. After the
operation is terminated, cool to the temperature of the room, make
the volume to fifty cubic centimeters, polarize, and determine the
rotation. The volume occupied by the zinc which is not dissolved, will
be about one-half cubic centimeter, hence the deviation should be
multiplied by the factor 2.475 in order to get the true deviation which
would have been produced by the pure liquor. We have then:
_A_ = the deviation direct.
_B_ = the deviation after inversion.
_C_ = the algebraic difference of the deviations.
The amount of sucrose, therefore, would be calculated by the formula of
Creydt,[59]
_C_ - 0.493_A_
_X_ = --------------;
0.827
for raffinose the formula would be
_A_ - _S_
_Y_ = ---------,
1.57
in which _S_ is the deviation due to the sucrose present. The solutions
inverted in the manner described are absolutely colorless. There is no
need of employing bone-black to secure the saccharimetric reading nor
does it present any uncertainty. It is thought by Courtonne that this
method will soon take the place of the method of Clerget on account of
the advantages above mentioned. The method will be somewhat improved by
adopting the following suggestions:
1. Instead of allowing any arbitrary number for the volume of the
undissolved zinc, decant the liquid, after inversion, into another
flask and wash repeatedly with hot water until all trace of sugar is
removed from the flask in which the inversion took place.
2. Instead of polarizing in a 200 millimeter tube make the observation
in a 500 millimeter tube, which will permit of the reading being made
without any correction whatever.
=96. Inversion by Means of Invertase.=—Instead of using acids for the
inversion of cane sugar the hydrolysis can be easily effected by means
of a ferment derived from yeast. A complete history of the literature
and characteristics of this ferment, together with a study of its
properties and the various methods of preparing it, has been given by
O’Sullivan and Tompson.[60] In the preparation of invertase, the method
found most effective is the following:
The yeast is allowed to liquify for at least a month in a fairly warm
room without stirring. At the end of this time the surface is removed
and any supernatant liquid poured away. The lower sedimentary part is
thrown on a quick-acting filter and allowed to drain for two days.
To the filtrate, alcohol of specific gravity 0.87 is gradually added
to the extent of one and a half times its volume, with continued and
vigorous stirring. The process of adding the alcohol and stirring
should require about half an hour, after which the mixture is allowed
to stand for twenty-four hours to allow the precipitated invertase
to settle. The supernatant liquid is poured away and the precipitate
washed several times on successive days by decantation with alcohol of
0.92 specific gravity. When the washings become nearly colorless the
precipitate is thrown on a filter, allowed to drain, and immediately
removed and mixed with a large bulk of alcohol of 0.92 specific
gravity. The precipitate is again collected, mixed thoroughly with its
own bulk of water, and some alcohol of 0.97 specific gravity, allowed
to stand for a few hours and thrown on a filter. The filtrate contains
the invertase.
=97. Determination of Activity of Invertase.=—The activity of a
solution of invertase, prepared as above, is measured by the number
of minutes required for it to reduce to zero the optical power of a
solution of 100 times its weight of cane sugar at a temperature of
15°.5. In order to facilitate the action of the invertase, a trace of
sulfuric acid is added to the solution. The manipulation is as follows:
Fifty grams of sucrose are dissolved in water and made to a volume of
nearly a quarter of a liter and placed in a bath maintained at 15°.5.
Half a gram of the invertase is added, the time noted, the solution
immediately made up to a quarter of a liter and well shaken. The
contents of the flask are poured rapidly into five beakers; the actual
quantity in each beaker is not necessarily the same. To each of these
beakers, in succession, are added the following amounts of decinormal
sulfuric acid, _viz._, one-tenth, three-tenths, six-tenths, one, and
one and four-tenths cubic centimeters. After an hour a small quantity
of the solution is taken from beaker No. 3 and the reaction of the
invertase stopped by adding a few drops of strong potassium hydroxid
and the time of adding this reagent noted. This solution is then read
in the polariscope and the percentage of sugar inverted is calculated
from the formula C₁₂H₂₂O₁₁ + H₂O = C₆H₁₂O₆ + C₆H₁₂O₆.
The calculation of the amount of cane sugar inverted is based on
the formula, (38.4 - _d_) ÷ 0.518 = _p_. In this formula _d_ equals
the divisions of the sugar scale read on the polariscope; _p_ the
percentage of cane sugar inverted; 38.4 the reading on the sugar scale
of the original sugar solution and 51.8 the total number of divisions
of the cane sugar scale that the polariscope reading would fall through
if all the sugar were inverted. The observation tubes used in the
polarization are only 100 millimeters in length. After stopping the
action of the invertase with potassium hydroxid the solution is allowed
to stand for some time before polarization inasmuch as the dextrose
formed appears to assume the state of birotation and some time is
required for it to reach its normal rotatory power. If the invertase
be used in the alcoholic solution a sufficient quantity should be
added to be equivalent to 0.01 of the sucrose present. The time which
the contents of beaker No. 3 will take to reach optical activity is
calculated in a manner described by O’Sullivan and Tompson, but too
long to be inserted here.[61] The five beakers mentioned above are
examined in succession and the amount of sulfuric acid best suited to
the maximum inversion thus determined. This quantity is then used in
subsequent hydrolyses with the given sample of invertase.
The action of invertase on sucrose is very rapid at the first and
becomes very much slower towards the end. At a temperature of 15°.5 it
is advisable to let the solution stand for forty-eight hours in order
to be sure that complete inversion has taken place. For this reason
the method by inversion by means of invertase is one of no great
practical importance, but it may often be useful to the analyst when
the employment of an acid is inadmissible.
=98. Inversion by Yeast.=—Owing to the difficulty of preparing
invertase, O’Sullivan and Thompson[62] propose to use yeast as the
hydrolytic agent, as first suggested by Kjeldahl. It is shown that in
the use of yeast it is not necessary to employ thymol or any other
antiseptic. The method of procedure is as follows: The cane sugar
solution of usual strength should not be alkaline, but, if possible,
should be exactly neutral. If there be any ferment suspected, the
temperature should be momentarily raised to 80° to destroy its
activity. The polariscopic reading of the solution is then taken at
15°.5 and the amount of copper reduced by the solution should also be
determined.
Fifty cubic centimeters of the solution are poured into a beaker and
raised to a temperature of 55° in a constant temperature bath. Some
brewers yeast amounting to about one-tenth of the total amount of sugar
to be inverted, pressed in a towel, is thrown into the hot solution and
the whole stirred until mixture is complete. The solution is left for
four hours in the water-bath, at the end of this time it is cooled to
15°.5, a little freshly precipitated aluminum hydroxid added, and the
volume made to 100 cubic centimeters. A portion of this solution is
filtered and its polariscopic reading observed. The solution is then
left till the next day, when another polariscopic reading is taken in
order to prove that inversion is complete. The copper reducing power is
also determined. The method of calculating the results is the same as
when invertase is used. The following formulas are employed.
_a_ = the number of divisions indicated by the polariscopic reading for
a 200 millimeter tube:
_aʹ_ = the same number after inversion:
_m_ = the number of the divisions of the polariscopic scale which 200
millimeters of the sugar solution containing one gram of cane sugar
per 100, alter at 15°.5 on being inverted: In the case of the ventzke
polarimeter scale, one gram of cane sugar in 100 cubic centimeters,
indicates +3.84 divisions and after inversion it gives -1.34 div. In
experiments of this kind, therefore, _m_ = 5.18.
_P_ = the weight of cane sugar present in 100 cubic centimeters of the
original solution: