=238. Sucrose and Invert Sugar.=—The chemical methods of procedure to
be followed in the case of a sample containing both sucrose and invert
sugar have been given in sufficient detail in preceding paragraphs
(=124, 171=). When, however, it is desirable to study further the
composition of the mixture, important changes in the method are
rendered imperative. While the estimation of the sucrose and the total
invert sugar, or the sum of the dextrose and levulose, is easy of
accomplishment the separate determination of the dextrose and levulose
is not so readily secured. In the latter case the total quantity of the
two sugars may be determined, and after the destruction or removal of
one of them the other be estimated in the usual way; or in the mixture
the levulose can be determined by the variation in its gyrodynat,
caused by changes of temperature.
=239. Optical Neutrality of Invert Sugar.=—The gyrodynat of levulose
decreases as the temperature rises (=107=) and at or near a temperature
of 87°.2, it becomes equal to that of dextrose, and, therefore, pure
invert sugar composed of equal molecules of levulose and dextrose is
optically neutral to polarized light at that temperature. On this fact
Chandler and Ricketts have based a method of analysis which excludes
any interference in polarization due to invert sugar.[196] To secure
the polarization at approximately a temperature of 87°, a water-bath
is placed between the nicols of an ordinary polariscope in such a way
as to hold a tubulated observation tube in the optical axis of the
instrument. The ends of the bath, in the prolongation of this axis, are
provided with clear glass disks. The space between the cover glasses
of the observation tube and the glass disks of the bath is occupied by
the water of the bath. When this is kept at a constant temperature it
does not interfere with the reading. The observation tube may be of
glass, but preferably is constructed of metal plated with platinum on
the inside. For the most exact work the length of the observation tube,
at 87°, is determined by measurement or calculation. The bath is heated
with alcohol lamps or other convenient means. The arrangement of the
apparatus is shown in Fig. 75.
In a mixture of sucrose and invert sugar any rotation of the plane
of polarized light at 87° is due to the sucrose alone. In a mixture
of dextrose and sucrose the polarization is determined, and, after
inversion, again determined at 87°. The latter number is due to
dextrose alone, and the difference between the two gives the rotation
due to sucrose.
[Illustration: FIG. 75.—CHANDLER AND RICKETTS’ POLARISCOPE.]
=240. Sucrose and Raffinose.=—In raw sugars made from beet molasses
considerable quantities of raffinose are found. The method of inversion
and polarization in such cases is described in paragraph =100=. In
making the inversion by the method proposed by Lindet (=95=), and
conducting the polarization on a laurent instrument, a slightly
different formula, given below, is used; _viz._:
_C_ - 0.4891_A_
_S_ = ---------------
0.810
_A_ - _S_
and _R_ = ----------,
1.54
in which the several letters refer to the same factors as are indicated
by them in the formula of Creydt. In the application of the formula
just given the normal weight of the mixed raw sugars used is 16.2
grams.[197]
=241. Optical Determination of Levulose.=—The determination of levulose
by optical methods alone is made possible by reason of the fact that
the gyrodynats of the sugars with which it is associated are not
sensibly affected by changes of temperature. The principle of the
process, as developed by the author, rests on the ascertainment of the
change in the gyrodynat of levulose when its rotation is observed at
widely separated temperatures.[198] The observation tube employed for
reading at low temperatures is provided with desiccating end tubes,
which prevent the deposition of moisture on the cover glasses. The
relations of this device to the optical parts of the apparatus are
illustrated in Fig. 76.
[Illustration: FIG. 76.—APPARATUS FOR POLARIMETRIC OBSERVATIONS AT LOW
TEMPERATURES.]
The protecting tubes are made of hard rubber and the desiccation is
secured by surrounding the space between the rubber and the perforated
metal axis with fragments of potash or calcium chlorid.
The details of the construction are shown in a horizontal section
through the center of the observation tube in Fig. 77. In this figure
the observation tube, made of glass or metal, is represented by _i_,
the metal jacket, open at the top in the =V= shape as described, by
_k_. The observation tube is closed by the heavy disk _b_ made of
non-polarizing glass. This disk is pressed against the end of the
observation tube by the rubber washer _a_, when the drying system about
to be described is screwed on to _k_. The apparatus for keeping the
cover glass dry is contained in the hard rubber tube _m_ and consists
of a perforated cylinder of brass _e_, supported at one end by the
perforated disk _c_ and at the outer ends by the arms _d_. It is closed
by a cover glass of non-polarizing glass _s_ and can be screwed on to
the system _h_ at _n_. The space _p_ is filled with coarse fragments
of caustic soda, potash, or calcium chlorid by removing the cover
glass _s_. The perforated disk _c_ prevents any of the fragments from
entering the axis of observation. When the cover glass _s_ is replaced,
it just touches the free end of the perforated metal tube preventing
any of the fragments of the drying material from falling into the
center at the outer end. When this drying tube is placed in position,
the contents of the observation tube _i_ can be kept at the temperature
of zero for an indefinite time without the deposition of a particle of
moisture either upon the glass _b_ or _s_.
[Illustration: FIG. 77.—CONSTRUCTION OF DESICATING TUBE.]
For determining the rotation at a high temperature the apparatus of
Chandler and Ricketts (=238=) may be used or the following device:
The polarizing apparatus shown above, Fig. 76, may be used after the
=V= shape box is removed from the stand, which is so constructed as
to receive a large box covered with asbestos felt an inch thick. The
observation tube is held within this box in the same way as in the one
just described so that the hot water extends not only the entire length
of the tube but also covers the cover glasses. In both cases the cover
glasses are made of heavier glass and are much larger in diameter than
found in the ordinary tubes for polariscopes. The protecting cylinders
of hard rubber are not needed at high temperatures but can be left on
without detriment.
The illustration, Fig. 78, shows the arrangement of the apparatus with
a silver tube in position, which can be filled and emptied without
removing it.
[Illustration: FIG. 78.—APPARATUS FOR POLARIZING AT HIGH TEMPERATURES.]
In practice the water is heated with a jet of steam and an even
temperature is secured by a mechanical stirrer kept slowly in motion.
With such a box it is easy to maintain a temperature for several
hours which will not vary more than half a degree. The temperature
for reading the hot solutions was fixed at 88°, this being nearly the
temperature at which a mixture of equal molecules of levulose and
dextrose is optically inactive. In every case the sugar solutions were
made up to the standard volume at the temperatures at which they were
to be read and thus the variations due to expansion or contraction
were avoided. When solutions are read at a high temperature, they must
be made with freshly boiled water so as to avoid the evolution of air
bubbles which may otherwise obscure the field of vision.
By means of the apparatus described it is easy for the analyst to make
a polarimetric reading at any temperature desired. In all cases the
observation tube should be left at least a half an hour and sometimes
longer in contact with the temperature control media before the reading
is made.
The appearance of the field of vision is usually a pretty fair index
of the point of time at which a constant temperature is established
throughout all parts of the system. Any variation in temperature
produces a distortion of the field of vision while a constant fixed
temperature will disclose the field of vision in its true shape and
distinctness of outline.
_Principles of the Calculation._—If 26.048 grams of pure sucrose be
dissolved in water and the volume made 100 cubic centimeters, it will
produce an angular rotation of 34°.68 when examined in a 200 millimeter
tube with polarized sodium monochromatic light. Upon the cane sugar
scale of an accurately graduated shadow instrument the reading will be
100 divisions corresponding to 100 per cent of pure sucrose.
In the complete inversion of the cane sugar the reaction which takes
place is represented by the following formula:
— +
C₁₂H₂₂O₁₁ + H₂0 = C₆H₁₂O₆ + C₆H₁₂O₆.
The minus and plus signs indicate that the resulting invert sugar is
a mixture of equal parts of levulose (_d_ fructose) and dextrose (_d_
glucose). We are not concerned here with the fact that a complete
inversion of cane sugar is a matter of great difficulty nor with the
danger which is always experienced of destroying a part of one of the
products of inversion. They are matters which may cause a variation
in the analytical data afterward, but do not affect the principles on
which the process is based.
In the inversion of 26.048 grams of cane sugar there are therefore
produced 13.71 grams of levulose and 13.71 grams of dextrose or, in
all, 27.42 grams of the mixed sugars.
The angular rotation which would be produced by 13.71 grams of
dextrose in a volume of 100 cubic centimeters and through a column 200
millimeters in length is, with sodium light, 14°.53 equivalent to 41.89
divisions of the cane sugar scale. The specific rotatory power of a
dextrose solution of the density given is almost exactly 53, and this
number is used in the calculations.
In a mixture of the two sugars under the conditions mentioned and at a
temperature of 0° the angular rotation observed is -15°.15 equivalent
to 43.37 divisions of the cane sugar scale.
The + rotation due to the dextrose is 14°.53. Therefore the total
negative rotation due to levulose at 0° is 15°.15 + 14°.53 =
29°.68. Hence the gyrodynat of levulose at 0° and in the degree of
concentration noted is readily calculated from the formula
29.68 × 100
[α]°_{D} = - ------------ = -108.24.
2 × 13.71
Since at 88° (_circa_) the mixture of levulose and dextrose is neutral
to polarized light, it follows that at that temperature the specific
rotatory power of levulose is equal to that of dextrose, _viz._, 53°.
[α]⁸⁸ °_{D} = - 53°.
The total variation in the specific rotatory power of levulose, between
zero and 88°, is 55°.24. The variation for each degree of temperature,
therefore, of the specific rotatory power of levulose is equal to
55.24 divided by 88, which is equal to 0.628. From these data it is
easy to calculate the specific rotatory power of levulose for any
given temperature. For instance, let it be required to determine the
gyrodynat of levulose at a temperature of 20°. It will be found equal
to 108.24 - 0.628 × 20 = 95.68. The required rotatory power is then
[_a_]²⁰ °_{D} = -95°.68.
In these calculations the influence of the presence of hydrochloric
acid upon the rotatory power of the levulose is neglected.
Since the variation in angular rotation in the mixture at different
temperatures is due almost wholly to the change in this property of the
levulose it follows that the variation for each degree of temperature
and each per cent of levulose can be calculated. Careful experiments
have shown that the variation in the rotatory power of levulose between
0° and 88° is represented by a straight line. For 13.71 grams per
100 cubic centimeters the variation for each degree of temperature
is equal to 43.37 ÷ 88 = 0.49 divisions on the cane sugar scale, or
15.15 ÷ 88 = 0°.1722 angular measure. If 13.71 grams of levulose in
100 cubic centimeters produce the deviations mentioned for each degree
of temperature, one gram would give the deviation obtained by the
following calculations:
For the cane sugar scale 0.49 ÷ 13.71 = 0°.0357 and for angular
rotation 0.1722 ÷ 13.71 = 0.01256.
The above data afford a simple formula for calculating the percentage
of levulose present from the variation observed in polarizing a
solution containing levulose, provided that the quantity of levulose
present is approximately fourteen grams per 100 cubic centimeters.
_Example._—Suppose in a given case the difference of reading between
a solution containing an unknown quantity of levulose at 0° and 88°
is equal to thirty divisions of the cane sugar scale. What weight of
levulose is present? We have already seen that one gram in 100 cubic
centimeters produces a variation of 0.0357 division for 1°. For 88°
this would amount to 3.1416 divisions. The total weight of levulose
present is therefore 30 ÷ 3.1416 = 9.549 grams. In the case given
26.048 grams of honey were taken for the examination. The percentage of
levulose was therefore 9.549 × 100 ÷ 26.048 = 36.66 per cent.
If it be inconvenient to determine the polarimetric observations at
temperatures so widely separated as 0° and 88° the interval may be
made less. In the above case if the readings had been made at 20° and
70° the total variation would have been only ⁵⁰/⁸⁸ of the one given,
_viz._, 17.05 divisions of the cane sugar scale. The calculation would
then have proceeded as follows:
0.0357 × 50 = 1.785.
Then, 17.05 ÷ 1.785 = 9.552 grams of levulose, from which the actual
percentage of levulose can be calculated as above.
With honeys the operation is to be conducted as follows:
Since honeys contain approximately twenty per cent of water and in the
dry substance have approximately forty-five per cent of levulose, about
38.50 grams of the honey should be taken to get approximately 13.8
grams of levulose.
In the actual determination the calculations may be based on
the factors above noted, but without respect to the degree of
concentration. If half the quantity of dextrose noted be present its
specific rotatory power is only reduced to about 52°.75, and this will
make but little difference in the results. In the case of honey 13.024
grams of the sample are conveniently used in the examination, half the
normal weight for the ventzke sugar scale. The error, however, due to
difference in concentration is quite compensated for by the ease of
clarification and manipulation. Alumina cream alone is used in the
clarification, thus avoiding the danger of heating the solution to a
high temperature in the presence of an excess of lead acetate.
An interesting fact is observed in cooling solutions of honey to
0°. The maximum left hand rotation is not reached as soon as the
temperature reaches 0° but only after it has been kept at that
temperature for two or three hours. The line representing the change in
rotatory power in solutions of honey between 10° and 88° is practically
straight but from 10° to 0°, if measured by the readings taken without
delay, it is decidedly curved; the reading being less at first than
it is afterwards. After three hours the 0° becomes sensibly constant
and then the whole line is nearly straight, but still with a slight
deficiency in the reading at the 0°. For this reason the computations
should be based on readings between 10° and 88° rather than on a
number covering the whole range of temperature. Nevertheless, if the
solution be kept at 10° for three hours before the final reading is
taken, no error of any practical magnitude is introduced.
The calculations given above, for the cane sugar scale, can also be
made in an exactly similar manner for angular rotation. The angular
variation produced by one gram of levulose for 1° of temperature
is 0°.01256. For 88° this would become 1°.10528. Suppose the total
observed angular deviation in a given case between 0° and 88° to be
10°.404, then the weight of levulose present is 10.404 ÷ 1.10528 =
9.413 grams.
In the case mentioned 26.048 grams of honey were taken for the
examination. The percentage of levulose present, therefore, was 9.413 ×
100 ÷ 26.048 = 36.13.
=241. General Formula for the Calculation of Percentage of
Levulose.=—Let _K_ = deviation in divisions of the cane sugar scale or
in angular rotation produced by one gram of levulose for 1° temperature.
Let _T_ and _tʹ_ = temperatures at which observations are made.
Let _R_ = observed deviation in rotation.
Let _W_ = weight of levulose obtained.
Let _L_ = per cent of levulose required.
_R_
Then _L_ = --------------- ÷ _W_.
_K_(_T_ - _tʹ_)
In most genuine honeys the value of _R_ between 0° and 88° is
approximately thirty divisions of the cane sugar scale or 10° angular
measure for 26.048 grams in 100 cubic centimeters, read in a 200
millimeter tube, or, for 13.024 grams in 100 cubic centimeters read in
a 400 millimeter tube.
The method of analysis outlined above has been applied in the
examination of a large number of honeys with most satisfactory results.
It can also be applied with equal facility to other substances
containing levulose.
=242. Sucrose and Dextrose.=—In mixtures these two sugars are easily
determined by optical processes, provided no other bodies sensibly
affecting the plane of polarized light be present. The total deviation
due to both sugars is determined in the usual way. The percentage
of sucrose is afterwards found by the inversion method (=92=). The
rotation, in the first instance due to the sucrose, is calculated from
the amount of this body found by inversion, and the residual rotation
is caused by the dextrose. The percentage of dextrose is easily
calculated by a simple proportion into which the numbers expressing the
gyrodynats of sucrose and dextrose enter. When the readings are made on
a ventzke scale the calculations are made as follows:
Weight of sample used 26.048 grams.
First polarization 88°.5
Polarization after inversion 10°.5
Temperature 20°.0
Percentage of sucrose 58.4
Rotation due to dextrose 30°.1
Percentage of dextrose:
66.5 : 53 = _x_ : 30.1; whence _x_ = 37.8.
The sample examined therefore contains 58.4 per cent of sucrose and
37.8 per cent of dextrose.
It is evident that the method just described is also applicable when
maltose, dextrin, or any other sugar or polarizing body, not sensibly
affected by the process of inversion to which the sucrose is subjected,
is substituted for dextrose. When, however, more than two optically
active bodies are present the purely polariscopic process is not
applicable. In such cases the chemical or the combined chemical and
optical methods described further on can be employed.
=243. Lactose in Milk.=—By reason of its definite gyrodynat lactose
in milk is quickly and accurately determined by optical methods, when
proper clarifying reagents are used to free the fluid of fat and
nitrogenous substances. Soluble albuminoids have definite levogyratory
powers and, if not entirely removed, serve to diminish the rotation due
to the lactose.
Milk casein precipitated by magnesium sulfate has the following
gyrodynatic numbers assigned to it:[199]
Dissolved in water [_a_]_{D} = -80°
” ” very dilute solution [_a_]_{D} = -87°.
” ” dilute sodium hydroxid solution [_a_]_{D} = -76°.
” ” strong potassium hydroxid solution [_a_]_{D} = -91°.
The hydrates of albumen have rotation powers which vary from [_a_]_{D}
= -71°.40 to [_a_]_{D} = -79°-05. From the chaotic state of knowledge
concerning the specific rotating power of the various albumens, it is
impossible to assign any number which will bear the test of criticism.
For the present, however, this number may be fixed at [_a_]_{D} = -70°
for the albumens which remain in solution in the liquids polarized for
milk sugar.[200]
Many reagents have been prepared for the removal of the disturbing
bodies from milk in order to make its polarization possible. Among
the precipitants which have been used in this laboratory may be
mentioned:[201]
(1) Saturated solution basic lead acetate, specific gravity 1.97:
(2) Nitric acid solution of mercuric nitrate diluted with an equal
volume of water: (=88.=)
(3) Acetic acid, specific gravity 1.040, containing twenty-nine per
cent acetic acid:
(4) Nitric acid, specific gravity 1.197, containing thirty per cent
nitric acid:
(5) Sulfuric acid, specific gravity 1.255, containing thirty-one per
cent sulfuric acid:
(6) Saturated solution of sodium chlorid:
(7) Saturated solution of magnesium sulfate:
(8) Solution of mercuric iodid in acetic acid, formula; potassium
iodid, 33.2 grams; mercuric chlorid, 13.5 grams; strong acetic acid,
20.0 cubic centimeters; water 640 cubic centimeters.
Alcohol, ether, and many solutions of mineral salts, hydrochloric and
other acids are also used as precipitants for albumen, but none of them
presents any advantages.
Experience has shown that the best results in polariscopic work are
secured by the use of either the mercuric iodid or the acid mercuric
nitrate for clarifying the milk. The latter reagent should be used
in quantities of about three cubic centimeters for each 100 of milk.
It is evident when it is desired to determine the residual nitrogen
in solution, the former reagent must be employed. The quantity of
albuminoid matter left in solution after clarification with mercurial
salts is so minute as to exert no sensible effect on the rotation of
the plane of polarized light produced by the lactose.
For purposes of calculation the gyrodynat of lactose in the ordinary
conditions of temperature and concentration may be represented by
[_a_]_{D} = 52°.5 (=107=).
_Polarization._—The proper weight of milk is placed in a sugar flask,
diluted with water, clarified with the mercuric salt, the volume
completed to the mark, and the contents shaken and poured on a filter.
The filtrate is polarized in tubes of convenient length. The observed
rotation may be expressed either in degrees of angular measurement or
of the sugar scale. The weight of milk used may be two or three times
that of the normal weight calculated for the instrument employed.
Instead of weighing the milk a corresponding volume determined by its
specific gravity may be delivered from a burette-pipette (p. 231).
For the laurent polariscope three times, and for the half-shadow
instruments for lamplight, twice the normal weight of milk should be
used. For approximately sixty cubic centimeters of milk the flask
should be marked at 105 cubic centimeters in compensation for the
volume of precipitated solids or the reading obtained from a 100 cubic
centimeter flask, decreased by one-twentieth.
For the laurent instrument the normal weight of lactose is determined
by the following proportions:
Gyrodynat of sucrose, 66.5: lactose: 52.5 = _x_: 16.19.
Whence _x_ = 20.51, that is, the number of grams of pure lactose in 100
cubic centimeters required to read 100 divisions of the sugar scale of
the instrument.
For the ventzke scale the normal quantity of lactose required to read
100 divisions is found from the following equation: